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Sample records for oncogene amplification detected

  1. Oncogene amplification detected by in situ hybridization in radiation induced rat skin tumors. [C-myc:a3

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    Yi Jin.

    1991-02-01

    Oncogene activation may play an important role in radiation induced carcinogenesis. C-myc oncogene amplification was detected by in situ hybridization in radiation-induced rat skin tumors, including squamous and basal cell carcinomas. In situ hybridization was performed with a biotinylated human c-myc third exon probe, visualized with an avidin-biotinylated alkaline phosphate detection system. No c-myc oncogene amplification was detected in normal rat skin at very early times after exposure to ionizing radiation, which is consistent with the view that c-myc amplification is more likely to be related to carcinogenesis than to normal cell proliferation. The incorporation of tritiated thymidine into the DNA of rat skin cells showed that the proliferation of epidermal cells reached a peak on the seventh day after exposure to ionizing radiation and then decreased. No connection between the proliferation of epidermal cell and c-myc oncogene amplification in normal or irradiated rat skin was found. The results indicated that c-myc amplification as measured by in situ hybridization was correlated with the Southern bolt results, but only some of the cancer cells were amplified. The c-myc positive cells were distributed randomly within regions of the tumor and exhibited a more uniform nuclear structure in comparison to the more vacuolated c-myc negative cells. No c-myc signal was detected in unirradiated normal skin or in irradiated skin cells near the tumors. C-myc amplification appears to be cell or cell cycle specific within radiation-induced carcinomas. 28 refs., 3 figs., 3 tabs.

  2. Amplification of cellular oncogenes in solid tumors

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    Ozkan Bagci

    2015-01-01

    Full Text Available The term gene amplification refers to an increase in copy number of a gene. Upregulation of gene expression through amplification is a general mechanism to increase gene dosage. Oncogene amplifications have been shown in solid human cancers and they are often associated with progression of cancer. Defining oncogene amplification is useful since it is used as a prognostic marker in clinical oncology nowadays, especially v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 2 (HER2 targeted agents are used in breast cancer patients with high level of HER2 overexpression as a therapeutic approach. However, patients without HER2 overexpression do not appear to benefit from these agents. We concluded that determination of oncogene amplification in solid tumors is an important factor in treatment of human cancers with many unknowns. We have referred to PubMed and some databases to prepare this article.

  3. Targeting MET Amplification as a New Oncogenic Driver

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    Kawakami, Hisato [Department of Medical Oncology, Kinki University Faculty of Medicine, 377-2 Ohno-higashi, Osaka-Sayama, Osaka 589-8511 (Japan); Okamoto, Isamu, E-mail: okamotoi@kokyu.med.kyushu-u.ac.jp [Department of Medical Oncology, Kinki University Faculty of Medicine, 377-2 Ohno-higashi, Osaka-Sayama, Osaka 589-8511 (Japan); Center for Clinical and Translational Research, Kyushu University Hospital, 3-1-1 Maidashi, Higashiku, Fukuoka 812-8582 (Japan); Okamoto, Wataru [Department of Medical Oncology, Kinki University Faculty of Medicine, 377-2 Ohno-higashi, Osaka-Sayama, Osaka 589-8511 (Japan); Division of Transrlational Research, Exploratory Oncology Research & Clinical Trial Center, National Cancer Center, 6-5-1 Kashiwanoha, Kashiwa, Chiba 277-8577 (Japan); Tanizaki, Junko [Department of Medical Oncology, Kinki University Faculty of Medicine, 377-2 Ohno-higashi, Osaka-Sayama, Osaka 589-8511 (Japan); Lowe Center for Thoracic Oncology, Dana-Farber Cancer Institute, HIM223, 450 Brookline Avenue, Boston, MA 02215 (United States); Nakagawa, Kazuhiko [Department of Medical Oncology, Kinki University Faculty of Medicine, 377-2 Ohno-higashi, Osaka-Sayama, Osaka 589-8511 (Japan); Nishio, Kazuto [Department of Genome Biology, Kinki University Faculty of Medicine, 377-2 Ohno-higashi, Osaka-Sayama, Osaka 589-8511 (Japan)

    2014-07-22

    Certain genetically defined cancers are dependent on a single overactive oncogene for their proliferation and survival, a phenomenon known as “oncogene addiction”. A new generation of drugs that selectively target such “driver oncogenes” manifests a clinical efficacy greater than that of conventional chemotherapy in appropriate genetically defined patients. MET is a proto-oncogene that encodes a receptor tyrosine kinase, and aberrant activation of MET signaling occurs in a subset of advanced cancers as result of various genetic alterations including gene amplification, polysomy, and gene mutation. Our preclinical studies have shown that inhibition of MET signaling either with the small-molecule MET inhibitor crizotinib or by RNA interference targeted to MET mRNA resulted in marked antitumor effects in cancer cell lines with MET amplification both in vitro and in vivo. Furthermore, patients with non-small cell lung cancer or gastric cancer positive for MET amplification have shown a pronounced clinical response to crizotinib. Accumulating preclinical and clinical evidence thus suggests that MET amplification is an “oncogenic driver” and therefore a valid target for treatment. However, the prevalence of MET amplification has not been fully determined, possibly in part because of the difficulty in evaluating gene amplification. In this review, we provide a rationale for targeting this genetic alteration in cancer therapy.

  4. Targeting MET Amplification as a New Oncogenic Driver

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    Hisato Kawakami

    2014-07-01

    Full Text Available Certain genetically defined cancers are dependent on a single overactive oncogene for their proliferation and survival, a phenomenon known as “oncogene addiction”. A new generation of drugs that selectively target such “driver oncogenes” manifests a clinical efficacy greater than that of conventional chemotherapy in appropriate genetically defined patients. MET is a proto-oncogene that encodes a receptor tyrosine kinase, and aberrant activation of MET signaling occurs in a subset of advanced cancers as result of various genetic alterations including gene amplification, polysomy, and gene mutation. Our preclinical studies have shown that inhibition of MET signaling either with the small-molecule MET inhibitor crizotinib or by RNA interference targeted to MET mRNA resulted in marked antitumor effects in cancer cell lines with MET amplification both in vitro and in vivo. Furthermore, patients with non-small cell lung cancer or gastric cancer positive for MET amplification have shown a pronounced clinical response to crizotinib. Accumulating preclinical and clinical evidence thus suggests that MET amplification is an “oncogenic driver” and therefore a valid target for treatment. However, the prevalence of MET amplification has not been fully determined, possibly in part because of the difficulty in evaluating gene amplification. In this review, we provide a rationale for targeting this genetic alteration in cancer therapy.

  5. Mutation and genomic amplification of the PIK3CA proto-oncogene in pituitary adenomas

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    Murat, C.B.; Braga, P.B.S.; Fortes, M.A.H.Z. [Laboratório de Endocrinologia Celular e Molecular (LIM-25), Faculdade de Medicina, Universidade de São Paulo, São Paulo, SP (Brazil); Bronstein, M.D. [Unidade de Neuroendocrinologia, Serviço de Endocrinologia, Hospital das Clínicas, Faculdade de Medicina, Universidade de São Paulo, São Paulo, SP (Brazil); Corrêa-Giannella, M.L.C.; Giorgi, R.R. [Laboratório de Endocrinologia Celular e Molecular (LIM-25), Faculdade de Medicina, Universidade de São Paulo, São Paulo, SP (Brazil)

    2012-07-13

    The tumorigenesis of pituitary adenomas is poorly understood. Mutations of the PIK3CA proto-oncogene, which encodes the p110-α catalytic subunit of PI3K, have been reported in various types of human cancers regarding the role of the gene in cell proliferation and survival through activation of the PI3K/Akt signaling pathway. Only one Chinese study described somatic mutations and amplification of the PIK3CA gene in a large series of pituitary adenomas. The aim of the present study was to determine genetic alterations of PIK3CA in a second series that consisted of 33 pituitary adenomas of different subtypes diagnosed by immunohistochemistry: 6 adrenocorticotropic hormone-secreting microadenomas, 5 growth hormone-secreting macroadenomas, 7 prolactin-secreting macroadenomas, and 15 nonfunctioning macroadenomas. Direct sequencing of exons 9 and 20 assessed by qPCR was employed to investigate the presence of mutations and genomic amplification defined as a copy number ≥4. Previously identified PIK3CA mutations (exon 20) were detected in four cases (12.1%). Interestingly, the Chinese study reported mutations only in invasive tumors, while we found a PIK3CA mutation in one noninvasive corticotroph microadenoma. PIK3CA amplification was observed in 21.2% (7/33) of the cases. This study demonstrates the presence of somatic mutations and amplifications of the PIK3CA gene in a second series of pituitary adenomas, corroborating the previously described involvement of the PI3K/Akt signaling pathway in the tumorigenic process of this gland.

  6. Complex Oncogenic Translocations with Gene Amplification are Initiated by Specific DNA Breaks in Lymphocytes

    OpenAIRE

    2009-01-01

    Chromosomal instability is a hallmark of many tumor types. Complex chromosomal rearrangements with associated gene amplification, known as complicons, characterize many hematologic and solid cancers. While chromosomal aberrations, including complicons, are useful diagnostic and prognostic cancer markers, their molecular origins are not known. Although accumulating evidence has implicated DNA double strand break repair in suppression of oncogenic genome instability, the genomic elements requir...

  7. Study of differential polymerase chain reaction of C-erbB-2 oncogene amplification in gastric cancer

    Institute of Scientific and Technical Information of China (English)

    1999-01-01

    AIM To study the significance of C-erbB-2 oncogene amplification in gastric cancer.METHODS C-erbB-2 oncogene amplification was examined by using differential polymerase chain reaction (dPCR) in surgical and endoscopic specimens of 83 cases of gastric cancer and 101 metastatic lymph nodes.RESULTS C-erbB-2 amplification was found in 28.9% (24/ 83) surgical specimens and 20.5% (17/ 83) endoscopic ones of gastric cancer patients. The amplification was significant in both types of specimens of advanced cancer cases (P<0.05) and surgical specimens with lymph node metastasis (P<0.01). The incidence of C-erbB-2 amplification in lymph nodes with metastasis was higher than in primary sites (surgical specimens, P<0.05). The patients with amplification tumors had poorer 5-year survival rates than those with unamplification ones in the early cancers and well to moderately differentiated adenocarcinomas (P<0.05). The same surgical samples were tested again by Southern blot hybridization to ascertain C-erbB-2 amplification, and the positive rate of C-erbB-2 amplification (15.7%) was lower than that of dPCR (28.9%, P<0.05).CONCLUSION Examining C-erbB-2 amplification by dPCR is a quick, simple, reliable and independent method, and is helpful in predicting prognosis and metastatic potential of gastric cancer.

  8. Genome-wide screening for genetic alterations in esophageal cancer by aCGH identifies 11q13 amplification oncogenes associated with nodal metastasis.

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    Jianming Ying

    Full Text Available BACKGROUND: Esophageal squamous cell carcinoma (ESCC is highly prevalent in China and other Asian countries, as a major cause of cancer-related mortality. ESCC displays complex chromosomal abnormalities, including multiple structural and numerical aberrations. Chromosomal abnormalities, such as recurrent amplifications and homozygous deletions, directly contribute to tumorigenesis through altering the expression of key oncogenes and tumor suppressor genes. METHODOLOGY/PRINCIPLE FINDINGS: To understand the role of genetic alterations in ESCC pathogenesis and identify critical amplification/deletion targets, we performed genome-wide 1-Mb array comparative genomic hybridization (aCGH analysis for 10 commonly used ESCC cell lines. Recurrent chromosomal gains were frequently detected on 3q26-27, 5p15-14, 8p12, 8p22-24, 11q13, 13q21-31, 18p11 and 20q11-13, with frequent losses also found on 8p23-22, 11q22, 14q32 and 18q11-23. Gain of 11q13.3-13.4 was the most frequent alteration in ESCC. Within this region, CCND1 oncogene was identified with high level of amplification and overexpression in ESCC, while FGF19 and SHANK2 was also remarkably over-expressed. Moreover, a high concordance (91.5% of gene amplification and protein overexpression of CCND1 was observed in primary ESCC tumors. CCND1 amplification/overexpression was also significantly correlated with the lymph node metastasis of ESCC. CONCLUSION: These findings suggest that genomic gain of 11q13 is the major mechanism contributing to the amplification. Novel oncogenes identified within the 11q13 amplicon including FGF19 and SHANK2 may play important roles in ESCC tumorigenesis.

  9. Complex oncogenic translocations with gene amplification are initiated by specific DNA breaks in lymphocytes.

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    Wright, Sarah M; Woo, Yong H; Alley, Travis L; Shirley, Bobbi-Jo; Akeson, Ellen C; Snow, Kathy J; Maas, Sarah A; Elwell, Rachel L; Foreman, Oded; Mills, Kevin D

    2009-05-15

    Chromosomal instability is a hallmark of many tumor types. Complex chromosomal rearrangements with associated gene amplification, known as complicons, characterize many hematologic and solid cancers. Whereas chromosomal aberrations, including complicons, are useful diagnostic and prognostic cancer markers, their molecular origins are not known. Although accumulating evidence has implicated DNA double-strand break repair in suppression of oncogenic genome instability, the genomic elements required for chromosome rearrangements, especially complex lesions, have not been elucidated. Using a mouse model of B-lineage lymphoma, characterized by complicon formation involving the immunoglobulin heavy chain (Igh) locus and the c-myc oncogene, we have now investigated the requirement for specific genomic segments as donors for complex rearrangements. We now show that specific DNA double-strand breaks, occurring within a narrow segment of Igh, are necessary to initiate complicon formation. By contrast, neither specific DNA breaks nor the powerful intronic enhancer Emu are required for complicon-independent oncogenesis. This study is the first to delineate mechanisms of complex versus simple instability and the first to identify specific chromosomal elements required for complex chromosomal aberrations. These findings will illuminate genomic cancer susceptibility and risk factors.

  10. Parametric Amplification For Detecting Weak Optical Signals

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    Hemmati, Hamid; Chen, Chien; Chakravarthi, Prakash

    1996-01-01

    Optical-communication receivers of proposed type implement high-sensitivity scheme of optical parametric amplification followed by direct detection for reception of extremely weak signals. Incorporates both optical parametric amplification and direct detection into optimized design enhancing effective signal-to-noise ratios during reception in photon-starved (photon-counting) regime. Eliminates need for complexity of heterodyne detection scheme and partly overcomes limitations imposed on older direct-detection schemes by noise generated in receivers and by limits on quantum efficiencies of photodetectors.

  11. Development of Peptide Nucleic Acid Probes for Detection of the HER2 Oncogene

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    Song, Young K.; Evangelista, Jennifer; Aschenbach, Konrad; Johansson, Peter; Wen, Xinyu; Chen, Qingrong; Lee, Albert; Hempel, Heidi; Gheeya, Jinesh S.; Getty, Stephanie; Gomez, Romel; Khan, Javed

    2013-01-01

    Peptide nucleic acids (PNAs) have gained much interest as molecular recognition tools in biology, medicine and chemistry. This is due to high hybridization efficiency to complimentary oligonucleotides and stability of the duplexes with RNA or DNA. We have synthesized 15/16-mer PNA probes to detect the HER2 mRNA. The performance of these probes to detect the HER2 target was evaluated by fluorescence imaging and fluorescence bead assays. The PNA probes have sufficiently discriminated between the wild type HER2 target and the mutant target with single base mismatches. Furthermore, the probes exhibited excellent linear concentration dependence between 0.4 to 400 fmol for the target gene. The results demonstrate potential application of PNAs as diagnostic probes with high specificity for quantitative measurements of amplifications or over-expressions of oncogenes. PMID:23593123

  12. Development of peptide nucleic acid probes for detection of the HER2 oncogene.

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    Belhu Metaferia

    Full Text Available Peptide nucleic acids (PNAs have gained much interest as molecular recognition tools in biology, medicine and chemistry. This is due to high hybridization efficiency to complimentary oligonucleotides and stability of the duplexes with RNA or DNA. We have synthesized 15/16-mer PNA probes to detect the HER2 mRNA. The performance of these probes to detect the HER2 target was evaluated by fluorescence imaging and fluorescence bead assays. The PNA probes have sufficiently discriminated between the wild type HER2 target and the mutant target with single base mismatches. Furthermore, the probes exhibited excellent linear concentration dependence between 0.4 to 400 fmol for the target gene. The results demonstrate potential application of PNAs as diagnostic probes with high specificity for quantitative measurements of amplifications or over-expressions of oncogenes.

  13. Binomial mitotic segregation of MYCN-carrying double minutes in neuroblastoma illustrates the role of randomness in oncogene amplification.

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    Gisela Lundberg

    Full Text Available BACKGROUND: Amplification of the oncogene MYCN in double minutes (DMs is a common finding in neuroblastoma (NB. Because DMs lack centromeric sequences it has been unclear how NB cells retain and amplify extrachromosomal MYCN copies during tumour development. PRINCIPAL FINDINGS: We show that MYCN-carrying DMs in NB cells translocate from the nuclear interior to the periphery of the condensing chromatin at transition from interphase to prophase and are preferentially located adjacent to the telomere repeat sequences of the chromosomes throughout cell division. However, DM segregation was not affected by disruption of the telosome nucleoprotein complex and DMs readily migrated from human to murine chromatin in human/mouse cell hybrids, indicating that they do not bind to specific positional elements in human chromosomes. Scoring DM copy-numbers in ana/telophase cells revealed that DM segregation could be closely approximated by a binomial random distribution. Colony-forming assay demonstrated a strong growth-advantage for NB cells with high DM (MYCN copy-numbers, compared to NB cells with lower copy-numbers. In fact, the overall distribution of DMs in growing NB cell populations could be readily reproduced by a mathematical model assuming binomial segregation at cell division combined with a proliferative advantage for cells with high DM copy-numbers. CONCLUSION: Binomial segregation at cell division explains the high degree of MYCN copy-number variability in NB. Our findings also provide a proof-of-principle for oncogene amplification through creation of genetic diversity by random events followed by Darwinian selection.

  14. HER-2/neu oncogene amplification and chromosome 17 aneusomy in endometrial carcinoma: correlation with oncoprotein expression and conventional pathological parameters.

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    Cianciulli, A M; Guadagni, F; Marzano, R; Benevolo, M; Merola, R; Giannarelli, D; Marandino, F; Vocaturo, G; Mariani, L; Mottolese, M

    2003-06-01

    The objective of the present study was to evaluate the correlation between HER-2 gene amplification and HER-2 protein overexpression in endometrial carcinoma using fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC). We also analyzed chromosome 17 aneusomy and the association between these biological parameters and conventional clinicopathological variables. FISH analysis was performed on 73 selected paraffin-embedded sections from endometrial carcinomas which previously had HER-2 status determined immunohistochemically using monoclonal antibodies (MoAb) 300G9 and CB11. Using a ratio of more than two oncogene signals/centromere to indicate amplification, a total of 42 out of the 73 endometrial tumors included in this study resulted positive by FISH where as protein overexpression was identified in 29 out of 73 with a concordance rate of 74.3%. However, when the mean signals/centromere per nucleus increased (ratio > 4 2 4 < or = 5 when we grouped the amplified cases on the basis of HER-2:CEP17 ratio. In conclusion, molecular characteristics provide objective data that may be useful in predicting prognosis in patients with endometrial cancer.

  15. Methylation profile and amplification of proto-oncogenes in rat pancreas induced with phytoestrogens

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    Lyn-Cook, B.D.; Blann, E.; Bo, J. [National Center for Toxicological Research, Jefferson, AR (United States)

    1995-01-01

    Specific gene hypermethylation has been shown in DNA from neonatal rats exposed to the phytoestrogens, coumestrol, and equol. The pancreas is an organ in which estrogen receptors have been shown to be present. Studies have correlated the development of acute pancreatitis with rising levels of human estrogen binding proteins. Neonatal rats were dosed with 10 or 100 {mu}g of coumestrol or equol on postnatal day (PND) 1-10. The animals were sacrificed at Day 15. The pancreas was excised and pancreatic acinar cells isolated for molecular analysis. DNA was isolated from the cells by lysis in TEN-9 buffer supplemented with proteinase K and 0.1% SDS. High molecular weight (HMW) DNA was digested with the methylated DNA specific restriction enzymes, Hpa II and Msp I, for determination of methylation profiles. Both coumestrol and equol at high doses caused hypermethylation of the c-H-ras proto-oncogene. No hypermethylation or hypomethylation was observed in the proto-oncogenes, c-myc or c-fos. Methylation is thought to be an epigenetic mechanism involved in the activation (hypomethylation) or inactivation (hypermethylation) of cellular genes which are known to play a role in carcinogenesis. Epidemiology studies have shown that equol may have anti-carcinogenic effects on some hormone-dependent cancers. Additional studies are needed to further understand the role of phytoestrogens and methylation in relation to pancreatic disorders. 15 refs., 4 figs.

  16. Loop-mediated isothermal amplification for detection of nucleic acids.

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    Tanner, Nathan A; Evans, Thomas C

    2014-01-06

    Sequence-specific isothermal nucleic acid amplification techniques are ideally suited for use in molecular diagnostic applications because they do not require thermal cycling equipment and the reactions are typically fast. One of the most widely cited isothermal techniques is termed loop-mediated isothermal amplification (LAMP). This protocol allows amplification times as fast as 5 to 10 min. Furthermore, various methodologies to detect amplification have been applied to LAMP to increase its utility for the point-of-care market. Basic LAMP protocols are provided herein for detection of specific DNA and RNA targets, along with a method to perform multiplex LAMP reactions, permitting even greater flexibility from this powerful technique.

  17. Significance of HER2 and C-MYC oncogene amplifications in breast cancer in atomic bomb survivors: associations with radiation exposure and histologic grade.

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    Miura, Shiro; Nakashima, Masahiro; Ito, Masahiro; Kondo, Hisayoshi; Meirmanov, Serik; Hayashi, Tomayoshi; Soda, Midori; Matsuo, Takeshi; Sekine, Ichiro

    2008-05-15

    It has been postulated that radiation induces breast cancers in atomic bomb (A-bomb) survivors. Oncogene amplification is an important mechanism during breast carcinogenesis and also serves as an indicator of genomic instability (GIN). The objective of this study was to clarify the association of oncogene amplification in breast cancer in A-bomb survivors with radiation exposure. In total, 593 breast cancers were identified in A-bomb survivors from 1968 to 1999, and the association between breast cancer incidence and A-bomb radiation exposure was evaluated. Invasive ductal cancers from 67 survivors and 30 nonsurvivors were analyzed for amplification of the HER2 and C-MYC genes by fluorescence in situ hybridization, and expression levels of hormone receptors were analyzed by immunostaining. The incidence rate increased significantly as exposure distance decreased from the hypocenter (hazard ratio per 1-km decrement, 1.47; 95% confidence interval [95% CI], 1.30-1.66). The incidence of HER2 and C-MYC amplification was increased significantly in the order of the control group, the distal group (P = .0238), and the proximal group (P = .0128). Multivariate analyses revealed that distance was a risk factor for the coamplification of C-MYC and HER2 in breast cancer in survivors (odds ratio per 1-km increment, 0.17; 95% CI, 0.01-0.63). The histologic grade of breast cancers became significantly higher in the order of the control group, the distal group, and the proximal group and was associated with oncogene amplifications. The current results suggested that A-bomb radiation may affect the development of oncogene amplification by inducing GIN and may be associated with a higher histologic grade in breast cancer among A-bomb survivors. (c) 2008 American Cancer Society.

  18. A dual amplification fluorescent strategy for sensitive detection of DNA methyltransferase activity based on strand displacement amplification and DNAzyme amplification.

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    Cui, Wanling; Wang, Lei; Jiang, Wei

    2016-03-15

    DNA methyltransferase (MTase) plays a critical role in many biological processes and has been regarded as a predictive cancer biomarker and a therapeutic target in cancer treatment. Sensitive detection of DNA MTase activity is essential for early cancer diagnosis and therapeutics. Here, we developed a dual amplification fluorescent strategy for sensitive detection of DNA MTase activity based on strand displacement amplification (SDA) and DNAzyme amplification. A trifunctional double-stranded DNA (dsDNA) probe was designed including a methylation site for DNA MTase recognition, a complementary sequence of 8-17 DNAzyme for synthesizing DNAzyme, and a nicking site for nicking enzyme cleavage. Firstly, the trifunctional dsDNA probe was methylated by DNA MTase to form the methylated dsDNA. Subsequently, HpaII restriction endonuclease specifically cleaved the residue of unmethylated dsDNA. Next, under the action of polymerase and nicking enzyme, the methylared dsDNA initiated SDA, releasing numbers of 8-17 DNAzymes. Finally, the released 8-17 DNAzymes triggered DNAzyme amplification reaction to induce a significant fluorescence enhancement. This strategy could detect DNA MTase activity as low as 0.0082U/mL. Additionally, the strategy was successfully applied for evaluating the inhibitions of DNA MTase using two anticancer drugs, 5-azacytidine and 5-aza-2'-deoxycytidine. The results indicate the proposed strategy has a potential application in early cancer diagnosis and therapeutics.

  19. Bacteriophage Amplification-Coupled Detection and Identification of Bacterial Pathogens

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    Cox, Christopher R.; Voorhees, Kent J.

    Current methods of species-specific bacterial detection and identification are complex, time-consuming, and often require expensive specialized equipment and highly trained personnel. Numerous biochemical and genotypic identification methods have been applied to bacterial characterization, but all rely on tedious microbiological culturing practices and/or costly sequencing protocols which render them impractical for deployment as rapid, cost-effective point-of-care or field detection and identification methods. With a view towards addressing these shortcomings, we have exploited the evolutionarily conserved interactions between a bacteriophage (phage) and its bacterial host to develop species-specific detection methods. Phage amplification-coupled matrix assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF-MS) was utilized to rapidly detect phage propagation resulting from species-specific in vitro bacterial infection. This novel signal amplification method allowed for bacterial detection and identification in as little as 2 h, and when combined with disulfide bond reduction methods developed in our laboratory to enhance MALDI-TOF-MS resolution, was observed to lower the limit of detection by several orders of magnitude over conventional spectroscopy and phage typing methods. Phage amplification has been combined with lateral flow immunochromatography (LFI) to develop rapid, easy-to-operate, portable, species-specific point-of-care (POC) detection devices. Prototype LFI detectors have been developed and characterized for Yersinia pestis and Bacillus anthracis, the etiologic agents of plague and anthrax, respectively. Comparable sensitivity and rapidity was observed when phage amplification was adapted to a species-specific handheld LFI detector, thus allowing for rapid, simple, POC bacterial detection and identification while eliminating the need for bacterial culturing or DNA isolation and amplification techniques.

  20. Detecting and Targeting Oncogenic Myc in Breast Cancer

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    2007-06-01

    cell acute lymphocytic leukaemia and multiple myeloma [14]. Amplification of c-Myc and/or deregulated expression is evident in many tumours including... multiple ChIP reactions [12], but this approach is expensive and labor intensive. Further, when dealing with biologically limited samples, such as...Oncology, Ontario Cancer Institute/Princess Margaret Hospital ; Departments of 4Medical Biophysics and 5Laboratory Medicine and Pathobiology, 6Computer

  1. Weak value amplification is suboptimal for estimation and detection.

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    Ferrie, Christopher; Combes, Joshua

    2014-01-31

    We show by using statistically rigorous arguments that the technique of weak value amplification does not perform better than standard statistical techniques for the tasks of single parameter estimation and signal detection. Specifically, we prove that postselection, a necessary ingredient for weak value amplification, decreases estimation accuracy and, moreover, arranging for anomalously large weak values is a suboptimal strategy. In doing so, we explicitly provide the optimal estimator, which in turn allows us to identify the optimal experimental arrangement to be the one in which all outcomes have equal weak values (all as small as possible) and the initial state of the meter is the maximal eigenvalue of the square of the system observable. Finally, we give precise quantitative conditions for when weak measurement (measurements without postselection or anomalously large weak values) can mitigate the effect of uncharacterized technical noise in estimation.

  2. LED-FISH: Fluorescence microscopy based on light emitting diodes for the molecular analysis of Her-2/neu oncogene amplification

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    Vollmer Ekkehard

    2008-12-01

    Full Text Available Abstract Light emitting diodes (LED, which are available as small monochromatic light sources with characteristic features such as maximum illumination power combined with minimum energy consumption and extremely long lifespan have already proved as a highly potential low-cost alternative for specific diagnostic applications in clinical medicine such as tuberculosis fluorescence microscopy. Likewise, the most reliable evaluation of Her-2/neu (c-erbB2 gene amplification, which has been established in the last few years for routine diagnosis in clinical pathology as determinant towards Herceptin-based treatment of patients with breast cancer, is based on fluorescence in situ hybridization (FISH and corresponding high priced fluorescence equipment. In order to test the possibility to utilize the advantages of low-cost LED technology on FISH analysis of c-erbB2 gene expression for routine diagnostic purposes, the applicability of a standard bright field Carl Zeiss Axiostar Plus microscope equipped with a Fraen AFTER* LED Fluorescence Microscope Kit for the detection of Her-2/neu gene signals was compared to an advanced Nikon Eclipse 80i fluorescence microscope in combination with a conventional 100W mercury vapor lamp. Both microscopes were fitted with the same Quicam FAST CCD digital camera to unequivocally compare the quality of the captured images. C-erbB2 gene expression was analyzed in 30 different human tissue samples of primary invasive breast cancer, following formalin fixation and subsequent paraffin-embedding. The Her2/neu gene signals (green were identifiable in the tumor cells in all cases and images of equal quality were captured under almost identical conditions by 480 nm (blue LED module equipped standard Axiostar microscope as compared to conventional fluorescence microscopy. In this first attempt, these monochromatic LED elements proved in principle to be suitable for the detection of Her-2/neu gene expression by FISH. Thus, our own

  3. Using {sup 18F} FDG PET/CT to Detect an occult Mesenchymal Tumor Causing Oncogenic Osteomalacia

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    Seo, Hyo Jung; Choi, Yun Jung; Kim, Hyun Jeong; Jeong, Yong Hyu; Cho, Arthur; Lee, Jae Hoon; Yun, Mijin; Lee, Jong Doo; Kang, Won Jun [Yonsei Univ. College of Medicine, Seoul (Korea, Republic of)

    2011-09-15

    Oncogenic osteomalacia is a rare paraneoplastic syndrome characterized by renal phosphate excretion, hypophosphatemia, and osteomalacia. This syndrome is often caused by tumors of mesenchymal origin. Patients with oncogenic osteomalacia have abnormal bone mineralization, resulting in a high frequency of fractures. Tumor resection is the treatment of choice, as it will often correct the metabolic imbalance. Although oncogenic osteomalacia is a potentially curable disease, diagnosis is difficult and often delayed because of the small size and sporadic location of the tumor. Bone scintigraphy and radiography best characterize osteoma lacia; magnetic resonance imaging findings are nonspecific. Here, we report a case of oncogenic osteomalacia secondary to a phosphaturic mesenchymal tumor that was successfully detected by {sup 18F} fluorodeoxyglucose positron emission tomography/computed tomography ({sup 18F} FDG PET/CT). This case illustrates the advantages of {sup 18F} FDG PET/CT in detecting the occult mesenchymal tumor that causes oncogenic osteomalacia.

  4. Rapid detection of Mycobacterium tuberculosis by recombinase polymerase amplification.

    Directory of Open Access Journals (Sweden)

    David S Boyle

    Full Text Available Improved access to effective tests for diagnosing tuberculosis (TB has been designated a public health priority by the World Health Organisation. In high burden TB countries nucleic acid based TB tests have been restricted to centralised laboratories and specialised research settings. Requirements such as a constant electrical supply, air conditioning and skilled, computer literate operators prevent implementation of such tests in many settings. Isothermal DNA amplification technologies permit the use of simpler, less energy intensive detection platforms more suited to low resource settings that allow the accurate diagnosis of a disease within a short timeframe. Recombinase Polymerase Amplification (RPA is a rapid, low temperature isothermal DNA amplification reaction. We report here RPA-based detection of Mycobacterium tuberculosis complex (MTC DNA in <20 minutes at 39 °C. Assays for two MTC specific targets were investigated, IS6110 and IS1081. When testing purified MTC genomic DNA, limits of detection of 6.25 fg (IS6110 and 20 fg (IS1081were consistently achieved. When testing a convenience sample of pulmonary specimens from suspected TB patients, RPA demonstrated superior accuracy to indirect fluorescence microscopy. Compared to culture, sensitivities for the IS1081 RPA and microscopy were 91.4% (95%CI: 85, 97.9 and 86.1% (95%CI: 78.1, 94.1 respectively (n = 71. Specificities were 100% and 88.6% (95% CI: 80.8, 96.1 respectively. For the IS6110 RPA and microscopy sensitivities of 87.5% (95%CI: 81.7, 93.2 and 70.8% (95%CI: 62.9, 78.7 were obtained (n = 90. Specificities were 95.4 (95% CI: 92.3,98.1 and 88% (95% CI: 83.6, 92.4 respectively. The superior specificity of RPA for detecting tuberculosis was due to the reduced ability of fluorescence microscopy to distinguish Mtb complex from other acid fast bacteria. The rapid nature of the RPA assay and its low energy requirement compared to other amplification technologies suggest RPA-based TB

  5. Amplification of Mitochondrial DNA for detection of Plasmodiumvivax in Balochistan.

    Science.gov (United States)

    Shahwani, Muhammad Naeem; Nisar, Samia; Aleem, Abdul; Panezai, Marina; Afridi, Sarwat; Malik, Shaukat Iqbal

    2017-05-01

    To access a new step using PCR to amplify the targeted mtDNA sequence for detecting specifically Plasmodium vivax and its co-infections, false positive and false negative results with Plasmodium falciparum. In this study we have standardized a new technical approach in which the target mitochondrial DNA sequence (mtDNA) was amplified by using a PCR technique as a tool to detect Plasmodium spp. Species specific primers were designed to hybridize with cytochrome c oxidase gene of P. vivax (cox I) and P. falciparum (cox III). Two hundred blood samples were collected on the basis of clinical symptoms which were initially examined through microscopic analysis after preparing Giemsa stained thick and thin blood smears. Afterwards genomic DNA was extracted from all samples and was then subjected to PCR amplification by using species specific primers and amplified segments were sequenced for confirmation of results. One-hundred and thirty-two blood samples were detected as positive for malaria by PCR, out of which 64 were found to be positive by PCR and 53 by both microscopy and PCR for P.vivax infection. Nine samples were found to be false negative, one P.vivax mono infection was declared as co infection by PCR and 3 samples identified as having P.falciparum gametes were confirmed as P.vivax by PCR amplification. Sensitivity and specificity were found to be 85% and 92% respectively. Results obtained through PCR method were comparatively better and reliable than microscopy.

  6. Multiplexed Recombinase Polymerase Amplification Assay To Detect Intestinal Protozoa.

    Science.gov (United States)

    Crannell, Zachary; Castellanos-Gonzalez, Alejandro; Nair, Gayatri; Mejia, Rojelio; White, A Clinton; Richards-Kortum, Rebecca

    2016-02-01

    This work describes a proof-of-concept multiplex recombinase polymerase amplification (RPA) assay with lateral flow readout that is capable of simultaneously detecting and differentiating DNA from any of the diarrhea-causing protozoa Giardia, Cryptosporidium, and Entamoeba. Together, these parasites contribute significantly to the global burden of diarrheal illness. Differential diagnosis of these parasites is traditionally accomplished via stool microscopy. However, microscopy is insensitive and can miss up to half of all cases. DNA-based diagnostics such as polymerase chain reaction (PCR) are far more sensitive; however, they rely on expensive thermal cycling equipment, limiting their availability to centralized reference laboratories. Isothermal DNA amplification platforms, such as the RPA platform used in this study, alleviate the need for thermal cycling equipment and have the potential to broaden access to more sensitive diagnostics. Until now, multiplex RPA assays have not been developed that are capable of simultaneously detecting and differentiating infections caused by different pathogens. We developed a multiplex RPA assay to detect the presence of DNA from Giardia, Cryptosporidium, and Entamoeba. The multiplex assay was characterized using synthetic DNA, where the limits-of-detection were calculated to be 403, 425, and 368 gene copies per reaction of the synthetic Giardia, Cryptosporidium, and Entamoeba targets, respectively (roughly 1.5 orders of magnitude higher than for the same targets in a singleplex RPA assay). The multiplex assay was also characterized using DNA extracted from live parasites spiked into stool samples where the limits-of-detection were calculated to be 444, 6, and 9 parasites per reaction for Giardia, Cryptosporidium, and Entamoeba parasites, respectively. This proof-of-concept assay may be reconfigured to detect a wide variety of targets by re-designing the primer and probe sequences.

  7. Detection of Entamoeba histolytica by Recombinase Polymerase Amplification

    Science.gov (United States)

    Nair, Gayatri; Rebolledo, Mauricio; White, A. Clinton; Crannell, Zachary; Richards-Kortum, R. Rebecca; Pinilla, A. Elizabeth; Ramírez, Juan David; López, M. Consuelo; Castellanos-Gonzalez, Alejandro

    2015-01-01

    Amebiasis is an important cause of diarrheal disease worldwide and has been associated with childhood malnutrition. Traditional microscopy approaches are neither sensitive nor specific for Entamoeba histolytica. Antigen assays are more specific, but many cases are missed unless tested by molecular methods. Although polymerase chain reaction (PCR) is effective, the need for sophisticated, expensive equipment, infrastructure, and trained personnel limits its usefulness, especially in the resource-limited, endemic areas. Here, we report development of a recombinase polymerase amplification (RPA) method to detect E. histolytica specifically. Using visual detection by lateral flow (LF), the test was highly sensitive and specific and could be performed without additional equipment. The availability of this inexpensive, sensitive, and field-applicable diagnostic test could facilitate rapid diagnosis and treatment of amebiasis in endemic regions. PMID:26123960

  8. Detection of Entamoeba histolytica by Recombinase Polymerase Amplification.

    Science.gov (United States)

    Nair, Gayatri; Rebolledo, Mauricio; White, A Clinton; Crannell, Zachary; Richards-Kortum, R Rebecca; Pinilla, A Elizabeth; Ramírez, Juan David; López, M Consuelo; Castellanos-Gonzalez, Alejandro

    2015-09-01

    Amebiasis is an important cause of diarrheal disease worldwide and has been associated with childhood malnutrition. Traditional microscopy approaches are neither sensitive nor specific for Entamoeba histolytica. Antigen assays are more specific, but many cases are missed unless tested by molecular methods. Although polymerase chain reaction (PCR) is effective, the need for sophisticated, expensive equipment, infrastructure, and trained personnel limits its usefulness, especially in the resource-limited, endemic areas. Here, we report development of a recombinase polymerase amplification (RPA) method to detect E. histolytica specifically. Using visual detection by lateral flow (LF), the test was highly sensitive and specific and could be performed without additional equipment. The availability of this inexpensive, sensitive, and field-applicable diagnostic test could facilitate rapid diagnosis and treatment of amebiasis in endemic regions.

  9. Hendra virus detection using Loop-Mediated Isothermal Amplification.

    Science.gov (United States)

    Foord, Adam J; Middleton, Deborah; Heine, Hans G

    2012-04-01

    Hendra virus (HeV) is a zoonotic paramyxovirus endemic in Australian Pteropus bats (fruit bats or flying foxes). Although bats appear to be unaffected by the virus, HeV can spread from fruit bats to horses, causing severe disease. Human infection results from close contact with the blood, body fluids and tissues of infected horses. HeV is a biosecurity level 4 (BSL-4) pathogen, with a high case-fatality rate in humans and horses. Current assays for HeV detection require complex instrumentation and are generally time consuming. The aim of this study was to develop a Loop-Mediated Isothermal Amplification (LAMP) assay to detect nucleic acid from all known HeV strains in horses without the requirement for complex laboratory equipment. A LAMP assay targeting a conserved region of the HeV P-gene was combined with a Lateral Flow Device (LFD) for detection of amplified product. All HeV isolates, the original HeV isolated in 1994 as well as the most recent isolates from 2011 were detected. Analytical sensitivity and specificity of the HeV-LAMP assay was equal to a TaqMan assay developed previously. Significantly, these assays detected HeV in horses before clinical signs were observed. The combined LAMP-LFD procedure is a sensitive method suitable for HeV diagnosis in a resource-limited situation or where rapid test results are critical.

  10. Detection of E6/E7 HPV oncogene transcripts as biomarker of cervical intaepithelial displasia

    Directory of Open Access Journals (Sweden)

    Mauro Carcheri

    2009-09-01

    Full Text Available It is widely accepted that only persistent infection with high risk types of Human Papillomavirus (HPV HR is a significant risk factor for the development of an invasive squamous cervical cancer. The overexpression of viral oncogenes E6/E7 of HPV is considered a necessary process for incurring in a malignant phenotype.A HPV infection can be identified by detection of HPV DNA in biological samples, but the DNAbased tests cannot delineate between transient or persistent and potentially transforming infection. Instead there is many evidence to suggest that detection of HPV gene expression may constitute a more specific approach to highlight a clinically significant infection. Especially seems that the detection of E6/E7 transcripts can be usefully used for identify the women with a persistent HPV infection that will can induce a future cervical cancer. The aim of our study is to investigate if the detection of oncogenic viral gene activity by detecting transcripts of the E6 and E7 genes can be most usefull of HPV-DNA test in the triage of ASCUS or low grade cervical lesions. Our results confirm that HPV E6/E7 mRNA test can be considered a promising method to stratify HPV positive women for risk of future high-grade cervical lesions or cervical intaepithelial neoplasia.

  11. Oncogene amplification in the proximal part of chromosome 6 in rat endometrial adenocarcinoma as revealed by combined BAC/PAC FISH, chromosome painting, zoo-FISH, and allelotyping.

    Science.gov (United States)

    Adamovic, Tatjana; Trossö, Fredrik; Roshani, Leyla; Andersson, Lars; Petersen, Greta; Rajaei, Saide; Helou, Khalil; Levan, Göran

    2005-10-01

    The inbred BDII rat is a valuable experimental model for the genetic analysis of endometrial adenocarcinoma (EAC). One common aberration detected by comparative genomic hybridization in rat EAC was gain/amplification affecting the proximal part of rat chromosome 6 (RNO6). We applied rat and mouse chromosome painting probes onto tumor cell metaphase preparations in order to detect and characterize gross RNO6 aberrations. In addition, the RNO6q11-q16 segment was analyzed by fluorescence in situ hybridization with probes representing 12 cancer-related genes in the region. The analysis revealed that seven tumors contained large RNO6-derived homogeneously staining regions (HSRs) in addition to several normal or near-normal RNO6 chromosomes. Five tumors (two of which also had HSRs) exhibited a selective increase of the RNO6q11-q16 segment, sometimes in conjunction with moderate amplification of one or a few genes. Most commonly, the amplification affected the region centered around band 6q16 and included the Mycn, Ddx1, and Rrm2 genes. A second region, centering around Slc8a1 and Xdh, also was affected by gene amplification but to a lesser extent. The aberrations in the proximal part of RNO6 were further analyzed using allelotyping of microsatellite markers in all tumors from animals that were heterozygous in the proximal RNO6 region. We could detect allelic imbalance (AI) in 12 of 20 informative tumors, 6 of which were in addition to those already analyzed by molecular cytogenetic methods as described. Our findings suggest that increase/amplification of genes in this chromosome region contribute to the development of this hormone-dependent tumor.

  12. Human minisatellite alleles detectable only after PCR amplification.

    Science.gov (United States)

    Armour, J A; Crosier, M; Jeffreys, A J

    1992-01-01

    We present evidence that a proportion of alleles at two human minisatellite loci is undetected by standard Southern blot hybridization. In each case the missing allele(s) can be identified after PCR amplification and correspond to tandem arrays too short to detect by hybridization. At one locus, there is only one undetected allele (population frequency 0.3), which contains just three repeat units. At the second locus, there are at least five undetected alleles (total population frequency 0.9) containing 60-120 repeats; they are not detected because these tandem repeats give very poor signals when used as a probe in standard Southern blot hybridization, and also cross-hybridize with other sequences in the genome. Under these circumstances only signals from the longest tandemly repeated alleles are detectable above the nonspecific background. The structures of these loci have been compared in human and primate DNA, and at one locus the short human allele containing three repeat units is shown to be an intermediate state in the expansion of a monomeric precursor allele in primates to high copy number in the longer human arrays. We discuss the implications of such loci for studies of human populations, minisatellite isolation by cloning, and the evolution of highly variable tandem arrays.

  13. One-pot isothermal DNA amplification Hybridisation and detection by a disc-based method

    OpenAIRE

    2014-01-01

    [EN] An integrated sensor comprising isothermal DNA amplification and in situ detection is presented. The method principle is based on recombinase polymerase amplification (RPA) and detection in the microarray format by compact disc technology as a high-throughput sensing platform. Primers were immobilised on the polycarbonate surface of digital versatile discs (DVD) and, after hemi-nested amplification, multiplexing identification of each tethered product was achieved by optical scanning wit...

  14. Mesenchymal transition and PDGFRA amplification/mutation are key distinct oncogenic events in pediatric diffuse intrinsic pontine gliomas.

    Directory of Open Access Journals (Sweden)

    Stephanie Puget

    Full Text Available Diffuse intrinsic pontine glioma (DIPG is one of the most frequent malignant pediatric brain tumor and its prognosis is universaly fatal. No significant improvement has been made in last thirty years over the standard treatment with radiotherapy. To address the paucity of understanding of DIPGs, we have carried out integrated molecular profiling of a large series of samples obtained with stereotactic biopsy at diagnosis. While chromosomal imbalances did not distinguish DIPG and supratentorial tumors on CGHarrays, gene expression profiling revealed clear differences between them, with brainstem gliomas resembling midline/thalamic tumours, indicating a closely-related origin. Two distinct subgroups of DIPG were identified. The first subgroup displayed mesenchymal and pro-angiogenic characteristics, with stem cell markers enrichment consistent with the possibility to grow tumor stem cells from these biopsies. The other subgroup displayed oligodendroglial features, and appeared largely driven by PDGFRA, in particular through amplification and/or novel missense mutations in the extracellular domain. Patients in this later group had a significantly worse outcome with an hazard ratio for early deaths, ie before 10 months, 8 fold greater that the ones in the other subgroup (p = 0.041, Cox regression model. The worse outcome of patients with the oligodendroglial type of tumors was confirmed on a series of 55 paraffin-embedded biopsy samples at diagnosis (median OS of 7.73 versus 12.37 months, p = 0.045, log-rank test. Two distinct transcriptional subclasses of DIPG with specific genomic alterations can be defined at diagnosis by oligodendroglial differentiation or mesenchymal transition, respectively. Classifying these tumors by signal transduction pathway activation and by mutation in pathway member genes may be particularily valuable for the development of targeted therapies.

  15. The oncogenic action of ionizing radiation on rat skin

    Energy Technology Data Exchange (ETDEWEB)

    Burns, F.J.

    1991-01-01

    Progress has occurred in several areas corresponding to the specific aims of the proposal: (1) Progression and multiple events in radiation carcinogenesis of rat skin as a function of LET; (2) cell cycle kinetics of irradiated rat epidermis as determined by double labeling and double emulsion autoradiography; (3) oncogene activation detected by in situ hybridization in radiation-induced rat skin tumors; (4) amplification of the c-myc oncogene in radiation-induced rat skin tumors as a function of LET; and (5) transformation of rat skin keratinocytes by ionizing radiation in combination with c-Ki-ras and c-myc oncogenes. 111 refs., 13 figs., 12 tabs.

  16. Isothermal amplification detection of nucleic acids by a double-nicked beacon.

    Science.gov (United States)

    Shi, Chao; Zhou, Meiling; Pan, Mei; Zhong, Guilin; Ma, Cuiping

    2016-03-01

    Isothermal and rapid amplification detection of nucleic acids is an important technology in environmental monitoring, foodborne pathogen detection, and point-of-care clinical diagnostics. Here we have developed a novel method of isothermal signal amplification for single-stranded DNA (ssDNA) detection. The ssDNA target could be used as an initiator, coupled with a double-nicked molecular beacon, to originate amplification cycles, achieving cascade signal amplification. In addition, the method showed good specificity and strong anti-jamming capability. Overall, it is a one-pot and isothermal strand displacement amplification method without the requirement of a stepwise procedure, which greatly simplifies the experimental procedure and decreases the probability of contamination of samples. With its advantages, the method would be very useful to detect nucleic acids in point-of-care or field use.

  17. Simultaneous detection, typing and quantitation of oncogenic human papillomavirus by multiplex consensus real-time PCR.

    Science.gov (United States)

    Jenkins, Andrew; Allum, Anne-Gry; Strand, Linda; Aakre, Randi Kersten

    2013-02-01

    A consensus multiplex real-time PCR test (PT13-RT) for the oncogenic human papillomavirus (HPV) types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 66 is described. The test targets the L1 gene. Analytical sensitivity is between 4 and 400 GU (genomic units) in the presence of 500 ng of human DNA, corresponding to 75,000 human cells. HPV types are grouped into multiplex groups of 3 or 4 resulting in the use of 4 wells per sample and permitting up to 24 samples per run (including controls) in a standard 96-well real-time PCR instrument. False negative results are avoided by (a) measuring sample DNA concentration to control that sufficient cellular material is present and (b) including HPV type 6 as a homologous internal control in order to detect PCR inhibition or competition from other (non-oncogenic) HPV types. Analysis time from refrigerator to report is 8 h, including 2.5 h hands-on time. Relative to the HC2 test, the sensitivity and specificity were respectively 98% and 83%, the lower specificity being attributable to the higher analytical sensitivity of PT13-RT. To assess type determination comparison was made with a reversed line-blot test. Type concordance was high (κ=0.79) with discrepancies occurring mostly in multiple-positive samples. Copyright © 2012 Elsevier B.V. All rights reserved.

  18. Comparison of variations detection between whole-genome amplification methods used in single-cell resequencing

    DEFF Research Database (Denmark)

    Hou, Yong; Wu, Kui; Shi, Xulian;

    2015-01-01

    BACKGROUND: Single-cell resequencing (SCRS) provides many biomedical advances in variations detection at the single-cell level, but it currently relies on whole genome amplification (WGA). Three methods are commonly used for WGA: multiple displacement amplification (MDA), degenerate-oligonucleoti...

  19. A universal assay for detection of oncogenic fusion transcripts by oligo microarray analysis

    Directory of Open Access Journals (Sweden)

    Ribeiro Franclim R

    2009-01-01

    Full Text Available Abstract Background The ability to detect neoplasia-specific fusion genes is important not only in cancer research, but also increasingly in clinical settings to ensure that correct diagnosis is made and the optimal treatment is chosen. However, the available methodologies to detect such fusions all have their distinct short-comings. Results We describe a novel oligonucleotide microarray strategy whereby one can screen for all known oncogenic fusion transcripts in a single experiment. To accomplish this, we combine measurements of chimeric transcript junctions with exon-wise measurements of individual fusion partners. To demonstrate the usefulness of the approach, we designed a DNA microarray containing 68,861 oligonucleotide probes that includes oligos covering all combinations of chimeric exon-exon junctions from 275 pairs of fusion genes, as well as sets of oligos internal to all the exons of the fusion partners. Using this array, proof of principle was demonstrated by detection of known fusion genes (such as TCF3:PBX1, ETV6:RUNX1, and TMPRSS2:ERG from all six positive controls consisting of leukemia cell lines and prostate cancer biopsies. Conclusion This new method bears promise of an important complement to currently used diagnostic and research tools for the detection of fusion genes in neoplastic diseases.

  20. Detection of FLT3 Oncogene Mutations in Acute Myeloid Leukemia Using Conformation Sensitive Gel Electrophoresis

    Directory of Open Access Journals (Sweden)

    Mamdooh Gari

    2008-11-01

    Full Text Available FLT3 (fms-related tyrosine kinase 3 is a receptor tyrosine kinase class III that is expressed on by early hematopoietic progenitor cells and plays an important role in hematopoietic stem cell proliferation, differentiation and survival. FLT3 is also expressed on leukemia blasts in most cases of acute myeloid leukemia (AML. In order to determine the frequency of FLT3 oncogene mutations, we analyzed genomic DNA of adult de novo acute myeloid leukemia (AML. Polymerase chain reaction (PCR and conformation-sensitive gel electrophoresis (CSGE were used for FLT3 exons 11, 14, and 15, followed by direct DNA sequencing. Two different types of functionally important FLT 3 mutations have been identified. Those mutations were unique to patients with inv(16, t(15:17 or t(8;21 and comprised fifteen cases with internal tandem duplication (ITD mutation in the juxtamembrane domain and eleven cases with point mutation (exon 20, Asp835Tyr. The high frequency of the flt3 proto-oncogene mutations in acute myeloid leukemia AML suggests a key role for the receptor function. The association of FLT3 mutations with chromosomal abnormalities invites speculation as to the link between these two changes in the pathogenesis of acute myeloid leukemiaAML. Furthermore, CSGE method has shown to be a rapid and sensitive screening method for detection of nucleotide alteration in FLT3 gene. Finally, this study reports, for the first time in Saudi Arabia, mutations in the human FLT3 gene in acute myeloid leukemia AML patients.

  1. Electrochemical detection of leukemia oncogenes using enzyme-loaded carbon nanotube labels

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Ai Cheng; Du, Dan; Chen, Baowei; Heng, Chew-Kiat; Lim, Tit-Meng; Lin, Yuehe

    2014-09-07

    Here we describe an ultrasensitive electrochemical nucleic acids assay amplified by carbon nanotubes (CNTs)-based labels for the detection of human acute lymphocytic leukemia (ALL) related p185 BCR-ABL fusion transcript. The carboxylated CNTs were functionalized with horseradish peroxidase (HRP) molecules and target-specific detection probes (DP) via diimide-activated amidation, and used to label and amplify target hybridization signal. The activity of captured HRP was monitored by square-wave voltammetry measuring the electroactive enzymatic product in the presence of 2-aminophenol and hydrogen peroxide substrate solution. The effect of DP and HRP loading of the CNT-based labels on its signal-to-noise ratio of electrochemical detection was studied systematically for the first time. Under optimized conditions, the signal-amplified assay achieved a detection limit of 83 fM targets oligonuecleotides and a 4-order wide dynamic range of target concentration. The resulting assay allowed a robust discrimination between the perfect match and a three-base mismatch sequence. When subjected to full-length (491 bp) DNA oncogene, the approach demonstrated a detection limit of approximately 33 pg of the target gene. The high sensitivity and specificity of assay enabled PCR-free detection of target transcripts in as little as 65 ng of mRNA extracted from positive ALL cell lines SUP-B15, in comparison to those obtained from negative cell lines HL-60. The approach holds promise for simple, low cost and ultrasensitive electrochemical nucleic acids detection in portable devices, point-of-care and early disease diagnostic applications.

  2. RNA extraction method is crucial for human papillomavirus E6/E7 oncogenes detection.

    Science.gov (United States)

    Fontecha, Nerea; Nieto, Maria Carmen; Andía, Daniel; Cisterna, Ramón; Basaras, Miren

    2017-03-09

    Human papillomavirus (HPV) DNA testing plays a main role in the management of cervical cancer, however to improve the specificity in cervical screening, there is a need to develop and validate different approaches that can identify women at risk for progressive disease. Nowadays, mRNA expression of viral E6 and E7 HPV oncogenes stands up as a potential biomarker to improve cervical screening. We aimed to validate a method for RNA extraction, detect HPV mRNA expression and, assess the relationship between E6/E7 mRNA expression and pathology of patients' lesions and progression. This study included 50 specimens that had been previously genotyped as HPV16, 18, 31, 33 and/or 45. Cervical swabs were extracted with three different RNA extraction methods -Nuclisens manual extraction kit (bioMérieux), High Pure Viral RNA Kit (Roche) and RNeasy Plus Mini kit (Qiagen)-, and mRNA was detected with NucliSens EasyQ HPV version 1 test (bioMérieux) afterwards. Association of oncogene expression with pathology and lesion progression was analyzed for each extraction method. E6/E7 mRNA positivity rate was higher in samples analyzed with bioMérieux (62%), followed by Roche (24%) and Qiagen (6%). Women with lesions and lesion progression showed a higher prevalence of viral RNA expression than women that had not lesions or with lesion persistence. While bioMérieux revealed a higher sensitivity (77.27%), Roche presented a higher PPV (75%) and an increased specificity (89.28%). Extraction methods based on magnetic beads provided better RNA yield than those based in columns. Both Nuclisens manual extraction kit (bioMérieux) and High Pure Viral RNA Kit (Roche) seemed to be adequate for E6/E7 mRNA detection. However, none of them revealed both high sensitivity and specificity values. Further studies are needed to obtain and validate a standard gold method for RNA expression detection, to be included as part of the routine cervical screening program.

  3. Isothermal Amplification Methods for the Detection of Nucleic Acids in Microfluidic Devices

    Directory of Open Access Journals (Sweden)

    Giuseppe Spoto

    2012-12-01

    Full Text Available Diagnostic tools for biomolecular detection need to fulfill specific requirements in terms of sensitivity, selectivity and high-throughput in order to widen their applicability and to minimize the cost of the assay. The nucleic acid amplification is a key step in DNA detection assays. It contributes to improving the assay sensitivity by enabling the detection of a limited number of target molecules. The use of microfluidic devices to miniaturize amplification protocols reduces the required sample volume and the analysis times and offers new possibilities for the process automation and integration in one single device. The vast majority of miniaturized systems for nucleic acid analysis exploit the polymerase chain reaction (PCR amplification method, which requires repeated cycles of three or two temperature-dependent steps during the amplification of the nucleic acid target sequence. In contrast, low temperature isothermal amplification methods have no need for thermal cycling thus requiring simplified microfluidic device features. Here, the use of miniaturized analysis systems using isothermal amplification reactions for the nucleic acid amplification will be discussed.

  4. Simultaneous detection of human papillomavirus integration and c-MYC gene amplification in cervical lesions: an emerging marker for the risk to progression.

    Science.gov (United States)

    Gimenes, Fabrícia; Souza, Raquel Pantarotto; de Abreu, André Luelsdorf Pimenta; Pereira, Monalisa Wolski; Consolaro, Marcia Edilaine Lopes; da Silva, Vânia Ramos Sela

    2016-04-01

    The persistence of high-risk oncogenic human papillomavirus (HR-HPV) infection and its integration into the host genome are key steps in the induction of malignant alterations. c-MYC chromosome region is a frequent localization for HPV insertion that has been observed in chromosome band 8q24 by fluorescence in situ hybridization (FISH). We report the HPV viral integration and amplification patterns of the c-MYC gene in cytological smears with FISH as a potential biomarker for the progression of squamous intraepithelial lesions (SIL). HPV detection and genotyping by polymerase chain reaction (PCR) and FISH analysis by "Vysis Cervical FISH Probe" kit (ABBOTT Molecular Inc.) were performed in 37 cervical samples including 8 NILM, 7 ASC-US, 7 LSIL, 3 ASC-H, 7 HSIL and 5 SCC. The results show concordance between FISH and PCR techniques for HPV detection. The majority of the samples contained HR-HPV, the majority being -16 and -18 genotypes. HPV integration as determined by FISH was most frequent in high-risk lesions. The c-MYC gene amplification was found only in HPV-positive samples and was detected primarily in high-risk lesions and in cells with an integrated form of HPV. HPV integration and c-MYC gene amplification detected by FISH could be an important biomarker for use in clinical practice to determine SIL with a risk of progression.

  5. Ultrasensitive detection of nucleic acids by template enhanced hybridization followed by rolling circle amplification and catalytic hairpin assembly.

    Science.gov (United States)

    Song, Weiling; Zhang, Qiao; Sun, Wenbo

    2015-02-11

    An ultrasensitive protocol for fluorescent detection of DNA is designed by combining the template enhanced hybridization process (TEHP) with Rolling Circle Amplification (RCA) and Catalytic Hairpin Assembly (CHA), showing a remarkable amplification efficiency.

  6. A cascade amplification strategy based on rolling circle amplification and hydroxylamine amplified gold nanoparticles enables chemiluminescence detection of adenosine triphosphate.

    Science.gov (United States)

    Wang, Ping; Zhang, Tonghuan; Yang, Taoyi; Jin, Nan; Zhao, Yanjun; Fan, Aiping

    2014-08-07

    A highly sensitive and selective chemiluminescent (CL) biosensor for adenosine triphosphate (ATP) was developed by taking advantage of the ATP-dependent enzymatic reaction (ATP-DER), the powerful signal amplification capability of rolling circle amplification (RCA), and hydroxylamine-amplified gold nanoparticles (Au NPs). The strategy relies on the ability of ATP, a cofactor of T4 DNA ligase, to trigger the ligation-RCA reaction. In the presence of ATP, the T4 DNA ligase catalyzes the ligation reaction between the two ends of the padlock probe, producing a closed circular DNA template that initiates the RCA reaction with phi29 DNA polymerase and dNTP. Therein, many complementary copies of the circular template can be generated. The ATP-DER is eventually converted into a detectable CL signal after a series of processes, including gold probe hybridization, hydroxylamine amplification, and oxidative gold metal dissolution coupled with a simple and sensitive luminol CL reaction. The CL signal is directly proportional to the ATP level. The results showed that the detection limit of the assay is 100 pM of ATP, which compares favorably with those of other ATP detection techniques. In addition, by taking advantage of ATP-DER, the proposed CL sensing system exhibits extraordinary specificity towards ATP and could distinguish the target molecule ATP from its analogues. The proposed method provides a new and versatile platform for the design of novel DNA ligation reaction-based CL sensing systems for other cofactors. This novel ATP-DER based CL sensing system may find wide applications in clinical diagnosis as well as in environmental and biomedical fields.

  7. Detection of genetically modified organisms (GMOs) using isothermal amplification of target DNA sequences.

    Science.gov (United States)

    Lee, David; La Mura, Maurizio; Allnutt, Theo R; Powell, Wayne

    2009-02-02

    The most common method of GMO detection is based upon the amplification of GMO-specific DNA amplicons using the polymerase chain reaction (PCR). Here we have applied the loop-mediated isothermal amplification (LAMP) method to amplify GMO-related DNA sequences, 'internal' commonly-used motifs for controlling transgene expression and event-specific (plant-transgene) junctions. We have tested the specificity and sensitivity of the technique for use in GMO studies. Results show that detection of 0.01% GMO in equivalent background DNA was possible and dilutions of template suggest that detection from single copies of the template may be possible using LAMP. This work shows that GMO detection can be carried out using LAMP for routine screening as well as for specific events detection. Moreover, the sensitivity and ability to amplify targets, even with a high background of DNA, here demonstrated, highlights the advantages of this isothermal amplification when applied for GMO detection.

  8. Application of a Non-amplification based Technology to Detect Invasive Fungal Pathogens

    Science.gov (United States)

    Hsu, Joe L.; Binkley, Jon; Clemons, Karl V.; Stevens, David A.; Nicolls, Mark R.; Holodniy, Mark

    2014-01-01

    Current diagnostic techniques for fungal diseases could be improved with respect to sensitivity, specificity and timeliness. To address this clinical need, we adapted a non-amplification based nucleic acid detection technology to identify fungal pathogens. We demonstrate a high-specificity, detection sensitivity, reproducibility and multiplex capacity for detecting fungal strains. PMID:24359934

  9. Application of a Non-amplification based Technology to Detect Invasive Fungal Pathogens

    OpenAIRE

    Hsu, Joe L.; Binkley, Jon; Clemons, Karl V.; Stevens, David A.; Nicolls, Mark R.; Holodniy, Mark

    2013-01-01

    Current diagnostic techniques for fungal diseases could be improved with respect to sensitivity, specificity and timeliness. To address this clinical need, we adapted a non-amplification based nucleic acid detection technology to identify fungal pathogens. We demonstrate a high-specificity, detection sensitivity, reproducibility and multiplex capacity for detecting fungal strains.

  10. Simple system for isothermal DNA amplification coupled to lateral flow detection.

    Directory of Open Access Journals (Sweden)

    Kristina Roskos

    Full Text Available Infectious disease diagnosis in point-of-care settings can be greatly improved through integrated, automated nucleic acid testing devices. We have developed an early prototype for a low-cost system which executes isothermal DNA amplification coupled to nucleic acid lateral flow (NALF detection in a mesofluidic cartridge attached to a portable instrument. Fluid handling inside the cartridge is facilitated through one-way passive valves, flexible pouches, and electrolysis-driven pumps, which promotes a compact and inexpensive instrument design. The closed-system disposable prevents workspace amplicon contamination. The cartridge design is based on standard scalable manufacturing techniques such as injection molding. Nucleic acid amplification occurs in a two-layer pouch that enables efficient heat transfer. We have demonstrated as proof of principle the amplification and detection of Mycobacterium tuberculosis (M.tb genomic DNA in the cartridge, using either Loop Mediated Amplification (LAMP or the Exponential Amplification Reaction (EXPAR, both coupled to NALF detection. We envision that a refined version of this cartridge, including upstream sample preparation coupled to amplification and detection, will enable fully-automated sample-in to answer-out infectious disease diagnosis in primary care settings of low-resource countries with high disease burden.

  11. Detection of biological molecules using boronate-based chemical amplification and optical sensors

    Energy Technology Data Exchange (ETDEWEB)

    Antwerp, W.P. van; Mastrototaro, J.J.; Lane, S.M.; Satcher, J.H. Jr.; Darrow, C.B.; Peyser, T.A.; Harder, J.

    1999-12-14

    Methods are provided for the determination of the concentration of biological levels of polyhydroxylated compounds, particularly glucose. The methods utilize an amplification system that is an analyte transducer immobilized in a polymeric matrix, where the system is implantable and biocompatible. Upon interrogation by an optical system, the amplification system produces a signal capable of detection external to the skin of the patient. Quantitation of the analyte of interest is achieved by measurement of the emitted signal.

  12. Rapid Salmonella detection using an acoustic wave device combined with the RCA isothermal DNA amplification method

    Directory of Open Access Journals (Sweden)

    Antonis Kordas

    2016-12-01

    Full Text Available Salmonella enterica serovar Typhimurium is a major foodborne pathogen that causes Salmonellosis, posing a serious threat for public health and economy; thus, the development of fast and sensitive methods is of paramount importance for food quality control and safety management. In the current work, we are presenting a new approach where an isothermal amplification method is combined with an acoustic wave device for the development of a label free assay for bacteria detection. Specifically, our method utilizes a Love wave biosensor based on a Surface Acoustic Wave (SAW device combined with the isothermal Rolling Circle Amplification (RCA method; various protocols were tested regarding the DNA amplification and detection, including off-chip amplification at two different temperatures (30 °C and room temperature followed by acoustic detection and on-chip amplification and detection at room temperature, with the current detection limit being as little as 100 Bacteria Cell Equivalents (BCE/sample. Our acoustic results showed that the acoustic ratio, i.e., the amplitude over phase change observed during DNA binding, provided the only sensitive means for product detection while the measurement of amplitude or phase alone could not discriminate positive from negative samples. The method's fast analysis time together with other inherent advantages i.e., portability, potential for multi-analysis, lower sample volumes and reduced power consumption, hold great promise for employing the developed assay in a Lab on Chip (LoC platform for the integrated analysis of Salmonella in food samples.

  13. Isothermal amplification using a chemical heating device for point-of-care detection of HIV-1.

    Science.gov (United States)

    Curtis, Kelly A; Rudolph, Donna L; Nejad, Irene; Singleton, Jered; Beddoe, Andy; Weigl, Bernhard; LaBarre, Paul; Owen, S Michele

    2012-01-01

    To date, the use of traditional nucleic acid amplification tests (NAAT) for detection of HIV-1 DNA or RNA has been restricted to laboratory settings due to time, equipment, and technical expertise requirements. The availability of a rapid NAAT with applicability for resource-limited or point-of-care (POC) settings would fill a great need in HIV diagnostics, allowing for timely diagnosis or confirmation of infection status, as well as facilitating the diagnosis of acute infection, screening and evaluation of infants born to HIV-infected mothers. Isothermal amplification methods, such as reverse-transcription, loop-mediated isothermal amplification (RT-LAMP), exhibit characteristics that are ideal for POC settings, since they are typically quicker, easier to perform, and allow for integration into low-tech, portable heating devices. In this study, we evaluated the HIV-1 RT-LAMP assay using portable, non-instrumented nucleic acid amplification (NINA) heating devices that generate heat from the exothermic reaction of calcium oxide and water. The NINA heating devices exhibited stable temperatures throughout the amplification reaction and consistent amplification results between three separate devices and a thermalcycler. The performance of the NINA heaters was validated using whole blood specimens from HIV-1 infected patients. The RT-LAMP isothermal amplification method used in conjunction with a chemical heating device provides a portable, rapid and robust NAAT platform that has the potential to facilitate HIV-1 testing in resource-limited settings and POC.

  14. Her2 expression and gene amplification is rarely detectable in patients with oral squamous cell carcinomas.

    Science.gov (United States)

    Hanken, Henning; Gaudin, Robert; Gröbe, Alexander; Fraederich, Meike; Eichhorn, Wolfgang; Smeets, Ralf; Simon, Ronald; Sauter, Guido; Grupp, Katharina; Izbicki, Jacob R; Sehner, Susanne; Heiland, Max; Blessmann, Marco

    2014-04-01

    Her2 (ErbB2) transforms cells when overexpressed and is an important therapeutic target in breast cancer. Contrary to breast cancer, studies on Her2 overexpression and gene amplification in squamous cell carcinomas of the head and neck region described largely different results. This study was undertaken to learn more on the prevalence and clinical significance of HER2 amplification and overexpression in squamous cell carcinomas of the head and neck. Her2 expression and gene amplification was analyzed by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) on two tissue microarrays composed of 427 squamous cell carcinomas of the head and neck region and 222 oral squamous cell carcinomas. Results were compared with clinicopathological features. Her2 expression and gene amplification was rarely detectable in squamous cell carcinomas of the head and neck region and unrelated to tumor phenotype or survival of the patients with oral squamous carcinoma. Our results demonstrate that Her2 protein and gene amplification was only detectable in a small subset of squamous cell carcinomas of the head and neck region as well as oral squamous cell carcinomas. However, it can be speculated that those few patients with Her2 overexpressing and gene amplificated tumors may possibly benefit from an anti-Her2 therapy. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  15. Tiny grains give huge gains: nanocrystal-based signal amplification for biomolecule detection.

    Science.gov (United States)

    Tong, Sheng; Ren, Binbin; Zheng, Zhilan; Shen, Han; Bao, Gang

    2013-06-25

    Nanocrystals, despite their tiny sizes, contain thousands to millions of atoms. Here we show that the large number of atoms packed in each metallic nanocrystal can provide a huge gain in signal amplification for biomolecule detection. We have devised a highly sensitive, linear amplification scheme by integrating the dissolution of bound nanocrystals and metal-induced stoichiometric chromogenesis, and demonstrated that signal amplification is fully defined by the size and atom density of nanocrystals, which can be optimized through well-controlled nanocrystal synthesis. Further, the rich library of chromogenic reactions allows implementation of this scheme in various assay formats, as demonstrated by the iron oxide nanoparticle linked immunosorbent assay (ILISA) and blotting assay developed in this study. Our results indicate that, owing to the inherent simplicity, high sensitivity and repeatability, the nanocrystal based amplification scheme can significantly improve biomolecule quantification in both laboratory research and clinical diagnostics. This novel method adds a new dimension to current nanoparticle-based bioassays.

  16. NAIMA: target amplification strategy allowing quantitative on-chip detection of GMOs

    Science.gov (United States)

    Morisset, Dany; Dobnik, David; Hamels, Sandrine; Žel, Jana; Gruden, Kristina

    2008-01-01

    We have developed a novel multiplex quantitative DNA-based target amplification method suitable for sensitive, specific and quantitative detection on microarray. This new method named NASBA Implemented Microarray Analysis (NAIMA) was applied to GMO detection in food and feed, but its application can be extended to all fields of biology requiring simultaneous detection of low copy number DNA targets. In a first step, the use of tailed primers allows the multiplex synthesis of template DNAs in a primer extension reaction. A second step of the procedure consists of transcription-based amplification using universal primers. The cRNA product is further on directly ligated to fluorescent dyes labelled 3DNA dendrimers allowing signal amplification and hybridized without further purification on an oligonucleotide probe-based microarray for multiplex detection. Two triplex systems have been applied to test maize samples containing several transgenic lines, and NAIMA has shown to be sensitive down to two target copies and to provide quantitative data on the transgenic contents in a range of 0.1–25%. Performances of NAIMA are comparable to singleplex quantitative real-time PCR. In addition, NAIMA amplification is faster since 20 min are sufficient to achieve full amplification. PMID:18710880

  17. NAIMA: target amplification strategy allowing quantitative on-chip detection of GMOs.

    Science.gov (United States)

    Morisset, Dany; Dobnik, David; Hamels, Sandrine; Zel, Jana; Gruden, Kristina

    2008-10-01

    We have developed a novel multiplex quantitative DNA-based target amplification method suitable for sensitive, specific and quantitative detection on microarray. This new method named NASBA Implemented Microarray Analysis (NAIMA) was applied to GMO detection in food and feed, but its application can be extended to all fields of biology requiring simultaneous detection of low copy number DNA targets. In a first step, the use of tailed primers allows the multiplex synthesis of template DNAs in a primer extension reaction. A second step of the procedure consists of transcription-based amplification using universal primers. The cRNA product is further on directly ligated to fluorescent dyes labelled 3DNA dendrimers allowing signal amplification and hybridized without further purification on an oligonucleotide probe-based microarray for multiplex detection. Two triplex systems have been applied to test maize samples containing several transgenic lines, and NAIMA has shown to be sensitive down to two target copies and to provide quantitative data on the transgenic contents in a range of 0.1-25%. Performances of NAIMA are comparable to singleplex quantitative real-time PCR. In addition, NAIMA amplification is faster since 20 min are sufficient to achieve full amplification.

  18. A novel method, digital genome scanning detects KRAS gene amplification in gastric cancers: involvement of overexpressed wild-type KRAS in downstream signaling and cancer cell growth

    Directory of Open Access Journals (Sweden)

    Yanagihara Kazuyoshi

    2009-06-01

    Full Text Available Abstract Background Gastric cancer is the third most common malignancy affecting the general population worldwide. Aberrant activation of KRAS is a key factor in the development of many types of tumor, however, oncogenic mutations of KRAS are infrequent in gastric cancer. We have developed a novel quantitative method of analysis of DNA copy number, termed digital genome scanning (DGS, which is based on the enumeration of short restriction fragments, and does not involve PCR or hybridization. In the current study, we used DGS to survey copy-number alterations in gastric cancer cells. Methods DGS of gastric cancer cell lines was performed using the sequences of 5000 to 15000 restriction fragments. We screened 20 gastric cancer cell lines and 86 primary gastric tumors for KRAS amplification by quantitative PCR, and investigated KRAS amplification at the DNA, mRNA and protein levels by mutational analysis, real-time PCR, immunoblot analysis, GTP-RAS pull-down assay and immunohistochemical analysis. The effect of KRAS knock-down on the activation of p44/42 MAP kinase and AKT and on cell growth were examined by immunoblot and colorimetric assay, respectively. Results DGS analysis of the HSC45 gastric cancer cell line revealed the amplification of a 500-kb region on chromosome 12p12.1, which contains the KRAS gene locus. Amplification of the KRAS locus was detected in 15% (3/20 of gastric cancer cell lines (8–18-fold amplification and 4.7% (4/86 of primary gastric tumors (8–50-fold amplification. KRAS mutations were identified in two of the three cell lines in which KRAS was amplified, but were not detected in any of the primary tumors. Overexpression of KRAS protein correlated directly with increased KRAS copy number. The level of GTP-bound KRAS was elevated following serum stimulation in cells with amplified wild-type KRAS, but not in cells with amplified mutant KRAS. Knock-down of KRAS in gastric cancer cells that carried amplified wild

  19. Electricity-free amplification and detection for molecular point-of-care diagnosis of HIV-1.

    Science.gov (United States)

    Singleton, Jered; Osborn, Jennifer L; Lillis, Lorraine; Hawkins, Kenneth; Guelig, Dylan; Price, Will; Johns, Rachel; Ebels, Kelly; Boyle, David; Weigl, Bernhard; LaBarre, Paul

    2014-01-01

    In resource-limited settings, the lack of decentralized molecular diagnostic testing and sparse access to centralized medical facilities can present a critical barrier to timely diagnosis, treatment, and subsequent control and elimination of infectious diseases. Isothermal nucleic acid amplification methods, including reverse transcription loop-mediated isothermal amplification (RT-LAMP), are well-suited for decentralized point-of-care molecular testing in minimal infrastructure laboratories since they significantly reduce the complexity of equipment and power requirements. Despite reduced complexity, however, there is still a need for a constant heat source to enable isothermal nucleic acid amplification. This requirement poses significant challenges for laboratories in developing countries where electricity is often unreliable or unavailable. To address this need, we previously developed a low-cost, electricity-free heater using an exothermic reaction thermally coupled with a phase change material. This heater achieved acceptable performance, but exhibited considerable variability. Furthermore, as an enabling technology, the heater was an incomplete diagnostic solution. Here we describe a more precise, affordable, and robust heater design with thermal standard deviation electricity-free heater and NALF-detection platform, we demonstrate sensitive and repeatable detection of HIV-1 with a ß-actin positive internal amplification control from processed sample to result in less than 80 minutes. Together, these elements are building blocks for an electricity-free platform capable of isothermal amplification and detection of a variety of pathogens.

  20. myc family oncogene amplification in tumor cell lines established from small cell lung cancer patients and its relationship to clinical status and course.

    Science.gov (United States)

    Johnson, B E; Ihde, D C; Makuch, R W; Gazdar, A F; Carney, D N; Oie, H; Russell, E; Nau, M M; Minna, J D

    1987-01-01

    44 small cell lung cancer cell lines established from 227 patients were studied for myc family DNA amplification (c-myc, N-myc, and L-myc). Two of 19 lines (11%) established from untreated patients' tumors had DNA amplification (one N-myc and one L-myc), compared with 11 of 25 (5 c-myc, 3 N-myc, and 3 L-myc) cell lines (44%) established from relapsed patients' tumors (P = 0.04). The 19 patients who had tumor cell lines established before chemotherapy treatment survived a median of 14 wk compared with 48 wk for the 123 extensive stage patients who did not have cell lines established (P less than 0.001). Relapsed patients whose cell lines had c-myc DNA amplification survived a shorter period (median of 33 wk) than patients whose cell lines did not have c-myc amplification (median of 53 wk; P = 0.04). We conclude that myc family DNA amplification is more common in tumor cell lines established from treated than untreated patients' tumors, and c-myc amplification in treated patients' tumor cell lines is associated with shortened survival. Images PMID:3034978

  1. Rapid Newcastle Disease Virus Detection Based on Loop-Mediated Isothermal Amplification and Optomagnetic Readout

    DEFF Research Database (Denmark)

    Tian, Bo; Ma, Jing; Zardán Gómez de la Torre, Teresa

    2016-01-01

    Rapid and sensitive diagnostic methods based on isothermal amplification are ideal substitutes for PCR in out-of-lab settings. However, there are bottlenecks in terms of establishing low-cost and user-friendly readout methods for isothermal amplification schemes. Combining the high amplification...... nanoparticles (MNPs) resulting in a dramatical increase in the hydrodynamic size of the MNPs. This increase was measured by an optomagnetic readout system and provided quantitative information on the amount of LAMP target sequence. Our assay resulted in a limit of detection of 10 aM of target sequence...... with a total assay time of 30 min. The assay has also been tested on clinical samples (vaccine and tissue specimens) with a performance comparable to real-time RT-PCR. By changing the LAMP primers, this strategy can serve as a general method for the detection of other DNA/RNA targets with high specificity...

  2. Real-time DNA Amplification and Detection System Based on a CMOS Image Sensor.

    Science.gov (United States)

    Wang, Tiantian; Devadhasan, Jasmine Pramila; Lee, Do Young; Kim, Sanghyo

    2016-01-01

    In the present study, we developed a polypropylene well-integrated complementary metal oxide semiconductor (CMOS) platform to perform the loop mediated isothermal amplification (LAMP) technique for real-time DNA amplification and detection simultaneously. An amplification-coupled detection system directly measures the photon number changes based on the generation of magnesium pyrophosphate and color changes. The photon number decreases during the amplification process. The CMOS image sensor observes the photons and converts into digital units with the aid of an analog-to-digital converter (ADC). In addition, UV-spectral studies, optical color intensity detection, pH analysis, and electrophoresis detection were carried out to prove the efficiency of the CMOS sensor based the LAMP system. Moreover, Clostridium perfringens was utilized as proof-of-concept detection for the new system. We anticipate that this CMOS image sensor-based LAMP method will enable the creation of cost-effective, label-free, optical, real-time and portable molecular diagnostic devices.

  3. Integrated platform with magnetic purification and rolling circular amplification for sensitive fluorescent detection of ochratoxin A.

    Science.gov (United States)

    Yao, Li; Chen, Yinji; Teng, Jun; Zheng, Wanli; Wu, Jingjing; Adeloju, Samuel B; Pan, Daodong; Chen, Wei

    2015-12-15

    In this article, we report the detection of ochratoxin A (OTA) with excellent sensitivity with the two-aspect signal amplification treatments. Combining the unique property of magnetic nanoparticles and the high efficiency of the in vitro amplification of rolling circular amplification (RCA), the competitive sensing protocol for ultrasensitive detection of OTA was achieved in about 80 min. The excellent magnetic separation treatment could effectively avoid the interference of background fluorescent noise in the sensing system while the RCA could tremendously increase the hybridization sequence for the quantum dot labeled probes and further increase the sensing response signal. Afterwards, two factors affecting the final detection limit, concentration of RCA components and RCA reaction time, were all systematically optimized for the best sensing performance. The response of the optimized protocol for OTA detection is highly linear over the wider range from 10(-3) to 10 ppb, which is 3 orders improvement in sensing range, and the limit of detection is calculated to be as low as 0.13 ppt, which is 10,000 folds improvement compared with the traditional methods. More importantly, given the selected aptamer, this universal signal amplification protocol could be widely applied to other fields by just change the recognition sequence of the aptamer.

  4. Cascaded multiple amplification strategy for ultrasensitive detection of HIV/HCV virus DNA.

    Science.gov (United States)

    Wang, Kun; Fan, Daoqing; Liu, Yaqing; Dong, Shaojun

    2017-01-15

    Ultrasensitive detection of HIV and HCV virus DNA is of great importance for early accurate diagnostics and therapy of HIV virus-infected patients. Herein, to our best knowledge, it is the first to use DNA cascaded multiple amplification strategy for ultrasensitive detection of HIV virus DNA with G-quadruplex-specific fluorescent or colorimetric probes as signal carriers. The developed strategy also exhibited universal applicability for HCV virus DNA detection. After reaction for about 4h, high sensitivity and specificity can be achieved at both fluorescent and colorimetric strategies (limit of detection (LOD) of 10 fM and 0.5pM were reached for fluorescent and colorimetric detection, respectively). And the single-based mismatched DNA even can be distinguished by naked eyes. It is believed that the cascaded multiple amplification strategy presents a huge advance in sensing platform and potential application in future clinical diagnosis.

  5. Molecular beacon probes combined with amplification by NASBA enable homogeneous, real-time detection of RNA

    NARCIS (Netherlands)

    Leone, G.; Schijndel, van H.; Gemen, van B.; Kramer, F.R.; Schoen, C.D.

    1998-01-01

    Molecular beacon probes can be employed in a NASBA amplicon detection system to generate a specific fluorescent signal concomitantly with amplification. A molecular beacon, designed to hybridize within the target sequence, was introduced into NASBA reactions that amplify the genomic RNA of potato le

  6. Amplification of RNA by NASBA allows direct detection of viable cells of Ralstonia solanacearum in potato

    NARCIS (Netherlands)

    Bentsink, L.; Leone, G.O.M.; Beckhoven, van J.R.C.M.; Schijndel, van H.B.; Gemen, van B.; Wolf, van der J.M.

    2002-01-01

    Aims: The objective of this study was to develop a Nucleic Acid Sequence Based Amplification (NASBA) assay, targeting 16S rRNA sequences, for direct detection of viable cells of Ralstonia solanacearum, the causal organism of bacterial wilt. The presence of intact 16S rRNA is considered to be a usefu

  7. Real-time isothermal detection of Shiga toxin-producing Escherichia coli using recombinase polymerase amplification

    Science.gov (United States)

    Shiga toxin (Stx) producing E. coli (STEC) are a major family of foodborne pathogens of immense public health, zoonotic and economic significance in the US and worldwide. To date, there are no published reports on use of recombinase polymerase amplification (RPA) for STEC detection. The primary goal...

  8. Development and application of loop-mediated isothermal amplification (LAMP) for detection of Plasmopara viticola

    NARCIS (Netherlands)

    Kong, X.; Qin, W.; Xiaoqing, X.; Kong, F.; Schoen, C.D.; Feng, J.; Wang, Z.; Zhang, H.

    2016-01-01

    A rapid LAMP (loop-mediated isothermal amplification) detection method was developed on the basis of the ITS sequence of P. viticola, the major causal agent of grape downy mildew. Among the 38 fungal and oomycete species tested, DNA isolated exclusively from P. viticola resulted in a specific

  9. Molecular beacon probes combined with amplification by NASBA enable homogeneous, real-time detection of RNA

    NARCIS (Netherlands)

    Leone, G.; Schijndel, van H.; Gemen, van B.; Kramer, F.R.; Schoen, C.D.

    1998-01-01

    Molecular beacon probes can be employed in a NASBA amplicon detection system to generate a specific fluorescent signal concomitantly with amplification. A molecular beacon, designed to hybridize within the target sequence, was introduced into NASBA reactions that amplify the genomic RNA of potato

  10. Molecular beacon probes combined with amplification by NASBA enable homogeneous, real-time detection of RNA

    NARCIS (Netherlands)

    Leone, G.; Schijndel, van H.; Gemen, van B.; Kramer, F.R.; Schoen, C.D.

    1998-01-01

    Molecular beacon probes can be employed in a NASBA amplicon detection system to generate a specific fluorescent signal concomitantly with amplification. A molecular beacon, designed to hybridize within the target sequence, was introduced into NASBA reactions that amplify the genomic RNA of potato le

  11. Nucleic acid sequence-based amplification with oligochromatography for detection of Trypanosoma brucei in clinical samples

    NARCIS (Netherlands)

    C.M. Mugasa; T. Laurent; G.J. Schoone; P.A. Kager; G.W. Lubega; H.D.F.H. Schallig

    2009-01-01

    Molecular tools, such as real-time nucleic acid sequence-based amplification (NASBA) and PCR, have been developed to detect Trypanosoma brucei parasites in blood for the diagnosis of human African trypanosomiasis (HAT). Despite good sensitivity, these techniques are not implemented in HAT control pr

  12. Detection of rotavirus using padlock probes and rolling circle amplification.

    Directory of Open Access Journals (Sweden)

    Anja Mezger

    Full Text Available Rotavirus infections are one of the most common reasons for hospitalizations due to gastrointestinal diseases. Rotavirus is often diagnosed by latex agglutination assay, chromatography immunoassay, or by electron microscopy, which are all quite insensitive. Reverse transcription polymerase chain reaction, on the other hand, is very sensitive to variations at the genomic level. We developed a novel assay based on a set of 58 different padlock probes with a detection limit of 1,000 copies. Twenty-two patient samples were analyzed and the assay showed high concordance with a PCR-based assay. In summary, we present a new assay for sensitive and variation tolerant detection of rotavirus.

  13. Novel bioluminescent quantitative detection of nucleic acid amplification in real-time.

    Directory of Open Access Journals (Sweden)

    Olga A Gandelman

    Full Text Available BACKGROUND: The real-time monitoring of polynucleotide amplification is at the core of most molecular assays. This conventionally relies on fluorescent detection of the amplicon produced, requiring complex and costly hardware, often restricting it to specialised laboratories. PRINCIPAL FINDINGS: Here we report the first real-time, closed-tube luminescent reporter system for nucleic acid amplification technologies (NAATs enabling the progress of amplification to be continuously monitored using simple light measuring equipment. The Bioluminescent Assay in Real-Time (BART continuously reports through bioluminescent output the exponential increase of inorganic pyrophosphate (PPi produced during the isothermal amplification of a specific nucleic acid target. BART relies on the coupled conversion of inorganic pyrophosphate (PPi produced stoichiometrically during nucleic acid synthesis to ATP by the enzyme ATP sulfurylase, and can therefore be coupled to a wide range of isothermal NAATs. During nucleic acid amplification, enzymatic conversion of PPi released during DNA synthesis into ATP is continuously monitored through the bioluminescence generated by thermostable firefly luciferase. The assay shows a unique kinetic signature for nucleic acid amplifications with a readily identifiable light output peak, whose timing is proportional to the concentration of original target nucleic acid. This allows qualitative and quantitative analysis of specific targets, and readily differentiates between negative and positive samples. Since quantitation in BART is based on determination of time-to-peak rather than absolute intensity of light emission, complex or highly sensitive light detectors are not required. CONCLUSIONS: The combined chemistries of the BART reporter and amplification require only a constant temperature maintained by a heating block and are shown to be robust in the analysis of clinical samples. Since monitoring the BART reaction requires only a

  14. Detection of FLT3 Oncogene Mutations in Acute Myeloid Leukemia Using Conformation Sensitive Gel Electrophoresis

    OpenAIRE

    2008-01-01

    FLT3 (fms-related tyrosine kinase 3) is a receptor tyrosine kinase class III that is expressed on by early hematopoietic progenitor cells and plays an important role in hematopoietic stem cell proliferation, differentiation and survival. FLT3 is also expressed on leukemia blasts in most cases of acute myeloid leukemia (AML). In order to determine the frequency of FLT3 oncogene mutations, we analyzed genomic DNA of adult de novo acute myeloid leukemia (AML). Polymerase chain reaction (PCR) and...

  15. Bioengineering bacteriophages to enhance the sensitivity of phage amplification-based paper fluidic detection of bacteria.

    Science.gov (United States)

    Alcaine, S D; Law, K; Ho, S; Kinchla, A J; Sela, D A; Nugen, S R

    2016-08-15

    Bacteriophage (phage) amplification is an attractive method for the detection of bacteria due to a narrow phage-host specificity, short amplification times, and the phages' ability to differentiate between viable and non-viable bacterial cells. The next step in phage-based bacteria detection is leveraging bioengineered phages to create low-cost, rapid, and easy-to-use detection platforms such as lateral flow assays. Our work establishes the proof-of-concept for the use of bioengineered T7 phage strains to increase the sensitivity of phage amplification-based lateral flow assays. We have demonstrated a greater than 10-fold increase in sensitivity using a phage-based protein reporter, maltose-binding protein, over the detection of replicated T7 phage viron itself, and a greater then 100-fold increase in sensitivity using a phage-based enzymatic reporter, alkaline phosphatase. This increase in sensitivity enabled us to detect 10(3)CFU/mL of Escherichia coli in broth after 7h, and by adding a filter concentration step, the ability to detect a regulatory relevant E. coli concentration of 100CFU/100mL in inoculated river water after 9h, where the current standard requires days for results. The combination of the paper fluidic format with phage-based detection provides a platform for the development of novel diagnostics that are sensitive, rapid, and easy to use.

  16. Rapid detection of Brucella spp. using loop-mediated isothermal amplification (LAMP).

    Science.gov (United States)

    Chen, Shouyi; Li, Xunde; Li, Juntao; Atwill, Edward R

    2013-01-01

    Brucella spp. are facultative intracellular bacteria that cause zoonotic disease of brucellosis worldwide. Livestock that are most vulnerable to brucellosis include cattle, goats, and pigs. Brucella spp. cause serious health problems to humans and animals and economic losses to the livestock industry. Traditional methods for detection of Brucella spp. take 48-72 h (Kumar et al., J Commun Dis 29:131-137, 1997; Barrouin-Melo et al., Res Vet Sci 83:340-346, 2007) that do not meet the food industry's need of rapid detection. Therefore, there is an urgent need of fast, specific, sensitive, and inexpensive method for diagnosing of Brucella spp. Loop-mediated isothermal amplification (LAMP) is a method to amplify nucleic acid at constant temperatures. Amplification can be detected by visual detection, fluorescent stain, turbidity, and electrophoresis. We targeted at the Brucella-specific gene omp25 and designed LAMP primers for detection of Brucella spp. Amplification of DNA with Bst DNA polymerase can be completed at 65 °C in 60 min. Amplified products can be detected by SYBR Green I stain and 2.0% agarose gel electrophoresis. The LAMP method is feasible for detection of Brucella spp. from blood and milk samples.

  17. Electrochemical genosensor for the rapid detection of GMO using loop-mediated isothermal amplification.

    Science.gov (United States)

    Ahmed, Minhaz Uddin; Saito, Masato; Hossain, M Mosharraf; Rao, S Ramachandara; Furui, Satoshi; Hino, Akihiro; Takamura, Yuzuru; Takagi, Masahiro; Tamiya, Eiichi

    2009-05-01

    In this study, we are reporting for the first time an efficient, accurate and inexpensive rapid detection system which employs the integration of isothermal amplification and subsequent analysis of unpurified amplicons by an electrochemical system. In our experiments, loop-mediated isothermal amplification (LAMP) with its higher efficiency than PCR was performed at a constant temperature (65 degrees C). Amplification products were combined with a redox active molecule Hoechst 33258 [H33258, 2'-(4-hydroxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5'-bi(1H-benzimidazole)] and analyzed by a DNA stick (DS) which is integrated with a disposable electrochemical printed (DEP) chip using linear sweep voltammetry (LSV). The DNA minor groove binding of the H33258 molecule causes a significant drop in the peak current intensity of the H33258 oxidation. The phenomenon of DNA binding induced by H33258, in addition to changes in the anodic current peak, was used to detect maize CBH 351 variety (StarLink). Since laborious probe immobilization was not required, and amplification and detection were performed on a single device, our biosensor eliminates potential cross-contamination. We believe that this type of sensor will have an unprecedented impact for environmental protection.

  18. Detection of genetically modified organisms (GMOs using isothermal amplification of target DNA sequences

    Directory of Open Access Journals (Sweden)

    La Mura Maurizio

    2009-02-01

    Full Text Available Abstract Background The most common method of GMO detection is based upon the amplification of GMO-specific DNA amplicons using the polymerase chain reaction (PCR. Here we have applied the loop-mediated isothermal amplification (LAMP method to amplify GMO-related DNA sequences, 'internal' commonly-used motifs for controlling transgene expression and event-specific (plant-transgene junctions. Results We have tested the specificity and sensitivity of the technique for use in GMO studies. Results show that detection of 0.01% GMO in equivalent background DNA was possible and dilutions of template suggest that detection from single copies of the template may be possible using LAMP. Conclusion This work shows that GMO detection can be carried out using LAMP for routine screening as well as for specific events detection. Moreover, the sensitivity and ability to amplify targets, even with a high background of DNA, here demonstrated, highlights the advantages of this isothermal amplification when applied for GMO detection.

  19. Detection of telomerase activity by combination of telomeric repeat amplification protocol and electrochemiluminescence assay

    Institute of Scientific and Technical Information of China (English)

    Xiao Ming Zhou; Li Jia

    2008-01-01

    A highly sensitive telomerase detection method that combines telomeric repeat amplification protocol (TRAP) and magnetic beads based electrochemiluminescence (ECL) assay has been developed. Briefly, telomerase recognizes biotinylated telomerase synthesis primer (B-TS) and synthesizes extension products, which then serve as the templates for PCR amplification using B-TS as the forward primer and Iris-(2'2'-bipyridyl) ruthenium (TBR) labeled ACX (TBR-ACX) as the reversed primer. The amplified product is captured on streptavidin-coated paramagnetic beads and detected by ECL. Telomerase positive HeLa cells were used to validate the feasibility of the method. The experimental results showed down to 10 cancer cells can be detected easily. The method is a useful tool for telomerase activity analysis due to its sensitivity, rapidity, safety, high throughput, and low cost. It can be used for screening a large amount of clinical samples.

  20. Use of signal-mediated amplification of RNA technology (SMART) to detect marine cyanophage DNA.

    Science.gov (United States)

    Hall, M J; Wharam, S D; Weston, A; Cardy, D L N; Wilson, W H

    2002-03-01

    Here, we describe the application of an isothermal nucleic acid amplification assay, signal-mediated amplification of RNA technology (SMART), to detect DNA extracted from marine cyanophages known to infect unicellular cyanobacteria from the genus Synechococcus. The SMART assay is based on the target-dependent production of multiple copies of an RNA signal, which is measured by an enzyme-linked oligosorbent assay. SMART was able to detect both synthetic oligonucleotide targets and genomic cyanophage DNA using probes designed against the portal vertex gene (g20). Specific signals were obtained for each cyanophage strain (S-PM2 and S-BnMI). Nonspecific genomic DNA did not produce false signals or inhibit the detection of a specific target. In addition, we found that extensive purification of target DNA may not be required since signals were obtained from crude cyanophage lysates. This is the first report of the SMART assay being used to discriminate between two similar target sequences.

  1. FLUORESCENCE IN SITU HYBRIDIZATION COMBINED WITH IMMUNOFLUORESCENT STAINING FOR RAPID DETECTION OF Nmyc AMPLIFICATION IN NEUROBLASTOMA

    Institute of Scientific and Technical Information of China (English)

    WANG Wei王伟; Marianne Ifversen; ZHAO Chun-ting赵春亭; WANG Hong-yi汪洪毅; ZHAO Hong-guo赵洪国

    2004-01-01

    Objective: To establish a method to improve the detection of disseminated tumor cells in bone marrow and peripheral blood samples of neuroblastoma patients and analysis of cytogenetic aberration. Methods: Immunofluorescent staining was performed using a cocktail of primary monoclonal neuroblastoma antibodies (14.G2a, 5.1H11). Fluorescence in situ hybridization was applied with fluorescent probes specific for Nmyc genes afterwards. A novel computer assisted scanning system for automatic search, image analysis and repositioning of these positive cells was developed. Fifty-six bone marrow and peripheral blood samples from 7 patients were evaluated by this method. Results: Fluorescence in situ hybridization can be combined with immunofluorescent staining in detecting Nmyc amplification in neuroblastoma patients. Fluorescence in situ hybridization results correlated well with data obtained by conventional cytogenetic procedures. Conclusion: The technique described allows search of tumor cells in the bone marrow as well as detection of Nmyc amplification in interphase nuclei.

  2. A simple molecular beacon with duplex-specific nuclease amplification for detection of microRNA.

    Science.gov (United States)

    Li, Yingcun; Zhang, Jiangyan; Zhao, Jingjing; Zhao, Likun; Cheng, Yongqiang; Li, Zhengping

    2016-02-01

    MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene activity, promoting or inhibiting cell proliferation, migration and apoptosis. Abnormal expression of miRNAs is associated with many diseases. Therefore, it is essential to establish a simple, rapid and sensitive miRNA detection method. In this paper, based on a simple molecular beacon (MB) and duplex-specific nuclease (DSN), we developed a target recycling amplification method for miRNA detection. By controlling the number of stem bases to 5, the MB probe used in this method can be prevented from hydrolysis by DSN without special modification. This assay is direct and simple to quantitatively detect miRNA with high sensitivity and specificity. The MB probe design provides a new strategy for nuclease-based amplification reaction.

  3. Optofluidic analysis system for amplification-free, direct detection of Ebola infection

    Science.gov (United States)

    Cai, H.; Parks, J. W.; Wall, T. A.; Stott, M. A.; Stambaugh, A.; Alfson, K.; Griffiths, A.; Mathies, R. A.; Carrion, R.; Patterson, J. L.; Hawkins, A. R.; Schmidt, H.

    2015-09-01

    The massive outbreak of highly lethal Ebola hemorrhagic fever in West Africa illustrates the urgent need for diagnostic instruments that can identify and quantify infections rapidly, accurately, and with low complexity. Here, we report on-chip sample preparation, amplification-free detection and quantification of Ebola virus on clinical samples using hybrid optofluidic integration. Sample preparation and target preconcentration are implemented on a PDMS-based microfluidic chip (automaton), followed by single nucleic acid fluorescence detection in liquid-core optical waveguides on a silicon chip in under ten minutes. We demonstrate excellent specificity, a limit of detection of 0.2 pfu/mL and a dynamic range of thirteen orders of magnitude, far outperforming other amplification-free methods. This chip-scale approach and reduced complexity compared to gold standard RT-PCR methods is ideal for portable instruments that can provide immediate diagnosis and continued monitoring of infectious diseases at the point-of-care.

  4. Ultrasensitive detection of microRNAs based on hairpin fluorescence probe assisted isothermal amplification.

    Science.gov (United States)

    Ma, Cuiping; Liu, Sen; Shi, Chao

    2014-08-15

    A hairpin fluorescence probe assisted isothermal amplification strategy was used for microRNAs (miRNAs) detection. The fluorescence hairpin probe was rationally designed by software NUPACK to reduce background signal. This isothermal amplification method consisted of two circuits. The amplification strategy not only could detect miRNA, but also amplified and reversely transcribed miRNA into DNA to enhance the stability of the target. The approach was ultrasensitive and as low as 8.5×10(-15)mol/L miR-Let-7a, corresponding to 8.5×10(-20)mol miR-Let-7a in 10µL, was able to be detected within 20min at 37°C. Moreover, successful detection of miR-Let-7a in a total RNA sample was also achieved. Thus, the rapid, simple, isothermal, and highly sensitive approach should be a promising tool for on-the-spot detection.

  5. Rapid detection of Bacillus anthracis by γ phage amplification and lateral flow immunochromatography.

    Science.gov (United States)

    Cox, Christopher R; Jensen, Kirk R; Mondesire, Roy R; Voorhees, Kent J

    2015-11-01

    New, rapid point-of-need diagnostic methods for Bacillus anthracis detection can enhance civil and military responses to accidental or deliberate dispersal of anthrax as a biological weapon. Current laboratory-based methods for clinical identification of B. anthracis require 12 to 120h, and are confirmed by plaque assay using the well-characterized γ typing phage, which requires an additional minimum of 24h for bacterial culture. To reduce testing time, the natural specificity of γ phage amplification was investigated in combination with lateral flow immunochromatography (LFI) for rapid, point-of-need B. anthracis detection. Phage-based LFI detection of B. anthracis Sterne was validated over a range of bacterial and phage concentrations with optimal detection achieved in as little as 2h from the onset of amplification with a threshold sensitivity of 2.5×10(4)cfu/mL. The novel use of γ phage amplification detected with a simple, inexpensive LFI assay provides a rapid, sensitive, highly accurate, and field-deployable method for diagnostic ID of B. anthracis in a fraction of the time required by conventional techniques, and without the need for extensive laboratory culture.

  6. Three different signal amplification strategies for the impedimetric sandwich detection of thrombin

    Energy Technology Data Exchange (ETDEWEB)

    Ocaña, Cristina; Valle, Manel del, E-mail: manel.delvalle@uab.cat

    2016-03-17

    In this work, we report a comparative study on three highly specific amplification strategies for the ultrasensitive detection of thrombin with the use of aptamer sandwich protocol. The protocol consisted on the use of a first thrombin aptamer immobilized on the electrode surface, the recognition of thrombin protein, and the reaction with a second biotinylated thrombin aptamer forming the sandwich. Through the exposed biotin end, three variants have been tested to amplify the electrochemical impedance signal. The strategies included (a) silver enhancement treatment, (b) gold enhancement treatment and (c) insoluble product produced by the combination of the enzyme horseradish peroxidase (HRP) and 3-amino-9-ethylcarbazole (AEC). The properties of the sensing surface were probed by electrochemical impedance measurements in the presence of the ferrocyanide/ferricyanide redox marker. Insoluble product strategy and silver enhancement treatment resulted in the lowest detection limit (0.3 pM), while gold enhancement method resulted in the highest reproducibility, 8.8% RSD at the pM thrombin concentration levels. Results of silver and gold enhancement treatment also permitted direct inspection by scanning electron microscopy (SEM). - Highlights: • Aptasensor to detect thrombin reaching the femtomolar level. • Biosensing protocol employs two thrombin aptamers in a sandwich capture scheme. • Use of second biotinylated aptamer allows many amplification and detection variants. • Precipitation reaction provides the highest signal amplification of ca. 3 times. • Double recognition event improves remarkably selectivity for thrombin detection.

  7. Oncogene abnormalities in a series of primary melanomas of the sinonasal tract: NRAS mutations and cyclin D1 amplification are more frequent than KIT or BRAF mutations.

    Science.gov (United States)

    Chraybi, Meriem; Abd Alsamad, Issam; Copie-Bergman, Christiane; Baia, Maryse; André, Jocelyne; Dumaz, Nicolas; Ortonne, Nicolas

    2013-09-01

    Primary malignant melanoma of sinonasal tract is a rare but severe form of melanoma. We retrospectively analyzed 17 cases and focused on the histologic presentation and the expression of c-Kit, epidermal growth factor receptor (EGFR), cyclin D1/Bcl-1, PS100, and HMB45 and searched for BRAF, NRAS, and KIT mutations that are known to be associated with melanoma subtypes, together with amplifications of KIT, cyclin D1, cyclin-dependent kinase 4, MDM2, and microphthalmia-associated transcription factor using quantitative polymerase chain reaction. In most cases (78%), an in situ component was evidenced. Invasive components were composed of diffuse areas of rhabdoid, epithelioid, or spindle cells and, in most cases, lacked inflammatory reaction, suggesting that an immune escape phenomenon probably develops when the disease progresses. EGFR was rarely and weakly expressed in the in situ component of 2 cases. None of the investigated case showed BRAF V600E, but 1 had a D594G mutation. NRAS mutations in exon 2 (G12D or G12A) were found in 3 cases (18%), and a KIT mutation in exon 11 (L576P), in 1, whereas c-Kit was expressed at the protein level in half of the cases. Amplifications of cyclin D1 were evidenced in 5 cases, confirmed in 3 by fluorescence in situ hybridization, but this was not always correlated with protein expression, found in 8 patients (62.5%), 3 having no significant amplification. In conclusion, primary malignant melanoma of sinonasal tract is not associated with BRAF V600E mutations. Instead, NRAS or KIT mutations and cyclin D1 amplification can be found in a proportion of cases, suggesting that primary malignant melanoma of sinonasal tract is heterogeneous at the molecular level and should not be sensitive to therapeutic approaches aiming at BRAF.

  8. Peripheral position of CCND1 and HER-2/neu oncogenes within chromosome territories in esophageal and gastric cancers non-related to amplification and overexpression

    Directory of Open Access Journals (Sweden)

    Lucimari Bizari

    2009-01-01

    Full Text Available Interphase chromosomes have been shown to occupy discrete regions of the nucleus denominated chromosome territories (CTs, their active genes being preferentially positioned on the surfaces of these CTs, where they are accessible to transcriptional machinery. By means of FISH (Fluorescence in situ Hybridization, we analyzed the CCND1 and HER-2/neu gene positions within the CTs and their relationship with gene amplification and protein over-expression in esophageal and gastric cancers. The CCND1 and HER-2/Neu genes were more often positioned at the periphery (mean frequency of 60%-83% of the CTs in tumor tissues of the esophagus and stomach. Moreover, this positioning revealed no association with either gene amplification or the protein over-expression status of these genes, although, in esophageal carcinoma, Kappa statistics showed a moderate agreement between amplification of the CCND1 gene (Kappa = 0.400 and its location within the CT, as well as with over-expression of the corresponding protein (Kappa = 0.444. Thus, our results suggest that gene positioning in interphase chromosomes does not follow a definitive pattern neither does it depend only on gene transcriptional activity. Apparently, this positioning could be both gene- and tissue-specific, and depends on other factors acting together, such as dense-gene, chromosome size, chromatin structure, and the level and stability of its expression.

  9. Sensitive detection of Escherichia coli O157:H7 based on cascade signal amplification in ELISA.

    Science.gov (United States)

    Shan, Shan; Liu, Daofeng; Guo, Qi; Wu, Songsong; Chen, Rui; Luo, Kai; Hu, Liming; Xiong, Yonghua; Lai, Weihua

    2016-09-01

    In this study, cascade signal amplification in ELISA involving double-antibody sandwich ELISA and indirectly competitive ELISA was established to sensitively detect Escherichia coli O157:H7. In the double-antibody sandwich ELISA, a complex was formed comprising anti-E. coli O157:H7 polyclonal antibody, E. coli O157:H7, biotinylated anti-E. coli O157:H7 monoclonal antibody, streptavidin, and biotinylated β-lactamase. Penicillin solution was then added into the ELISA well and hydrolyzed by β-lactamase. Afterward, the penicillin solution was transferred to indirectly competitive ELISA. The concentration of penicillin can be sensitively detected in indirectly competitive ELISA. In the cascade signal amplification system, increasing the amount of added E. coli O157:H7 resulted in more β-lactamase and less penicillin. The detection sensitivity of E. coli O157:H7, which was 20cfu/mL with the cascade signal amplification in ELISA, was 1,000-fold higher than that of traditional ELISA. Furthermore, the novel method can be used to detect E. coli O157:H7 in milk (2cfu/g). Therefore, this new signaling strategy will facilitate analyses of highly sensitive foodborne pathogens.

  10. An exo probe-based recombinase polymerase amplification assay for the rapid detection of porcine parvovirus.

    Science.gov (United States)

    Wang, Jian-Chang; Liu, Li-Bing; Han, Qing-An; Wang, Jin-Feng; Yuan, Wan-Zhe

    2017-10-01

    Recombinase polymerase amplification (RPA), an isothermal amplification technology, has been developed as an alternative to PCR in pathogen detection. A real-time RPA assay (rt-RPA) was developed to detect the porcine parvovirus (PPV) using primers and exo probe specific for the VP2 gene. The amplification was performed at 39°C for 20min. There was no cross-reaction with other pathogens tested. Using the recombinant plasmid pPPV-VP2 as template, the analytical sensitivity was 103 copies. The assay performance was evaluated by testing 115 field samples by rt-RPA and a real-time PCR assay. The diagnostic agreement between assays was 100%, and PPV DNA was detected in 94 samples. The R(2) value of rt-RPA and real-time PCR was 0.909 by linear regression analysis. The developed rt-RPA assay provides a useful alternative tool for rapid, simple and reliable detection of PPV in diagnostic laboratories and at point-of-care, especially in remote and rural areas. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Label- and amplification-free electrochemical detection of bacterial ribosomal RNA.

    Science.gov (United States)

    Henihan, Grace; Schulze, Holger; Corrigan, Damion K; Giraud, Gerard; Terry, Jonathan G; Hardie, Alison; Campbell, Colin J; Walton, Anthony J; Crain, Jason; Pethig, Ronald; Templeton, Kate E; Mount, Andrew R; Bachmann, Till T

    2016-07-15

    Current approaches to molecular diagnostics rely heavily on PCR amplification and optical detection methods which have restrictions when applied to point of care (POC) applications. Herein we describe the development of a label-free and amplification-free method of pathogen detection applied to Escherichia coli which overcomes the bottleneck of complex sample preparation and has the potential to be implemented as a rapid, cost effective test suitable for point of care use. Ribosomal RNA is naturally amplified in bacterial cells, which makes it a promising target for sensitive detection without the necessity for prior in vitro amplification. Using fluorescent microarray methods with rRNA targets from a range of pathogens, an optimal probe was selected from a pool of probe candidates identified in silico. The specificity of probes was investigated on DNA microarray using fluorescently labeled 16S rRNA target. The probe yielding highest specificity performance was evaluated in terms of sensitivity and a LOD of 20 pM was achieved on fluorescent glass microarray. This probe was transferred to an EIS end point format and specificity which correlated to microarray data was demonstrated. Excellent sensitivity was facilitated by the use of uncharged PNA probes and large 16S rRNA target and investigations resulted in an LOD of 50 pM. An alternative kinetic EIS assay format was demonstrated with which rRNA could be detected in a species specific manner within 10-40min at room temperature without wash steps.

  12. Bioagent detection using miniaturized NMR and nanoparticle amplification : final LDRD report.

    Energy Technology Data Exchange (ETDEWEB)

    Clewett, C. F. M.; Adams, David Price; Fan, Hongyou; Williams, John D.; Sillerud, Laurel O.; Alam, Todd Michael; Aldophi, Natalie L. (New Mexico Resonance, Albuquerque, NM); McDowell, Andrew F. (New Mexico Resonance, Albuquerque, NM)

    2006-11-01

    This LDRD program was directed towards the development of a portable micro-nuclear magnetic resonance ({micro}-NMR) spectrometer for the detection of bioagents via induced amplification of solvent relaxation based on superparamagnetic nanoparticles. The first component of this research was the fabrication and testing of two different micro-coil ({micro}-coil) platforms: namely a planar spiral NMR {micro}-coil and a cylindrical solenoid NMR {micro}-coil. These fabrication techniques are described along with the testing of the NMR performance for the individual coils. The NMR relaxivity for a series of water soluble FeMn oxide nanoparticles was also determined to explore the influence of the nanoparticle size on the observed NMR relaxation properties. In addition, The use of commercially produced superparamagnetic iron oxide nanoparticles (SPIONs) for amplification via NMR based relaxation mechanisms was also demonstrated, with the lower detection limit in number of SPIONs per nanoliter (nL) being determined.

  13. Rapid and sensitive detection of canine parvovirus type 2 by recombinase polymerase amplification.

    Science.gov (United States)

    Wang, Jianchang; Liu, Libing; Li, Ruiwen; Wang, Jinfeng; Fu, Qi; Yuan, Wanzhe

    2016-04-01

    A novel recombinase polymerase amplification (RPA)-based method for detection of canine parvovirus type 2 (CPV-2) was developed. Sensitivity analysis showed that the detection limit of RPA was 10 copies of CPV-2 genomic DNA. RPA amplified both CPV-2a and -2b DNA but did not amplify the template of other important dog viruses (CCoV, PRV or CDV), demonstrating high specificity. The method was further validated with 57 canine fecal samples. An outstanding advantage of RPA is that it is an isothermal reaction and can be performed in a water bath, making RPA a potential alternative method for CPV-2 detection in resource-limited settings.

  14. Direct detection of potato leafroll virus in potato tubers by immunocapture and the isothermal nucleic acid amplification method NASBA

    NARCIS (Netherlands)

    Leone, G.; Schijndel, van H.B.; Gemen, van B.; Schoen, C.D.

    1997-01-01

    NASBA, an isothermal amplification method for nucleic acids, was applied to the detection of RNA of potato leafroll virus (PLRV) in a single enzymatic reaction at 41 °C. A set of primers was selected from the coat protein open reading frame sequence of PLRV to allow amplification of viral RNA. The

  15. Detection of HIV cDNA Point Mutations with Rolling-Circle Amplification Arrays

    Directory of Open Access Journals (Sweden)

    Zhongwei Wu

    2010-01-01

    Full Text Available In this paper we describe an isothermal rolling-circle amplification (RCA protocol to detect gene point mutations on chips. The method is based on an allele-specific oligonucleotide circularization mediated by a special DNA ligase. The probe is circularized when perfect complementary sequences between the probe oligonucleotide and HIV cDNA gene. Mismatches around the ligation site can prevent probe circularization. The circularized probe (C-probe can be amplified by rolling circle amplification to generate multimeric singlestranded DNA (ssDNA under isothermal conditions. There are four sequence regions to bind respectively with fluorescent probe, RCA primer, solid probe and HIV cDNA template in the C-probe which we designed. These ssDNA products are hybridized with fluorescent probes and solid probes which are immobilized on a glass slide composing a regular microarray pattern. The fluorescence signals can be monitored by a scanner in the presence of HIV cDNA templates, whereas the probes cannot be circularized and signal of fluorescence cannot be found. The RCA array has capability of high-throughput detection of the point mutation and the single-nucleotide polymorphism (SNP.The development of C-probe-based technologies offers a promising prospect for situ detection, microarray, molecular diagnosis, single nucleotide polymorphism, and whole genome amplification.

  16. Chemiluminescence imaging for microRNA detection based on cascade exponential isothermal amplification machinery.

    Science.gov (United States)

    Xu, Yongjie; Li, Dandan; Cheng, Wei; Hu, Rong; Sang, Ye; Yin, Yibing; Ding, Shijia; Ju, Huangxian

    2016-09-14

    A novel G-quadruplex DNAzyme-driven chemiluminescence (CL) imaging method was developed for ultrasensitive and specific detection of miRNA based on the cascade exponential isothermal amplification reaction (EXPAR) machinery. A structurally tailored hairpin probe switch was designed to selectively recognise miRNA and form hybridisation products to trigger polymerase and nicking enzyme machinery, resulting in the generation of product I, which was complementary to a region of the functional linear template. Then, the response of the functional linear template to the generated product I further activated the exponential isothermal amplification machinery, leading to synthesis of numerous horseradish peroxidase mimicking DNAzyme units for CL signal transduction. The amplification paradigm generated a linear response from 10 fM to 100 pM, with a low detection limit of 2.91 fM, and enabled discrimination of target miRNA from a single-base mismatched target. The developed biosensing platform demonstrated the advantages of isothermal, homogeneous, visual detection for miRNA assays, offering a promising tool for clinical diagnosis.

  17. Rapid and visual detection of Listeria monocytogenes based on nanoparticle cluster catalyzed signal amplification.

    Science.gov (United States)

    Zhang, Lisha; Huang, Ru; Liu, Weipeng; Liu, Hongxing; Zhou, Xiaoming; Xing, Da

    2016-12-15

    Foodborne pathogens pose a significant threat to human health worldwide. The identification of foodborne pathogens needs to be rapid, accurate and convenient. Here, we constructed a nanoparticle cluster (NPC) catalyzed signal amplification biosensor for foodborne pathogens visual detection. In this work, vancomycin (Van), a glycopeptide antibiotic for Gram-positive bacteria, was used as the first molecular recognition agent to capture Listeria monocytogenes (L. monocytogenes). Fe3O4 NPC modified aptamer, was used as the signal amplification nanoprobe, specifically recognize to the cell wall of L. monocytogenes. As vancomycin and aptamer recognize L. monocytogenes at different sites, the sandwich recognition showed satisfied specificity. Compared to individual Fe3O4 nanoparticle (NP), NPC exhibit collective effect-enhanced catalytic activity for the color reaction of chromogenic substrate. The change in absorbance or color could represent the concentration of target. Using the Fe3O4 NPC-based signal amplification method, L. monocytogenes whole cells could be directly assayed within a linear range of 5.4×10(3)-10(8) cfu/mL and a visual limit of detection of 5.4×10(3) cfu/mL. Fe3O4 NPC-based method was more sensitive than the Fe3O4 NP-based method. All these attractive characteristics of highly sensitivity, visual and labor-saving, make the biosensor possess a potential application for foodborne pathogenic bacteria detection. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Detection of HIV cDNA point mutations with rolling-circle amplification arrays.

    Science.gov (United States)

    Wu, Lingwei; Liu, Quanjun; Wu, Zhongwei; Lu, Zuhong

    2010-01-27

    In this paper we describe an isothermal rolling-circle amplification (RCA) protocol to detect gene point mutations on chips. The method is based on an allele-specific oligonucleotide circularization mediated by a special DNA ligase. The probe is circularized when perfect complementary sequences between the probe oligonucleotide and HIV cDNA gene. Mismatches around the ligation site can prevent probe circularization. The circularized probe (C-probe) can be amplified by rolling circle amplification to generate multimeric singlestranded DNA (ssDNA) under isothermal conditions. There are four sequence regions to bind respectively with fluorescent probe, RCA primer, solid probe and HIV cDNA template in the C-probe which we designed. These ssDNA products are hybridized with fluorescent probes and solid probes which are immobilized on a glass slide composing a regular microarray pattern. The fluorescence signals can be monitored by a scanner in the presence of HIV cDNA templates, whereas the probes cannot be circularized and signal of fluorescence cannot be found. The RCA array has capability of high-throughput detection of the point mutation and the single-nucleotide polymorphism (SNP).The development of C-probe-based technologies offers a promising prospect for situ detection, microarray, molecular diagnosis, single nucleotide polymorphism, and whole genome amplification.

  19. Label-free and highly sensitive electrochemical detection of E. coli based on rolling circle amplifications coupled peroxidase-mimicking DNAzyme amplification.

    Science.gov (United States)

    Guo, Yuna; Wang, Yu; Liu, Su; Yu, Jinghua; Wang, Hongzhi; Wang, Yalin; Huang, Jiadong

    2016-01-15

    In this work, a simple, label-free, low cost electrochemical biosensor for highly sensitive and selective detection of Escherichia coli has been developed on the basis of rolling circle amplification (RCA) coupled peroxidase-mimicking DNAzyme amplification. A aptamer-primer probe (APP) containing anti-E. coli aptamer and a primer sequence complementary to a circular probe, which includes two G-quadruplex units, is used for recognizing target and triggering RCA-based polymerase elongation. Due to RCA coupled DNAzyme amplification strategy, the presence of target E. coli leads to the formation of numerous G-quadruplex oligomers on electrode, which folds into G-quadruplex/hemin complexs with the help of K(+) and hemin, thus generating extremely strong catalytic activity toward H2O2 and giving a remarkably strong electrochemical response. As far as we know, this work is the first time that RCA coupled peroxidase-mimicking DNAzyme amplification technique have been integrated into electrochemical assay for detecting pathogenic bacteria. Under optimal conditions, the proposed biosensor exhibits ultrahigh sensitivity toward E. coli with detection limits of 8cfumL(-1) and a detection range of 5 orders of magnitude. Besides, our biosensor also shows high selectivity toward target E. coli and has the advantages in its rapidness, low cost, simplified operations without the need of electrochemical labeling steps and additional labile reagents. Hence, the RCA coupled peroxidase-mimicking DNAzyme amplification-based electrochemical method might create a useful and practical platform for detecting E. coli and related food safety analysis and clinical diagnosis. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. Sensitive Method for Biomolecule Detection Utilizing Signal Amplification with Porphyrin Nanoparticles.

    Science.gov (United States)

    Gibson, Lauren E; Wright, David W

    2016-06-07

    Disease diagnosis requires identification of biomarkers that occur in small quantities, making detection a difficult task. Effective diagnosis is an even greater challenge in low-resource areas of the world. Methods must be simple, stable, and sensitive so that tests can be easily administered and withstand uncontrolled environmental conditions. One approach to this issue is development of stable signal amplification strategies. In this work, we applied the nanocrystal-based signal amplification method to tetra(4-carboxyphenyl)porphyrin nanoparticles (TCPP NPs). The dissolution of the nanoparticle into thousands of porphyrin molecules results in amplified detection of the biomarker. By using nanoparticles as the signal-generating moiety, stability of the detection method is increased relative to commonly used enzyme-based assays. Additionally, the inherent fluorescent signal of TCPP molecules can be measured after nanoparticle dissolution. The ability to directly read the TCPP fluorescent signal increases assay simplicity by reducing the steps required for the test. This detection method was optimized by detecting rabbit IgG and then was applied to the detection of the malarial biomarker Plasmodium falciparum histidine-rich protein II (pfHRPII) from a complex matrix. The results for both biomarkers were assays with low picomolar limits of detection.

  1. Detection of Mycobacterium ulcerans by the loop mediated isothermal amplification method.

    Directory of Open Access Journals (Sweden)

    Anthony Ablordey

    Full Text Available BACKGROUND: Buruli ulcer (BU caused by Mycobacterium ulcerans (M. ulcerans has emerged as an important public health problem in several rural communities in sub-Saharan Africa. Early diagnosis and prompt treatment are important in preventing disfiguring complications associated with late stages of the disease progression. Presently there is no simple and rapid test that is appropriate for early diagnosis and use in the low-resource settings where M. ulcerans is most prevalent. METHODOLOGY: We compared conventional and pocket warmer loop mediated isothermal amplification (LAMP methods (using a heat block and a pocket warmer respectively as heat source for amplification reaction for the detection of M. ulcerans in clinical specimens. The effect of purified and crude DNA preparations on the detection rate of the LAMP assays were also investigated and compared with that of IS2404 PCR, a reference assay for the detection of M. ulcerans. Thirty clinical specimens from suspected BU cases were examined by LAMP and IS2404 PCR. PRINCIPAL FINDINGS: The lower detection limit of both LAMP methods at 60°C was 300 copies of IS2404 and 30 copies of IS2404 for the conventional LAMP at 65°C. When purified DNA extracts were used, both the conventional LAMP and IS2404 PCR concordantly detected 21 positive cases, while the pocket warmer LAMP detected 19 cases. Nine of 30 samples were positive by both the LAMP assays as well as IS2404 PCR when crude extracts of clinical specimens were used. CONCLUSION/SIGNIFICANCE: The LAMP method can be used as a simple and rapid test for the detection of M. ulcerans in clinical specimens. However, obtaining purified DNA, as well as generating isothermal conditions, remains a major challenge for the use of the LAMP method under field conditions. With further improvement in DNA extraction and amplification conditions, the pwLAMP could be used as a point of care diagnostic test for BU.

  2. Intrachromosomal amplification of chromosome 21 (iAMP21 detected by ETV6/RUNX1 FISH screening in childhood acute lymphoblastic leukemia: a case report

    Directory of Open Access Journals (Sweden)

    Daniela Ribeiro Ney Garcia

    2013-01-01

    Full Text Available Chromosome abnormalities that usually define high-risk acute lymphoblastic leukemia are the t(9;22/ breakpoint cluster region protein-Abelson murine leukemia viral oncogene homolog 1, hypodiploid with < 44 chromosomes and 11q23/ myeloid/lymphoid leukemia gene rearrangements. The spectrum of acute lymphoblastic leukemia genetic abnormalities is nevertheless rapidly expanding. Therefore, newly described chromosomal aberrations are likely to have an impact on clinical care in the near future. Recently, the rare intrachromosomal amplification of chromosome 21 started to be considered a high-risk chromosomal abnormality. It occurs in approximately 2-5% of pediatric patients with B-cell precursor acute lymphoblastic leukemia. This abnormality is associated with a poor outcome. Hence, an accurate detection of this abnormality is expected to become very important in the choice of appropriate therapy. In this work the clinical and molecular cytogenetic evaluation by fluorescence in situ hybridization of a child with B-cell precursor acute lymphoblastic leukemia presenting the rare intrachromosomal amplification of chromosome 21 is described.

  3. An accurate assay for HCV based on real-time fluorescence detection of isothermal RNA amplification.

    Science.gov (United States)

    Wu, Xuping; Wang, Jianfang; Song, Jinyun; Li, Jiayan; Yang, Yongfeng

    2016-09-01

    Hepatitis C virus (HCV) is one of the common reasons of liver fibrosis and hepatocellular carcinoma (HCC). Early, rapid and accurate HCV RNA detection is important to prevent and control liver disease. A simultaneous amplification and testing (SAT) assay, which is based on isothermal amplification of RNA and real-time fluorescence detection, was designed to optimize routine HCV RNA detection. In this study, HCV RNA and an internal control (IC) were amplified and analyzed simultaneously by SAT assay and detection of fluorescence using routine real-time PCR equipment. The assay detected as few as 10 copies of HCV RNA transcripts. We tested 705 serum samples with SAT, among which 96.4% (680/705) showed consistent results compared with routine real-time PCR. About 92% (23/25) discordant samples were confirmed to be same results as SAT-HCV by using a second real-time PCR. The sensitivity and specificity of SAT-HCV assay were 99.6% (461/463) and 100% (242/242), respectively. In conclusion, the SAT assay is an accurate test with a high specificity and sensitivity which may increase the detection rate of HCV. It is therefore a promising tool to diagnose HCV infection.

  4. GMO detection in food and feed through screening by visual loop-mediated isothermal amplification assays.

    Science.gov (United States)

    Wang, Cong; Li, Rong; Quan, Sheng; Shen, Ping; Zhang, Dabing; Shi, Jianxin; Yang, Litao

    2015-06-01

    Isothermal DNA/RNA amplification techniques are the primary methodology for developing on-spot rapid nucleic acid amplification assays, and the loop-mediated isothermal amplification (LAMP) technique has been developed and applied in the detection of foodborne pathogens, plant/animal viruses, and genetically modified (GM) food/feed contents. In this study, one set of LAMP assays targeting on eight frequently used universal elements, marker genes, and exogenous target genes, such as CaMV35S promoter, FMV35S promoter, NOS, bar, cry1Ac, CP4 epsps, pat, and NptII, were developed for visual screening of GM contents in plant-derived food samples with high efficiency and accuracy. For these eight LAMP assays, their specificity was evaluated by testing commercial GM plant events and their limits of detection were also determined, which are 10 haploid genome equivalents (HGE) for FMV35S promoter, cry1Ac, and pat assays, as well as five HGE for CaMV35S promoter, bar, NOS terminator, CP4 epsps, and NptII assays. The screening applicability of these LAMP assays was further validated successfully using practical canola, soybean, and maize samples. The results suggested that the established visual LAMP assays are applicable and cost-effective for GM screening in plant-derived food samples.

  5. Rapid,sensitive detection of Vibrio anguillarum using loop-mediated isothermal amplification

    Institute of Scientific and Technical Information of China (English)

    高宏伟; 李富花; 张晓军; 王兵; 相建海

    2010-01-01

    Vibrio anguillarum is an important bacterial pathogen of aquatic organisms and a significant problem in aquatic farming.The rapid detection and identification of V.anguillarum,and other pathogens that infect marine organisms,is crucial to effective disease management.In this study,we developed a loop-mediated amplification (LAMP) assay to detect V.anguillarum in an hour in a single tube without the need for thermal cycling.Conserved regions of the metalloproteinase (empA) gene of V.anguillarum served as the...

  6. Microarray analysis after RNA amplification can detect pronounced differences in gene expression using limma

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    Orengo Christine

    2006-10-01

    Full Text Available Abstract Background RNA amplification is necessary for profiling gene expression from small tissue samples. Previous studies have shown that the T7 based amplification techniques are reproducible but may distort the true abundance of targets. However, the consequences of such distortions on the ability to detect biological variation in expression have not been explored sufficiently to define the true extent of usability and limitations of such amplification techniques. Results We show that expression ratios are occasionally distorted by amplification using the Affymetrix small sample protocol version 2 due to a disproportional shift in intensity across biological samples. This occurs when a shift in one sample cannot be reflected in the other sample because the intensity would lie outside the dynamic range of the scanner. Interestingly, such distortions most commonly result in smaller ratios with the consequence of reducing the statistical significance of the ratios. This becomes more critical for less pronounced ratios where the evidence for differential expression is not strong. Indeed, statistical analysis by limma suggests that up to 87% of the genes with the largest and therefore most significant ratios (p -20 in the unamplified group have a p-value below 10e-20 in the amplified group. On the other hand, only 69% of the more moderate ratios (10e-20 -10 in the unamplified group have a p-value below 10e-10 in the amplified group. Our analysis also suggests that, overall, limma shows better overlap of genes found to be significant in the amplified and unamplified groups than the Z-scores statistics. Conclusion We conclude that microarray analysis of amplified samples performs best at detecting differences in gene expression, when these are large and when limma statistics are used.

  7. Rapid and sensitive detection of Listeria monocytogenes by loop-mediated isothermal amplification.

    Science.gov (United States)

    Tang, Meng-Jun; Zhou, Sheng; Zhang, Xiao-Yan; Pu, Jun-Hua; Ge, Qing-Lian; Tang, Xiu-Jun; Gao, Yu-Shi

    2011-12-01

    Loop-mediated isothermal amplification (LAMP) was designed for detection of Listeria monocytogenes, which is an important food-borne kind of pathogenic bacteria causing human and animal disease. The primers set for the hlyA gene consist of six primers targeting eight regions on specific gene. The LAMP assay could be performed within 40 min at 65°C in a water bath. Amplification products were visualized by calcein and manganous ion and agarose gel electrophoresis. Sensitivity of the LAMP assay for detection of L. monocytogenes in pure cultures was 2.0 CFU per reaction. The LAMP assay was 100-fold higher sensitive than that of the conventional PCR assay. Taking this way, 60 chicken samples were investigated for L. monocytogenes. The accuracy of LAMP was shown to be 100% when compared to the "gold standard" culture-biotechnical, while the PCR assay failed to detect L. monocytogenes in two of the positive samples. It is shown that LAMP assay can be used as a sensitive, rapid, and simple detection tool for the detection of L. monocytogenes and will facilitate the surveillance for contamination of L. monocytogenes in food.

  8. Development of a loop-mediated isothermal amplification assay for detection of Phytophthora sojae.

    Science.gov (United States)

    Dai, Ting-Ting; Lu, Chen-Chen; Lu, Jing; Dong, SuoMeng; Ye, WenWu; Wang, YuanChao; Zheng, XiaoBo

    2012-09-01

    Phytophthora sojae is a devastating pathogen that causes soybean Phytophthora root rot. This study reports the development of a loop-mediated isothermal amplification (LAMP) assay targeting the A3aPro element for visual detection of P. sojae. The A3aPro-LAMP assay efficiently amplified the target element in Phytophthora spp., Pythium spp., and true fungi isolates. Magnesium pyrophosphate resulting from the LAMP of P. sojae could be detected by real-time measurement of turbidity. Phytophthora sojae DNA products were visualized as a ladder-like banding pattern on 2% gel electrophoresis. A positive colour (sky blue) was only observed in the presence of P. sojae with the addition of hydroxynaphthol blue prior to amplification, whereas none of other isolates showed a colour change. The detection limit of the A3aPro-specific LAMP assay for P. sojae was 10 pg μL(-1) of genomic DNA per reaction. The assay also detected P. sojae from diseased soybean tissues and residues. These results suggest that the A3aPro-LAMP assay reported here can be used for the visual detection of P. sojae in plants and production fields.

  9. Rapid PCR amplification protocols decrease the turn-around time for detection of antibiotic resistance genes in Gram-negative pathogens.

    Science.gov (United States)

    Geyer, Chelsie N; Hanson, Nancy D

    2013-10-01

    A previously designed end-point multiplex PCR assay and singleplex assays used to detect β-lactamase genes were evaluated using rapid PCR amplification methodology. Amplification times were 16-18 minutes with an overall detection time of 1.5 hours. Rapid PCR amplifications could decrease the time required to identify resistance mechanisms in Gram-negative organisms.

  10. Ligase chain reaction amplification for sensitive electrochemiluminescent detection of single nucleotide polymorphisms

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Ying; Yang, Mengli; Xiang, Yun, E-mail: yunatswu@swu.edu.cn; Yuan, Ruo, E-mail: yuanruo@swu.edu.cn; Chai, Yaqin

    2013-09-24

    Graphical abstract: -- Highlights: •Ligase chain reaction amplification (LCR) is employed to sensitively detect single nucleotide polymorphisms. •During LCR, the mutant target gene is recycled and duplicated exponentially to achieve dramatic signal amplification. •The method shows a selectivity factor of 10{sup 3} toward the mutant target gene against the interfering wild target gene. -- Abstract: Single nucleotide polymorphisms are the most common type of genetic variations among human beings and can serve as biomarkers for various types of diseases. In this work, based on ligase chain reaction amplification for the production of massive hemin/G-quadruplex DNAzymes to quench the electrochemiluminescent (ECL) emission of quantum dots (QDs), a universal and sensitive single nucleotide polymorphism detection method is described. During the ligase chain reaction process, the mutant K-ras target gene is recycled and exponentially duplicated, leading to the attachment of numerous G-rich sequences on the QD-embedded sensing surface. Upon the addition of the assistant sequences and hemin, numerous hemin/G-quadruplex DNAzymes are formed, which consume the dissolved oxygen in the detection buffer and result in significant quenching of QD ECL emission for sensitive single nucleotide polymorphism determination. The developed method shows a linear range of 50 fM to 50 pM and an estimated detection limit of 45 fM for the mutant K-ras gene. The proposed strategy also exhibits high selectivity towards the mutant K-ras gene against the co-existence of 10{sup 3}-fold excess of the wild-type K-ras gene, which makes our method a useful addition to the alternatives for single nucleotide polymorphism monitoring.

  11. A Novel Ultrasensitive ECL Sensor for DNA Detection Based on Nicking Endonuclease-Assisted Target Recycling Amplification, Rolling Circle Amplification and Hemin/G-Quadruplex

    Directory of Open Access Journals (Sweden)

    Fukang Luo

    2015-01-01

    Full Text Available In this study, we describe a novel universal and highly sensitive strategy for the electrochemiluminescent (ECL detection of sequence specific DNA at the aM level based on Nt.BbvCI (a nicking endonuclease-assisted target recycling amplification (TRA, rolling circle amplification (RCA and hemin/G-quadruplex. The target DNAs can hybridize with self-assembled capture probes and assistant probes to form “Y” junction structures on the electrode surface, thus triggering the execution of a TRA reaction with the aid of Nt.BbvCI. Then, the RCA reaction and the addition of hemin result in the production of numerous hemin/G-quadruplex, which consume the dissolved oxygen in the detection buffer and result in a significant ECL quenching effect toward the O2/S2O82− system. The proposed strategy combines the amplification ability of TRA, RCA and the inherent high sensitivity of the ECL technique, thus enabling low aM (3.8 aM detection for sequence-specific DNA and a wide linear range from 10.0 aM to 1.0 pM. At the same time, this novel strategy shows high selectivity against single-base mismatch sequences, which makes our novel universal and highly sensitive method a powerful addition to specific DNA sequence detection.

  12. Detection of Bar Transgenic Sugarcane with a Rapid and Visual Loop-Mediated Isothermal Amplification Assay

    Science.gov (United States)

    Zhou, Dinggang; Wang, Chunfeng; Li, Zhu; Chen, Yun; Gao, Shiwu; Guo, Jinlong; Lu, Wenying; Su, Yachun; Xu, Liping; Que, Youxiong

    2016-01-01

    Genetic engineering offers an attractive alternative in sugarcane breeding for increasing cane and sugar yields as well as disease and insect resistance. Bar transgenic sugarcane employing the herbicide tolerance is a useful agronomical trait in weed control. In this study, a loop-mediated isothermal amplification (LAMP) assay for rapid detection of the bar gene in transgenic sugarcane has been developed and evaluated. A set of six primers was designed for LAMP-based amplification of the bar gene. The LAMP reaction conditions were optimized as follows: 5.25 mM of Mg2+, 6:1 ratio of inner vs. outer primer, and 6.0 U of Bst DNA polymerase in a reaction volume of 25.0 μL. The detection limit of the recombinant plasmid 1Ac0229 was as low as 10 copies in the developed LAMP, which was 10-fold higher sensitive than that of conventional PCR. In 100 putative transgenic lines, the bar gene was detected in 100/100 cases (100%) by LAMP and 97/100 cases (97%) by conventional PCR, respectively. In conclusion, the developed LAMP assay is visual, rapid, sensitive, reliable, and cost-effective for detection of the bar specific transgenic sugarcane. PMID:27014303

  13. In situ DNA amplification with magnetic primers for the electrochemical detection of food pathogens.

    Science.gov (United States)

    Lermo, A; Campoy, S; Barbé, J; Hernández, S; Alegret, S; Pividori, M I

    2007-04-15

    A sensitive and selective genomagnetic assay for the electrochemical detection of food pathogens based on in situ DNA amplification with magnetic primers has been designed. The performance of the genomagnetic assay was firstly demonstrated for a DNA synthetic target by its double-hybridization with both a digoxigenin probe and a biotinylated capture probe, and further binding to streptavidin-modified magnetic beads. The DNA sandwiched target bound on the magnetic beads is then separated by using a magneto electrode based on graphite-epoxy composite. The electrochemical detection is finally achieved by an enzyme marker, anti-digoxigenin horseradish peroxidase (HRP). The novel strategy was used for the rapid and sensitive detection of polymerase chain reaction (PCR) amplified samples. Promising resultants were also achieved for the DNA amplification directly performed on magnetic beads by using a novel magnetic primer, i.e., the up PCR primer bound to magnetic beads. Moreover, the magneto DNA biosensing assay was able to detect changes at single nucleotide polymorphism (SNP) level, when stringent hybridization conditions were used. The reliability of the assay was tested for Salmonella spp., the most important pathogen affecting food safety.

  14. Rapid and specific detection of Yam mosaic virus by reverse-transcription recombinase polymerase amplification.

    Science.gov (United States)

    Silva, Gonçalo; Bömer, Moritz; Nkere, Chukwuemeka; Kumar, P Lava; Seal, Susan E

    2015-09-15

    Yam mosaic virus (YMV; genus Potyvirus) is considered to cause the most economically important viral disease of yams (Dioscorea spp.) in West Africa which is the dominant region for yam production globally. Yams are a vegetatively propagated crop and the use of virus-free planting material forms an essential component of disease control. Current serological and PCR-based diagnostic methods for YMV are time consuming involving a succession of target detection steps. In this study, a novel assay for specific YMV detection is described that is based on isothermal reverse transcription-recombinase polymerase amplification (RT-exoRPA). This test has been shown to be reproducible and able to detect as little as 14 pg/μl of purified RNA obtained from an YMV-infected plant, a sensitivity equivalent to that obtained with the reverse transcription-polymerase chain reaction (RT-PCR) in current general use. The RT-exoRPA assay has, however, several advantages over the RT-PCR; positive samples can be detected in less than 30 min, and amplification only requires a single incubation temperature (optimum 37°C). These features make the RT-exoRPA assay a promising candidate for adapting into a field test format to be used by yam breeding programmes or certification laboratories. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. Hybridization chain reaction amplification for highly sensitive fluorescence detection of DNA with dextran coated microarrays.

    Science.gov (United States)

    Chao, Jie; Li, Zhenhua; Li, Jing; Peng, Hongzhen; Su, Shao; Li, Qian; Zhu, Changfeng; Zuo, Xiaolei; Song, Shiping; Wang, Lianhui; Wang, Lihua

    2016-07-15

    Microarrays of biomolecules hold great promise in the fields of genomics, proteomics, and clinical assays on account of their remarkably parallel and high-throughput assay capability. However, the fluorescence detection used in most conventional DNA microarrays is still limited by sensitivity. In this study, we have demonstrated a novel universal and highly sensitive platform for fluorescent detection of sequence specific DNA at the femtomolar level by combining dextran-coated microarrays with hybridization chain reaction (HCR) signal amplification. Three-dimensional dextran matrix was covalently coated on glass surface as the scaffold to immobilize DNA recognition probes to increase the surface binding capacity and accessibility. DNA nanowire tentacles were formed on the matrix surface for efficient signal amplification by capturing multiple fluorescent molecules in a highly ordered way. By quantifying microscopic fluorescent signals, the synergetic effects of dextran and HCR greatly improved sensitivity of DNA microarrays, with a detection limit of 10fM (1×10(5) molecules). This detection assay could recognize one-base mismatch with fluorescence signals dropped down to ~20%. This cost-effective microarray platform also worked well with samples in serum and thus shows great potential for clinical diagnosis.

  16. Development of Loop-Mediated Isothermal Amplification (LAMP Assays for Rapid Detection of Ehrlichia ruminantium

    Directory of Open Access Journals (Sweden)

    Geysen Dirk

    2010-11-01

    Full Text Available Abstract Background The rickettsial bacterium Ehrlichia ruminantium is the causative agent of heartwater, a potential zoonotic disease of ruminants transmitted by ticks of the genus Amblyomma. The disease is distributed in nearly all of sub-Saharan Africa and some islands of the Caribbean, from where it threatens the American mainland. This report describes the development of two different loop-mediated isothermal amplification (LAMP assays for sensitive and specific detection of E. ruminantium. Results Two sets of LAMP primers were designed from the pCS20 and sodB genes. The detection limits for each assay were 10 copies for pCS20 and 5 copies for sodB, which is at least 10 times higher than that of the conventional pCS20 PCR assay. DNA amplification was completed within 60 min. The assays detected 16 different isolates of E. ruminantium from geographically distinct countries as well as two attenuated vaccine isolates. No cross-reaction was observed with genetically related Rickettsiales, including zoonotic Ehrlichia species from the USA. LAMP detected more positive samples than conventional PCR but less than real-time PCR, when tested with field samples collected in sub-Saharan countries. Conclusions Due to its simplicity and specificity, LAMP has the potential for use in resource-poor settings and also for active screening of E. ruminantium in both heartwater-endemic areas and regions that are at risk of contracting the disease.

  17. Detection of Clostridium perfringens alpha toxin gene in lambs by loop mediated isothermal amplification

    Directory of Open Access Journals (Sweden)

    B. Radhika

    2016-01-01

    Full Text Available Aim: The loop mediated isothermal amplification (LAMP was standardized for rapid detection of Clostridium perfringens. Materials and Methods: A total of 120 fecal samples were collected from enterotoxemia suspected lambs were used for screening of C. perfringens cpa gene by LAMP. The specificity of the LAMP amplified products was tested by digesting with restriction enzyme XmnI for alpha toxin gene. Results: Out of 120 samples screened 112 (93.3% samples were positive by both LAMP and polymerase chain reaction (PCR for detection of cpa gene which indicated the equal sensitivity of both the tests. The enzyme produced single cut in 162 base pair amplified product of alpha toxin gene at 81 base pair resulting in a single band in gel electrophoresis. Conclusion: Both LAMP and PCR for detection of cpa gene indicated the equal sensitivity of both the tests. Standardization of LAMP reaction for amplification of epsilon and beta toxin genes will help to identify the C. perfringens toxin types from the clinical samples. The test could be a suitable alternative to the PCR in detection of toxin types without the help of sophisticated machinery like thermal cycler. Considering its simplicity in operation and high sensitivity, there is the potential use of this technique in clinical diagnosis and surveillance of infectious diseases.

  18. Rapid detection of Opisthorchis viverrini copro-DNA using loop-mediated isothermal amplification (LAMP).

    Science.gov (United States)

    Arimatsu, Yuji; Kaewkes, Sasithorn; Laha, Thewarach; Hong, Sung-Jong; Sripa, Banchob

    2012-03-01

    Opisthorchis viverrini and other foodborne trematode infections are major health problem in Thailand, the Lao People's Democratic Republic, Vietnam and Cambodia. Differential diagnosis of O. viverrini based on the microscopic observation of parasite eggs is difficult in areas where Clonorchis sinensis and minute intestinal flukes coexist. We therefore established a rapid, sensitive and specific method for detecting O. viverrini infection from the stool samples using the loop-mediated isothermal amplification (LAMP) method. A total of five primers from seven regions were designed to target the internal transcribed spacer 1 (ITS1) in ribosomal DNA for specific amplification. Hydroxy naphthol blue (HNB) was more effective to detect the LAMP product compared to the Real-time LAMP and turbidity assay for its simple and distinct detection. The LAMP assay specifically amplified O. viverrini ITS1 but not C. sinensis and minute intestinal flukes with the limit of detection around 10(-3)ng DNA/μL. The sensitivity of the LAMP was 100% compared to egg positive samples. While all microscopically positive samples were positive by LAMP, additionally 5 of 13 (38.5%) microscopically negative samples were also LAMP positive. The technique has great potential for differential diagnosis in endemic areas with mixed O. viverrini and intestinal fluke infections. As it is an easy and simple method, the LAMP is potentially applicable for point-of-care diagnosis.

  19. Detection of Yersinia enterocolitica in milk powders by cross-priming amplification combined with immunoblotting analysis.

    Science.gov (United States)

    Zhang, Hongwei; Feng, Shaolong; Zhao, Yulong; Wang, Shuo; Lu, Xiaonan

    2015-12-02

    Yersinia enterocolitica (Y. enterocolitica) is frequently isolated from a wide variety of foods and can cause human yersiniosis. Biochemical and culture-based assays are common detection methods, but require a long incubation time and easily misidentify Y. enterocolitica as other non-pathogenic Yersinia species. Alternatively, cross-priming amplification (CPA) under isothermal conditions combined with immunoblotting analysis enables a more sensitive detection in a relatively short time period. A set of specific displacement primers, cross primers and testing primers was designed on the basis of six specific sequences in Y. enterocolitica 16S-23S rDNA internal transcribed spacer. Under isothermal condition, amplification and hybridization were conducted simultaneously at 63°C for 60 min. The specificity of CPA was tested for 96 different bacterial strains and 165 commercial milk powder samples. Two red lines were developed on BioHelix Express strip for all of the Y. enterocolitica strains, and one red line was shown for non-Y. enterocolitica strains. The limit of detection of CPA was 10(0)fg for genomic DNA (1000 times more sensitive than PCR assay), 10(1) CFU/ml for pure bacterial culture, and 10(0) CFU per 100 g milk powder with pre-enrichment at 37°C for 24 h. CPA combined with immunoblotting analysis can achieve highly specific and sensitive detection of Y. enterocolitica in milk powder in 90 min after pre-enrichment.

  20. Emerging Loop-Mediated Isothermal Amplification-Based Microchip and Microdevice Technologies for Nucleic Acid Detection.

    Science.gov (United States)

    Safavieh, Mohammadali; Kanakasabapathy, Manoj K; Tarlan, Farhang; Ahmed, Minhaz U; Zourob, Mohammed; Asghar, Waseem; Shafiee, Hadi

    2016-03-14

    Rapid, sensitive, and selective pathogen detection is of paramount importance in infectious disease diagnosis and treatment monitoring. Currently available diagnostic assays based on polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) are time-consuming, complex, and relatively expensive, thus limiting their utility in resource-limited settings. Loop-mediated isothermal amplification (LAMP) technique has been used extensively in the development of rapid and sensitive diagnostic assays for pathogen detection and nucleic acid analysis and hold great promise for revolutionizing point-of-care molecular diagnostics. Here, we review novel LAMP-based lab-on-a-chip (LOC) diagnostic assays developed for pathogen detection over the past several years. We review various LOC platforms based on their design strategies for pathogen detection and discuss LAMP-based platforms still in development and already in the commercial pipeline. This review is intended as a guide to the use of LAMP techniques in LOC platforms for molecular diagnostics and genomic amplifications.

  1. Dual Electrophoresis Detection System for Rapid and Sensitive Immunoassays with Nanoparticle Signal Amplification

    Science.gov (United States)

    Zhang, Fangfang; Ma, Junjie; Watanabe, Junji; Tang, Jinlong; Liu, Huiyu; Shen, Heyun

    2017-02-01

    An electrophoretic technique was combined with an enzyme-linked immunosorbent assay (ELISA) system to achieve a rapid and sensitive immunoassay. A cellulose acetate filter modified with polyelectrolyte multilayer (PEM) was used as a solid substrate for three-dimensional antigen-antibody reactions. A dual electrophoresis process was used to induce directional migration and local condensation of antigens and antibodies at the solid substrate, avoiding the long diffusion times associated with antigen-antibody reactions in conventional ELISAs. The electrophoretic forces drove two steps in the ELISA process, namely the adsorption of antigen, and secondary antibody-labelled polystyrene nanoparticles (NP-Ab). The total time needed for dual electrophoresis-driven detection was just 4 min, nearly 2 h faster than a conventional ELISA system. Moreover, the rapid NP-Ab electrophoresis system simultaneously achieved amplification of the specific signal and a reduction in noise, leading to a more sensitive NP-Ab immunoassay with a limit of detection (LOD) of 130 fM, and wide range of detectable concentrations from 0.13 to 130 pM. These results suggest that the combination of dual electrophoresis detection and NP-Ab signal amplification has great potential for future immunoassay systems.

  2. Detection of bar transgenic sugarcane with a rapid and visual loop-mediated isothermal amplification assay

    Directory of Open Access Journals (Sweden)

    Dinggang eZhou

    2016-03-01

    Full Text Available Genetic engineering offers an attractive alternative in sugarcane breeding for increasing cane and sugar yields as well as disease and insect resistance. Bar transgenic sugarcane employing the herbicide tolerance is a useful agronomical trait in weed control. In this study, a loop-mediated isothermal amplification (LAMP assay for rapid detection of the bar gene in transgenic sugarcane has been developed and evaluated. A set of six primers was designed for LAMP-based amplification of the bar gene. The LAMP reaction conditions were optimized as follows: 5.25 mM of Mg2+, 6:1 ratio of inner vs outer primer, and 6.0 U of Bst DNA polymerase in a reaction volume of 25.0 μL. The detection limit of the recombinant plasmid 1Ac0229 was as low as 10 copies in the developed LAMP, which was ten-fold higher sensitive than that of conventional PCR. In 100 putative transgenic lines, the bar gene was detected in 100/100 cases (100% by LAMP and 97/100 cases (97% by conventional PCR, respectively. In conclusion, the developed LAMP assay is visual, rapid, sensitive, reliable and cost-effective for detection of the bar specific transgenic sugarcane.

  3. Detection of Staphylococcus aureus in Milk Using Real-time Fluorescence Loop-mediated Isothermal Amplification

    Directory of Open Access Journals (Sweden)

    Ying Yu

    2015-07-01

    Full Text Available Staphylococcus aureus is a kind of worldwide food-borne pathogen. Recently, S. aureus has gained considerable attention because of the increasing alimentary toxicosis incidence. In this study, a Real-Time fluorescence Loop-Mediated isothermal Amplification (RT-LAMP was developed to detect S. aureus rapidly. The heat-stable nuclease (nuc gene of S. aureus, the target sequence, was selected to design four special primers. A rapid detection method for S. aureus was initially established under optimum reaction conditions. The assay, performed for 40 min at 61°C, did not show cross reactivity with other bacterial species. The specificity and sensitivity of RT-LAMP for detecting S. aureus were 100% and 8.0 CFU/mL, respectively. Results indicated that RT-LAMP was a potential field-usable molecular tool for detecting S. aureus This method can be an alternative to conventional LAMP in clinical applications and operational programs.

  4. Development of a nested polymerase chain reaction method to detect oncogenic Marek's disease virus from feather tips.

    Science.gov (United States)

    Murata, Shiro; Chang, Kyung-Soo; Lee, Sung-Il; Konnai, Satoru; Onuma, Misao; Ohashi, Kazuhiko

    2007-09-01

    For the easy survey of Marek's disease virus (MDV), feather tip-derived DNA from MDV-infected chickens can be used because feather tips are easy to collect and feather follicle epithelium is known to be the only site of productive replication of cell-free MDV. To develop a diagnostic method to differentiate highly virulent strains of MDV from the attenuated MDV vaccine strain, CVI988, which is widely used, nested polymerase chain reaction (PCR) was performed to detect a segment of the meq gene in feather tip samples of chickens experimentally infected with MDV. In chickens infected with Md5, a strain of oncogenic MDV, the meq gene was consistently detected, whereas the L-meq gene, in which a 180-base pair (180-bp) sequence is inserted into the meq gene, was detected in CVI988-infected chickens. Moreover, the meq gene was mainly detected even in chickens co-infected with both Md5 and CVI988. These results suggest that this method is appropriate for the surveillance of the highly virulent MDV infection in the field.

  5. Detection of total and hemolysin-producing Vibrio parahaemolyticus in shellfish using multiplex PCR amplification of tl, tdh and trh.

    Science.gov (United States)

    Bej, A K; Patterson, D P; Brasher, C W; Vickery, M C; Jones, D D; Kaysner, C A

    1999-06-01

    Vibrio parahaemolyticus is an important human pathogen which can cause gastroenteritis when consumed in raw or partially-cooked seafood. A multiplex PCR amplification-based detection of total and virulent strains of V. parahaemolyticus was developed by targeting thermolabile hemolysin encoded by tl, thermostable direct hemolysin encoded by tdh, and thermostable direct hemolysin-related trh genes. Following optimization using oligonucleotide primers targeting tl, tdh and trh genes, the multiplex PCR was applied to V. parahaemolyticus from 27 clinical, 43 seafood, 15 environmental, 7 strains obtained from various laboratories and 19 from oyster plants. All 111 V. parahaemolyticus isolates showed PCR amplification of the tl gene; however, only 60 isolates showed amplification of tdh, and 43 isolates showed amplification of the trh gene. Also, 18 strains showed amplification of the tdh gene, but these strains did not show amplification of the trh gene. However, one strain exhibited amplification for the trh but not the tdh gene, suggesting both genes need to be targeted in a PCR amplification reaction to detect all hemolysin-producing strains of this pathogen. The multiplex PCR approach was successfully used to detect various strains of V parahaemolyticus in seeded oyster tissue homogenate. Sensitivity of detection for all three target gene segments was at least between 10(1)-10(2) cfu per 10 g of alkaline peptone water enriched seeded oyster tissue homogenate. This high level of sensitivity of detection of this pathogen within 8 h of pre-enrichment is well within the action level (10(4) cfu per 1 g of shell stock) suggested by the National Seafood Sanitation Program guideline. Compared to conventional microbiological culture methods, this multiplex PCR approach is rapid and reliable for accomplishing a comprehensive detection of V. parahaemolyticus in shellfish.

  6. Next-generation sequence detects ARAP3 as a novel oncogene in papillary thyroid carcinoma

    Directory of Open Access Journals (Sweden)

    Wang QX

    2016-11-01

    Full Text Available Qing-Xuan Wang, En-Dong Chen, Ye-Feng Cai, Yi-Li Zhou, Zhou-Ci Zheng, Ying-Hao Wang, Yi-Xiang Jin, Wen-Xu Jin, Xiao-Hua Zhang, Ou-Chen Wang Department of Oncology, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang Province, China Purpose: Thyroid cancer is the most frequent malignancies of the endocrine system, and it has became the fastest growing type of cancer worldwide. Much still remains unknown about the molecular mechanisms of thyroid cancer. Studies have found that some certain relationship between ARAP3 and human cancer. However, the role of ARAP3 in thyroid cancer has not been well explained. This study aimed to investigate the role of ARAP3 gene in papillary thyroid carcinoma. Methods: Whole exon sequence and whole genome sequence of primary papillary thyroid carcinoma (PTC samples and matched adjacent normal thyroid tissue samples were performed and then bioinformatics analysis was carried out. PTC cell lines (TPC1, BCPAP, and KTC-1 with transfection of small interfering RNA were used to investigate the functions of ARAP3 gene, including cell proliferation assay, colony formation assay, migration assay, and invasion assay. Results: Using next-generation sequence and bioinformatics analysis, we found ARAP3 genes may play an important role in thyroid cancer. Downregulation of ARAP3 significantly suppressed PTC cell lines (TPC1, BCPAP, and KTC-1, cell proliferation, colony formation, migration, and invasion. Conclusion: This study indicated that ARAP3 genes have important biological implications and may act as a potentially drugable target in PTC. Keywords: papillary thyroid carcinoma, next-generation sequence, ARAP3, oncogene

  7. Development of Rapid Isothermal Amplification Assays for Detection of Phytophthora spp. in Plant Tissue.

    Science.gov (United States)

    Miles, Timothy D; Martin, Frank N; Coffey, Michael D

    2015-02-01

    Several isothermal amplification techniques recently have been developed that are tolerant of inhibitors present in many plant extracts, which can reduce the need for obtaining purified DNA for running diagnostic assays. One such commercially available technique that has similarities with real-time polymerase chain reaction (PCR) for designing primers and a labeled probe is recombinase polymerase amplification (RPA). This technology was used to develop two simple and rapid approaches for detection of Phytophthora spp.: one genus-specific assay multiplexed with a plant internal control and the other species-specific assays for Phytophthora ramorum and P. kernoviae. All assays were tested for sensitivity (ranging from 3 ng to 1 fg of DNA) and specificity using DNA extracted from more than 136 Phytophthora taxa, 21 Pythium spp., 1 Phytopythium sp., and a wide range of plant species. The lower limit of linear detection using purified DNA was 200 to 300 fg of DNA in all pathogen RPA assays. Six different extraction buffers were tested for use during plant tissue maceration and the assays were validated in the field by collecting 222 symptomatic plant samples from over 50 different hosts. Only 56 samples were culture positive for Phytophthora spp. whereas 91 were positive using the Phytophthora genus-specific RPA test and a TaqMan real-time PCR assay. A technique for the generation of sequencing templates from positive RPA amplifications to confirm species identification was also developed. These RPA assays have added benefits over traditional technologies because they are rapid (results can be obtained in as little as 15 min), do not require DNA extraction or extensive training to complete, use less expensive portable equipment than PCR-based assays, and are significantly more specific than current immunologically based methods. This should provide a rapid, field-deployable capability for pathogen detection that will facilitate point-of-sample collection processing

  8. Chip-based device for parallel sorting, amplification, detection, and identification of nucleic acid subsequences

    Energy Technology Data Exchange (ETDEWEB)

    Beer, Neil Reginald; Colston, Jr, Billy W.

    2016-08-09

    An apparatus for chip-based sorting, amplification, detection, and identification of a sample having a planar substrate. The planar substrate is divided into cells. The cells are arranged on the planar substrate in rows and columns. Electrodes are located in the cells. A micro-reactor maker produces micro-reactors containing the sample. The micro-reactor maker is positioned to deliver the micro-reactors to the planar substrate. A microprocessor is connected to the electrodes for manipulating the micro-reactors on the planar substrate. A detector is positioned to interrogate the sample contained in the micro-reactors.

  9. Rapid identification of bio-molecules applied for detection of biosecurity agents using rolling circle amplification.

    Directory of Open Access Journals (Sweden)

    Jenny Göransson

    Full Text Available Detection and identification of pathogens in environmental samples for biosecurity applications are challenging due to the strict requirements on specificity, sensitivity and time. We have developed a concept for quick, specific and sensitive pathogen identification in environmental samples. Target identification is realized by padlock- and proximity probing, and reacted probes are amplified by RCA (rolling-circle amplification. The individual RCA products are labeled by fluorescence and enumerated by an instrument, developed for sensitive and rapid digital analysis. The concept is demonstrated by identification of simili biowarfare agents for bacteria (Escherichia coli and Pantoea agglomerans and spores (Bacillus atrophaeus released in field.

  10. Loop-mediated isothermal amplification (LAMP): A novel rapid detection platform for pathogens.

    Science.gov (United States)

    Li, Yanmei; Fan, Penghui; Zhou, Shishui; Zhang, Li

    2017-03-18

    Foodborne bacterial infections and diseases have been considered to be a major threat for public health in the worldwide. Increased incidence of human diseases caused by foodborne pathogens have been correlated with growing world population and mobility. Loop-mediated isothermal amplification (LAMP) has been regarded as an innovative gene amplification technology and emerged as an alternative to PCR-based methodologies in both clinical laboratory and food safety testing. Nowadays, LAMP has been applied to detection and identification on pathogens from microbial diseases, as it showed significant advantage in high sensitivity, specificity and rapidity. The high sensitivity of LAMP enables detection of the pathogens in sample materials even without time consuming sample preparation. An overview of LAMP mainly containing the development history, reaction principle and its application to four kind of foodborne pathogens detection are presented in this paper. As concluded, with the advantages of rapidity, simplicity, sensitivity, specificity and robustness, LAMP is capable of applications for clinical diagnosis as well as surveillance of infection diseases. Moreover, the main purpose of this paper is to provide theoretical basis for the clinical application of LAMP technology.

  11. Development of loop-mediated isothermal amplification for rapid detection of Entamoeba histolytica

    Institute of Scientific and Technical Information of China (English)

    Windell L Rivera; Vanissa A Ong

    2013-01-01

    Objective: To develop a loop-mediated isothermal amplification (LAMP) assay for the detection of Entamoeba histolytica (E. histolytica), the causative agent of amebiasis. Methods: The LAMP primer set was designed from E. histolytica hemolysin gene HLY6. Genomic DNA of E. histolytica trophozoites strain HK9 was used to optimize the LAMP mixture and conditions. Amplification of DNA in the LAMP mixture was monitored through visual inspection for turbidity of the LAMP mix as well as addition of fluorescent dye. Results: Positive LAMP reactions turned turbid while negative ones remained clear. Upon addition of a fluorescent dye, all positive reactions turned green while the negative control remained orange under ambient light. After elecrophoresis in 1.5%agarose gels, a ladder of multiple bands of different sizes can be observed in positive samples while no bands were detected in the negative control. The sensitivity of the assay was found to be 5 parasites per reaction which corresponds to approximately 15.8 ng/μL DNA. The specificity of the assay was verified by the absence of amplified products when DNA from other gastrointestinal parasites such as the morphologically similar but non-pathogenic species, Entamoeba dispar, and other diarrhea-causing organisms such as Blastocystis hominis and Escherichia coli were used. Conclusions: The LAMP assay we have developed enables the detection of E. histolytica with rapidity and ease, therefore rendering it is suitable for laboratory and field diagnosis of amebiasis.

  12. Integrated biochip for PCR-based DNA amplification and detection on capacitive biosensors

    Science.gov (United States)

    Moschou, D.; Vourdas, N.; Filippidou, M. K.; Tsouti, V.; Kokkoris, G.; Tsekenis, G.; Zergioti, I.; Chatzandroulis, S.; Tserepi, A.

    2013-05-01

    Responding to an increasing demand for LoC devices to perform bioanalytical protocols for disease diagnostics, the development of an integrated LoC device consisting of a μPCR module integrated with resistive microheaters and a biosensor array for disease diagnostics is presented. The LoC is built on a Printed Circuit Board (PCB) platform, implementing both the amplification of DNA samples and DNA detection/identification on-chip. The resistive microheaters for PCR and the wirings for the sensor read-out are fabricated by means of standard PCB technology. The microfluidic network is continuous-flow, designed to perform 30 PCR cycles with heated zones at constant temperatures, and is built onto the PCB utilizing commercial photopatternable polyimide layers. Following DNA amplification, the product is driven in a chamber where a Si-based biosensor array is placed for DNA detection through hybridization. The sensor array is tested for the detection of mutations of the KRAS gene, responsible for colon cancer.

  13. A chronocoulometric aptasensor based on gold nanoparticles as a signal amplification strategy for detection of thrombin.

    Science.gov (United States)

    Jiao, Xiao Xia; Chen, Jing Rong; Zhang, Xi Yuan; Luo, Hong Qun; Li, Nian Bing

    2013-10-15

    A sensitive chronocoulometric aptasensor for the detection of thrombin has been developed based on gold nanoparticle amplification. The functional gold nanoparticles, loaded with link DNA (LDNA) and report DNA (RDNA), were immobilized on an electrode by thrombin aptamers performing as a recognition element and capture probe. LDNA was complementary to the thrombin aptamers and RDNA was noncomplementary, but could combine with [Ru(NH₃)₆]³⁺ (RuHex) cations. Electrochemical signals obtained by RuHex that bound quantitatively to the negatively charged phosphate backbone of DNA via electrostatic interactions were measured by chronocoulometry. In the presence of thrombin, the combination of thrombin and thrombin aptamers and the release of the functional gold nanoparticles could induce a significant decrease in chronocoulometric signal. The incorporation of gold nanoparticles in the chronocoulometric aptasensor significantly enhanced the sensitivity. The performance of the aptasensor was further increased by the optimization of the surface density of aptamers. Under optimum conditions, the chronocoulometric aptasensor exhibited a wide linear response range of 0.1-18.5 nM with a detection limit of 30 pM. The results demonstrated that this nanoparticle-based amplification strategy offers a simple and effective approach to detect thrombin.

  14. Development of a loop-mediated isothermal amplification method for detecting virulent Rhodococcus equi.

    Science.gov (United States)

    Kinoshita, Yuta; Niwa, Hidekazu; Higuchi, Tohru; Katayama, Yoshinari

    2016-09-01

    Rhodococcus equi is the most important causative bacterium of severe pneumonia in foals. We report herein the development of a specific loop-mediated isothermal amplification (LAMP) assay, which targets a gene encoding vapA for detecting virulent R. equi The detection limit of the LAMP assay was 10(4) colony forming units (CFU)/mL, which was equal to 10 CFU/reaction. The clinical efficacy of the LAMP assay was compared with those of 2 published PCR-based methods: nested PCR and quantitative real-time (q)PCR. Agreements between bacterial culture, which is the gold standard for detection of R. equi, and each of the 3 molecular tests were measured by calculating a kappa coefficient. The kappa coefficients of the LAMP (0.760), nested PCR (0.583), and qPCR (0.888) indicated substantial agreement, moderate agreement, and almost perfect agreement, respectively. Although the clinical efficacy of LAMP was not the best among the 3 methods tested, LAMP could be more easily introduced into less well-equipped clinics because it does not require special equipment (such as a thermocycler) for gene amplification. Veterinary practitioners could diagnose R. equi pneumonia more quickly by using LAMP and could use the results to select an appropriate initial treatment. © 2016 The Author(s).

  15. Ternary surface monolayers for ultrasensitive (zeptomole) amperometric detection of nucleic acid hybridization without signal amplification.

    Science.gov (United States)

    Wu, Jie; Campuzano, Susana; Halford, Colin; Haake, David A; Wang, Joseph

    2010-11-01

    A ternary surface monolayer, consisting of coassembled thiolated capture probe, mercaptohexanol and dithiothreitol, is shown to offer dramatic improvements in the signal-to-noise characteristics of electrochemical DNA hybridization biosensors based on common self-assembled monolayers. Remarkably low detection limits down to 40 zmol (in 4 μL samples) as well as only 1 CFU Escherichia coli per sensor are thus obtained without any additional amplification step in connection to the commonly used horseradish peroxidase/3,3',5,5'-tetramethylbenzidine system. Such dramatic improvements in the detection limits (compared to those of common binary alkanethiol interfaces and to those of most electrochemical DNA sensing strategies without target or signal amplification) are attributed primarily to the remarkably higher resistance to nonspecific adsorption. This reflects the highly compact layer (with lower pinhole density) produced by the coupling of the cyclic- and linear-configuration "backfillers" that leads to a remarkably low background noise even in the presence of complex sample matrixes. A wide range of surface compositions have been investigated, and the ternary mixed monolayer has been systematically optimized. Detailed impedance spectroscopy and cyclic voltammetric studies shed useful insights into the surface coverage. The impressive sensitivity and high specificity of the simple developed methodology indicate great promise for a wide range of nucleic acid testing, including clinical diagnostics, biothreat detection, food safety, and forensic analysis.

  16. Rapid detection of IHNV by molecular padlock recognition and surface-associated isothermal amplification

    Science.gov (United States)

    McCarthy, Erik L.; Egeler, Teressa J.; Bickerstaff, Lee E.; Pereira da Cunha, Mauricio; Millard, Paul J.

    2005-11-01

    RNA sequences derived from infectious hematopoeitic necrosis virus (IHNV) could be detected using a combination of surface-associated molecular padlock DNA probes (MPP) and rolling circle amplification (RCA) in microcapillary tubes. DNA oligonucleotides with base sequences identical to RNA obtained from IHNV were recognized by MPP. Circularized MPP were then captured on the inner surface of glass microcapillary tubes by immobilized DNA oligonucleotide primers. Extension of the immobilized primers by isothermal RCA gave rise to DNA concatamers, which were in turn bound by the fluorescent reporter SYBR Green II nucleic acid stain, and measured by microfluorimetry. Surface-associated molecular padlock technology, combined with isothermal RCA, exhibited high selectivity and sensitivity without thermal cycling. This technology is applicable to direct RNA and DNA detection, permitting detection of a variety of viral or bacterial pathogens.

  17. Signal-amplification detection of small molecules by use of Mg2+-dependent DNAzyme.

    Science.gov (United States)

    Guo, Zhijun; Wang, Jiahai; Wang, Erkang

    2013-05-01

    Because small molecules can be beneficial or toxic in biology and the environment, specific and sensitive detection of small molecules is one of the most important objectives of the scientific community. In this study, new signal amplification assays for detection of small molecules based on Mg(2+)-dependent DNAzyme were developed. A cleavable DNA substrate containing a ribonucleotide, the ends of which were labeled with black hole quencher (BHQ) and 6-carboxyfluorescein (FAM), was used for fluorescence detection. When the small molecule of interest is added to the assay solution, the Mg(2+)-dependent DNAzyme is activated, facilitating hybridization between the Mg(2+)-dependent DNAzyme and the DNA substrate. Binding of the substrate to the DNAzyme structure results in hydrolytic cleavage of the substrate in the presence of Mg(2+) ions. The fluorescence signal was amplified by continuous cleavage of the enzyme substrate. Ochratoxin A (OTA) and adenosine triphosphate (ATP) were used as model analytes in these experiments. This method can detect OTA specifically with a detection limit as low as 140 pmol L(-1) and detect ATP specifically with a detection limit as low as 13 nmol L(-1). Moreover, this method is potentially extendable to detection of other small molecules which are able to dissociate the aptamer from the DNAzyme, leading to activation of the DNAzyme.

  18. Rapid detection of the Klebsiella pneumoniae carbapenemase (KPC) gene by loop-mediated isothermal amplification (LAMP).

    Science.gov (United States)

    Nakano, Ryuichi; Nakano, Akiyo; Ishii, Yoshikazu; Ubagai, Tsuneyuki; Kikuchi-Ueda, Takane; Kikuchi, Hirotoshi; Tansho-Nagakawa, Shigeru; Kamoshida, Go; Mu, Xiaoqin; Ono, Yasuo

    2015-03-01

    Klebsiella pneumoniae carbapenemases (KPC), which are associated with resistance to carbapenem, have recently spread worldwide and have become a global concern. It is necessary to detect KPC-producing organisms in clinical settings to be able to control the spread of this resistance. We have developed a loop-mediated isothermal amplification (LAMP) method for rapid detection of KPC producers. LAMP primer sets were designed to recognize the homologous regions of blaKPC-2 to blaKPC-17 and could amplify blaKPC rapidly. The specificity and sensitivity of the primers in the LAMP reactions for blaKPC detection were determined. This LAMP assay was able to specifically detect KPC producers at 68 °C, and no cross-reactivity was observed for other types of β-lactamase (class A, B, C, or D) producers. The detection limit for this assay was found to be 10(0) CFU per tube, in 25 min, which was 10-fold more sensitive than a PCR assay for blaKPC detection. Then, the sensitivity of the LAMP reactions for blaKPC detection in human specimens (sputum samples, urine samples, fecal samples and blood samples) was analyzed; it was observed that the LAMP assay had almost the same sensitivity in these samples as when using purified DNA. The LAMP assay is easy to perform and rapid. It may therefore be routinely applied for detection of KPC producers in the clinical laboratory.

  19. A cascade signal amplification strategy for surface enhanced Raman spectroscopy detection of thrombin based on DNAzyme assistant DNA recycling and rolling circle amplification.

    Science.gov (United States)

    Gao, Fenglei; Du, Lili; Tang, Daoquan; Lu, Yao; Zhang, Yanzhuo; Zhang, Lixian

    2015-04-15

    A sensitive protocol for surface enhanced Raman spectroscopy (SERS) detection of thrombin is designed with R6G-Ag NPs as a signal tag by combining DNAzyme assistant DNA recycling and rolling circle amplification (RCA). Molecular beacon (MB) as recognition probe immobilizes on the glass slides and performs the amplification procedure. After thrombin-induced structure-switching DNA hairpins of probe 1, the DNAzyme is liberated from the caged structure, which hybridizes with the MB for cleavage of the MB in the presence of cofactor Zn(2+) and initiates the DNA recycling process, leading to the cleavage of a large number of MB and the generation of numerous primers for triggering RCA reaction. The long amplified RCA product which contained hundreds of tandem-repeat sequences, which can bind with oligonucleotide functionalized Ag NPs reporters. The attached signal tags can be easily read out by SERS. Because of the cascade signal amplification, these newly designed protocols provides a sensitive SERS detection of thrombin down to the femolar level (2.3fM) with a linear range of 5 orders of magnitude (from 10(-14) to 10(-9)M) and have high selectivity toward its target protein. The proposed method is expected to be a good clinical tool for the diagnosis of a thrombotic disease.

  20. A sensitive colorimetric assay system for nucleic acid detection based on isothermal signal amplification technology.

    Science.gov (United States)

    Hu, Bo; Guo, Jing; Xu, Ying; Wei, Hua; Zhao, Guojie; Guan, Yifu

    2017-08-01

    Rapid and accurate detection of microRNAs in biological systems is of great importance. Here, we report the development of a visual colorimetric assay which possesses the high amplification capabilities and high selectivity of the rolling circle amplification (RCA) method and the simplicity and convenience of gold nanoparticles used as a signal indicator. The designed padlock probe recognizes the target miRNA and is circularized, and then acts as the template to extend the target miRNA into a long single-stranded nucleotide chain of many tandem repeats of nucleotide sequences. Next, the RCA product is hybridized with oligonucleotides tagged onto gold nanoparticles. This interaction leads to the aggregation of gold nanoparticles, and the color of the system changes from wine red to dark blue according to the abundance of miRNA. A linear correlation between fluorescence and target oligonucleotide content was obtained in the range 0.3-300 pM, along with a detection limit of 0.13 pM (n = 7) and a RSD of 3.9% (30 pM, n = 9). The present approach provides a simple, rapid, and accurate visual colorimetric assay that allows sensitive biodetection and bioanalysis of DNA and RNA nucleotides of interest in biologically important samples. Graphical abstract The colorimetric assay system for analyzing target oligonucleotides.

  1. Single-use, electricity-free amplification device for detection of HIV-1.

    Science.gov (United States)

    Curtis, Kelly A; Rudolph, Donna L; Morrison, Daphne; Guelig, Dylan; Diesburg, Steven; McAdams, David; Burton, Robert A; LaBarre, Paul; Owen, Michele

    2016-11-01

    Early and accurate diagnosis of HIV is key for the reduction of transmission and initiation of patient care. The availability of a rapid nucleic acid test (NAT) for use at the point-of-care (POC) will fill a gap in HIV diagnostics, improving the diagnosis of acute infection and HIV in infants born to infected mothers. In this study, we evaluated the performance of non-instrumented nucleic acid amplification, single-use disposable (NINA-SUD) devices for the detection of HIV-1 in whole blood using reverse-transcription, loop-mediated isothermal amplification (RT-LAMP) with lyophilized reagents. The NINA-SUD heating device harnesses the heat from an exothermic chemical reaction initiated by the addition of saline to magnesium iron powder. Reproducibility was demonstrated between NINA-SUD units and comparable, if not superior, performance for detecting clinical specimens was observed as compared to the thermal cycler. The stability of the lyophilized HIV-1 RT-LAMP reagents was also demonstrated following storage at -20, 4, 25, and 30°C for up to one month. The single-use, disposable NAT minimizes hands-on time and has the potential to facilitate HIV-1 testing in resource-limited settings or at the POC. Published by Elsevier B.V.

  2. Rapid Detection of Listeria by Bacteriophage Amplification and SERS-Lateral Flow Immunochromatography.

    Science.gov (United States)

    Stambach, Nicholas R; Carr, Stephanie A; Cox, Christopher R; Voorhees, Kent J

    2015-12-14

    A rapid Listeria detection method was developed utilizing A511 bacteriophage amplification combined with surface-enhanced Raman spectroscopy (SERS) and lateral flow immunochromatography (LFI). Anti-A511 antibodies were covalently linked to SERS nanoparticles and printed onto nitrocellulose membranes. Antibody-conjugated SERS nanoparticles were used as quantifiable reporters. In the presence of A511, phage-SERS nanoparticle complexes were arrested and concentrated as a visible test line, which was interrogated quantitatively by Raman spectroscopy. An increase in SERS intensity correlated to an increase in captured phage-reporter complexes. SERS limit of detection was 6 × 10(6) pfu·mL(-1), offering detection below that obtainable by the naked eye (LOD 6 × 10(7) pfu·mL(-1)). Phage amplification experiments were carried out at a multiplicity of infection (MOI) of 0.1 with 4 different starting phage concentrations monitored over time using SERS-LFI and validated by spot titer assay. Detection of L. monocytogenes concentrations of 1 × 10(7) colony forming units (cfu)·mL(-1), 5 × 10(6) cfu·mL(-1), 5 × 10(5) cfu·mL(-1) and 5 × 10(4) cfu·mL(-1) was achieved in 2, 2, 6, and 8 h, respectively. Similar experiments were conducted at a constant starting phage concentration (5 × 10(5) pfu·mL(-1)) with MOIs of 1, 2.5, and 5 and were detected in 2, 4, and 5 h, respectively.

  3. Rapid Detection of Listeria by Bacteriophage Amplification and SERS-Lateral Flow Immunochromatography

    Directory of Open Access Journals (Sweden)

    Nicholas R. Stambach

    2015-12-01

    Full Text Available A rapid Listeria detection method was developed utilizing A511 bacteriophage amplification combined with surface-enhanced Raman spectroscopy (SERS and lateral flow immunochromatography (LFI. Anti-A511 antibodies were covalently linked to SERS nanoparticles and printed onto nitrocellulose membranes. Antibody-conjugated SERS nanoparticles were used as quantifiable reporters. In the presence of A511, phage-SERS nanoparticle complexes were arrested and concentrated as a visible test line, which was interrogated quantitatively by Raman spectroscopy. An increase in SERS intensity correlated to an increase in captured phage-reporter complexes. SERS limit of detection was 6 × 106 pfu·mL−1, offering detection below that obtainable by the naked eye (LOD 6 × 107 pfu·mL−1. Phage amplification experiments were carried out at a multiplicity of infection (MOI of 0.1 with 4 different starting phage concentrations monitored over time using SERS-LFI and validated by spot titer assay. Detection of L. monocytogenes concentrations of 1 × 107 colony forming units (cfu·mL−1, 5 × 106 cfu·mL−1, 5 × 105 cfu·mL−1 and 5 × 104 cfu·mL−1 was achieved in 2, 2, 6, and 8 h, respectively. Similar experiments were conducted at a constant starting phage concentration (5 × 105 pfu·mL−1 with MOIs of 1, 2.5, and 5 and were detected in 2, 4, and 5 h, respectively.

  4. Loop-mediated isothermal amplification (LAMP method for rapid detection of Trypanosoma brucei rhodesiense.

    Directory of Open Access Journals (Sweden)

    Zablon Kithinji Njiru

    Full Text Available Loop-mediated isothermal amplification (LAMP of DNA is a novel technique that rapidly amplifies target DNA under isothermal conditions. In the present study, a LAMP test was designed from the serum resistance-associated (SRA gene of Trypanosoma brucei rhodesiense, the cause of the acute form of African sleeping sickness, and used to detect parasite DNA from processed and heat-treated infected blood samples. The SRA gene is specific to T. b. rhodesiense and has been shown to confer resistance to lysis by normal human serum. The assay was performed at 62 degrees C for 1 h, using six primers that recognised eight targets. The template was varying concentrations of trypanosome DNA and supernatant from heat-treated infected blood samples. The resulting amplicons were detected using SYTO-9 fluorescence dye in a real-time thermocycler, visual observation after the addition of SYBR Green I, and gel electrophoresis. DNA amplification was detected within 35 min. The SRA LAMP test had an unequivocal detection limit of one pg of purified DNA (equivalent to 10 trypanosomes/ml and 0.1 pg (1 trypanosome/ml using heat-treated buffy coat, while the detection limit for conventional SRA PCR was approximately 1,000 trypanosomes/ml. The expected LAMP amplicon was confirmed through restriction enzyme RsaI digestion, identical melt curves, and sequence analysis. The reproducibility of the SRA LAMP assay using water bath and heat-processed template, and the ease in results readout show great potential for the diagnosis of T. b. rhodesiense in endemic regions.

  5. Detection of Anaplasma platys in dogs using real-time loop-mediated isothermal amplification.

    Science.gov (United States)

    Li, Hua-tao; Sun, Ling-suang; Chen, Zhong-ming; Hu, Jing-si; Ye, Cun-dong; Jia, Kun; Wang, Heng; Yuan, Li-guo; Zhang, Gui-hong; Li, Shoujun

    2014-03-01

    Anaplasma platys is a parasite of canine platelets that causes infectious cyclic thrombocytopenia. In this study, a novel real-time loop-mediated isothermal amplification (RT-LAMP) method was developed to detect A. platys. RT-LAMP primer sets were designed using a citrate synthase gene sequence and the assay was performed at 63 °C for 30 min. No cross-reactivity was observed with other Anaplasma or Ehrlichia spp. and the method exhibited a similar level of sensitivity in detecting the organism in 58 canine blood samples to that of a nested PCR. This RT-LAMP is a rapid and potentially cost-effective method of diagnosing A. platys infection in dogs. Copyright © 2014. Published by Elsevier Ltd.

  6. Bacteriophage amplification assay for detection of Listeria spp. using virucidal laser treatment

    Directory of Open Access Journals (Sweden)

    I.C. Oliveira

    2012-09-01

    Full Text Available A protocol for the bacteriophage amplification technique was developed for quantitative detection of viable Listeria monocytogenes cells using the A511 listeriophage with plaque formation as the end-point assay. Laser and toluidine blue O (TBO were employed as selective virucidal treatment for destruction of exogenous bacteriophage. Laser and TBO can bring a total reduction in titer phage (ca. 10(8 pfu/mL without affecting the viability of L. monocytogenes cells. Artificially inoculated skimmed milk revealed mean populations of the bacteria as low as between 13 cfu/mL (1.11 log cfu/mL, after a 10-h assay duration. Virucidal laser treatment demonstrated better protection of Listeria cells than the other agents previously tested. The protocol was faster and easier to perform than standard procedures. This protocol constitutes an alternative for rapid, sensitive and quantitative detection of L. monocytogenes.

  7. Ultrasensitive detection of microRNA through rolling circle amplification on a DNA tetrahedron decorated electrode.

    Science.gov (United States)

    Miao, Peng; Wang, Bidou; Meng, Fanyu; Yin, Jian; Tang, Yuguo

    2015-03-18

    MicroRNAs are a class of evolutionally conserved, small noncoding RNAs involved in the regulation of gene expression and affect a variety of biological processes including cellular differentiation, immunological response, tumor development, and so on. Recently, microRNAs have been identified as promising disease biomarkers. In this work, we have fabricated a novel electrochemical method for ultrasensitive detection of microRNA. Generally, a DNA tetrahedron decorated gold electrode is employed as the recognition interface. Then, hybridizations between DNA tetrahedron, microRNA, and primer probe initiate rolling circle amplification (RCA) on the electrode surface. Silver nanoparticles attached to the RCA products provide significant electrochemical signals and a limit of detection as low as 50 aM is achieved. Moreover, homology microRNA family members with only one or two mismatches can be successfully distinguished. Therefore, this proposed method reveals great advancements toward improved disease diagnosis and prognosis.

  8. Experimental amplification of an entangled photon: what if the detection loophole is ignored?

    CERN Document Server

    Pomarico, Enrico; Sekatski, Pavel; Zbinden, Hugo; Gisin, Nicolas

    2011-01-01

    The experimental verification of quantum features, such as entanglement, at large scales is extremely challenging because of environment-induced decoherence. Indeed, measurement techniques for demonstrating the quantumness of multiparticle systems in the presence of losses are difficult to define and, if not sufficiently accurate, they provide wrong conclusions. We present a Bell test where one photon of an entangled pair is amplified and then detected by threshold detectors, whose signals undergo postselection. The amplification is performed by a classical machine, which produces a fully separable micro-macro state. However, by adopting such a technique, one can surprisingly observe a violation of the CHSH inequality. This is due to the fact that ignoring the detection loophole, opened by the postselection and the system losses, can lead to misinterpretations, such as claiming the micro-macro entanglement in a setup where evidently there is not. By using threshold detectors and postselection, one can only in...

  9. Rapid detection of Salmonella Typhi by loop-mediated isothermal amplification (LAMP) method.

    Science.gov (United States)

    Abdullah, J; Saffie, N; Sjasri, F A R; Husin, A; Abdul-Rahman, Z; Ismail, A; Aziah, I; Mohamed, M

    2014-01-01

    An in-house loop-mediated isothermal amplification (LAMP) reaction was established and evaluated for sensitivity and specificity in detecting the presence of Salmonella Typhi (S. Typhi) isolates from Kelantan, Malaysia. Three sets of primers consisting of two outer and 4 inner were designed based on locus STBHUCCB_38510 of chaperone PapD of S. Typhi genes. The reaction was optimised using genomic DNA of S. Typhi ATCC7251 as the template. The products were visualised directly by colour changes of the reaction. Positive results were indicated by green fluorescence and negative by orange colour. The test was further evaluated for specificity, sensitivity and application on field samples. The results were compared with those obtained by gold standard culture method and Polymerase Chain Reaction (PCR). This method was highly specific and -10 times more sensitive in detecting S. Typhi compared to the optimised conventional polymerase chain reaction (PCR) method.

  10. Rapid detection of Salmonella Typhi by loop-mediated isothermal amplification (LAMP method

    Directory of Open Access Journals (Sweden)

    J. Abdullah

    2014-12-01

    Full Text Available An in-house loop-mediated isothermal amplification (LAMP reaction was established and evaluated for sensitivity and specificity in detecting the presence of Salmonella Typhi (S. Typhi isolates from Kelantan, Malaysia. Three sets of primers consisting of two outer and 4 inner were designed based on locus STBHUCCB_38510 of chaperone PapD of S. Typhi genes. The reaction was optimised using genomic DNA of S. Typhi ATCC7251 as the template. The products were visualised directly by colour changes of the reaction. Positive results were indicated by green fluorescence and negative by orange colour. The test was further evaluated for specificity, sensitivity and application on field samples. The results were compared with those obtained by gold standard culture method and Polymerase Chain Reaction (PCR. This method was highly specific and -10 times more sensitive in detecting S. Typhi compared to the optimised conventional polymerase chain reaction (PCR method.

  11. Development of a Recombinase Polymerase Amplification Assay for Rapid Detection of the Mycobacterium avium subsp. paratuberculosis.

    Science.gov (United States)

    Hansen, Sören; Schäfer, Jenny; Fechner, Kim; Czerny, Claus-Peter; Abd El Wahed, Ahmed

    2016-01-01

    The detection of Mycobacterium avium subsp. paratuberculosis (MAP) infections in ruminants is crucial to control spread among animals and to humans. Cultivation of MAP is seen as the gold standard for detection, although it is very time consuming and labour intensive. In addition, several PCR assays have been developed to detect MAP in around 90 minutes, but these assays required highly sophisticated equipment as well as lengthy and complicated procedure. In this study, we have developed a rapid assay for the detection of MAP based on the recombinase polymerase amplification (RPA) assay targeting a MAP specific region, the IS900 gene. The detection limit was 16 DNA molecules in 15 minutes as determined by the probit analysis on eight runs of the plasmid standard. Cross reactivity with other mycobacterial and environmentally associated bacterial strains was not observed. The clinical performance of the MAP RPA assay was tested using 48 MAP-positive and 20 MAP-negative blood, sperm, faecal and tissue samples. All results were compared with reads of a highly sensitive real-time PCR assay. The specificity of the MAP RPA assay was 100%, while the sensitivity was 89.5%. The RPA assay is quicker and much easier to handle than real-time PCR. All RPA reagents were cold-chain independent. Moreover, combining RPA assay with a simple extraction protocol will maximize its use at point of need for rapid detection of MAP.

  12. The detection of Plasmodiophora brassicae using loop-mediated isothermal DNA amplification

    Directory of Open Access Journals (Sweden)

    Joanna Kaczmarek

    2014-12-01

    Full Text Available Plasmodiophora brassicae, the cause of clubroot, is a very serious problem preventing from successful and profitable cultivation of oilseed rape in Poland. The pathogen was found in all main growing areas of oilseed rape; it also causes considerable problems in growing of vegetable brassicas. The aim of this work was to elaborate fast, cheap and reliable screening method to detect P. brassicae. To achieve this aim the Loop-mediated isothermal DNA amplification (LAMP technique has been elaborated. The set of three primer pairs was designed using LAMP software. The detection was performed with the GspSSD polymerase, isolated from bacteria Geobacillus sp., with strand displacement activity. DNA extraction from clubbed roots obtained from farmers’ fields of oilseed rape infected by P. brassicae was done using a modified CTAB method. The reaction was performed for 60 min at 62oC. The visual detection was done using CFX96 Real Time PCR Detection System (BioRad or Gerie II Amplicatior (Optigen. The detection with LAMP proved its usefulness; it was easy, fast and accurate and independent of plant age. The detection limit was 5 spores per 1 µl of the spore suspension, so LAMP was less sensitive than quantitative PCR tests reported in the literature. However, the method is cheap and simple, so it is a good alternative, when it comes to practical use and the assessment of numerous samples.

  13. Application of protein misfolding cyclic amplification to detection of prions in anaerobic digestate.

    Science.gov (United States)

    Gilroyed, Brandon H; Braithwaite, Shannon L; Price, Luke M; Reuter, Tim; Czub, Stefanie; Graham, Catherine; Balachandran, Arumuga; McAllister, Tim A; Belosevic, Miodrag; Neumann, Norman F

    2015-11-01

    The exceptional physio-chemical resistance of prions to established decontamination procedures poses a challenge to assessing the suitability of applied inactivation methods. Prion detection is limited by the sensitivity level of Western blotting or by the cost and time factors of bioassays. In addition, prion detection assays can be limited by either the unique or complex nature of matrices associated with environmental samples. To investigate anaerobic digestion (AD) as a practical and economical approach for potential conversion of specified risk materials (SRM) into value added products (i.e., renewable energy), challenges associated with detection of prions in a complex matrix need to be overcome to determine potential inactivation. Protein misfolding cyclic amplification (PMCA) assay, with subsequent Western blot visualization, was used to detect prions within the AD matrix. Anaerobic digestate initially inhibited the PMCA reaction and/or Western blot detection. However, at concentrations of ≤1% of anaerobic digestate, 263K scrapie prions could be amplified and semi-quantitatively detected. Infectious 263K prions were also proven to be bioavailable in the presence of high concentrations of digestate (10-90%). Development of the PMCA application to digestate provides extremely valuable insight into the potential degradation and/or fate of prions in complex biological matrices without requiring expensive and time-consuming bioassays.

  14. Detection of Papaya leaf distortion mosaic virus by reverse-transcription loop-mediated isothermal amplification.

    Science.gov (United States)

    Shen, Wentao; Tuo, Decai; Yan, Pu; Li, Xiaoying; Zhou, Peng

    2014-01-01

    Papaya leaf distortion mosaic virus (PLDMV) can infect transgenic papaya resistant to a related pathogen, Papaya ringspot virus (PRSV), posing a substantial threat to papaya production in China. Current detection methods, however, are unable to be used for rapid detection in the field. Here, a reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the detection of PLDMV, using a set of four RT-LAMP primers designed based on the conserved sequence of PLDMV CP. The RT-LAMP method detected specifically PLDMV and was highly sensitive, with a detection limit of 1.32×10(-6) μg of total RNA per reaction. Indeed, the reaction was 10 times more sensitive than one-step RT-PCR, while also requiring significantly less time and equipment. The effectiveness of RT-LAMP and one-step RT-PCR in detecting the virus were compared using 90 field samples of non-transgenic papaya and 90 field samples of commercialized PRSV-resistant transgenic papaya from Hainan Island. None of the non-transgenic papaya tested positive for PLDMV using either method. In contrast, 19 of the commercialized PRSV-resistant transgenic papaya samples tested positive by RT-LAMP assay, and 6 of those tested negative by RT-PCR. Therefore, the PLDMV-specific RT-LAMP is a simple, rapid, sensitive, and cost-effective tool in the field diagnosis and control of PLDMV.

  15. Reverse transcription loop-mediated isothermal amplification assay for rapid detection of Papaya ringspot virus.

    Science.gov (United States)

    Shen, Wentao; Tuo, Decai; Yan, Pu; Yang, Yong; Li, Xiaoying; Zhou, Peng

    2014-08-01

    Papaya ringspot virus (PRSV) and Papaya leaf distortion mosaic virus (PLDMV), which causes disease symptoms similar to PRSV, threaten commercial production of both non-transgenic-papaya and PRSV-resistant transgenic papaya in China. A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay to detect PLDMV was developed previously. In this study, the development of another RT-LAMP assay to distinguish among transgenic, PRSV-infected and PLDMV-infected papaya by detection of PRSV is reported. A set of four RT-LAMP primers was designed based on the highly conserved region of the P3 gene of PRSV. The RT-LAMP method was specific and sensitive in detecting PRSV, with a detection limit of 1.15×10(-6)μg of total RNA per reaction. Indeed, the reaction was 10 times more sensitive than one-step RT-PCR. Field application of the RT-LAMP assay demonstrated that samples positive for PRSV were detected only in non-transgenic papaya, whereas samples positive for PLDMV were detected only in commercialized PRSV-resistant transgenic papaya. This suggests that PRSV remains the major limiting factor for non-transgenic-papaya production, and the emergence of PLDMV threatens the commercial transgenic cultivar in China. However, this study, combined with the earlier development of an RT-LAMP assay for PLDMV, will provide a rapid, sensitive and cost-effective diagnostic power to distinguish virus infections in papaya.

  16. Rapid isothermal detection of Phytophthora species on plant samples using recombinase polymerase amplification

    Science.gov (United States)

    Recently several isothermal amplification techniques have been developed that are extremely tolerant towards inhibitors present in many plant extracts. Recombinase polymerase amplification (RPA) assays for the genus Phytophthora have been developed which provide a simple and rapid method to macerate...

  17. Application of hyperbranched rolling circle amplification for direct detection of mycobacterium tuberculosis in clinical sputum specimens.

    Directory of Open Access Journals (Sweden)

    Yang Liu

    Full Text Available BACKGROUND: Global tuberculosis (TB control is encumbered by the lack of a rapid and simple detection method for diagnosis, especially in low-resource areas. An isothermal amplification method, hyperbranched rolling circle amplification (HRCA, was optimized to detect Mycobacterium tuberculosis (Mtb in clinical sputum specimens. METHODS: A clinical validation study was performed to assess the diagnostic accuracy of HRCA. In order to analyze the detection limit of HRCA under optimal conditions, the method was initially used to detect purified H37Rv strain DNA and culture suspensions. Next, three strains of Mycobacterium tuberculosis complex (MTC and eight strains of non-tuberculosis mycobacterium (NTM were analyzed in order to evaluate specificity. Sputum specimens from 136 patients with diagnosed pulmonary TB, 38 lung cancer patients, and 34 healthy donors were tested by HRCA to validate the clinical application of HRCA for the rapid detection of Mtb. RESULTS: The detection limit of HRCA for purified H37Rv DNA and culture suspensions was 740 aM and 200cfu/ml, respectively. The results of all MTC strains were positive in contrast to the NTM specimens which were all negative. The detection sensitivity for the 136 sputum specimens from TB patients was 77.2% (105/136, which was slightly lower than that of quantitative real-time PCR(79.4%, 108/136 and culture (80.9%,110/136. The sensitivity of all three methods was statistically higher than smear microscopy (44.9%, 61/136. The overall specificity of HRCA was 98.6% (71/72 which was similar to that of quantitative real-time PCR (qRT-PCR and smear/culture methods (100%, 72/72. CONCLUSIONS: Use of the HRCA assay for detection of Mtb within clinical sputum specimens was demonstrated to be highly sensitive and specific. Moreover, the performance of HRCA is simple and cost-effective compared with qRT-PCR and is less time consuming than culture. Therefore, HRCA is a promising TB diagnostic tool that can be

  18. Detection of Vibrio cholerae by isothermal cross-priming amplification combined with nucleic acid detection strip analysis.

    Science.gov (United States)

    Zhang, Xia; Du, Xin-Jun; Guan, Chun; Li, Ping; Zheng, Wen-Jie; Wang, Shuo

    2015-08-01

    Vibrio cholerae is a water- and food-borne human pathogen, and V. cholerae serotypes O1 and O139 have attracted attention because of their severe pathogenesis. However, non-O1, non-O139 cholera vibrios (NCVs) were also recently recognized as having virulence properties. In this study, we developed a cross-priming amplification (CPA) method for the detection of all serotypes of V. cholerae. The specificity of the CPA method was tested using a panel of 60 different bacterial strains. All of the V. cholerae strains showed positive results, and 41 other types of bacteria gave negative results. The limit of detection of the CPA method was 79.28 fg of genomic DNA, 4.2 × 10(2) CFU/ml for bacteria in pure culture, and 5.6 CFU per 25 g of sample with pre-enrichment. This method showed a higher sensitivity than the loop-mediated isothermal amplification (LAMP) method did and was more convenient to perform. These results indicate that the CPA method can be used for the rapid preliminary screening of V. cholerae. Copyright © 2015 Elsevier Ltd. All rights reserved.

  19. Amplification of resonant Rayleigh light scattering response using immunogold colloids for detection of lysozyme.

    Science.gov (United States)

    Truong, Phuoc Long; Choi, Seung Phill; Sim, Sang Jun

    2013-10-25

    A strategy for attomolar-level detection of small molecule-size proteins is reported based on Rayleigh light scattering spectroscopy of individual nanoplasmonic aptasensors by exploiting the outstanding characteristics of gold colloids to amplify the nontransparent resonant signal at ultralow analyte concentrations. The fabrication method utilizes thiol-mediated adsorption of a DNA aptamer on the immobilized Au nanoparticle surface, the interfacial binding characteristics of the aptamer with its target molecules, and the antibody-antigen interaction through plasmonic resonance coupling of the Au nanoparticles. Using lysozyme as a model analyte for disease detection, the detection limit of the aptasensor is ∼7 × 10(3) aM, corresponding to the LSPR λmax shift of ∼2.25 nm. Up to a 380% increase in the localized resonant λmax shift is demonstrated upon antibody binding to the analyte compared to the primary response during signal amplification using immunogold colloids. This enhancement leads to a limit of detection of ∼7 aM, which is an improvement of three orders of magnitude. The results demonstrate substantial promise for developing coupled plasmonic nanostructures for ultrasensitive detection of various biological and chemical analytes. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Loop-mediated isothermal amplification targeting 18S ribosomal DNA for rapid detection of Acanthamoeba.

    Science.gov (United States)

    Yang, Hye-Won; Lee, Yu-Ran; Inoue, Noboru; Jha, Bijay Kumar; Danne, Dinzouna-Boutamba Sylvatrie; Kim, Hong-Kyun; Lee, Junhun; Goo, Youn-Kyoung; Kong, Hyun-Hee; Chung, Dong-Il; Hong, Yeonchul

    2013-06-01

    Amoebic keratitis (AK) caused by Acanthamoeba is one of the most serious corneal infections. AK is frequently misdiagnosed initially as viral, bacterial, or fungal keratitis, thus ensuring treatment delays. Accordingly, the early detection of Acanthamoeba would contribute significantly to disease management and selection of an appropriate anti-amoebic therapy. Recently, the loop-mediated isothermal amplification (LAMP) method has been applied to the clinical diagnosis of a range of infectious diseases. Here, we describe a rapid and efficient LAMP-based method targeting Acanthamoeba 18S rDNA gene for the detection of Acanthamoeba using clinical ocular specimens in the diagnosis of AK. Acanthamoeba LAMP assays detected 11 different strains including all AK-associated species. The copy number detection limit for a positive signal was 10 DNA copies of 18S rDNA per reaction. No cross-reactivity with the DNA of fungi or other protozoa was observed. The sensitivity of LAMP assay was higher than those of Nelson primer PCR and JDP primer PCR. In the present study, LAMP assay based on directly heat-treated samples was found to be as efficient at detecting Acanthamoeba as DNA extracted using a commercial kit, whereas PCR was only effective when commercial kit-extracted DNA was used. This study showed that the devised Acanthamoeba LAMP assay could be used to diagnose AK in a simple, sensitive, and specific manner.

  1. Rapid detection of Staphylococcus aureus by loop-mediated isothermal amplification.

    Science.gov (United States)

    Wang, Xin-Ru; Wu, Li-Fen; Wang, Yan; Ma, Ying-Ying; Chen, Feng-Hua; Ou, Hong-Ling

    2015-01-01

    Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA), is a major bacterial pathogen associated with nosocomial and community-acquired S. aureus infections all over the world. A rapid detection assay for staphylococcal gene of nuc and mecA is needed. In this study, a rapid identification assay based on the loop-mediated isothermal amplification (LAMP) method was established. PCR and LAMP assays were used to detect Staphylococcus aureus and other related species for nuc and mecA. With optimization of the primers and reaction temperature, the LAMP successfully amplified the genes under isothermal conditions at 62 °C within 60 min, of which the results were identical with those of the conventional PCR methods. The detection limits of the LAMP for nuc and mecA were 1.47 and 14.7 pg/μl DNA per tube, respectively, by naked eye inspections, while the detection limits of the PCR for nuc and mecA were 14.7 pg/μl and 147 pg/μl DNA, respectively. Finally, The LAMP method was then applied to clinical blood plaque samples. The LAMP and PCR demonstrated identical results for the plaque samples with the culture assay. Together, the LAMP offers an alternative detection assay for nuc and mecA with a great advantage of the sensitivity and rapidity.

  2. Centrifugal loop-mediated isothermal amplification microdevice for rapid, multiplex and colorimetric foodborne pathogen detection.

    Science.gov (United States)

    Oh, Seung Jun; Park, Byung Hyun; Jung, Jae Hwan; Choi, Goro; Lee, Doh C; Kim, Do Hyun; Seo, Tae Seok

    2016-01-15

    We present a centrifugal microfluidic device which enables multiplex foodborne pathogen identification by loop-mediated isothermal amplification (LAMP) and colorimetric detection using Eriochrome Black T (EBT). Five identical structures were designed in the centrifugal microfluidic system to perform the genetic analysis of 25 pathogen samples in a high-throughput manner. The sequential loading and aliquoting of the LAMP cocktail, the primer mixtures, and the DNA sample solutions were accomplished by the optimized zigzag-shaped microchannels and RPM control. We targeted three kinds of pathogenic bacteria (Escherichia coli O157:H7, Salmonella typhimurium and Vibrio parahaemolyticus) and detected the amplicons of the LAMP reaction by the EBT-mediated colorimetric method. For the limit-of-detection (LOD) test, we carried out the LAMP reaction on a chip with serially diluted DNA templates of E. coli O157:H7, and could observe the color change with 380 copies. The used primer sets in the LAMP reaction were specific only to the genomic DNA of E. coli O157:H7, enabling the on-chip selective, sensitive, and high-throughput pathogen identification with the naked eyes. The entire process was completed in 60min. Since the proposed microsystem does not require any bulky and expensive instrumentation for end-point detection, our microdevice would be adequate for point-of-care (POC) testing with high simplicity and high speed, providing an advanced genetic analysis microsystem for foodborne pathogen detection.

  3. Challenging loop-mediated isothermal amplification (LAMP) technique for molecular detection ofToxoplasma gondii

    Institute of Scientific and Technical Information of China (English)

    Shirzad Fallahi; Zahra Arab Mazar; Mehrdad Ghasemian; Ali Haghighi

    2015-01-01

    Objective:To compare analytical sensitivity and specificity of a newly described DNA amplification technique, LAMP and nested PCR assay targeting the RE and B1 genes for the detection ofToxoplasma gondii (T. gondii)DNA.Methods: The analytical sensitivity of LAMP and nested-PCR was obtained against10-fold serial dilutions ofT. gondii DNA ranging from 1 ng to 0.01 fg. DNA samples of other parasites and human chromosomal DNA were used to determine the specificity of molecular assays.Results:After testing LAMP and nested-PCR in duplicate, the detection limit of RE-LAMP, B1-LAMP, RE-nested PCR and B1-nested PCR assays was one fg, 100 fg, 1 pg and 10 pg ofT. gondii DNA respectively. All the LAMP assays and nested PCRs were 100% specific. The RE-LAMP assay revealed the most sensitivity for the detection ofT. gondii DNA.Conclusions:The obtained results demonstrate that the LAMP technique has a greater sensitivity for detection ofT. gondii. Furthermore, these findings indicate that primers based on the RE are more suitable than those based on the B1 gene. However, the B1-LAMP assay has potential as a diagnostic tool for detection ofT. gondii.

  4. Rapid, sensitive, and specific detection of Clostridium tetani by loop-mediated isothermal amplification assay.

    Science.gov (United States)

    Jiang, Dongneng; Pu, Xiaoyun; Wu, Jiehong; Li, Meng; Liu, Ping

    2013-01-01

    Tetanus is a specific infectious disease, which is often associated with catastrophic events such as earthquakes, traumas, and war wounds. The obligate anaerobe Clostridium tetani is the pathogen that causes tetanus. Once the infection of tetanus progresses to an advanced stage within the wounds of limbs, the rates of amputation and mortality increase manifold. Therefore, it is necessary to devise a rapid and sensitive point-of-care detection method for C. tetani so as to ensure an early diagnosis and clinical treatment of tetanus. In this study, we developed a detection method for C. tetani using loop-mediated isothermal amplification (LAMP) assay, wherein the C. tetani tetanus toxin gene was used as the target gene. The method was highly specific and sensitive, with a detection limit of 10 colony forming units (CFU)/ml, and allowed quantitative analysis. While detecting C. tetani in clinical samples, it was found that the LAMP results completely agreed with those of the traditional API 20A anaerobic bacteria identification test. As compared with the traditional API test and PCR assay, LAMP detection of C. tetani is simple and rapid, and the results can be identified through naked-eye observation. Therefore, it is an ideal and rapid point-of-care testing method for tetanus.

  5. Rapid detection of squash leaf curl virus by loop-mediated isothermal amplification.

    Science.gov (United States)

    Kuan, Cheng-Ping; Wu, Min-Tze; Lu, Yi-Lin; Huang, Hung-Chang

    2010-10-01

    A loop-mediated isothermal amplification (LAMP) assay was employed to develop a simple and efficient system for the detection of squash leaf curl virus (SLCV) in diseased plants of squash (Cucurbita pepo) and melon (Cucumis melo). Completion of LAMP assay required 30-60 min under isothermal conditions at 65 degrees C by employing a set of four primers targeting SLCV. Although the sensitivity of the LAMP assay and the polymerase chain reaction (PCR) assay was comparable at high virus concentrations, the LAMP assay was by a 10-fold dilution factor more sensitive than the PCR assay for the detection of SLCV in diseased plants. No reaction was detected in the tissues of healthy plants by either the LAMP or the PCR. The LAMP products can be visualized by staining directly in the tube with SYBR Safe DNA gel stain dye. The sensitivity of the SYBR Safe DNA gel stain is similar to analysis by gel electrophoresis. Although both the LAMP and the PCR methods were capable of detecting SLCV in infected tissues of squash and melon, the LAMP method would be more useful than the PCR method for detection of SLCV infection in cucurbitaceous plants because it is more rapid, simple, accurate and sensitive.

  6. Direct detection of Marek's disease virus in poultry dust by loop-mediated isothermal amplification.

    Science.gov (United States)

    Woźniakowski, Grzegorz; Samorek-Salamonowicz, Elżbieta

    2014-11-01

    Marek's disease virus (MDV) is a serious concern for poultry production and represents a unique herpesvirus model. MDV can be shed by doubly infected chickens despite vaccination. The fully infectious MDV particles are produced in the feather follicle epithelium (FFE), and MDV remains infectious for many months in fine skin particles and feather debris. Molecular biology methods including PCR and real-time PCR have been shown to be valuable for the detection of MDV DNA in farm dust. Recently, loop-mediated isothermal amplification (LAMP) was found to be useful in the detection of MDV in feathers and internal organs of infected chickens. LAMP is also less affected by the inhibitors present in DNA samples. Taking into account the advantages of LAMP, direct detection of MDV DNA in poultry dust has been conducted in this research. The detection of MDV DNA was possible in 11 out of the 12 examined dust samples without DNA extraction. The DNA was retrieved from dust samples by dilution and incubation at 95 °C for 5 min. The direct detection of MDV DNA in the dust was possible within 30 min using a water bath and UV light. The results were confirmed by electrophoresis and melting curve analysis of the LAMP products. Our results show that LAMP may be used to test for the presence of virulent MDV in poultry farm dust without DNA extraction.

  7. Sensitive detection of multiple mycotoxins by SPRi with gold nanoparticles as signal amplification tags.

    Science.gov (United States)

    Hu, Weihua; Chen, Hongming; Zhang, Huanhuan; He, Guangli; Li, Xin; Zhang, Xiaoxing; Liu, Yang; Li, Chang Ming

    2014-10-01

    Detection of multiple toxic mycotoxins is of importance in food quality control. Surface plasmon resonance imaging (SPRi) is an advanced tool for simultaneously multiple detections with accuracy; however, it suffers from limited sensitivity due to the instrumental constraint and small sizes of mycotoxins with only one epitope for an insensitive competitive immunoassay. In this work a gold nanoparticle (AuNP)-enhanced SPRi chip is designed to sensitively detect multiple mycotoxins using a competitive immunoassay format. The sensing surface is constructed by uniformly attaching dense mycotoxin antigens on poly[oligo(ethylene glycol) methacrylate-co-glycidyl methacrylate] (POEGMA-co-GMA) brush modified SPRi gold chip. After competitive binding in a sample solution containing respective monoclonal antibodies, secondary antibody-conjugated AuNPs are employed to bind with the captured monoclonal antibodies for further amplification of the SPRi signal. Highly specific and sensitive simultaneous detection is achieved for three typical mycotoxins including Aflatoxin B1 (AFB1), Ochratoxin A (OTA) and Zearalenone (ZEN) with low detection limits of 8, 30 and 15 pg mL(-1) and dynamic ranges covering three orders of magnitude.

  8. Novel reverse transcription loop-mediated isothermal amplification for rapid detection of foot-and-mouth disease virus

    DEFF Research Database (Denmark)

    Dukes, J.P.; King, D.P.; Alexandersen, Søren

    2006-01-01

    Speed is paramount in the diagnosis of foot-and-mouth disease (FMD) and simplicity is required if a test is to be deployed in the field. The development of a one-step, reverse transcription loop-mediated amplification (RT-LAMP) assay enables FMD virus (FMDV) to be detected in under an hour...... in a single tube without thermal cycling. A fragment of the 3D RNA polymerase gene of the virus is amplified at 65 degrees C in the presence of a primer mixture and both reverse transcriptase and Bst DNA polymerase. Compared with real-time RT-PCR, RT-LAMP was consistently faster, and ten copies of FMDV...... transcript were detected in twenty-two minutes. Amplification products were detected by visual inspection, agarose gel electrophoresis, or in real-time by the addition of a fluorescent dye. The specificity of the reaction was demonstrated by the absence of amplification of RNA from other viruses that cause...

  9. Detection of Tumor Suppressor Gene and Oncogene in SO-Rb_(50) Human Retinoblastoma Cell Line

    Institute of Scientific and Technical Information of China (English)

    1993-01-01

    Retinoblastoma (Rb) is the most common malignant'cancer of eye. So-Rb_(50) is the first Rb cell line established in China in 1988. It has passed to the 387th passage now. We collected cells of the 327th passage of SO-Rb_(50), purified its genomic DNA and detected it with Rb and c-myc cDNA probes respectively (normal human white blood cells DNA was the control). We found the Rb gene was deleted while c-myc gene was amplified three times. This provides a basis for further study of the regulation of tumor ...

  10. Extraction, amplification and detection of DNA in microfluidic chip-based assays

    KAUST Repository

    Wu, Jinbo

    2013-12-20

    This review covers three aspects of PCR-based microfluidic chip assays: sample preparation, target amplification, and product detection. We also discuss the challenges related to the miniaturization and integration of each assay and make a comparison between conventional and microfluidic schemes. In order to accomplish these essential assays without human intervention between individual steps, the micro-components for fluid manipulation become critical. We therefore summarize and discuss components such as microvalves (for fluid regulation), pumps (for fluid driving) and mixers (for blending fluids). By combining the above assays and microcomponents, DNA testing of multi-step bio-reactions in microfluidic chips may be achieved with minimal external control. The combination of assay schemes with the use of micro-components also leads to rapid methods for DNA testing via multi-step bioreactions. Contains 259 references.

  11. Detection of Streptococcus mutans using padlock probe based on Rolling Circle Amplification (RCA

    Directory of Open Access Journals (Sweden)

    Mônica Moreira

    2015-02-01

    Full Text Available The aim of this study was to develop and evaluate a padlock probe based on the Rolling Circle Amplification (RCA, which targeted to 16S-23S rDNA region of S. mutans. The specificity of developed padlock probe was tested for DNA within a panel strains, including S. mutans isolated from the saliva and reference strains of the genus Streptococcus, as well as total DNA samples of biofilm and saliva. The results were positive either for DNA samples of S. mutans or DNA samples recovered from the biofilm and saliva revealing the specificity of designed padlock probe. The padlock probe based on the RCA was proved to be an effective, reproducible method for S. mutansdetection and demonstrated the possibility of a rapid detection and accurate identification of S. mutans infection.

  12. Sensitive and rapid detection of Giardia lamblia infection in pet dogs using loop-mediated isothermal amplification.

    Science.gov (United States)

    Li, Jie; Wang, Peiyuan; Zhang, Aiguo; Zhang, Ping; Alsarakibi, Muhamd; Li, Guoqing

    2013-04-01

    Giardia lamblia is recognized as one of the most prevalent parasites in dogs. The present study aimed to establish a loop-mediated isothermal amplification (LAMP) assay for rapid and specific detection of G. lamblia from dogs. The fecal samples were collected and prepared for microscopic analysis, and then the genomic DNA was extracted directly from purified cysts. The concentration of DNA samples of G. lamblia were diluted by 10-fold serially ranging from 10(-1) to 10(-5) ng/µl for LAMP and PCR assays. The LAMP assay allows the amplification to be finished within 60 min under isothermal conditions of 63℃ by employing 6 oligonucleotide primers designed based on G. lamblia elongation factor 1 alpha (EF1α) gene sequence. Our tests showed that the specific amplification products were obtained only with G. lamblia, while no amplification products were detected with DNA of other related protozoans. Sensitivity evaluation indicated that the LAMP assay was sensitive 10 times more than PCR. It is concluded that LAMP is a rapid, highly sensitive and specific DNA amplification technique for detection of G. lamblia, which has implications for effective control and prevention of giardiasis.

  13. Highly Sensitive Loop-Mediated Isothermal Amplification for the Detection of Leptospira

    Directory of Open Access Journals (Sweden)

    Hua-Wei Chen

    2015-01-01

    Full Text Available Leptospirosis is a worldwide zoonosis caused by an infection with the pathogenic species of Leptospira. We have developed a loop-mediated isothermal amplification (LAMP assay to detect the DNA of Leptospira spp. Six sets of primers targeting the gene of the subsurface protein, lipL32, were evaluated for their detection sensitivity. The best primer set detected less than 25 copies of lipL32 per reaction of both plasmid DNA template and purified leptospiral genomic DNA. By combining primers targeting lipL32 with the previously published primer set targeting lipL41, the sensitivity of the assay was improved to 12 copies of L. interrogans. The specificity of the LAMP assay was evaluated by using the genomic DNA from other clinically encountered bacterial species such as different strains of Orientia tsutsugamushi, Rickettsia typhi, Rickettsia conorii, Rickettsia rickettsii, Coxiella burnetii, and Bartonella bacilliformis. These genomic DNA samples were all negative in our LAMP assay. The sensitivity of the LAMP assay was very similar to that of quantitative real time PCR. Several detection methods for the amplified product of LAMP assay were performed to demonstrate the simplicity of the assay. In summary, our results have suggested that this assay is rapid, robust, and easy to perform and has the potential to be used in endemic locations.

  14. Detection of Brucellosis in Sika Deer ( Cervus nippon ) through Loop-mediated Isothermal Amplification (LAMP).

    Science.gov (United States)

    Liu, Qianhong; Wei, Jie; Sun, Qingsong; Wang, Ben; Wang, Yuting; Hu, Ying; Wu, Wenrong

    2017-03-20

    Brucellosis (Brucella bovis) in sika deer ( Cervus nippon ) can cause enormous losses to stag breeding, especially in areas in which stag breeding has become an important industry. It also poses a threat to humans because it is a zoonotic disease. Use of the loop-mediated isothermal amplification (LAMP) assay has been poorly described in the diagnosis of brucellosis in deer. We developed a LAMP assay targeting the omp25 gene sequence to detect brucellosis (Brucella bovis) in sika deer. The reaction can be completed in 60 min at 63 C and, with a detection limit of 17 pg, it was more sensitive than conventional PCR, with its detection limit of 1.7 ng. No cross-reactivity was observed with four bacteria: Escherichia coli , Salmonella enterica subsp. enterica, Clostridium pasteurianum , and Pseudomonas aeruginosa . We used 263 samples of blood to evaluate the reaction. The percentage of agreement between LAMP and PCR reached 91%; relative specificity reached 87%, and relative sensitivity reached 100%. The results indicate LAMP can be a simple and rapid diagnostic tool for detecting brucellosis in sika deer, particularly in the field, where it is essential to control brucellosis in deer with a rapid and accurate diagnosis for removal of positive animals.

  15. A dumbell probe-mediated rolling circle amplification strategy for highly sensitive transcription factor detection.

    Science.gov (United States)

    Li, Chunxiang; Qiu, Xiyang; Hou, Zhaohui; Deng, Keqin

    2015-02-15

    Highly sensitive detection of transcription factors (TF) is essential to proteome and genomics research as well as clinical diagnosis. We describe herein a novel fluorescent-amplified strategy for ultrasensitive, quantitative, and inexpensive detection of TF. The strategy consists of a hairpin DNA probe containing a TF binding sequence for target TF, a dumbbell-shaped probe, a primer DNA probe designed partly complementary to hairpin DNA probe, and a dumbbell probe. In the presence of target TF, the binding of the TF with hairpin DNA probe will prohibit the hybridization of the primer DNA probe with the "stem" and "loop" region of the hairpin DNA probe, then the unhybridized region of the primer DNA will hybridize with dumbbell probe, subsequently promote the ligation reaction and the rolling circle amplification (RCA), finally, the RCA products are quantified via the fluorescent intensity of SYBR Green I (SG). Using TATA-binding protein (TBP) as a model transcription factor, the proposed assay system can specifically detect TBP with a detection limit as low as 40.7 fM, and with a linear range from 100 fM to 1 nM. Moreover, this assay related DNA probe does not involve any modification and the whole assay proceeds in one tube, which makes the assay simple and low cost. It is expected to become a powerful tool for bioanalysis and clinic diagnostic application.

  16. Endpoint Visual Detection of Three Genetically Modified Rice Events by Loop-Mediated Isothermal Amplification

    Directory of Open Access Journals (Sweden)

    Qing Zhu

    2012-11-01

    Full Text Available Genetically modified (GM rice KMD1, TT51-1, and KF6 are three of the most well known transgenic Bt rice lines in China. A rapid and sensitive molecular assay for risk assessment of GM rice is needed. Polymerase chain reaction (PCR, currently the most common method for detecting genetically modified organisms, requires temperature cycling and relatively complex procedures. Here we developed a visual and rapid loop-mediated isothermal amplification (LAMP method to amplify three GM rice event-specific junction sequences. Target DNA was amplified and visualized by two indicators (SYBR green or hydroxy naphthol blue [HNB] within 60 min at an isothermal temperature of 63 °C. Different kinds of plants were selected to ensure the specificity of detection and the results of the non-target samples were negative, indicating that the primer sets for the three GM rice varieties had good levels of specificity. The sensitivity of LAMP, with detection limits at low concentration levels (0.01%–0.005% GM, was 10- to 100-fold greater than that of conventional PCR. Additionally, the LAMP assay coupled with an indicator (SYBR green or HNB facilitated analysis. These findings revealed that the rapid detection method was suitable as a simple field-based test to determine the status of GM crops.

  17. Development of a loop-mediated isothermal amplification assay for detection of Trichomonas vaginalis.

    Science.gov (United States)

    Reyes, John Carlo B; Solon, Juan Antonio A; Rivera, Windell L

    2014-07-01

    A loop-mediated isothermal amplification (LAMP) assay targeting the 2-kbp repeated DNA species-specific sequence was developed for detection of Trichomonas vaginalis, the causative agent of trichomoniasis. The analytical sensitivity and specificity of the LAMP assay were evaluated using pooled genital swab and urine specimens, respectively, spiked with T. vaginalis trophozoites. Genital secretion and urine did not inhibit the detection of the parasite. The sensitivity of the LAMP was 10-1000 times higher than the PCR performed. The detection limit of LAMP was 1 trichomonad for both spiked genital swab and urine specimens. Also, LAMP did not exhibit cross-reactivity with closely-related trichomonads, Trichomonas tenax and Pentatrichomonas hominis, and other enteric and urogenital microorganisms, Entamoeba histolytica, Candida albicans, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. This is the first report of a LAMP assay for the detection of T. vaginalis and has prospective application for rapid diagnosis and control of trichomoniasis. Copyright © 2014 Elsevier Inc. All rights reserved.

  18. Loop-Mediated Isothermal Amplification for Detection of Staphylococcus aureus in Dairy Cow Suffering from Mastitis

    Directory of Open Access Journals (Sweden)

    Zhang Tie

    2012-01-01

    Full Text Available To develop a rapid detection method of Staphylococcus aureus using loop-mediated isothermal amplification (LAMP, four specific primers were designed according to six distinct sequences of the nuc gene. In addition, the specificity and sensitivity of LAMP were verified and compared with those of PCR. Results showed that the LAMP reaction was completed within 45 min at 62.5°C, and ladder bands were appeared in LAMP products analyzed by gel electrophoresis. After adding 1x SYBR Green l, the positive reaction tube showed green color and the negative reaction tube remained orange, indicating that the LAMP has high specificity. The minimal detectable concentration of LAMP was 1×102 CFU/mL and that of PCR was 1×104 CFU/mL, indicating that the LAMP was 100 times more sensitive than the PCR. The LAMP method for detection of Staphylococcus aureus has many advantages, such as simple operation, high sensitivity, high specificity, and rapid analysis. Therefore, this method is more suitable for the rapid on-site detection of Staphylococcus aureus.

  19. Loop-mediated isothermal amplification method for differentiation and rapid detection of Taenia species.

    Science.gov (United States)

    Nkouawa, Agathe; Sako, Yasuhito; Nakao, Minoru; Nakaya, Kazuhiro; Ito, Akira

    2009-01-01

    Rapid detection and differentiation of Taenia species are required for the control and prevention of taeniasis and cysticercosis in areas where these diseases are endemic. Because of the lower sensitivity and specificity of the conventional diagnosis based on microscopical examination, molecular tools are more reliable for differential diagnosis of these diseases. In this study, we developed and evaluated a loop-mediated isothermal amplification (LAMP) assay for differential diagnosis of infections with Taenia species with cathepsin L-like cysteine peptidase (clp) and cytochrome c oxidase subunit 1 (cox1) genes. LAMP with primer sets to the cox1 gene could differentiate between three species, and LAMP with primer sets to the clp gene could differentiate Taenia solium from Taenia saginata/Taenia asiatica. Restriction enzyme digestion of the LAMP products from primer set Tsag-clp allowed the differentiation of Taenia saginata from Taenia asiatica. We demonstrated the high specificity of LAMP by testing known parasite DNA samples extracted from proglottids (n = 100) and cysticerci (n = 68). LAMP could detect one copy of the target gene or five eggs of T. asiatica and T. saginata per gram of feces, showing sensitivity similar to that of PCR methods. Furthermore, LAMP could detect parasite DNA in all taeniid egg-positive fecal samples (n = 6). Due to the rapid, simple, specific, and sensitive detection of Taenia species, the LAMP assays are valuable tools which might be easily applicable for the control and prevention of taeniasis and cysticercosis in countries where these diseases are endemic.

  20. Development of loop-mediated isothermal amplification for rapid detection of avian leukosis virus subgroup A.

    Science.gov (United States)

    Wang, Yongqiang; Kang, Zhonghui; Gao, Yulong; Qin, Liting; Chen, Lei; Wang, Qi; Li, Jiukuan; Gao, Honglei; Qi, Xiaole; Lin, Huan; Wang, Xiaomei

    2011-04-01

    This study aimed to establish a loop-mediated isothermal amplification (LAMP) method for distinguishing avian leukosis virus (ALV) subgroup A from other subgroups of the virus. On the basis of the results of sequence comparison and the sequence characteristics of ALV subgroups, a LAMP method was designed to target the gp85 segment for detection of ALV-A. Under optimal reaction conditions, ALV-A LAMP produced neither cross-reactions with other major subgroups (including subgroups J, B, C, and E) nor nonspecific reactions with other common avian infectious diseases. A sensitivity test showed that this method can detect 20 copies of proviral nucleic acid sequence within 45 min, which is 100 times more sensitive than the conventional polymerase chain reaction (PCR). This method can detect subgroup A virus rapidly and the results can be assessed based on color changes. The whole reaction process can be performed without opening the lid of the reaction tube, which reduces the possibility of contamination greatly and simplifies the detection process, indicating the considerable potential of this method for in situ application in the future.

  1. Development of Loop-Mediated Isothermal Amplification for Detection of Leifsonia xyli subsp. xyli in Sugarcane

    Directory of Open Access Journals (Sweden)

    Jing Liu

    2013-01-01

    Full Text Available Ratoon stunt, caused by the xylem-limited coryneform bacterium Leifsonia xyli subsp. xyli (Lxx, is a deep bacteriosis and prevalent in most of sugarcane-producing countries. Based on loop-mediated isothermal amplification (LAMP, we developed a method for detecting Lxx. The major advantages of the LAMP method are visual judgment by color and time saving with only 60 min for identification of Lxx and without the need for costly PCR apparatus and gel scanner. In the present study, positive and negative samples detected by the LAMP method were clearly distinguishable. When total DNA extracted from internode juice was used as the template, the sensitivity of LAMP was 10 times higher than that of the conventional PCR detection. The LAMP assay is a highly specific, rapid, and sensitive method for the diagnosis of ratoon stunt caused by Lxx in sugarcane. This is the first report of LAMP-based assay for the detection of Lxx in sugarcane.

  2. A simple identification method of saliva by detecting Streptococcus salivarius using loop-mediated isothermal amplification.

    Science.gov (United States)

    Nakanishi, Hiroaki; Ohmori, Takeshi; Hara, Masaaki; Takada, Aya; Shojo, Hideki; Adachi, Noboru; Saito, Kazuyuki

    2011-01-01

    We previously reported that detection of Streptococcus salivarius is feasible for proving the presence of saliva in a forensic sample. Here, a simple and rapid method for the detection of S. salivarius in forensic samples was developed that uses loop-mediated isothermal amplification (LAMP). The LAMP primer set was designed using S. salivarius-specific sequences of glucosyltransferase K. To simplify the procedure, the sample was prepared by boiling and mutanolysin treatment only, and the entire analytical process was completed within 2.5 h. The cut-off value was set at 0.1 absorbance units, measured at 660 nm, upon termination of the reaction. S. salivarius was identified in all saliva samples, but was not detected in other body fluids or on the skin surface. Using this method, S. salivarius was successfully detected in various mock forensic samples. We therefore suggest that this approach is useful for the identification of saliva in forensic practice. © 2010 American Academy of Forensic Sciences.

  3. A novel dendritic surfactant for enhanced microcystin-LR detection by double amplification in a quartz crystal microbalance biosensor.

    Science.gov (United States)

    Xia, Yuetong; Zhang, Jianping; Jiang, Long

    2011-08-01

    Enhanced sensitivity for the hepatotoxin microcystin-LR (MC-LR) was achieved in a quartz crystal microbalance (QCM) system via double amplification. For primary amplification, an innovative interface on the QCM was obtained as a matrix by the vesicle layer formed by our synthetic dendritic surfactant, bis (amidoethyl-carbamoylethyl) octadecylamine (C18N3). The vesicle matrix was then functionalised by an optimised concentration of monoclonal antibodies against MC-LR (anti-MC-LR) to detect the analyte. The results showed that a detection limit of 100 ng/mL was achieved by primary amplification. To achieve higher sensitivity, secondary amplification was implemented with anti-MC-LR gold nanoparticle (AuNPs) conjugates as probes, which lowered the detection limit for MC-LR to 1 ng/mL (the maximum concentration recommended by the World Health Organization [WHO] in drinking water for humans). The QCM immunosensor reported here has advantages such as high sensitivity, portability, simplicity, and cost-effectiveness for MC-LR detection. It would be uniquely superior compared with current MC-LR detection techniques for on-the-spot water detection. Furthermore, the methodology described here is also potentially significant in many fields for the routine monitoring of environmental and food safety.

  4. Development of RNA-FISH Assay for Detection of Oncogenic FGFR3-TACC3 Fusion Genes in FFPE Samples

    Science.gov (United States)

    Kojima, Takahiro; Nishimura, Kouichi; Kandori, Shuya; Kawahara, Takashi; Yoshino, Takayuki; Ueno, Satoshi; Iizumi, Yuichi; Mitsuzuka, Koji; Arai, Yoichi; Tsuruta, Hiroshi; Habuchi, Tomonori; Kobayashi, Takashi; Matsui, Yoshiyuki; Ogawa, Osamu; Sugimoto, Mikio; Kakehi, Yoshiyuki; Nagumo, Yoshiyuki; Tsutsumi, Masakazu; Oikawa, Takehiro; Kikuchi, Koji; Nishiyama, Hiroyuki

    2016-01-01

    Introduction and Objectives Oncogenic FGFR3-TACC3 fusions and FGFR3 mutations are target candidates for small molecule inhibitors in bladder cancer (BC). Because FGFR3 and TACC3 genes are located very closely on chromosome 4p16.3, detection of the fusion by DNA-FISH (fluorescent in situ hybridization) is not a feasible option. In this study, we developed a novel RNA-FISH assay using branched DNA probe to detect FGFR3-TACC3 fusions in formaldehyde-fixed paraffin-embedded (FFPE) human BC samples. Materials and Methods The RNA-FISH assay was developed and validated using a mouse xenograft model with human BC cell lines. Next, we assessed the consistency of the RNA-FISH assay using 104 human BC samples. In this study, primary BC tissues were stored as frozen and FFPE tissues. FGFR3-TACC3 fusions were independently detected in FFPE sections by the RNA-FISH assay and in frozen tissues by RT-PCR. We also analyzed the presence of FGFR3 mutations by targeted sequencing of genomic DNA extracted from deparaffinized FFPE sections. Results FGFR3-TACC3 fusion transcripts were identified by RNA-FISH and RT-PCR in mouse xenograft FFPE tissues using the human BC cell lines RT112 and RT4. These cell lines have been reported to be fusion-positive. Signals for FGFR3-TACC3 fusions by RNA-FISH were positive in 2/60 (3%) of non-muscle-invasive BC (NMIBC) and 2/44 (5%) muscle-invasive BC (MIBC) patients. The results of RT-PCR of all 104 patients were identical to those of RNA-FISH. FGFR3 mutations were detected in 27/60 (45%) NMIBC and 8/44 (18%) MIBC patients. Except for one NMIBC patient, FGFR3 mutation and FGFR3-TACC3 fusion were mutually exclusive. Conclusions We developed an RNA-FISH assay for detection of the FGFR3-TACC3 fusion in FFPE samples of human BC tissues. Screening for not only FGFR3 mutations, but also for FGFR3-TACC3 fusion transcripts has the potential to identify additional patients that can be treated with FGFR inhibitors. PMID:27930669

  5. Detection of Staphylococcus aureus by polymerase chain reaction amplification of the nuc gene.

    Science.gov (United States)

    Brakstad, O G; Aasbakk, K; Maeland, J A

    1992-07-01

    Synthetic oligonucleotide primers of 21 and 24 bases, respectively, were used in the polymerase chain reaction (PCR) to amplify a sequence of the nuc gene, which encodes the thermostable nuclease of Staphylococcus aureus. A DNA fragment of approximately 270 bp was amplified from lysed S. aureus cells or isolated DNA. The PCR product was detected by agarose gel electrophoresis or Southern blot analysis by using a 33-mer internal nuc gene hybridization probe. With S. aureus cells the lower detection limit was less than 10 CFU, and with the isolated target the lower detection limit was 0.69 pg of DNA. The primers recognized 90 of 90 reference or clinical S. aureus strains. Amplification was not recorded when 80 strains representing 16 other staphylococcal species were tested or when 20 strains representing 9 different nonstaphylococcal species were tested. Some of the non-S. aureus staphylococci produced thermostable nucleases but were PCR negative. The PCR product was generated when in vitro-cultured S. aureus was used to prepare simulated clinical specimens of blood, urine, cerebrospinal fluid, or synovial fluid. No PCR product was generated when the sterile body fluids were tested. However, the sensitivity of the PCR was reduced when S. aureus in blood or urine was tested in comparison with that when bacteria in saline were tested. With the bacteria in blood, the detection limit of the PCR was 10(3) CFU. A positive PCR result was recorded when a limited number of clinical samples from wounds verified to be infected with S. aureus were tested, while the PCR product was not detected in materials from infections caused by other bacteria. Generation of PCR products was not affected by exposure of S. aureus to bactericidal agents, including cloxacillin and gentamicin, prior to testing, but was affected by exposure to UV radiation. The PCR for amplification of the nuc gene has potential for the rapid diagnosis of S. aureus infections by direct testing of clinical

  6. Quantum dots based electrochemiluminescent immunosensor by coupling enzymatic amplification for ultrasensitive detection of clenbuterol

    Energy Technology Data Exchange (ETDEWEB)

    Yao, Xun; Yan, Panpan; Tang, Qinghui [College of Chemistry, Chemical Engineering and Materials Science, Soochow University, Suzhou 215123 (China); The Key Lab of Health Chemistry and Molecular Diagnosis of Suzhou, Suzhou 215123 (China); Deng, Anping, E-mail: denganping@suda.edu.cn [College of Chemistry, Chemical Engineering and Materials Science, Soochow University, Suzhou 215123 (China); The Key Lab of Health Chemistry and Molecular Diagnosis of Suzhou, Suzhou 215123 (China); Li, Jianguo, E-mail: lijgsd@suda.edu.cn [College of Chemistry, Chemical Engineering and Materials Science, Soochow University, Suzhou 215123 (China); The Key Lab of Health Chemistry and Molecular Diagnosis of Suzhou, Suzhou 215123 (China)

    2013-10-10

    Graphical abstract: -- Highlights: •An ultrasensitive ECL immunosensor of CdSe QDs for clenbuterol determination is developed. •The CdSe QDs showed great biocompatibility and could be easier to make direct use of such QDs in the region of biological system. •Enzymatic amplification strategy was proposed by combining the coreactant and pAb/GaRIgG-HRP. •Enzymatic amplification increased ECL emission and extended the analyte in presence of substrate. •It provided a method for detecting clenbuterol and enlarged the usage of QDs in ECL biosensing. -- Abstract: An ultrasensitive electrochemiluminescence (ECL) immunosensor based on CdSe quantum dots (QDs) has been designed for the detection of clenbuterol. The immunosensor was fabricated by layer by layer and characterized with atomic force microscopic images (AFM) and electrochemical impedance spectra (EIS). In oxygen-saturated pH = 9.0 Tris-HCl buffer, a strong ECL emission of QDs could be observed during the cathodic process due to the H{sub 2}O{sub 2} product from electrochemical reduction of dissolved oxygen. Upon the formation of immunocomplex, the second antibody labeled with horseradish peroxidase was simply immobilized on the electrode surface. The ECL emission decreased since steric hindrance of the immunocomplex slowed down the electron-transfer speed of dissolved oxygen, and also could be greatly amplified by an enzymatic cycle to consume the self-produced coreactant. Using clenbuterol as model analyte, the ECL intensity was determined by the concentration of competitive immunoassay of clenbuterol with a wide calibration in the range of 0.05 ng mL{sup −1} to 1000 ng mL{sup −1}, and a low detection limit was 0.02 ng mL{sup −1}. The immunosensor shows good stability and fabrication reproducibility. It was applied to detecting practical samples with the satisfactory results. This immunosensing strategy opens a new avenue for detection of residue and application of QDs in ECL biosensing.

  7. Chromosomal Rainbows detect Oncogenic Rearrangements of Signaling Molecules in Thyroid Tumors

    Energy Technology Data Exchange (ETDEWEB)

    O' Brien, Benjamin; Jossart, Gregg H.; Ito, Yuko; Greulich-Bode, Karin M.; Weier, Jingly F.; Munne, Santiago; Clark, Orlo H.; Weier, Heinz-Ulrich G.

    2010-08-19

    Altered signal transduction can be considered a hallmark of many solid tumors. In thyroid cancers the receptor tyrosine kinase (rtk) genes NTRK1 (Online Mendelian Inheritance in Man = OMIM *191315, also known as 'TRKA'), RET ('Rearranged during Transfection protooncogene', OMIM *164761) and MET (OMIM *164860) have been reported as activated, rearranged or overexpressed. In many cases, a combination of cytogenetic and molecular techniques allows elucidation of cellular changes that initiate tumor development and progression. While the mechanisms leading to overexpression of the rtk MET gene remain largely unknown, a variety of chromosomal rearrangements of the RET or NTKR1 gene could be demonstrated in thyroid cancer. Abnormal expressions in these tumors seem to follow a similar pattern: the rearrangement translocates the 3'-end of the rtk gene including the entire catalytic domain to an expressed gene leading to a chimeric RNA and protein with kinase activity. Our research was prompted by an increasing number of reports describing translocations involving ret and previously unknown translocation partners. We developed a high resolution technique based on fluorescence in situ hybridization (FISH) to allow rapid screening for cytogenetic rearrangements which complements conventional chromosome banding analysis. Our technique applies simultaneous hybridization of numerous probes labeled with different reporter molecules which are distributed along the target chromosome allowing the detection of cytogenetic changes at near megabase-pair (Mbp) resolution. Here, we report our results using a probe set specific for human chromosome 10, which is altered in a significant portion of human thyroid cancers (TC's). While rendering accurate information about the cytogenetic location of rearranged elements, our multi-locus, multi-color analysis was developed primarily to overcome limitations of whole chromosome painting (WCP) and chromosome banding

  8. Rapid, highly sensitive and highly specific gene detection by combining enzymatic amplification and DNA chip detection simultaneously

    Directory of Open Access Journals (Sweden)

    Koji Hashimoto

    2016-05-01

    Full Text Available We have developed a novel gene detection method based on the loop-mediated isothermal amplification (LAMP reaction and the DNA dissociation reaction on the same DNA chip surface to achieve a lower detection limit, broader dynamic range and faster detection time than are attainable with a conventional DNA chip. Both FAM- and thiol-labeled DNA probe bound to the complementary sequence accompanying Dabcyl was immobilized on the gold surface via Au/thiol bond. The LAMP reaction was carried out on the DNA probe fixed gold surface. At first, Dabcyl molecules quenched the FAM fluorescence. According to the LAMP reaction, the complementary sequence with Dabcyl was competitively reacted with the amplified targeted sequence. As a result, the FAM fluorescence increased owing to dissociation of the complementary sequence from the DNA probe. The simultaneous reaction of LAMP and DNA chip detection was achieved, and 103 copies of the targeted gene were detected within an hour by measuring fluorescence intensity of the DNA probe.

  9. An integrated closed-tube 2-plex PCR amplification and hybridization assay with switchable lanthanide luminescence based spatial detection.

    Science.gov (United States)

    Lahdenperä, Susanne; Spangar, Anni; Lempainen, Anna-Maija; Joki, Laura; Soukka, Tero

    2015-06-21

    Switchable lanthanide luminescence is a binary probe technology that inherently enables a high signal modulation in separation-free detection of DNA targets. A luminescent lanthanide complex is formed only when the two probes hybridize adjacently to their target DNA. We have now further adapted this technology for the first time in the integration of a 2-plex polymerase chain reaction (PCR) amplification and hybridization-based solid-phase detection of the amplification products of the Staphylococcus aureus gyrB gene and an internal amplification control (IAC). The assay was performed in a sealed polypropylene PCR chip containing a flat-bottom reaction chamber with two immobilized capture probe spots. The surface of the reaction chamber was functionalized with NHS-PEG-azide and alkyne-modified capture probes for each amplicon, labeled with a light harvesting antenna ligand, and covalently attached as spots to the azide-modified reaction chamber using a copper(i)-catalyzed azide-alkyne cycloaddition. Asymmetric duplex-PCR was then performed with no template, one template or both templates present and with a europium ion carrier chelate labeled probe for each amplicon in the reaction. After amplification europium fluorescence was measured by scanning the reaction chamber as a 10 × 10 raster with 0.6 mm resolution in time-resolved mode. With this assay we were able to co-amplify and detect the amplification products of the gyrB target from 100, 1000 and 10,000 copies of isolated S. aureus DNA together with the amplification products from the initial 5000 copies of the synthetic IAC template in the same sealed reaction chamber. The addition of 10,000 copies of isolated non-target Escherichia coli DNA in the same reaction with 5000 copies of the synthetic IAC template did not interfere with the amplification or detection of the IAC. The dynamic range of the assay for the synthetic S. aureus gyrB target was three orders of magnitude and the limit of detection of 8 p

  10. Combining isothermal rolling circle amplification and electrochemiluminescence for highly sensitive point mutation detection

    Science.gov (United States)

    Su, Qiang; Zhou, Xiaoming

    2008-12-01

    Many pathogenic and genetic diseases are associated with changes in the sequence of particular genes. We describe here a rapid and highly efficient assay for the detection of point mutation. This method is a combination of isothermal rolling circle amplification (RCA) and high sensitive electrochemluminescence (ECL) detection. In the design, a circular template generated by ligation upon the recognition of a point mutation on DNA targets was amplified isothermally by the Phi29 polymerase using a biotinylated primer. The elongation products were hybridized with tris (bipyridine) ruthenium (TBR)-tagged probes and detected in a magnetic bead based ECL platform, indicating the mutation occurrence. P53 was chosen as a model for the identification of this method. The method allowed sensitive determination of the P53 mutation from wild-type and mutant samples. The main advantage of RCA-ECL is that it can be performed under isothermal conditions and avoids the generation of false-positive results. Furthermore, ECL provides a faster, more sensitive, and economical option to currently available electrophoresis-based methods.

  11. Real-time PCR with internal amplification control for detecting tuberculosis: method design and validation.

    Science.gov (United States)

    Flores, E; Rodríguez, J C; Garcia-Pachón, E; Soto, J L; Ruiz, M; Escribano, I; Royo, G

    2009-08-01

    Real-time PCR has been a major development in the diagnosis of tuberculosis. However, most tests do not include an internal amplification control (IAC), which therefore limits it clinical application. In this study a new, easy to perform real-time PCR test with IAC was designed and validated in clinical samples. The primers amplified a 163-bp fragment of IS6110 of Mycobacterium tuberculosis and the IAC was designed with a fragment of a different microorganism (Chlamydia trachomatis). The interassay and intraassay variation of this test were very low (0.45-1.65% and 0.18-1.80%, respectively). The detection accuracy was validated in 50 samples (25 urine, 25 sputum) with different concentrations of M. tuberculosis, 18 clinical isolates of non-tuberculous mycobacteria and 148 samples with clinical suspicion of pulmonary tuberculosis. The specificity was 100%. The detection limit of this PCR test without IAC was approximately 15 bacteria and with IAC approximately 32 bacteria. This real-time PCR with IAC assay can improve the detection of M. tuberculosis and contribute to standardization of this diagnostic technique.

  12. A real-time loop-mediated isothermal amplification assay for rapid detection of Shigella species.

    Science.gov (United States)

    Liew, P S; Teh, C S J; Lau, Y L; Thong, K L

    2014-12-01

    Shigellosis is a foodborne illness caused by the genus Shigella and is an important global health issue. The development of effective techniques for rapid detection of this pathogen is essential for breaking the chain of transmission. Therefore, we have developed a novel loop-mediated isothermal amplification (LAMP) assay targeting the invasion plasmid antigen H (ipaH) gene to rapidly detect Shigella species. This assay could be performed in 90 min at an optimal temperature of 64ºC, with endpoint results visualized directly. Notably, the method was found to be more sensitive than conventional PCR. Indeed, the detection limit for the LAMP assay on pure bacterial cultures was 5.9 x 10(5) CFU/ml, while PCR displayed a limit of 5.9 x 10(7) CFU/ml. In spiked lettuce samples, the sensitivity of the LAMP assay was 3.6 x 10(4) CFU/g, whereas PCR was 3.6 x 10(5) CFU/g. Overall, the assay accurately identified 32 Shigella spp. with one enteroinvasive Escherichia coli displaying positive reaction while the remaining 32 non-Shigella strains tested were negative.

  13. Isothermal rolling circle amplification of virus genomes for rapid antigen detection and typing.

    Science.gov (United States)

    Brasino, Michael D; Cha, Jennifer N

    2015-08-01

    In this work, isothermal rolling circle amplification (RCA) of the multi-kilobase genome of engineered filamentous bacteriophage is used to report the presence and identification of specific protein analytes in solution. First, bacteriophages were chosen as sensing platforms because peptides or antibodies that bind medically relevant targets can be isolated through phage display or expressed as fusions to their p3 and p8 coat proteins. Second, the circular, single-stranded genome contained within the phage serves as a natural large DNA template for a RCA reaction to rapidly generate exponential amounts of double stranded DNA in a single isothermal step that can be easily detected using low-cost fluorescent nucleic acid stains. Amplifying the entire phage genome also provides high detection sensitivities. Furthermore, since the sequence of the viral DNA can be easily modified with multiple restriction enzyme sites, a simple DNA digest can be applied to detect and identify multiple antigens simultaneously. The methods developed here will lead to protein sensors that are highly scalable to produce, can be run without complex biological equipment and do not require the use of multiple antibodies or high-cost fluorescent DNA probes or nucleotides.

  14. Rapid detection of European orthobunyaviruses by reverse transcription loop-mediated isothermal amplification assays.

    Science.gov (United States)

    Camp, Jeremy V; Nowotny, Norbert

    2016-10-01

    The development of reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) assays are described herein for the detection of two orthobunyaviruses (Bunyaviridae), which represent the two main serogroups found in mosquitoes in Central Europe. The RT-LAMP assays were optimized for the detection of Ťahyňa virus (a California encephalitis group virus found in Aedes sp or Ochlerotatus sp mosquitoes) and Batai virus (also called Čalovo virus, a Bunyamwera group virus found in Anopheles maculipennis s.l. mosquitoes) nucleic acid using endemic European virus isolates. The sensitivity of the RT-LAMP assays was determined to be comparable to that of conventional tests, with a limit of detectionisothermal conditions using very simple equipment. Furthermore, it was possible to proceed with the assays without nucleic acid extraction, albeit at a 100-fold loss of sensitivity. The RT-LAMP assays are a sensitive, cost-efficient method for both arbovirus surveillance as well as diagnostic laboratories to detect the presence of these endemic orthobunyaviruses.

  15. Weak signal amplification and detection by higher-order sensory neurons.

    Science.gov (United States)

    Jung, Sarah N; Longtin, Andre; Maler, Leonard

    2016-04-01

    Sensory systems must extract behaviorally relevant information and therefore often exhibit a very high sensitivity. How the nervous system reaches such high sensitivity levels is an outstanding question in neuroscience. Weakly electric fish (Apteronotus leptorhynchus/albifrons) are an excellent model system to address this question because detailed background knowledge is available regarding their behavioral performance and its underlying neuronal substrate. Apteronotus use their electrosense to detect prey objects. Therefore, they must be able to detect electrical signals as low as 1 μV while using a sensory integration time of weak signals are extracted and amplified by the nervous system is not yet understood. We studied the responses of cells in the early sensory processing areas, namely, the electroreceptor afferents (EAs) and pyramidal cells (PCs) of the electrosensory lobe (ELL), the first-order electrosensory processing area. In agreement with previous work we found that EAs cannot encode very weak signals with a spike count code. However, PCs can encode prey mimic signals by their firing rate, revealing a huge signal amplification between EAs and PCs and also suggesting differences in their stimulus encoding properties. Using a simple leaky integrate-and-fire (LIF) model we predict that the target neurons of PCs in the midbrain torus semicircularis (TS) are able to detect very weak signals. In particular, TS neurons could do so by assuming biologically plausible convergence rates as well as very simple decoding strategies such as temporal integration, threshold crossing, and combining the inputs of PCs.

  16. Rapid and sensitive detection of Bordetella bronchiseptica by loop-mediated isothermal amplification (LAMP

    Directory of Open Access Journals (Sweden)

    Hui Zhang

    2013-10-01

    Full Text Available Bordetella bronchiseptica causes acute and chronic respiratory infections in diverse animal species and occasionally in humans. In this study, we described the establishment of a simple, sensitive and cost-efficient loop-mediated isothermal amplification (LAMP assay for the detection of B. bronchiseptica. A set of primers towards a 235 bp region within the flagellum gene of B. bronchiseptica was designed with online software.. The specificity of the LAMP assay was examined by using 6 porcine pathogens and 100 nasal swabs collected from healthy pigs and suspect infected pigs. The results indicated that positive reactions were confirmed for all B. bronchiseptica and no cross-reactivity was observed from other non-B. bronchiseptica. In sensitivity evaluations, the technique successfully detected a serial dilutions of extracted B. bronchiseptica DNA with a detection limit of 9 copies, which was 10 times more sensitive than that of PCR. Compared with conventional PCR, the higher sensitivity of LAMP method and no need for the complex instrumentation make this LAMP assay a promising alternative for the diagnosis of B. bronchiseptica in rural areas and developing countries where there lacks of complex laboratory services.

  17. Entropy Beacon: A Hairpin-Free DNA Amplification Strategy for Efficient Detection of Nucleic Acids.

    Science.gov (United States)

    Lv, Yifan; Cui, Liang; Peng, Ruizi; Zhao, Zilong; Qiu, Liping; Chen, Huapei; Jin, Cheng; Zhang, Xiao-Bing; Tan, Weihong

    2015-12-01

    Here, we propose an efficient strategy for enzyme- and hairpin-free nucleic acid detection called an entropy beacon (abbreviated as Ebeacon). Different from previously reported DNA hybridization/displacement-based strategies, Ebeacon is driven forward by increases in the entropy of the system, instead of free energy released from new base-pair formation. Ebeacon shows high sensitivity, with a detection limit of 5 pM target DNA in buffer and 50 pM in cellular homogenate. Ebeacon also benefits from the hairpin-free amplification strategy and zero-background, excellent thermostability from 20 °C to 50 °C, as well as good resistance to complex environments. In particular, based on the huge difference between the breathing rate of a single base pair and two adjacent base pairs, Ebeacon also shows high selectivity toward base mutations, such as substitution, insertion, and deletion and, therefore, is an efficient nucleic acid detection method, comparable to most reported enzyme-free strategies.

  18. Early detection of Mycobacterium tuberculosis complex in BACTEC MGIT cultures using nucleic acid amplification.

    Science.gov (United States)

    Lin, S Y; Hwang, S C; Yang, Y C; Wang, C F; Chen, Y H; Chen, T C; Lu, P L

    2016-06-01

    We evaluated the application of nucleic acid amplification (NAA) in liquid cultures for the early detection of Mycobacterium tuberculosis. The Cobas TaqMan MTB test, IS6110 real-time PCR, and hsp65 PCR-restriction fragment length polymorphism (RFLP) analysis were used to detect BACTEC MGIT 960 (MGIT) cultures on days 3, 5, 7, and 14. The procedure was initially tested with a reference strain, H37Rv (ATCC 27294). Subsequently, 200 clinical specimens, including 150 Acid Fast bacillus (AFB) smear-positive and 50 AFB smear-negative samples, were examined. The Cobas TaqMan MTB test and IS6110-based PCR analysis were able to detect M. tuberculosis after 1 day when the inoculum of H37Rv was >3 x 10(-2) CFU/ml. After a 5-day incubation in the MGIT system, all three NAA assays had a positive detection regardless of the inoculum size. After a 1-day incubation of the clinical specimens in the MGIT system, the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for the Cobas TaqMan MTB assay were 70.2%, 100%, 100%, and 82.3% respectively. For IS6110-based PCR analysis, these values were 63.1%, 100%, 100%, and 78.9%, and were 88.1%, 100%, 100%, and 92.1% respectively for hsp65 PCR-RFLP analysis. After a 3-day incubation, the specificity and PPV were 100% for all three NAA tests; the Cobas TaqMan MTB assay had the best sensitivity (97.6%) and NPV (98.3%). The sensitivity, specificity, PPV, and NPV for conventional culture analysis were 98.8%, 100%, 100%, and 99.1%. Thus, NAA may be useful for the early detection of M. tuberculosis after 3 days in MGIT.

  19. Colorimetric Detection of 23 Human Papillomavirus Genotypes by Loop-Mediated Isothermal Amplification.

    Science.gov (United States)

    Lin, Junxiao; Ma, Biao; Fang, Jiehong; Wang, Ye; He, Haizhen; Lin, Wei; Su, Wei; Zhang, Mingzhou

    2017-03-01

    Human papillomavirus (HPV) infection is linked to cervical cancer. With the technological development of molecular biology and epidemiology, detection and treatment of HPV has become an important mean to prevent cervical cancer. A simple, rapid, and sensitive colorimetric loop-mediated isothermal amplification (LAMP) method was established herein to detect 23 HPV genotypes. The sequences of the primers for the LAMP reaction were located in the L1 gene of the HPV genome. As it is a fluorescent dye, calcein was added before the reaction. The reaction was run under isothermal conditions at 65°C for 40 minutes. A positive reaction was indicated by a color change from yellow to fluorescent green. The fluorescence curve diagram represents the monitoring of real time quantitative instrument. 450 cervical swab samples from patients with single infections of 23 different HPV genotypes were examined to evaluate the specificity. The results revealed no cross-reaction with other HPV genotypes. A serial dilution of a cloned plasmid containing 23 HPV L1 gene sequences was employed to evaluate the sensitivity. Different HPV subtypes have different detection capability. The sensitivity of different HPV subtypes tested by LAMP assay was in the range from 1.0 x10 to 4.0 x 103 copies per reaction. The LAMP assay and the RDB (reverse dot blot) were compared for detecting and genotyping HPV among the 450 clinical samples. There were 385 (85.6%) and 375 (83.3%) HPV positive specimens detected by LAMP and RDB, respectively, as well as 306 (68.0%) and 296 (65.8%) for HR-HPV positive specimens. The agreement between the LAMP and RDB assays was 93.3% (κ = 0.75) for HPV positivity and 94.7% (κ = 0.88) for HR-HPV positivity. It was concluded that this colorimetric LAMP assay had potential application for the rapid screening of the HPV infection in resource-limited hospitals or rural clinics.

  20. Multiplex ligation-dependent probe amplification for the detection of chromosomal gains and losses in formalin-fixed tissue.

    NARCIS (Netherlands)

    Dijk, M.C.R.F. van; Rombout, P.D.M.; Sprenger, S.H.E.; Straatman, H.M.P.M.; Bernsen, M.R.; Ruiter, D.J.; Jeuken, J.W.M.

    2005-01-01

    Molecular analysis on formalin-fixed paraffin-embedded tissue is of increasing importance in diagnostic histopathology and tumor research. Multiplex ligation-dependent probe amplification (MLPA) is a technique that can be used for detection of copy number alterations of up to 45 different DNA sequen

  1. Development of one-step Loop-Mediated Isothermal Amplification (LAMP) for the detection of norovirus in oysters

    Science.gov (United States)

    The aim of this study was to develop a simple and rapid technique for detecting human norovirus (NoV). The loop-mediated isothermal amplification (LAMP) technique was evaluated and found to be sensitive, highly specific, and useful for routine oyster testing. Reverse transcription-LAMP (RT-LAMP) pri...

  2. Loop-mediated isothermal amplification for detection of the tomato and potato late blight pathogen, Phytophthora infestans

    Science.gov (United States)

    Aims: To design and validate a colorimetric loop-mediated isothermal amplification assay for rapid detection of P. infestans DNA. Methods and Results: Two sets of LAMP primers were designed and evaluated for their sensitivity and specificity for P. infestans. ITSII primers targeted a portion of the ...

  3. On-chip electrical detection of parallel loop-mediated isothermal amplification with DG-BioFETs for the detection of foodborne bacterial pathogens

    Science.gov (United States)

    The use of field effect transistors (FETs) as the transduction element for the detection of DNA amplification reactions will enable portable and inexpensive nucleic acid analysis. Transistors used as biological sensors,or BioFETs, minimize the cost and size of detection platforms by leveraging fabri...

  4. Detection of Goss's Wilt Pathogen Clavibacter michiganensis subsp. nebraskensis in Maize by Loop-Mediated Amplification.

    Science.gov (United States)

    Yasuhara-Bell, Jarred; de Silva, Asoka; Heuchelin, Scott A; Chaky, Jennifer L; Alvarez, Anne M

    2016-03-01

    The Goss's wilt pathogen, Clavibacter michiganensis subsp. nebraskensis, can cause considerable losses in maize (Zea mays) production. Diagnosis of Goss's wilt currently is based on symptomology and identification of C. michiganensis subsp. nebraskensis, following isolation on a semiselective medium and/or serological testing. In an effort to provide a more efficient identification method, a loop-mediated amplification (LAMP) assay was developed to detect the tripartite ATP-independent periplasmic (TRAP)-type C4-dicarboxylate transport system large permease component and tested using strains of C. michiganensis subsp. nebraskensis, all other C. michiganensis subspecies and several genera of nontarget bacteria. Only strains of C. michiganensis subsp. nebraskensis reacted positively with the LAMP assay. The LAMP assay was then used to identify bacterial isolates from diseased maize. 16S rDNA and dnaA sequence analyses were used to confirm the identity of the maize isolates and validate assay specificity. The Cmm ImmunoStrip assay was included as a presumptive identification test of C. michiganensis subsp. nebraskensis at the species level. The Cmn-LAMP assay was further tested using symptomatic leaf tissue. The Cmn-LAMP assay was run in a hand-held real-time monitoring device (SMART-DART) and performed equally to in-lab quantitative polymerase chain reaction equipment. The Cmn-LAMP assay accurately identified C. michiganensis subsp. nebraskensis and has potential as a field test. The targeted sequence also has potential application in other molecular detection platforms.

  5. Thermal Contrast Amplification Reader Yielding 8-Fold Analytical Improvement for Disease Detection with Lateral Flow Assays.

    Science.gov (United States)

    Wang, Yiru; Qin, Zhenpeng; Boulware, David R; Pritt, Bobbi S; Sloan, Lynne M; González, Iveth J; Bell, David; Rees-Channer, Roxanne R; Chiodini, Peter; Chan, Warren C W; Bischof, John C

    2016-12-06

    There is an increasing need for highly sensitive and quantitative diagnostics at the point-of-care. The lateral flow immunoassay (LFA) is one of the most widely used point-of-care diagnostic tests; however, LFAs generally suffer from low sensitivity and lack of quantification. To overcome these limitations, thermal contrast amplification (TCA) is a new method that is based on the laser excitation of gold nanoparticles (GNPs), the most commonly used visual signature, to evoke a thermal signature. To facilitate the clinical translation of the TCA technology, we present the development of a TCA reader, a platform technology that significantly improves the limit of detection and provides quantification of disease antigens in LFAs. This TCA reader provides enhanced sensitivity over visual detection by the human eye or by a colorimetric reader (e.g., BD Veritor System Reader). More specifically, the TCA reader demonstrated up to an 8-fold enhanced analytical sensitivity and quantification among LFAs for influenza, malaria, and Clostridium difficile. Systematic characterization of the laser, infrared camera, and other components of the reader and their integration into a working reader instrument are described. The development of the TCA reader enables simple, highly sensitive quantification of LFAs at the point-of-care.

  6. Pre-symptomatic detection of prions by cyclic amplification of protein misfolding.

    Science.gov (United States)

    Soto, Claudio; Anderes, Laurence; Suardi, Silvia; Cardone, Franco; Castilla, Joaquin; Frossard, Marie-Jose; Peano, Sergio; Saa, Paula; Limido, Lucia; Carbonatto, Michaela; Ironside, James; Torres, Juan-Maria; Pocchiari, Maurizio; Tagliavini, Fabrizio

    2005-01-31

    Transmissible spongiform encephalopathies (TSEs) are neurodegenerative disorders affecting humans and animals. At present, it is not possible to recognize individuals incubating the disease before the clinical symptoms appear. We investigated the effectiveness of the "Protein Misfolding Cyclic Amplification" (PMCA) technology to detect the protease-resistance disease-associated prion protein (PrP(res)) in pre-symptomatic stages. PMCA allowed detection of PrP(res) in the brain of pre-symptomatic hamsters, enabling a clear identification of infected animals as early as two weeks after inoculation. Furthermore, PMCA was able to amplify minute quantities of PrP(res) from a variety of experimental and natural TSEs. Finally, PMCA allowed the demonstration of PrP(res) in an experimentally infected cow 32 month post-inoculation, that did not show clinical signs and was negative by standard Western blot analysis. Our findings indicate that PMCA may be useful for the development of an ultra-sensitive diagnostic test to minimize the risk of further propagation of TSEs.

  7. Fluorogenic Detection of Duck Tembusu Virus( DTMUV ) by Loop-mediated Isothermal Amplification(LAMP)

    Institute of Scientific and Technical Information of China (English)

    Zhang; Lin; Wang; Bin; Zhang; Wei; Zhang; Xiumei

    2014-01-01

    This study was to develop an efficient and simple method for the detection of duck Tembusu virus( DTMUV) by loop-mediated isothermal amplification( LAMP). Six pairs of LAMP primers were designed according to the conserved region of the DTMUV E gene sequence in Gen Bank,which were then used for the optimization of various reaction components and reaction system of specific LAMP for DTMUV. Further the fluorescent reagent SYBR Green I and a certain proportion of calcium and manganese ion were used to determin the color development of products for visible analysis instead of agarose gel electrophoresis. The results showed that the sensitivity SYBR Green I as the fluorescent reagent was 10 copies viruses per μL,which is 100 times higher than normal PCR method,while the detection limit of combined use of calcium and manganese ion was 1 000 copies viruses per μL. Although the sensitivity of mixture of calcium and manganese ion is lower than SYBR Green I,it can avoid the aerosol contamination. The fluorogenic analysis-based LAMP system established in our study has a high sensitivity and avoid the cross contamination,which is of huge potential in research institutions,grass-roots laboratories and field testing and can provide effective means to completely curb the occurrence and spreading of DTMUV.

  8. Loop-Mediated Isothermal Amplification Assay for the Rapid Detection of Staphylococcus aureus

    Directory of Open Access Journals (Sweden)

    King Ting Lim

    2013-01-01

    Full Text Available Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA, is an important human pathogen that produces a variety of toxins and causes a wide range of infections, including soft-tissue infections, bacteremia, and staphylococcal food poisoning. A loop-mediated isothermal amplification (LAMP assay targeting the arcC gene of S. aureus was developed and evaluated with 119 S. aureus and 25 non-S. aureus strains. The usefulness of the assay was compared with the PCR method that targets spa and arcC genes. The optimal temperature for the LAMP assay was 58.5°C with a detection limit of 2.5 ng/μL and 102 CFU/mL when compared to 12.5 ng/μL and 103 CFU/mL for PCR (spa and arcC. Both LAMP and PCR assays were 100% specific, 100% sensitive, 100% positive predictive value (PPV, and 100% negative predictive value (NPV. When tested on 30 spiked blood specimens (21 MRSA, eight non-S. aureus and one negative control, the performance of LAMP and PCR was comparable: 100% specific, 100% sensitive, 100% PPV, and 100% NPV. In conclusion, the LAMP assay was equally specific with a shorter detection time when compared to PCR in the identification of S. aureus. The LAMP assay is a promising alternative method for the rapid identification of S. aureus and could be used in resource-limited laboratories and fields.

  9. Highly sensitive chemiluminescence technology for protein detection using aptamer-based rolling circle amplification platform

    Institute of Scientific and Technical Information of China (English)

    Zhi-Juan Cao; Qian-Wen Peng; Xue Qiu; Cai-Yun Liu; Jian-Zhong Lu

    2011-01-01

    A robust, selective and highly sensitive chemiluminescent (CL) platform for protein assay was presented in this paper. This novel CL approach utilized rolling circle amplification (RCA) as a signal enhancement technique and the 96-well plate as the immobilization and separation carrier. Typically, the antibody immobilized on the surface of 96-well plate was sandwiched with the protein target and the aptamer-primer sequence. This aptamer-primer sequence was then employed as the primer of RCA. Based on this design, a number of the biotinylated probes and streptavidin-horseradish peroxidase (SA-HRP) were captured on the plate, and the CL signal was amplified. In summary, our results demonstrated a robust biosensor with a detection limit of 10 fM that is easy to be established and utilized, and devoid of light source. Therefore, this new technique .will broaden the perspective for future development of DNA-based biosensors for the detection of other protein biomarkers related to clinical diseases, by taking advantages of high sensitivity and selectivity.

  10. Loop-mediated isothermal amplification (LAMP: Early detection of Toxoplasma gondii infection in mice

    Directory of Open Access Journals (Sweden)

    Kong Qing-Ming

    2012-01-01

    Full Text Available Abstract Background Toxoplasmosis is a widespread zoonotic parasitic disease that occurs in both animals and humans. Traditional molecular assays are often difficult to perform, especially for the early diagnosis of Toxoplasma gondii infections. Here, we established a novel loop-mediated isothermal amplification targeting the 529 bp repeat element (529 bp-LAMP to detect T. gondii DNA in blood samples of experimental mice infected with tachyzoites of the RH strain. Findings The assay was performed with Bst DNA polymerase at 65°C for 1 h. The detection limit of the 529 bp-LAMP assay was as low as 0.6 fg of T. gondii DNA. The sensitivity of this assay was 100 and 1000 fold higher than that of the LAMP targeting B1 gene (B1-LAMP and nested PCR targeting 529 bp repeat element (529 bp-nested PCR, respectively. The specificity of the 529 bp-LAMP assay was determined using the DNA samples of Trypanosoma evansi, Plasmodium falciparum, Paragonimus westermani, Schistosoma japonicum, Fasciola hepatica and Angiostrongylus cantonensis. No cross-reactivity with the DNA of any parasites was found. The assay was able to detect T. gondii DNA in all mouse blood samples at one day post infection (dpi. Conclusions We report the following findings: (i The detection limit of the 529 bp-LAMP assay is 0.6 fg of T. gondii DNA; (ii The assay does not involve any cross-reactivity with the DNA of other parasites; (iii This is the first report on the application of the LAMP assay for early diagnosis of toxoplasmosis in blood samples from experimentally infected mice. Due to its simplicity, sensitivity and cost-effectiveness for common use, we suggest that this assay should be used as an early diagnostic tool for health control of toxoplasmosis.

  11. Specific detection of Pectobacterium carotovorum by loop-mediated isothermal amplification.

    Science.gov (United States)

    Yasuhara-Bell, Jarred; Marrero, Glorimar; De Silva, Asoka; Alvarez, Anne M

    2016-12-01

    Potatoes are an important agroeconomic crop worldwide and maceration diseases caused by pectolytic bacterial pathogens result in significant pre- and post-harvest losses. Pectobacterium carotovorum shares a common host range with other Pectobacterium spp. and other members of the Enterobacteriaceae, such as Dickeya spp. As these pathogens cannot be clearly differentiated on the basis of the symptoms they cause, improved methods of identification are critical for the determination of sources of contamination. Current standardized methods for the differentiation of pectolytic species are time consuming and require trained personnel, as they rely on traditional bacteriological practices that do not always produce conclusive results. In this growing world market, there is a need for rapid diagnostic tests that can differentiate between pectolytic pathogens, as well as separate them from non-pectolytic enteric bacteria associated with soft rots of potato. An assay has been designed previously to detect the temperate pathogen Pectobacterium atrosepticum, but there is currently no recognized rapid assay for the detection of the tropical/subtropical counterpart, Pectobacterium carotovorum. This report describes the development of a loop-mediated isothermal amplification (LAMP) assay that detects P. carotovorum with high specificity. The assay was evaluated using all known species of Pectobacterium and only showed positive reactions for P. carotovorum. This assay was also tested against 15 non-target genera of plant-associated bacteria and did not produce any false positives. The LAMP assay described here can be used as a rapid test for the differentiation of P. carotovorum from other pectolytic pathogens, and its gene target can be the basis for the development of other molecular-based detection assays.

  12. Detection of Salmonella invA by isothermal and chimeric primer-initiated amplification of nucleic acids (ICAN) in Zambia.

    Science.gov (United States)

    Isogai, Emiko; Makungu, Chitwambi; Yabe, John; Sinkala, Patson; Nambota, Andrew; Isogai, Hiroshi; Fukushi, Hideto; Silungwe, Manda; Mubita, Charles; Syakalima, Michelo; Hang'ombe, Bernard Mudenda; Kozaki, Shunji; Yasuda, Jun

    2005-01-01

    The isothermal and chimeric primer-initiated amplification of nucleic acids (ICAN) is a new isothermal DNA amplification method composed of exo Bca DNA polymerase, RNaseH and DNA-RNA chimeric primers. We detected invA of Salmonella from chicken carcasses, egg yolk and cattle fecal samples. Fifty-three of 59 isolates were invA-positive in ICAN-chromatostrip detection. The result was consistent with those obtained by standard PCR. Salmonella invA was detected in 12 of 14 carcass rinses by ICAN, while in 7 of 14 rinses by standard PCR. These results indicate that ICAN is an efficient, sensitive and simple system to detect invA of Salmonella species in developing countries such as Zambia.

  13. Development of a PCR-compatible enrichment medium for Yersinia enterocolitica: amplification precision and dynamic detection range during cultivation.

    Science.gov (United States)

    Knutsson, Rickard; Fontanesi, Massimo; Grage, Halfdan; Rådström, Peter

    2002-02-05

    A Yersinia PCR-Compatible Enrichment (YPCE) medium was developed, which removes the necessity for sample pretreatment before PCR-based detection of Yersinia enterocolitica. The medium was designed through a sequence of independent screening and factorial design experiments to study the PCR inhibition and growth characteristics of medium components. The compatibility of the YPCE medium was evaluated using real-time PCR. The real-time PCR assay, based on the fluorescent double-stranded DNA binding dye SYBR green, generated approximately a 4-log linear range of amplification and in the range of 10(5)-10(8) (CFU/ml), the coefficient of variation or = 10(6) (CFU/ml), the DNA amplification was influenced and a change in the log-linear slope leading to a lower amplification efficiency was observed. To study the dynamic detection range and relative amplification precision during enrichment. Y. enterocolitica and background flora were inoculated at various concentrations. It was possible to detect inoculation concentrations of 10(1) (CFU/ml) Y. enterocolitica in the presence of at least an inoculation concentration of 10(3) (CFU/ml) of an undefined background flora and the optimal conditions for sample withdrawal was in the range of 9 to 18 h enrichment. The YPCE medium can, especially for swab samples, form part of a simple analysis procedure allowing high throughput PCR.

  14. Field Evaluation of a High Throughput Loop Mediated Isothermal Amplification Test for the Detection of Asymptomatic Plasmodium Infections in Zanzibar

    OpenAIRE

    Aydin-Schmidt, Berit; Morris, Ulrika; Ding, Xavier C; Jovel, Irina; Mwinyi I Msellem; Bergman, Daniel; Islam, Atiqul; Ali, Abdullah S; Polley, Spencer; Gonzalez, Iveth J.; Mårtensson, Andreas; Björkman, Anders

    2017-01-01

    Background New field applicable diagnostic tools are needed for highly sensitive detection of residual malaria infections in pre-elimination settings. Field performance of a high throughput DNA extraction system for loop mediated isothermal amplification (HTP-LAMP) was therefore evaluated for detecting malaria parasites among asymptomatic individuals in Zanzibar. Methods HTP-LAMP performance was evaluated against real-time PCR on 3008 paired blood samples collected on filter papers in a commu...

  15. Toehold-mediated nonenzymatic amplification circuit on graphene oxide fluorescence switching platform for sensitive and homogeneous microRNA detection

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Ru; Liao, Yuhui; Zhou, Xiaoming, E-mail: zhouxm@scnu.edu.cn; Xing, Da, E-mail: xingda@scnu.edu.cn

    2015-08-12

    A novel graphene oxide (GO) fluorescence switch-based homogenous system has been developed to solve two problems that are commonly encountered in conventional GO-based biosensors. First, with the assistance of toehold-mediated nonenzymatic amplification (TMNA), the sensitivity of this system greatly surpasses that of previously described GO-based biosensors, which are always limited to the nM range due to the lack of efficient signal amplification. Second, without enzymatic participation in amplification, the unreliability of detection resulting from nonspecific desorption of DNA probes on the GO surface by enzymatic protein can be avoided. Moreover, the interaction mechanism of the double-stranded TMNA products contains several single-stranded toeholds at two ends and GO has also been explored with combinations of atomic force microscopy imaging, zeta potential detection, and fluorescence assays. It has been shown that the hybrids can be anchored to the surface of GO through the end with more unpaired bases, and that the other end, which has weaker interaction with GO, can escape GO adsorption due to the robustness of the central dsDNA structures. We verified this GO fluorescence switch-based detection system by detecting microRNA 21, an overexpressed non-encoding gene in a variety of malignant cells. Rational design of the probes allowed the isothermal nonenzymatic reaction to achieve more than 100-fold amplification efficiency. The detection results showed that our strategy has a detection limit of 10 pM and a detection range of four orders of magnitude. - Highlights: • This paper explored the interaction mechanism of TMNA products with GO surface. • This homogeneous and isothermal system permits a detection limit of 10 pM for microRNA. • This nonenzymatic strategy can avoid nonspecific desorption caused by enzyme protein. • The interaction model can be used to explore the application ability of nonenzymatic circuit.

  16. Field Evaluation of a High Throughput Loop Mediated Isothermal Amplification Test for the Detection of Asymptomatic Plasmodium Infections in Zanzibar

    OpenAIRE

    Aydin-Schmidt, Berit; Morris, Ulrika; Ding, Xavier C; Jovel, Irina; Msellem, Mwinyi I; Bergman, Daniel; Islam, Atiqul; Ali, Abdullah S.; Polley, Spencer; Gonzalez, Iveth J.; Mårtensson, Andreas; Björkman, Anders

    2017-01-01

    Background New field applicable diagnostic tools are needed for highly sensitive detection of residual malaria infections in pre-elimination settings. Field performance of a high throughput DNA extraction system for loop mediated isothermal amplification (HTP-LAMP) was therefore evaluated for detecting malaria parasites among asymptomatic individuals in Zanzibar. Methods HTP-LAMP performance was evaluated against real-time PCR on 3008 paired blood samples collected on filter papers in a commu...

  17. Detection of BCR-ABL Fusion mRNA Using Reverse Transcriptase Loop-mediated Isothermal Amplification

    Energy Technology Data Exchange (ETDEWEB)

    Dugan, L C; Hall, S; Kohlgruber, A; Urbin, S; Torres, C; Wilson, P

    2011-12-08

    RT-PCR is commonly used for the detection of Bcr-Abl fusion transcripts in patients diagnosed with chronic myelogenous leukemia, CML. Two fusion transcripts predominate in CML, Br-Abl e13a2 and e14a2. They have developed reverse transcriptase isothermal loop-mediated amplification (RT-LAMP) assays to detect these two fusion transcripts along with the normal Bcr transcript.

  18. Loop‑mediated Isothermal Amplification assay (LAMP) based detection of Pasteurella multocida in cases of haemorrhagic septicaemia and fowl cholera.

    Science.gov (United States)

    Bhimani, Mayurkumar; Bhanderi, Bharat; Roy, Ashish

    2015-01-01

    Twenty two isolates of Pasteurella multocida were obtained from different tissues of dead birds and animals (cattle, buffalo, sheep, and goat) suspected of fowl cholera and haemorrhagic septicaemia. The isolates were confirmed as P. multocida by various biochemical tests and PM PCR. An attempt was made to standardize Loop mediated isothermal amplification (LAMP) using newly designed primer sequences of KMT1 gene. Loop mediated isothermal amplification was conducted using 6 sets of primers at 65°C for 30 minutes and the result was confirmed by visual observation using SYBR green fluorescence dye as marker of positive reaction under UV transilluminator. On electrophoretic analysis of the products on 2% agarose gel, a ladder like pattern was observed, which suggested a positive amplification, whereas no amplification was observed in negative controls. Additionally, product of positive reaction yielded a green fluorescence following addition of SYBR green under UV transilluminator. It was observed that LAMP is a more sensitive test than polymerase chain reaction (PCR), as the former could detect DNA to lower limit of 22.8 pg/µl, while the latter could detect DNA to lower limit of 2.28 ng/ µl, thus LAMP could detect 100 times lesser concentration of DNA in comparison to PCR. Loop mediated isothermal amplification is a rather newer molecular technique, which can be used for rapid detection of infectious agent at field level and which does not require sophisticated instrument, i.e. thermal cycler. Furthermore, unlike the conventional PCR technique, LAMP requires lesser time to perform and result can be read visually.

  19. The Use of Polymerase Chain Reaction Amplification for the Detection of Viruses and Bacteria in Severe Community-Acquired Pneumonia.

    Science.gov (United States)

    Siow, Wen Ting; Koay, Evelyn Siew-Chuan; Lee, Chun Kiat; Lee, Hong Kai; Ong, Venetia; Ngerng, Wang Jee; Lim, Hui Fang; Tan, Adeline; Tang, Julian Wei-Tze; Phua, Jason

    2016-01-01

    Pathogens are often not identified in severe community-acquired pneumonia (CAP), and the few studies using polymerase chain reaction (PCR) techniques for virus detection are from temperate countries. This study assesses if PCR amplification improves virus and bacteria detection, and if viral infection contributes to mortality in severe CAP in a tropical setting, where respiratory pathogens have less well-defined seasonality. In this cohort study of patients with severe CAP in an intensive care unit, endotracheal aspirates for intubated patients and nasopharyngeal swabs for non-intubated patients were sent for PCR amplification for respiratory viruses. Blood, endotracheal aspirates for intubated patients, and sputum for non-intubated patients were analysed using a multiplex PCR system for bacteria. Out of 100 patients, using predominantly cultures, bacteria were identified in 42 patients; PCR amplification increased this number to 55 patients. PCR amplification identified viruses in 32 patients. In total, only bacteria, only viruses, and both bacteria and viruses were found in 37, 14, and 18 patients, respectively. The commonest viruses were influenza A H1N1/2009 and rhinovirus; the commonest bacterium was Streptococcus pneumoniae. Hospital mortality rates for patients with no pathogens, bacterial infection, viral infection, and bacterial-viral co-infection were 16.1, 24.3, 0, and 5.6%, respectively (p = 0.10). On multivariable analysis, virus detection was associated with lower mortality (adjusted odds ratio 0.12, 95% confidence interval 0.2-0.99; p = 0.049). Viruses and bacteria were detected in 7 of 10 patients with severe CAP with the aid of PCR amplification. Viral infection appears to be independently associated with lower mortality. © 2016 S. Karger AG, Basel.

  20. Development of a quantitative fluorescence single primer isothermal amplification-based method for the detection of Salmonella.

    Science.gov (United States)

    Wang, Jianchang; Li, Rui; Hu, Lianxia; Sun, Xiaoxia; Wang, Jinfeng; Li, Jing

    2016-02-16

    Food-borne disease caused by Salmonella has long been, and continues to be, an important global public health problem, necessitating rapid and accurate detection of Salmonella in food. Real time PCR is the most recently developed approach for Salmonella detection. Single primer isothermal amplification (SPIA), a novel gene amplification technique, has emerged as an attractive microbiological testing method. SPIA is performed under a constant temperature, eliminating the need for an expensive thermo-cycler. In addition, SPIA reactions can be accomplished in 30 min, faster than real time PCR that usually takes over 2h. We developed a quantitative fluorescence SPIA-based method for the detection of Salmonella. Using Salmonella Typhimurium genomic DNA as template and a primer targeting Salmonella invA gene, we showed the detection limit of SPIA was 2.0 × 10(1)fg DNA. Its successful amplification of different serotypic Salmonella genomic DNA but not non-Salmonella bacterial DNA demonstrated the specificity of SPIA. Furthermore, this method was validated with artificially contaminated beef. In conclusion, we showed high sensitivity and specificity of SPIA in the detection of Salmonella, comparable to real time PCR. In addition, SPIA is faster and more cost-effective (non-use of expensive cyclers), making it a potential alternative for field detection of Salmonella in resource-limited settings that are commonly encountered in developing countries.

  1. Loop-mediated isothermal amplification for the detection of goose circovirus

    Directory of Open Access Journals (Sweden)

    Woźniakowski Grzegorz

    2012-06-01

    Full Text Available Abstract Background Goose circovirus (GCV presents an immunosuppressive problem in production of geese. The infection’s clinical symptoms include growth retardation or feathering disorders but the infection process may remain non-symptomatic what makes the infected birds more susceptible for secondary viral, bacterial and fungal infections. Diagnosis of GCV infection is made by histopathological examination, dot blot hybridization, polymerase chain reaction (PCR and real-time PCR. However these techniques require application of thermocyclers and qualified staff which may be cost-consuming for some diagnostic units. The aim of this study was to develop loop-mediated isothermal amplification assay (LAMP as a simple method of GCV detection. Results The presented study has shown LAMP as a rapid tool of detecting DNA of goose circovirus (GCV as soon in 30 min time. The method used three sets of primers: two outer primers (F3 and B3, two inner primers (FIP and BIP and two loop primers (FL and BL to accelerate the reaction. The optimum reaction temperature and the time were 61°C for 30 min, respectively. The results were analysed using SYBR Green dye and GelRedTM solutions. Thirty-eight isolates of GCV collected from geese flocks in Poland were examined. For comparison, real-time polymerase chain reaction with F3 and B3 primers and SYBR Green dye was conducted. The obtained results have shown GCV-LAMP as a sensitive, rapid and specific assay and alternative for PCR-based methods. Conclusions The developed technique due to its simplicity may be applied by any veterinary laboratory or even mobile diagnostics units for the routine detection of GCV.

  2. Loop-mediated isothermal amplification for the detection of goose circovirus

    Science.gov (United States)

    2012-01-01

    Background Goose circovirus (GCV) presents an immunosuppressive problem in production of geese. The infection’s clinical symptoms include growth retardation or feathering disorders but the infection process may remain non-symptomatic what makes the infected birds more susceptible for secondary viral, bacterial and fungal infections. Diagnosis of GCV infection is made by histopathological examination, dot blot hybridization, polymerase chain reaction (PCR) and real-time PCR. However these techniques require application of thermocyclers and qualified staff which may be cost-consuming for some diagnostic units. The aim of this study was to develop loop-mediated isothermal amplification assay (LAMP) as a simple method of GCV detection. Results The presented study has shown LAMP as a rapid tool of detecting DNA of goose circovirus (GCV) as soon in 30 min time. The method used three sets of primers: two outer primers (F3 and B3), two inner primers (FIP and BIP) and two loop primers (FL and BL) to accelerate the reaction. The optimum reaction temperature and the time were 61°C for 30 min, respectively. The results were analysed using SYBR Green dye and GelRedTM solutions. Thirty-eight isolates of GCV collected from geese flocks in Poland were examined. For comparison, real-time polymerase chain reaction with F3 and B3 primers and SYBR Green dye was conducted. The obtained results have shown GCV-LAMP as a sensitive, rapid and specific assay and alternative for PCR-based methods. Conclusions The developed technique due to its simplicity may be applied by any veterinary laboratory or even mobile diagnostics units for the routine detection of GCV. PMID:22695123

  3. Electrochemical branched-DNA assay for polymerase chain reaction-free detection and quantification of oncogenes in messenger RNA.

    Science.gov (United States)

    Lee, Ai-Cheng; Dai, Ziyu; Chen, Baowei; Wu, Hong; Wang, Jun; Zhang, Aiguo; Zhang, Lurong; Lim, Tit-Meng; Lin, Yuehe

    2008-12-15

    We describe a novel electrochemical branched-DNA (bDNA) assay for polymerase chain reaction (PCR)-free detection and quantification of p185 BCR-ABL leukemia fusion transcripts in the population of messenger ribonucleic acid (mRNA) extracted from cell lines. The bDNA amplifier carrying high loading of alkaline phosphatase (ALP) tracers was used to amplify the target signal. The targets were captured on microplate well surfaces through cooperative sandwich hybridization prior to the labeling of bDNA. The activity of captured ALP was monitored by square-wave voltammetric (SWV) analysis of the electroactive enzymatic product in the presence of 1-naphthyl phosphate. The voltammetric characteristics of substrate and enzymatic product as well as the parameters of SWV analysis were systematically optimized. A detection limit of 1 fM (1 x 10(-19) mol of target transcripts in 100 microL) and a 3-order-wide dynamic range of target concentration were achieved by the electrochemical bDNA assay. Such limit corresponded to approximately 17 fg of the p185 BCR-ABL fusion transcripts. The specificity and sensitivity of assay enabled direct detection of target transcripts in as little as 4.6 ng of mRNA population without PCR amplification. In combination with the use of a well-quantified standard, the electrochemical bDNA assay was capable of direct use for a PCR-free quantitative analysis of target transcripts in mRNA population. A mean transcript copy number of 62,900/ng of mRNA was determined, which was at least 50-fold higher than that of real-time quantitative PCR (qPCR). The finding was consistent with the underestimation of targets by qPCR reported earlier. In addition, the unique design based on bDNA technology increases the assay specificity as only the p185 BCR-ABL fusion transcripts will respond to the detection. The approach thus provides a simple, sensitive, accurate, and quantitative tool alternative to the qPCR for early disease diagnosis.

  4. [Detection of the Zaire Subtype of the Ebola Virus by Isothermal Multiple Self-matching Initiated Amplification].

    Science.gov (United States)

    Li, Xinna; Nie, Kai; Wang, Ji; Zhang, Dan; Guan, Li; Liu, Jun; Ke, Yuehua; Zhou, Hangyu; Ma, Xuejun

    2016-01-01

    Given the Ebola outbreak in West Africa and the risks of spread to other regions, a rapid, sensitive and simple method for the detection of the Ebola virus (EBOV) is of great significance for the prevention and control of Ebola. We developed a simple colorimetric isothermal multiple self-matching initiated amplification (IMSA) for rapid detection of the Zaire subtype of the Ebola virus (EBOV-Z). This method employed six primers that recognized seven sites of the EBOV-Z nucleoprotein gene for amplification of nucleic acids under isothermal conditions at 63 degrees C for 1 h. Amplification products were detected through visual inspection of color change by pre-addition of hydroxyl naphthol blue dye. Relative sensitivity was validated by detection of serial tenfold dilutions of virus-like particles containing the partial EBOV-Z nucleoprotein gene and mock clinical sample. Specificity of IMSA was validated by detection of the plasma of 30 healthy volunteers, the dengue virus, and Japanese encephalitis virus. IMSA had comparable sensitivity to Reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and cross-reaction with human plasma or other viruses was not observed. Reverse transcription-isothermal multiple self-matching initiated amplification (RT-IMSA) was also evaluated and compared in parallel with the commercial RT-qPCR kit for detection of EBOV-suspected samples of human blood in Sierra Leone. Sensitivity and specificity of the RT-IMSA was 91.4% and 100%, respectively. These data suggest that RT-IMSA is a valuable tool for the detection of the EBOV with the distinct advantages of simplicity and low cost compared with RT-qPCR.

  5. Loop-mediated isothermal amplification: rapid detection of Angiostrongylus cantonensis infection in Pomacea canaliculata

    Directory of Open Access Journals (Sweden)

    Zhuo MingMing

    2011-10-01

    Full Text Available Abstract Background Angiostrongylus cantonensis is a zoonotic parasite that causes eosinophilic meningitis in humans. The most common source of infection with A. cantonensis is the consumption of raw or undercooked mollusks (e.g., snails and slugs harbouring infectious third-stage larvae (L3. However, the parasite is difficult to identify in snails. The purpose of this study was to develop a quick, simple molecular method to survey for A. cantonensis in intermediate host snails. Findings We used a loop-mediated isothermal amplification (LAMP assay, which was performed using Bst DNA polymerase. Reactions amplified the A. cantonensis 18S rRNA gene and demonstrated high sensitivity; as little as 1 fg of DNA was detected in the samples. Furthermore, no cross-reactivity was found with other parasites such as Toxoplasma gondii, Plasmodium falciparum, Schistosoma japonicum, Clonorchis sinensis, Paragonimus westermani and Anisakis. Pomacea canaliculata snails were exposed to A. cantonensis first-stage larvae (L1 in the laboratory, and L3 were observed in the snails thirty-five days after infection. All nine samples were positive as determined by the LAMP assay for A. cantonensis, which was identified as positive by using PCR and microscopy, this demonstrates that LAMP is sensitive and effective for diagnosis. Conclusions LAMP is an appropriate diagnostic method for the routine identification of A. cantonensis within its intermediate host snail P. canaliculata because of its simplicity, sensitivity, and specificity. It holds great promise as a useful monitoring tool for A. cantonensis in endemic regions.

  6. Electrical detection of dsDNA and polymerase chain reaction amplification.

    Science.gov (United States)

    Salm, Eric; Liu, Yi-Shao; Marchwiany, Daniel; Morisette, Dallas; He, Yiping; Razouk, Laila; Bhunia, Arun K; Bashir, Rashid

    2011-12-01

    Food-borne pathogens and food safety-related outbreaks have come to the forefront over recent years. Estimates on the annual cost of sicknesses, hospitalizations, and deaths run into the billions of dollars. There is a large body of research on detection of food-borne pathogens; however, the widely accepted current systems are limited by costly reagents, lengthy time to completion, and expensive equipment. Our aim is to develop a label-free method for determining a change in DNA concentration after a PCR assay. We first used impedance spectroscopy to characterize the change in concentration of purified DNA in deionized water within a microfluidic biochip. To adequately measure the change in DNA concentration in PCR solution, it was necessary to go through a purification and precipitation step to minimize the effects of primers, PCR reagents, and excess salts. It was then shown that the purification and precipitation of the fully amplified PCR reaction showed results similar to the control tests performed with DNA in deionized water. We believe that this work has brought label free electrical biosensors for PCR amplification one step closer to reality.

  7. Prion Amplification and Hierarchical Bayesian Modeling Refine Detection of Prion Infection

    Science.gov (United States)

    Wyckoff, A. Christy; Galloway, Nathan; Meyerett-Reid, Crystal; Powers, Jenny; Spraker, Terry; Monello, Ryan J.; Pulford, Bruce; Wild, Margaret; Antolin, Michael; Vercauteren, Kurt; Zabel, Mark

    2015-02-01

    Prions are unique infectious agents that replicate without a genome and cause neurodegenerative diseases that include chronic wasting disease (CWD) of cervids. Immunohistochemistry (IHC) is currently considered the gold standard for diagnosis of a prion infection but may be insensitive to early or sub-clinical CWD that are important to understanding CWD transmission and ecology. We assessed the potential of serial protein misfolding cyclic amplification (sPMCA) to improve detection of CWD prior to the onset of clinical signs. We analyzed tissue samples from free-ranging Rocky Mountain elk (Cervus elaphus nelsoni) and used hierarchical Bayesian analysis to estimate the specificity and sensitivity of IHC and sPMCA conditional on simultaneously estimated disease states. Sensitivity estimates were higher for sPMCA (99.51%, credible interval (CI) 97.15-100%) than IHC of obex (brain stem, 76.56%, CI 57.00-91.46%) or retropharyngeal lymph node (90.06%, CI 74.13-98.70%) tissues, or both (98.99%, CI 90.01-100%). Our hierarchical Bayesian model predicts the prevalence of prion infection in this elk population to be 18.90% (CI 15.50-32.72%), compared to previous estimates of 12.90%. Our data reveal a previously unidentified sub-clinical prion-positive portion of the elk population that could represent silent carriers capable of significantly impacting CWD ecology.

  8. Detection of the quarantine species Thrips palmi by loop-mediated isothermal amplification.

    Directory of Open Access Journals (Sweden)

    Arnika Przybylska

    Full Text Available Thrips palmi (from the order Thysanoptera is a serious insect pest of various crops, including vegetables, fruits and ornamental plants, causing significant economic losses. Its presence constitutes a double threat; not only does T. palmi feed on the plants, it is also a vector for several plant viruses. T. palmi originated in Asia, but has spread to North and Central America, Africa, Oceania and the Caribbean in recent decades. This species has been sporadically noted in Europe and is under quarantine regulation in the European Union. For non-specialists its larval stages are indistinguishable morphologically from another widespread and serious insect pest Frankliniella occidentalis (a non-quarantine species in the European Union as well as other frequently occurring thrips. In this study, we have developed a loop-mediated isothermal amplification protocol to amplify rDNA regions of T. palmi. The results were consistent whether isolated DNA or crushed insects were used as template, indicating that the DNA isolation step could be omitted. The described method is species-specific and sensitive and provides a rapid diagnostic tool to detect T. palmi in the field.

  9. LOOP-MEDIATED ISOTHERMAL AMPLIFICATION (LAMP FOR THE DETECTION OF SALMONELLA SPP. ISOLATED FROM DIFFERENT FOOD TYPES

    Directory of Open Access Journals (Sweden)

    Kostas Papanotas

    2012-08-01

    Full Text Available The objective of this study was the application and evaluation of a loop-mediated isothermal amplification (LAMP method for the detection of Salmonella spp. strains isolated from food samples. Salmonella specific invA gene sequences (50 strains, 15 serotypes were amplified at 65oC in 60 min. All of the strains of Salmonella subsp. Enterica were shown to be positive using the LAMP reaction assay, whereas, all other bacteria, virus and yeasts tested in this study were negative. LAMP products could be visually detected under day light or ultraviolet light, while the specific amplification of the DNA of Salmonella strains generated ladder-like pattern bands on agarose gel. LAMP is suitable for the sensitive, rapid, and inexpensive detection of Salmonella spp. in food analytical laboratories.

  10. Quantum dot layer-by-layer assemblies as signal amplification labels for ultrasensitive electronic detection of uropathogens.

    Science.gov (United States)

    Xiang, Yun; Zhang, Haixia; Jiang, Bingying; Chai, Yaqin; Yuan, Ruo

    2011-06-01

    The preparation and use of a new class of signal amplification label, quantum dot (QD) layer-by-layer (LBL) assembled polystyrene microsphere composite, for amplified ultrasensitive electronic detection of uropathogen-specific DNA sequences is described. The target DNA is sandwiched between the capture probes immobilized on the magnetic beads and the signaling probes conjugated to the QD LBL assembled polystyrene beads. Because of the dramatic signal amplification by the numerous QDs involved in each single DNA binding event, subfemtomolar level detection of uropathogen-specific DNA sequences is achieved, which makes our strategy among the most sensitive electronic approach for nucleic acid-based monitoring of pathogens. Our signal amplified detection scheme could be readily expanded to monitor other important biomolecules (e.g., proteins, peptides, amino acids, cells, etc.) in ultralow levels and thus holds great potential for early diagnosis of disease biomarkers.

  11. Rapid detection of sacbrood virus (SBV by one-step reverse transcription loop-mediated isothermal amplification assay

    Directory of Open Access Journals (Sweden)

    Jin-Long Yang

    2012-02-01

    Full Text Available Abstract Background Sacbrood virus (SBV primarily infects honeybee broods, and in order to deal with the problem cost effective detection methods are required. Findings A one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP assay was developed for the rapid identification of SBV. The data demonstrated that, in a simple water bath, SBV RNA could be detected as early as 20 min at 65°C, and a positive amplification reaction was visible to the naked eye due to a color change brought on by the addition of nucleic acid stain SYBR Green. Conclusions The current study presents a method for the rapid and simple detection of SBV by RT-LAMP with high sensitivity and analytic specificity.

  12. Copper sulfide nanoparticle-decorated graphene as a catalytic amplification platform for electrochemical detection of alkaline phosphatase activity.

    Science.gov (United States)

    Peng, Juan; Han, Xiao-Xia; Zhang, Qing-Chun; Yao, Hui-Qin; Gao, Zuo-Ning

    2015-06-01

    Copper sulfide nanoparticle-decorated graphene sheet (CuS/GR) was successfully synthesized and used as a signal amplification platform for electrochemical detection of alkaline phosphatase activity. First, CuS/GR was prepared through a microwave-assisted hydrothermal approach. The CuS/GR nanocomposites exhibited excellent electrocatalytic activity toward the oxidation of ALP hydrolyzed products such as 1-naphthol, which produced a current response. Thus, a catalytic amplification platform based on CuS/GR nanocomposite for electrochemical detection of ALP activity was designed using 1-naphthyl phosphate as a model substrate. The current response increased linearly with ALP concentration from 0.1 to 100 U L(-1) with a detection limit of 0.02 U L(-1). The assay was applied to estimate ALP activity in human serum samples with satisfactory results. This strategy may find widespread and promising applications in other sensing systems that involves ALP.

  13. Establishment of an accurate and fast detection method using molecular beacons in loop-mediated isothermal amplification assay

    Science.gov (United States)

    Liu, Wei; Huang, Simo; Liu, Ningwei; Dong, Derong; Yang, Zhan; Tang, Yue; Ma, Wen; He, Xiaoming; Ao, Da; Xu, Yaqing; Zou, Dayang; Huang, Liuyu

    2017-01-01

    This study established a constant-temperature fluorescence quantitative detection method, combining loop-mediated isothermal amplification (LAMP) with molecular beacons. The advantages of LAMP are its convenience and efficiency, as it does not require a thermocycler and results are easily visualized by the naked eye. However, a major disadvantage of current LAMP techniques is the use of indirect evaluation methods (e.g., electrophoresis, SYBR Green I dye, precipitation, hydroxynaphthol blue dye, the turbidimetric method, calcein/Mn2+ dye, and the composite probe method), which cannot distinguish between the desired products and products of nonspecific amplification, thereby leading to false positives. Use of molecular beacons avoids this problem because molecular beacons produce fluorescence signals only when binding to target DNA, thus acting as a direct indicator of amplification products. Our analyses determined the optimal conditions for molecular beacons as an evaluation tool in LAMP: beacon length of 25–45 bp, beacon concentration of 0.6–1 pmol/μL, and reaction temperature of 60–65 °C. In conclusion, we validated a novel molecular beacon loop-mediated isothermal amplification method (MB-LAMP), realizing the direct detection of LAMP product. PMID:28059137

  14. Direct detection of microRNAs using isothermal amplification and molecular beacon with excellent sensitivity and specificity

    Science.gov (United States)

    Zhang, Wancun; Zhang, Qi; Qian, Zhiyu; Gu, Yueqing

    2017-02-01

    MicroRNAs (miRNAs) play important roles in a wide range of biological processes, including proliferation, development, metabolism, immunological response, tumorigenesis, and viral infection. The detection of miRNAs is imperative for gaining a better understanding of the functions of these biomolecules and has great potential for the early diagnosis of human disease as well as the discovery of new drugs through the use of miRNAs as targets. In this article, we develop a highly sensitive, and specific miRNA assay based on the two-stage isothermal amplification reactions and molecular beacon. The two-stage isothermal amplification reactions involves two templates and two-stage amplification reactions under isothermal conditions. The first template enables the amplification of miRNA, and the second template enables the conversion of miRNA to the reporter oligonucleotide(Y). Importantly, different miRNAs can be converted to the same Y seperately, which can hybridize with the same set of molecular beacon to generate fluorescent signals. This assay is highly sensitive and specific with a detection limit of 1 fM and can even discriminate single-nucleotide differences. Moreover, in combination with the specific templates, this method can be applied for multiplex miRNA assay by simply using the same molecular beacon. This method has potential to become a promising miRNA quantification method in biomedical research and clinical diagnosis.

  15. Detection of quasiresonant amplification of planetary waves and their connection to northern hemisphere summer heat extremes

    Science.gov (United States)

    Kornhuber, Kai; Coumou, Dim; Petri, Stefan; Petoukhov, Vladimir

    2014-05-01

    Several recent northern hemisphere (NH) summer heat extremes have been linked to anomalous patterns of mid-latitudinal planetary waves , e.g. the European heat wave in 2003, the Russian Heat wave and Pakistani floods in 2010 and the US heat wave in 2011(Lau and Kim 2012, Black et al 2004, Petoukhov et al 2013). The NH large-scale circulation patterns in those years were characterized by persistent longitudinal planetary-scale high-amplitude waves of relative high wavenumber (6-8). A common mechanism that could lead to the observed high-amplitude planetary waves was proposed by Petukhov et al. (Petukhov et al 2013). Under certain conditions, free synoptic waves can be 'trapped' in a midlatitudinal waveguide while their amplitudes are amplified by a quasiresonant response to thermal and orographic forcing. We have searched the available reanalysis data for the emergence of waveguides for particular planetary waves and will present preliminary results of this analysis. Using spectral analysis, we quantify the planetary wave field in terms of wavenumber, amplitude, phase and eastward phase-propagation. We will present statistics of these wave quantities for periods with and without waveguides. With those conditions explicitly implemented in code we should be able to detect and point out the periods in time the requirements for amplification were met. By doing so the connection of actual summer month heat extremes to quasiresonance events can be assessed statistically. Black E., Blackburn M., Hoskins B. and Methven J.; 2004: Factors contributing to the summer 2003 European heatwave 217-23 Lau W. K. M. and Kim K.-M.; 2012: The 2010 Pakistan Flood and Russian Heat Wave: Teleconnection of Hydrometeorological Extremes J. Hydrometeorol. 13 392-403 Online: http://journals.ametsoc.org/doi/abs/10.1175/JHM-D-11-016.1 Petoukhov V., Rahmstorf S., Petri S. and Schellnhuber H .J.;2013: Quasi-resonant amplification of atmospheric planetary waves as a mechanism for recent Northern

  16. Two-stage sample-to-answer system based on nucleic acid amplification approach for detection of malaria parasites.

    Science.gov (United States)

    Liu, Qing; Nam, Jeonghun; Kim, Sangho; Lim, Chwee Teck; Park, Mi Kyoung; Shin, Yong

    2016-08-15

    Rapid, early, and accurate diagnosis of malaria is essential for effective disease management and surveillance, and can reduce morbidity and mortality associated with the disease. Although significant advances have been achieved for the diagnosis of malaria, these technologies are still far from ideal, being time consuming, complex and poorly sensitive as well as requiring separate assays for sample processing and detection. Therefore, the development of a fast and sensitive method that can integrate sample processing with detection of malarial infection is desirable. Here, we report a two-stage sample-to-answer system based on nucleic acid amplification approach for detection of malaria parasites. It combines the Dimethyl adipimidate (DMA)/Thin film Sample processing (DTS) technique as a first stage and the Mach-Zehnder Interferometer-Isothermal solid-phase DNA Amplification (MZI-IDA) sensing technique as a second stage. The system can extract DNA from malarial parasites using DTS technique in a closed system, not only reducing sample loss and contamination, but also facilitating the multiplexed malarial DNA detection using the fast and accurate MZI-IDA technique. Here, we demonstrated that this system can deliver results within 60min (including sample processing, amplification and detection) with high sensitivity (malaria in low-resource settings.

  17. Multiplex detection of microRNAs by combining molecular beacon probes with T7 exonuclease-assisted cyclic amplification reaction.

    Science.gov (United States)

    Liu, Yacui; Zhang, Jiangyan; Tian, Jingxiao; Fan, Xiaofei; Geng, Hao; Cheng, Yongqiang

    2017-01-01

    A simple, highly sensitive, and specific assay was developed for the homogeneous and multiplex detection of microRNAs (miRNAs) by combining molecular beacon (MB) probes and T7 exonuclease-assisted cyclic amplification. An MB probe with five base pairs in the stem region without special modification can effectively prevent the digestion by T7 exonuclease. Only in the presence of target miRNA is the MB probe hybridized with the target miRNA, and then digested by T7 exonuclease in the 5' to 3' direction. At the same time, the target miRNA is released and subsequently initiates the nuclease-assisted cyclic digestion process, generating enhanced fluorescence signal significantly. The results show that the combination of T7 exonuclease-assisted cyclic amplification reaction and MB probe possesses higher sensitivity for miRNA detection. Moreover, multiplex detection of miRNAs was successfully achieved by designing two MB probes labeled with FAM and Cy3, respectively. As a result, the method opens a new pathway for the sensitive and multiplex detection of miRNAs as well as clinical diagnosis. Graphical Abstract A simple, highly sensitive, and specific assay was developed for the detection of microRNAs by combining molecular beacon probes with T7 exonuclease-assisted cyclic amplification reaction.

  18. A fully integrated paperfluidic molecular diagnostic chip for the extraction, amplification, and detection of nucleic acids from clinical samples.

    Science.gov (United States)

    Rodriguez, Natalia M; Wong, Winnie S; Liu, Lena; Dewar, Rajan; Klapperich, Catherine M

    2016-02-21

    Paper diagnostics have successfully been employed to detect the presence of antigens or small molecules in clinical samples through immunoassays; however, the detection of many disease targets relies on the much higher sensitivity and specificity achieved via nucleic acid amplification tests (NAAT). The steps involved in NAAT have recently begun to be explored in paper matrices, and our group, among others, has reported on paper-based extraction, amplification, and detection of DNA and RNA targets. Here, we integrate these paper-based NAAT steps into a single paperfluidic chip in a modular, foldable system that allows for fully integrated fluidic handling from sample to result. We showcase the functionality of the chip by combining nucleic acid isolation, isothermal amplification, and lateral flow detection of human papillomavirus (HPV) 16 DNA directly from crude cervical specimens in less than 1 hour for rapid, early detection of cervical cancer. The chip is made entirely of paper and adhesive sheets, making it low-cost, portable, and disposable, and offering the potential for a point-of-care molecular diagnostic platform even in remote and resource-limited settings.

  19. Aptamer-based colorimetric detection of proteins using a branched DNA cascade amplification strategy and unmodified gold nanoparticles.

    Science.gov (United States)

    Chang, Chia-Chen; Chen, Chen-Yu; Chuang, Tsung-Liang; Wu, Tzu-Heng; Wei, Shu-Chen; Liao, Hongen; Lin, Chii-Wann

    2016-04-15

    A branched DNA amplification strategy was employed to design a colorimetric aptameric biosensor using unmodified gold nanoparticles (AuNPs). First, a programmed DNA dendritic nanostructure was formed using two double-stranded substrate DNAs and two single-stranded auxiliary DNAs as assembly components via a target-assisted cascade amplification reaction, and it was then captured by DNA sensing probe-stabilized AuNPs. The release of sensing probes from AuNPs led to the formation of unstable AuNPs, promoting salt-induced aggregation. By integrating the signal amplification capacity of the branched DNA cascade reaction and unmodified AuNPs as a sensing indicator, this amplified colorimetric sensing strategy allows protein detection with high sensitivity (at the femtomole level) and selectivity. The limit of detection of this approach for VEGF was lower than those of other aptamer-based detection methods. Moreover, this assay provides modification-free and enzyme-free protein detection without sophisticated instrumentation and might be generally applicable to the detection of other protein targets in the future.

  20. Signal amplification strategies for DNA and protein detection based on polymeric nanocomposites and polymerization: A review

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Shaohong; Yuan, Liang; Hua, Xin; Xu, Lingling; Liu, Songqin, E-mail: liusq@seu.edu.cn

    2015-06-02

    Highlights: • We review the innovative advances in polymer-based signal amplification. • Conceptual connectivity between different amplified methodologies is illustrated. • Examples explain the mechanisms of polymers/polymerizations-based amplification. • Several elegant applications are summarized that illustrate underlying concept. - Abstract: Demand is increasing for ultrasensitive bioassays for disease diagnosis, environmental monitoring and other research areas. This requires novel signal amplification strategies to maximize the signal output. In this review, we focus on a series of significant signal amplification strategies based on polymeric nanocomposites and polymerization. Some common polymers are used as carriers to increase the local concentration of signal probes and/or biomolecules on their surfaces or in their interiors. Some polymers with special fluorescence and optical properties can efficiently transfer the excitation energy from a single site to the whole polymer backbone. This results in superior fluorescence signal amplification due to the resulting collective effort (integration of signal). Recent polymerization-based signal amplification strategies that employ atom transfer radical polymerization (ATRP) and photo-initiated polymerization are also summarized. Several distinctive applications of polymers in ultrasensitive bioanalysis are highlighted.

  1. A sensitive lateral flow biosensor for Escherichia coli O157:H7 detection based on aptamer mediated strand displacement amplification

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Wei [College of Food Science and Engineering, Ocean University of China, Qingdao 266003 (China); Zhao, Shiming [Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530 (China); Mao, Yiping [Yueyang Institute for Food and Drug Control, Yueyang 430198 (China); Fang, Zhiyuan [Affiliated Tumor Hospital of Guangzhou Medical University, Guangzhou 510095 (China); Lu, Xuewen [Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530 (China); Zeng, Lingwen, E-mail: zeng6@yahoo.com [Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530 (China)

    2015-02-25

    Highlights: • Limit of detection as low as 10 CFU mL{sup −1}Escherichia coli O157:H7. • No need of antibodies and substituted with aptamers. • Isothermal strand displacement amplification for signal amplification. • Results observed by the naked eye. • Great potential application in the area of food control. - Abstract: Foodborne diseases caused by pathogens are one of the major problems in food safety. Convenient and sensitive point-of-care rapid diagnostic tests for food-borne pathogens have been a long-felt need of clinicians. Commonly used methods for pathogen detection rely on conventional culture-based tests, antibody-based assays and polymerase chain reaction (PCR)-based techniques. These methods are costly, laborious and time-consuming. Herein, we present a simple and sensitive aptamer based biosensor for rapid detection of Escherichia coli O157:H7 (E. coli O157:H7). In this assay, two different aptamers specific for the outmembrane of E. coli O157:H7 were used. One of the aptamers was used for magnetic bead enrichment, and the other was used as a signal reporter for this pathogen, which was amplified by isothermal strand displacement amplification (SDA) and further detected by a lateral flow biosensor. Only the captured aptamers on cell membrane were amplified, limitations of conventional DNA amplification based method such as false-positive can be largely reduced. The generated signals (red bands on the test zone of a lateral flow strip) can be unambiguously read out by the naked eye. As low as 10 colony forming units (CFU) of E. coli O157:H7 were detected in this study. Without DNA extraction, the reduced handling and simpler equipment requirement render this assay a simple and rapid alternative to conventional methods.

  2. Point of care nucleic acid detection of viable pathogenic bacteria with isothermal RNA amplification based paper biosensor

    Science.gov (United States)

    Liu, Hongxing; Xing, Da; Zhou, Xiaoming

    2014-09-01

    Food-borne pathogens such as Listeria monocytogenes have been recognized as a major cause of human infections worldwide, leading to substantial health problems. Food-borne pathogen identification needs to be simpler, cheaper and more reliable than the current traditional methods. Here, we have constructed a low-cost paper biosensor for the detection of viable pathogenic bacteria with the naked eye. In this study, an effective isothermal amplification method was used to amplify the hlyA mRNA gene, a specific RNA marker in Listeria monocytogenes. The amplification products were applied to the paper biosensor to perform a visual test, in which endpoint detection was performed using sandwich hybridization assays. When the RNA products migrated along the paper biosensor by capillary action, the gold nanoparticles accumulated at the designated Test line and Control line. Under optimized experimental conditions, as little as 0.5 pg/μL genomic RNA from Listeria monocytogenes could be detected. The whole assay process, including RNA extraction, amplification, and visualization, can be completed within several hours. The developed method is suitable for point-of-care applications to detect food-borne pathogens, as it can effectively overcome the false-positive results caused by amplifying nonviable Listeria monocytogenes.

  3. A novel CMOS image sensor system for quantitative loop-mediated isothermal amplification assays to detect food-borne pathogens.

    Science.gov (United States)

    Wang, Tiantian; Kim, Sanghyo; An, Jeong Ho

    2017-02-01

    Loop-mediated isothermal amplification (LAMP) is considered as one of the alternatives to the conventional PCR and it is an inexpensive portable diagnostic system with minimal power consumption. The present work describes the application of LAMP in real-time photon detection and quantitative analysis of nucleic acids integrated with a disposable complementary-metal-oxide semiconductor (CMOS) image sensor. This novel system works as an amplification-coupled detection platform, relying on a CMOS image sensor, with the aid of a computerized circuitry controller for the temperature and light sources. The CMOS image sensor captures the light which is passing through the sensor surface and converts into digital units using an analog-to-digital converter (ADC). This new system monitors the real-time photon variation, caused by the color changes during amplification. Escherichia coli O157 was used as a proof-of-concept target for quantitative analysis, and compared with the results for Staphylococcus aureus and Salmonella enterica to confirm the efficiency of the system. The system detected various DNA concentrations of E. coli O157 in a short time (45min), with a detection limit of 10fg/μL. The low-cost, simple, and compact design, with low power consumption, represents a significant advance in the development of a portable, sensitive, user-friendly, real-time, and quantitative analytic tools for point-of-care diagnosis.

  4. Human telomerase RNA component (hTERC gene amplification detected by FISH in precancerous lesions and carcinoma of the larynx

    Directory of Open Access Journals (Sweden)

    Liu Yu

    2012-03-01

    Full Text Available Abstract Background Gain of 3q26 is frequently observed in squamous cell carcinomas of mucosal origin, including those originating in the head and neck region. The human telomerase RNA component (hTERC gene, which is located on chromosome 3q26, encodes for an RNA subunit of telomerase that maintains the length of telomeres through cellular divisions, and is activated in malignant diseases. The present study was designed to detect hTERC amplification in laryngeal lesions and evaluate whether this might serve as a supportive biomarker in histopathological analysis for in the diagnosis of laryngeal lesions. Methods Fluorescent in situ hybridization (FISH was applied on formalin-fixed paraffin-embedded blocks of 93 laryngeal specimens, including 14 normal epithelium (NE, 15 mild dysplasia (Md, 18 moderate dysplasia (MD, 16 severe dysplasia (SD, 9 carcinoma in situ (CIS, and 21 invasive carcinoma (IC. Results By histopathologic examination, hTERC amplification rates in NE, Md, MD, SD, CIS and IC cases were 0% (0/14, 13.33% (2/15, 72.22% (13/18, 81.25% (13/16, 100% (9/9 and 100% (21/21, respectively. Amplification of hTERC was significantly associated with histopathologic diagnosis (P Conclusions The hTERC amplification is important in the development of laryngeal squamous cell carcinoma (LSCC. FISH detection of hTERC amplification may provide an effective approach in conjunction with histopathologic evaluation for differential diagnosis of laryngeal lesions. Virtual Slides The virtual slide(s for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/2226606266791985

  5. Rapid Point-of-Care Isothermal Amplification Assay for the Detection of Malaria without Nucleic Acid Purification.

    Science.gov (United States)

    Modak, Sayli S; Barber, Cheryl A; Geva, Eran; Abrams, William R; Malamud, Daniel; Ongagna, Yhombi Serge Yvon

    2016-01-01

    Malaria remains one of the most prevalent infectious diseases and results in significant mortality. Isothermal amplification (loop-mediated isothermal amplification) is used to detect malarial DNA at levels of ~1 parasite/µL blood in ≤30 minutes without the isolation of parasite nucleic acid from subject's blood or saliva. The technique targets the mitochondrial cytochrome oxidase subunit 1 gene and is capable of distinguishing Plasmodium falciparum from Plasmodium vivax. Malarial diagnosis by the gold standard microscopic examination of blood smears is generally carried out only after moderate-to-severe symptoms appear. Rapid diagnostic antigen tests are available but generally require infection levels in the range of 200-2,000 parasites/µL for a positive diagnosis and cannot distinguish if the disease has been cleared due to the persistence of circulating antigen. This study describes a rapid and simple molecular assay to detect malarial genes directly from whole blood or saliva without DNA isolation.

  6. Rapid detection of herpes simplex virus in clinical samples by flow cytometry after amplification in tissue culture.

    OpenAIRE

    McSharry, J J; Costantino, R; McSharry, M B; Venezia, R A; Lehman, J M

    1990-01-01

    Murine monoclonal antibodies (MAbs) against herpes simplex virus type 1 and 2 (HSV-1 and -2, respectively) nuclear antigens were used to identify cells infected with HSV-1 or -2 by indirect immunofluorescence in conjunction with flow cytometry after virus amplification of MRC-5 cell monolayers. The results indicate that MAbs Q1, Q2, and H-640 detect HSV-1- and HSV-2-infected cells, MAb SD-1 detects HSV-2- but not HSV-1-infected cells, and MAb 58-S detects HSV-1- but not HSV-2-infected cells. ...

  7. Detection of MYCN Gene Amplification in Neuroblastoma by Fluorescence In Situ Hybridization: A Pediatric Oncology Group Study

    Directory of Open Access Journals (Sweden)

    Prasad Mathew

    2001-01-01

    Full Text Available To assess the utility of fluorescence in situ hybridization (FISH for analysis of MYCN gene amplification in neuroblastoma, we compared this assay with Southern blot analysis using tumor specimens collected from 232 patients with presenting characteristics typical of this disease. The FISH technique identified MYCN amplification in 47 cases, compared with 39 by Southern blotting, thus increasing the total number of positive cases by 21%. The major cause of discordancy was a low fraction of tumor cells (≤30% replacement in clinical specimens, which prevented an accurate estimate of MYCN copy number by Southern blotting. With FISH, by contrast, it was possible to analyze multiple interphase nuclei of tumor cells, regardless of the proportion of normal peripheral blood, bone marrow, or stromal cells in clinical samples. Thus, FISH could be performed accurately with very small numbers of tumor cells from touch preparations of needle biopsies. Moreover, this procedure allowed us to discern the heterogeneous pattern of MYCN amplification that is characteristic of neuroblastoma. We conclude that FISH improves the detection of MYCN gene amplification in childhood neuroblastomas in a clinical setting, thus facilitating therapeutic decisions based on the presence or absence of this prognostically important biologic marker.

  8. Detection and identification of opportunistic Exophiala species using the rolling circle amplification of ribosomal internal transcribed spacers.

    Science.gov (United States)

    Najafzadeh, M J; Dolatabadi, S; Saradeghi Keisari, M; Naseri, A; Feng, P; de Hoog, G S

    2013-09-01

    Deep infections by melanized fungi deserve special attention because of a potentially fatal, cerebral or disseminated course of disease in otherwise healthy patients. Timely diagnostics are a major problem with these infections. Rolling circle amplification (RCA) is a sensitive, specific and reproducible isothermal DNA amplification technique for rapid molecular identification of microorganisms. RCA-based diagnostics are characterized by good reproducibility, with few amplification errors compared to PCR. The method is applied here to species of Exophiala known to cause systemic infections in humans. The ITS rDNA region of five Exophiala species (E. dermatitidis, E. oligosperma, E. spinifera, E. xenobiotica, and E. jeanselmei) was sequenced and aligned in view of designing specific padlock probes to be used for the detection of single nucleotide polymorphisms (SNPs) of the Exophiala species concerned. The assay proved to successfully amplify DNA of the target fungi at the level of species; while no cross-reactivity was observed. Amplification products were visualized on 1% agarose gels to verify the specificity of probe-template binding. Amounts of reagents were minimized to avoid the generation of false positive results. The sensitivity of RCA may help to improve early diagnostics of these difficult to diagnose infections.

  9. Visual Detection of Potato leafroll virus by One-step Reverse Transcription Loop-Mediated Isothermal Amplification of DNA with Hydroxynaphthol Blue Dye

    NARCIS (Netherlands)

    Ahmadi, S.; Almasi, A.M.; Fatehi, F.; Struik, P.C.; Moradi, A.

    2013-01-01

    Loop-mediated isothermal amplification (LAMP) assay is a novel technique for amplifying DNA under constant temperature, with high specificity, sensitivity, rapidity and efficiency. We applied reverse transcription loop-mediated isothermal amplification (RT-LAMP) to visually detect Potato leafroll vi

  10. Rapid and sensitive detection of Listeria ivanovii by loop-mediated isothermal amplification of the smcL gene.

    Directory of Open Access Journals (Sweden)

    Yi Wang

    Full Text Available A loop-mediated isothermal amplification (LAMP assay for rapid and sensitive detection of the L. ivanovii strains had been developed and evaluated in this study. Oligonucleotide primers specific for L. ivanovii species were designed corresponding to smcL gene sequences. The primers set comprise six primers targeting eight regions on the species-specific gene smcL. The LAMP assay could be completed within 1 h at 64°C in a water bath. Amplification products were directly observed by the Loopamp Fluorescent Detection Reagent (FD or detected by agarose gel electrophoresis. Moreover, the LAMP reactions were also detected by real-time measurement of turbidity. The exclusivity of 77 non-L. ivanovii and the inclusivity of 17 L. ivanovii were both 100% in the assay. Sensitivity of the LAMP assay was 250 fg DNA and 16 CFU per reaction for detection of L. ivanovii in pure cultures and simulated human stool. The LAMP assay was 10 and 100-fold more sensitive than quantitative PCR (qPCR and conventional PCR assays,respectively. When applied to human stool samples spiked with low level (8 CFU/0.5 g of L. ivanovii strains, the new LAMP assay described here achieved positive detection after 6 hours enrichment. In conclusion, the new LAMP assay in this study can be used as a valuable, rapid and sensitive detection tool for the detection of L. ivanovii in field, medical and veterinary laboratories.

  11. The application of loop-mediated isothermal amplification for detection of common pathogenic bacteria in lower respiratory tract infections

    Institute of Scientific and Technical Information of China (English)

    陈愉生

    2014-01-01

    Objective To investigate the spectrum of common pathogenic bacteria of low respiratory tract infection by loop-mediated isothermal amplification(LAMP)of nucleic acid test and to prove the clinical significance of this method.Methods A total of 289 qualified sputum samples from patients with lower respiratory tract infections in Fujian Province were detected by LAMP technique,and then the distribution of pathogenic bacteria was analyzed.The positive cases(the patients whose specific3

  12. Sensitive and rapid detection of Paragonimus westermani infection in humans and animals by loop-mediated isothermal amplification (LAMP).

    Science.gov (United States)

    Chen, M X; Ai, L; Zhang, R L; Xia, J J; Wang, K; Chen, S H; Zhang, Y N; Xu, M J; Li, X; Zhu, X Q; Chen, J X

    2011-05-01

    In the present study, a loop-mediated isothermal amplification (LAMP) assay was developed and validated for the detection of Paragonimus westermani adults, metacercariae, and eggs in human and animal samples. The LAMP amplification can be finished in 45 min under isothermal condition at 60°C by employing a set of four species-specific primer mixtures and the results can be checked by naked-eye visualization. No amplification products were detected with deoxyribunucleic acid (DNA) of related trematode species including Fasciola hepatica, Fasciola gigantica, Clonorchis sinensis, Opisthorchis viverrini, Schistosoma mansoni, and Schistosoma japonicum. The method was further validated by examining P. westermani DNA in intermediate hosts including freshwater crabs and crayfish, as well as in sputum and pleural fluid samples from patients of paragonimiasis. These results indicated that the LAMP assay was highly specific, sensitive, and rapid, and it was approximately 100 times more sensitive than conventional specific PCR. The LAMP assay established in this study provides a rapid and sensitive tool for the detection of P. westermani DNA in freshwater crabs, crayfish, sputum, and pleural fluid samples, which has important implications for effective control of human paragonimiasis.

  13. Improvement and Evaluation of Loop-Mediated Isothermal Amplification for Rapid Detection of Toxoplasma gondii Infection in Human Blood Samples

    Science.gov (United States)

    Sun, Xi-meng; Ji, Yong-sheng; Liu, Xian-yong; Xiang, Mei; He, Guang; Xie, Li; Suo, Jing-xia; Suo, Xun

    2017-01-01

    Loop-mediated isothermal amplification (LAMP), an attractive DNA amplification method, was developed as a valuable tool for the rapid detection of Toxoplasma gondii. In this study, species-specific LAMP primers were designed by targeting the AF146527 sequence, which was a conserved sequence of 200- to 300-fold repetitive 529 bp fragment of T.gondii. LAMP reaction system was optimized so that it could detect the minimal DNA sample such as a single tachyzoite or 10 copies of recombinant plasmid. No cross-reactivity was found when using DNA from other parasites as templates. Subsequently, a total of 200 human blood samples were directly investigated by two diagnostic methods, LAMP and conventional PCR. Fourteen of 200 (7%) samples were positive for Toxoplasma by LAMP (the primers developed in this study), whereas only 5 of 200 (2.5%) were proved positive by conventional PCR. The procedure of the LAMP assay was very simple, as the reaction would be carried out in a single tube under isothermal conditions at 64°C and the result would be read out with 1 h (as early as 35 min with loop primers). Thus, this method has the advantages of rapid amplification, simple operation, and easy detection and would be useful for rapid and reliable clinical diagnosis of acute toxoplasmosis, especially in developing countries. PMID:28056092

  14. Simultaneous amplification of two bacterial genes: more reliable method of Helicobacter pylori detection in microbial rich dental plaque samples.

    Science.gov (United States)

    Chaudhry, Saima; Idrees, Muhammad; Izhar, Mateen; Butt, Arshad Kamal; Khan, Ayyaz Ali

    2011-01-01

    Polymerase Chain reaction (PCR) assay is considered superior to other methods for detection of Helicobacter pylori (H. pylori) in oral cavity; however, it also has limitations when sample under study is microbial rich dental plaque. The type of gene targeted and number of primers used for bacterial detection in dental plaque samples can have a significant effect on the results obtained as there are a number of closely related bacterial species residing in plaque biofilm. Also due to high recombination rate of H. pylori some of the genes might be down regulated or absent. The present study was conducted to determine the frequency of H. pylori colonization of dental plaque by simultaneously amplifying two genes of the bacterium. One hundred dental plaque specimens were collected from dyspeptic patients before their upper gastrointestinal endoscopy and presence of H. pylori was determined through PCR assay using primers targeting two different genes of the bacterium. Eighty-nine of the 100 samples were included in final analysis. With simultaneous amplification of two bacterial genes 51.6% of the dental plaque samples were positive for H. pylori while this prevalence increased to 73% when only one gene amplification was used for bacterial identification. Detection of H. pylori in dental plaque samples is more reliable when two genes of the bacterium are simultaneously amplified as compared to one gene amplification only.

  15. Signal-on electrochemical detection of antibiotics at zeptomole level based on target-aptamer binding triggered multiple recycling amplification.

    Science.gov (United States)

    Wang, Hongzhi; Wang, Yu; Liu, Su; Yu, Jinghua; Guo, Yuna; Xu, Ying; Huang, Jiadong

    2016-06-15

    In the work, a signal-on electrochemical DNA sensor based on multiple amplification for ultrasensitive detection of antibiotics has been reported. In the presence of target, the ingeniously designed hairpin probe (HP1) is opened and the polymerase-assisted target recycling amplification is triggered, resulting in autonomous generation of secondary target. It is worth noting that the produced secondary target could not only hybridize with other HP1, but also displace the Helper from the electrode. Consequently, methylene blue labeled HP2 forms a "close" probe structure, and the increase of signal is monitored. The increasing current provides an ultrasensitive electrochemical detection for antibiotics down to 1.3 fM. To our best knowledge, such work is the first report about multiple recycling amplification combing with signal-on sensing strategy, which has been utilized for quantitative determination of antibiotics. It would be further used as a general strategy associated with more analytical techniques toward the detection of a wide spectrum of analytes. Thus, it holds great potential for the development of ultrasensitive biosensing platform for the applications in bioanalysis, disease diagnostics, and clinical biomedicine.

  16. Development of a multiplex loop-mediated isothermal amplification method for the simultaneous detection of Salmonella spp. and Vibrio parahaemolyticus

    Science.gov (United States)

    Liu, Ningwei; Zou, Dayang; Dong, Derong; Yang, Zhan; Ao, Da; Liu, Wei; Huang, Liuyu

    2017-01-01

    Rapid detection of food-borne pathogens is important in the food industry, to monitor and prevent the spread of these pathogens through contaminated food products. We therefore established a multiplex real-time loop-mediated isothermal amplification (LAMP) assay to simultaneously detect and distinguish Salmonella spp. and Vibrio parahaemolyticus DNA in a single reaction. Two target sequences, one specific for Salmonella and the other specific for Vibrio parahaemolyticus, were amplified by specific LAMP primers in the same reaction tube. After amplification at 65 °C for 60 min, the amplified products were subjected to melting curve analysis and thus could be distinguished based on the different melting temperatures (Tm values) of the two specifically amplified products. The specificity of the multiplex LAMP assay was evaluated using 19 known bacterial strains, including one V. parahaemolyticus and seven Salmonella spp. strains. The multiplex LAMP showed 100% inclusivity and exclusivity, and a detection limit similar to that of multiplex PCR. In addition, we observed and corrected preferential amplification induced by what we call LAMP selection in the multiplex LAMP reaction. In conclusion, our assay was rapid, specific, and quantitative, making it a useful tool for the food industry. PMID:28349967

  17. A lab-on-a-chip system with integrated sample preparation and loop-mediated isothermal amplification for rapid and quantitative detection of Salmonella spp. in food samples

    DEFF Research Database (Denmark)

    Sun, Yi; Than Linh, Quyen; Hung, Tran Quang;

    2015-01-01

    amplification (LAMP) for rapid and quantitative detection of Salmonella spp. in food samples. The whole diagnostic procedures including DNA isolation, isothermal amplification, and real-time detection were accomplished in a single chamber. Up to eight samples could be handled simultaneously and the system...... was capable to detect Salmonella at concentration of 50 cells per test within 40 min. The simple design, together with high level of integration, isothermal amplification, and quantitative analysis of multiple samples in short time will greatly enhance the practical applicability of the LOC system for rapid...

  18. Detection of MDM2/CDK4 amplification in lipomatous soft tissue tumors from formalin-fixed, paraffin-embedded tissue: comparison of multiplex ligation-dependent probe amplification (MLPA) and fluorescence in situ hybridization (FISH).

    Science.gov (United States)

    Creytens, David; van Gorp, Joost; Ferdinande, Liesbeth; Speel, Ernst-Jan; Libbrecht, Louis

    2015-02-01

    In this study, the detection of MDM2 and CDK4 amplification was evaluated in lipomatous soft tissue tumors using multiplex ligation-dependent probe amplification (MLPA), a PCR-based technique, in comparison with fluorescence in situ hybridization (FISH). These 2 techniques were evaluated in a series of 77 formalin-fixed, paraffin-embedded lipomatous tumors (27 benign adipose tumors, 28 atypical lipomatous tumors/well-differentiated liposarcomas, 18 dedifferentiated liposarcomas, and 4 pleomorphic liposarcomas). Using MLPA, with a cut-off ratio of >2, 36/71 samples (22 atypical lipomatous tumors/well-differentiated liposarcomas, and 14 dedifferentiated liposarcomas) showed MDM2 and CDK4 amplification. Using FISH as gold standard, MLPA showed a sensitivity of 90% (36/40) and a specificity of 100% (31/31) in detecting amplification of MDM2 and CDK4 in lipomatous soft tissue tumors. In case of high-level amplification (MDM2-CDK4/CEP12 ratio >5), concordance was 100%. Four cases of atypical lipomatous tumor/well-differentiated liposarcoma (4/26, 15%) with a low MDM2 and CDK4 amplification level (MDM2-CDK4/CEP12 ratio ranging between 2 and 2.5) detected by FISH showed no amplification by MLPA, although gain of MDM2 and CDK4 (ratios ranging between 1.6 and 1.9) was seen with MLPA. No amplification was detected in benign lipomatous tumors and pleomorphic liposarcomas. Furthermore, there was a very high concordance between the ratios obtained by FISH and MLPA. In conclusion, MLPA proves to be an appropriate and straightforward technique for screening MDM2/CDK4 amplification in lipomatous tumors, especially when a correct cut-off value and reference samples are chosen, and could be considered a good alternative to FISH to determine MDM2 and CDK4 amplification in liposarcomas. Moreover, because MLPA, as a multiplex technique, allows simultaneous detection of multiple chromosomal changes of interest, it could be in the future a very reliable and fast molecular analysis on

  19. Sensitivity of APTIMA HPV E6/E7 mRNA test in comparison with hybrid capture 2 HPV DNA test for detection of high risk oncogenic human papillomavirus in 396 biopsy confirmed cervical cancers.

    Science.gov (United States)

    Basu, Partha; Banerjee, Dipanwita; Mittal, Srabani; Dutta, Sankhadeep; Ghosh, Ishita; Chowdhury, Nilarun; Abraham, Priya; Chandna, Puneet; Ratnam, Sam

    2016-07-01

    The sensitivity of E6/E7 mRNA-based Aptima HPV test (AHPV; Hologic, Inc.) for detection of cervical cancer has been reported based on only a small number of cases. We determined the sensitivity of AHPV in comparison with the DNA-based Hybrid Capture 2 HPV test (HC2; Qiagen) for the detection of oncogenic HPV in a large number of cervical cancers at the time of diagnosis using cervical samples obtained in ThinPrep (Hologic). Samples yielding discordant results were genotyped using Linear Array assay (LA; Roche). Of 396 cases tested, AHPV detected 377 (sensitivity, 95.2%; 95%CI: 93.1-97.3), and HC2 376 (sensitivity, 94.9%; 95%CI: 92.7-97.1) with an agreement of 97.2% (kappa 0.7; 95%CI: 0.54-0.87). Among six AHPV+/HC2- cases, LA identified oncogenic HPV types in four including a type 73 and was negative in two. Among five AHPV-/HC2+ cases, LA detected oncogenic HPV types in two including a type 73 and was negative in three. Of 14 AHPV-/HC2- cases, 13 were genotyped. LA detected oncogenic HPV types in six, non-oncogenic types in three, and was negative in four. This is the largest study to demonstrate the sensitivity of AHPV for the detection of invasive cervical cancer and this assay showed equal sensitivity to HC2.

  20. Sensitive and specific colorimetric DNA detection by invasive reaction coupled with nicking endonuclease-assisted nanoparticles amplification.

    Science.gov (United States)

    Zou, Bingjie; Cao, Xiaomei; Wu, Haiping; Song, Qinxin; Wang, Jianping; Kajiyama, Tomoharu; Kambara, Hideki; Zhou, Guohua

    2015-04-15

    Colorimetric DNA detection is preferable to methods in clinical molecular diagnostics, because no expensive equipment is required. Although many gold nanoparticle-based colorimetric DNA detection strategies have been developed to analyze DNA sequences of interest, few of them can detect somatic mutations due to their insufficient specificity. In this study, we proposed a colorimetric DNA detection method by coupling invasive reaction with nicking endonuclease-assisted nanoparticles amplification (IR-NEANA). A target DNA firstly produces many flaps by invasive reaction. Then the flaps are converted to targets of nicking reaction-assisted nanoparticles amplification by ligation reaction to produce the color change of AuNPs, which can be observed by naked eyes. The detection limit of IR-NEANA was determined as 1pM. Most importantly, the specificity of the method is high enough to pick up as low as 1% mutant from a large amount of wild-type DNA backgrounds. The EGFR gene mutated at c.2573 T>G in 9 tissue samples from non-small cell lung cancer patients were successfully detected by using IR-NEANA, suggesting that our proposed method can be used to detect somatic mutations in biological samples.

  1. A novel, sensitive and label-free loop-mediated isothermal amplification detection method for nucleic acids using luminophore dyes.

    Science.gov (United States)

    Roy, Sharmili; Wei, Sim Xiao; Ying, Jean Liew Zhi; Safavieh, Mohammadali; Ahmed, Minhaz Uddin

    2016-12-15

    Electrochemiluminescence (ECL) has been widely rendered for nucleic acid testing. Here, we integrate loop-mediated isothermal amplification (LAMP) with ECL technique for DNA detection and quantification. The target LAMP DNA bound electrostatically with [Ru(bpy)3](+2) on the carbon electrode surface, and an ECL reaction was triggered by tripropylamine (TPrA) to yield luminescence. We illustrated this method as a new and highly sensitive strategy for the detection of sequence-specific DNA from different meat species at picogram levels. The proposed strategy renders the signal amplification capacities of TPrA and combines LAMP with inherently high sensitivity of the ECL technique, to facilitate the detection of low quantities of DNA. By leveraging this technique, target DNA of Sus scrofa (pork) meat was detected as low as 1pg/µL (3.43×10(-1)copies/µL). In addition, the proposed technique was applied for detection of Bacillus subtilis DNA samples and detection limit of 10pg/µL (2.2×10(3)copies/µL) was achieved. The advantages of being isothermal, sensitive and robust with ability for multiplex detection of bio-analytes makes this method a facile and appealing sensing modality in hand-held devices to be used at the point-of-care (POC).

  2. Development of a rapid and specific loop-mediated isothermal amplification detection method that targets Marek's disease virus meq gene.

    Science.gov (United States)

    Wei, Xiuying; Shi, Xingming; Zhao, Yan; Zhang, Jing; Wang, Mei; Liu, Changjun; Cui, Hongyu; Hu, Shunlei; Quan, Yanming; Chen, Hongyan; Wang, Yunfeng

    2012-08-01

    A rapid, sensitive and specific loop-mediated isothermal amplification (LAMP) method was developed and evaluated for the detection of Marek's disease virus (MDV) by amplification of conserved MDV meq gene sequences. LAMP is an innovative technique that allows the rapid detection of targeted nucleic acid sequences under isothermal conditions without the need for complex instrumentation. In this study, meq gene sequences were amplified successfully from different MDV strains by LAMP within 60min and no cross-reactivity was observed in a panel of related viruses that were associated with diseases of chickens. The detection limit of LAMP was 3.2 copies/million cells compared with 320 copies/million cells required for conventional PCR. Positive detection rates were assessed using either LAMP or PCR by examination of feather follicles that were collected from chickens infected experimentally with either strain J-1 (n=20) or strain Md5 (n=17), In addition to these samples, three isolates that were suspected to have been infected in the clinic were also tested. Results showed that the positive detection rate for LAMP was 95% (38/40), compared with 87.5% (35/40) and 90% (38/40) for strains J-1 and Md5 by PCR, respectively. These results indicated that the LAMP assay was more sensitive, rapid and specific than conventional PCR for the detection of MDV. This easy-to-perform technique will be useful for the detection of MDV and will aid in the establishment of disease control protocols.

  3. [Colorimetric detection of human influenza A H1N1 virus by reverse transcription loop mediated isothermal amplification].

    Science.gov (United States)

    Nie, Kai; Wang, Da-Yan; Qin, Meng; Gao, Rong-Bao; Wang, Miao; Zou, Shu-Mei; Han, Feng; Zhao, Xiang; Li, Xi-Yan; Shu, Yue-Long; Ma, Xue-Jun

    2010-03-01

    A simple, rapid and sensitive colorimetric Reverse Transcription Loop Mediated Isothermal Amplification (RT-LAMP) method was established to detect human influenza A H1N1 virus. The method employed a set of six specially designed primers that recognized eight distinct sequences of the HA gene for amplification of nucleic acid under isothermal conditions at 65 degrees C for one and half hour. The amplification process of RT-LAMP was monitored by the addition of HNB (Hydroxy naphthol blue) dye prior to amplification. A positive reaction was indicated by a color change from violet to sky blue and confirmed by agarose electrophoresis. The specificity of the RT-LAMP assay was validated by cross-reaction with different swine and human influenza virus including human seasonal influenza A /H1N1 A /H3N2, influenza B and swine A /H1N1. The sensitivity of this assay was evaluated by serial dilutions of RNA molecules from in vitro transcription of human influenza A H1N1 HA gene. The assay was further evaluated with 30 clinical specimens with suspected pandemic influenza A H1N1 virus infection in parallel with RT-PCR detection and 26 clinical specimens with seasonal influenza virus infection. Our results showed that the RT-LAMP was able to achieve a sensitivity of 60 RNA copies with high specificity, and detection rate was comparable to that of the RT-PCR with the clinical samples of pandemic influenza A H1N1 infection. The RT-LAMP reaction with HNB could also be measured at 650nm in a microplate reader for quantitative analysis. Thus, we concluded that this colorimetric RT-LAMP assay had potential for the rapid screening of the human influenza A H1N1 virus infection in National influenza monitoring network laboratories and sentinel hospitals of provincial and municipal region in China.

  4. On-Chip Isothermal Nucleic Acid Amplification on Flow-Based Chemiluminescence Microarray Analysis Platform for the Detection of Viruses and Bacteria.

    Science.gov (United States)

    Kunze, A; Dilcher, M; Abd El Wahed, A; Hufert, F; Niessner, R; Seidel, M

    2016-01-01

    This work presents an on-chip isothermal nucleic acid amplification test (iNAAT) for the multiplex amplification and detection of viral and bacterial DNA by a flow-based chemiluminescence microarray. In a principle study, on-chip recombinase polymerase amplification (RPA) on defined spots of a DNA microarray was used to spatially separate the amplification reaction of DNA from two viruses (Human adenovirus 41, Phi X 174) and the bacterium Enterococcus faecalis, which are relevant for water hygiene. By establishing the developed assay on the microarray analysis platform MCR 3, the automation of isothermal multiplex-amplification (39 °C, 40 min) and subsequent detection by chemiluminescence imaging was realized. Within 48 min, the microbes could be identified by the spot position on the microarray while the generated chemiluminescence signal correlated with the amount of applied microbe DNA. The limit of detection (LOD) determined for HAdV 41, Phi X 174, and E. faecalis was 35 GU/μL, 1 GU/μL, and 5 × 10(3) GU/μL (genomic units), which is comparable to the sensitivity reported for qPCR analysis, respectively. Moreover the simultaneous amplification and detection of DNA from all three microbes was possible. The presented assay shows that complex enzymatic reactions like an isothermal amplification can be performed in an easy-to-use experimental setup. Furthermore, iNAATs can be potent candidates for multipathogen detection in clinical, food, or environmental samples in routine or field monitoring approaches.

  5. High-density SNP arrays improve detection of HER2 amplification and polyploidy in breast tumors

    DEFF Research Database (Denmark)

    Hansen, Thomas V. O.; Vikesaa, Jonas; Buhl, Sine S

    2015-01-01

    ) arrays can provide additional diagnostic power to assess HER2 gene status. METHODS: DNA from 65 breast tumor samples previously diagnosed by HER2 IHC and FISH analysis were blinded and examined for HER2 copy number variation employing SNP array analysis. RESULTS: SNP array analysis identified 24 (37......%) samples with selective amplification or imbalance of the HER2 region in the q-arm of chromosome 17. In contrast, only 15 (23%) tumors were found to have HER2 amplification by IHC and FISH analysis. In total, there was a discrepancy in 19 (29%) samples between SNP array and IHC/FISH analysis. In 12...

  6. The isothermal amplification detection of double-stranded DNA based on a double-stranded fluorescence probe.

    Science.gov (United States)

    Shi, Chao; Shang, Fanjin; Pan, Mei; Liu, Sen; Ma, Cuiping

    2016-06-15

    Here we have developed a novel method of isothermal amplification detection of double-stranded DNA (dsDNA) based on double-stranded fluorescence probe (ds-probe). Target dsDNA repeatedly generated single-stranded DNA (ssDNA) with polymerase and nicking enzyme. The ds-probe as a primer hybridized with ssDNA and extended to its 5'-end. The displaced ssDNA served as a new detection target to initiate above-described reaction. Meanwhile, the extended ds-probe could dynamically dissociate from ssDNA and self-hybridize, converting into a turn-back structure to initiate another amplification reaction. In particular, the ds-probe played a key role in the entire experimental process, which not only was as a primer but also produced the fluorescent signal by an extension and displacement reaction. Our method could detect the pBluescript II KS(+) plasmid with a detection limit of 2.3 amol, and it was also verified to exhibit a high specificity, even one-base mismatch. Overall, it was a true isothermal dsDNA detection strategy with a strongly anti-jamming capacity and one-pot, only requiring one ds-probe, which greatly reduced the cost and the probability of contamination. With its advantages, the approach of dsDNA detection will offer a promising tool in the field of point-of-care testing (POCT).

  7. Simultaneous Identification and Susceptibility Determination to Multiple Antibiotics of Staphylococcus aureus by Bacteriophage Amplification Detection Combined with Mass Spectrometry.

    Science.gov (United States)

    Rees, Jon C; Pierce, Carrie L; Schieltz, David M; Barr, John R

    2015-07-01

    The continued advance of antibiotic resistance in clinically relevant bacterial strains necessitates the development and refinement of assays that can rapidly and cost-effectively identify bacteria and determine their susceptibility to a panel of antibiotics. A methodology is described herein that exploits the specificity and physiology of the Staphylococci bacteriophage K to identify Staphylococcus aureus (S. aureus) and determine its susceptibility to clindamycin and cefoxitin. The method uses liquid chromatography-mass spectrometry to monitor the replication of bacteriophage after it is used to infect samples thought to contain S. aureus. Amplification of bacteriophage K indicates the sample contains S. aureus, for it is only in the presence of a suitable host that bacteriophage K can amplify. If bacteriophage amplification is detected in samples containing the antibiotics clindamycin or cefoxitin, the sample is deemed to be resistant to these antibiotics, respectively, for bacteriophage can only amplify in a viable host. Thus, with a single work flow, S. aureus can be detected in an unknown sample and susceptibility to clindamycin and cefoxitin can be ascertained. This Article discusses implications for the use of bacteriophage amplification in the clinical laboratory.

  8. Human genome: proto-oncogenes and proretroviruses.

    Science.gov (United States)

    Kisselev, L L; Chumakov, I M; Zabarovsky, E R; Prassolov, V S; Mett, V L; Berditchevsky, F B; Tret'yakov, L D

    1985-01-01

    A brief review of the studies undertaken at the Laboratory for Molecular Bases of Oncogenesis (Institute of Molecular Biology, Moscow) till middle of 1984 is presented. The human genome contains multiple dispersed nucleotide sequences related to the proto-oncogene mos and to proretroviral sequences in tight juxtaposition to each other. From sequencing appropriate cloned fragments of human DNA in phage and plasmid vectors it follows that one of these regions, NV-1, is a pseudogene of proto-mos with partial duplications and two Alu elements intervening its coding sequence, and the other, CL-1, seems to be also a mos-related gene with a deletion of the internal part of the structural gene. CL-1 is flanked by a proretroviral-like sequence including tRNAiMet binding site and U5 (part of the long terminal repeat). The proretroviral-like sequences are transcribed in 21-35S poly(A)+RNA abundant in normal and malignant human cells. Two hypotheses are proposed: endogenous retroviruses take part in amplification of at least some proto-oncogenes; proto-oncogenes are inactivated via insertion of movable genetic elements and conversion into pseudogenes. Potential oncogenicity of a normal human genome undergoes two controversial influences: it increases due to proto-oncogene amplification and decreases due to inactivation of some of them.

  9. Copy number and loss of heterozygosity detected by SNP array of formalin-fixed tissues using whole-genome amplification.

    Science.gov (United States)

    Stokes, Angela; Drozdov, Ignat; Guerra, Eliete; Ouzounis, Christos A; Warnakulasuriya, Saman; Gleeson, Michael J; McGurk, Mark; Tavassoli, Mahvash; Odell, Edward W

    2011-01-01

    The requirement for large amounts of good quality DNA for whole-genome applications prohibits their use for small, laser capture micro-dissected (LCM), and/or rare clinical samples, which are also often formalin-fixed and paraffin-embedded (FFPE). Whole-genome amplification of DNA from these samples could, potentially, overcome these limitations. However, little is known about the artefacts introduced by amplification of FFPE-derived DNA with regard to genotyping, and subsequent copy number and loss of heterozygosity (LOH) analyses. Using a ligation adaptor amplification method, we present data from a total of 22 Affymetrix SNP 6.0 experiments, using matched paired amplified and non-amplified DNA from 10 LCM FFPE normal and dysplastic oral epithelial tissues, and an internal method control. An average of 76.5% of SNPs were called in both matched amplified and non-amplified DNA samples, and concordance was a promising 82.4%. Paired analysis for copy number, LOH, and both combined, showed that copy number changes were reduced in amplified DNA, but were 99.5% concordant when detected, amplifications were the changes most likely to be 'missed', only 30% of non-amplified LOH changes were identified in amplified pairs, and when copy number and LOH are combined ∼50% of gene changes detected in the unamplified DNA were also detected in the amplified DNA and within these changes, 86.5% were concordant for both copy number and LOH status. However, there are also changes introduced as ∼20% of changes in the amplified DNA are not detected in the non-amplified DNA. An integrative network biology approach revealed that changes in amplified DNA of dysplastic oral epithelium localize to topologically critical regions of the human protein-protein interaction network, suggesting their functional implication in the pathobiology of this disease. Taken together, our results support the use of amplification of FFPE-derived DNA, provided sufficient samples are used to increase power

  10. A sensitive SERS assay for detecting proteins and nucleic acids using a triple-helix molecular switch for cascade signal amplification.

    Science.gov (United States)

    Ye, Sujuan; Wu, Yanying; Zhang, Wen; Li, Na; Tang, Bo

    2014-08-25

    A novel surface-enhanced Raman scattering (SERS) detection system is developed for proteins and nucleic acids based on a triple-helix molecular switch for multiple cycle signal amplification, achieving high sensitivity, universality, rapid analysis, and high selectivity.

  11. The detection of circulating tumor cells expressing E6/E7 HR-HPV oncogenes in peripheral blood in cervical cancer patients after radical hysterectomy.

    Science.gov (United States)

    Weismann, P; Weismanova, E; Masak, L; Mlada, K; Keder, D; Ferancikova, Z; Vizvaryova, M; Konecny, M; Zavodna, K; Kausitz, J; Benuska, J; Repiska, V

    2009-01-01

    The aim of this study was to establish the sensitive, specific and clinically acceptable method for detection of tumor cells (TCs) circulating in peripheral blood (PB) of cervical cancer patients without the clinically detectable risk of disease progression. The 7.5 ml of PB of healthy donor was spiked with 5 to 100 cells from SiHa or HeLa cell lines. The spiked tumor cells were collected without gradient centrifugation, by standard gradient centrifugation or by modified gradient centrifugation combined with immunomagnetic separation using EpCAM antibody with affinity for epithelial cell adhesion molecule. The number of collected TCs was determined by EpCAM-FITC-staining and their viability was detected by nested RT-PCR amplifying E6/E7 HR-HPV 16 or HR-HPV 18 oncogenes. For the technical validation of this approach the TCs separation and RT-PCRs were repeated several times. The recovery of viable TCs was reproducibly higher using modified gradient centrifugation combined with immunomagnetic separation in comparison with standard approach. The recovery of TCs in low number of spiked TCs (range from 5 - 20 TCs in 7.5 ml of PB) using modified gradient centrifugation was not reproducible. The recovery of TCs in higher number of spiked TCs (25 TCs and more in 7.5 ml of PB) was reproducible with average recovery about 50 %. The sensitivity of nested RT-PCR amplifying E6/E7 oncogenes was decisively influenced by the number of recovered TCs and the amount of cDNA introduced to RT-PCR, as well. Using this approach we were allowed to detect circulating TCs (CTCs) in cervical cancer patients without metastases, thus this procedure might become a tool to early estimation of disease progression. According to our knowledge, this is the first report describing the use of EpCAM antibody for CTCs detection in cervical cancer patients.

  12. Novel Methodology for Rapid Detection of KRAS Mutation Using PNA-LNA Mediated Loop-Mediated Isothermal Amplification.

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    Masahiro Itonaga

    Full Text Available Detecting point mutation of human cancer cells quickly and accurately is gaining in importance for pathological diagnosis and choice of therapeutic approach. In the present study, we present novel methodology, peptide nucleic acid-locked nucleic acid mediated loop-mediated isothermal amplification (PNA-LNA mediated LAMP, for rapid detection of KRAS mutation using advantages of both artificial DNA and LAMP. PNA-LNA mediated LAMP reactions occurred under isothermal temperature conditions of with 4 primary primers set for the target regions on the KRAS gene, clamping PNA probe that was complimentary to the wild type sequence and LNA primers complementary to the mutated sequences. PNA-LNA mediated LAMP was applied for cDNA from 4 kinds of pancreatic carcinoma cell lines with or without KRAS point mutation. The amplified DNA products were verified by naked-eye as well as a real-time PCR equipment. By PNA-LNA mediated LAMP, amplification of wild type KRAS DNA was blocked by clamping PNA probe, whereas, mutant type KRAS DNA was significantly amplified within 50 min. Mutant alleles could be detected in samples which diluted until 0.1% of mutant-to-wild type ratio. On the other hand, mutant alleles could be reproducibly with a mutant-to-wild type ratio of 30% by direct sequencing and of 1% by PNA-clamping PCR. The limit of detection (LOD of PNA-LNA mediated LAMP was much lower than the other conventional methods. Competition of LNA clamping primers complementary to two different subtypes (G12D and G12V of mutant KRAS gene indicated different amplification time depend on subtypes of mutant cDNA. PNA-LNA mediated LAMP is a simple, rapid, specific and sensitive methodology for the detection of KRAS mutation.

  13. Rapid and specific detection of the thermostable direct hemolysin gene in Vibrio parahaemolyticus by loop-mediated isothermal amplification.

    Science.gov (United States)

    Nemoto, Jiro; Sugawara, Chiyo; Akahane, Kenji; Hashimoto, Keiji; Kojima, Tadashi; Ikedo, Masanari; Konuma, Hirotaka; Hara-Kudo, Yukiko

    2009-04-01

    Several investigators have reported that thermostable direct hemolysin (TDH) and TDH-related hemolysin are important virulence factors of Vibrio parahaemolyticus, but it has been difficult to detect these factors rapidly in seafood and other environmental samples. A novel nucleic acid amplification method, termed the loop-mediated isothermal amplification (LAMP), which amplifies DNA with high specificity and rapidity under isothermal conditions, was applied. In this study, we designed tdh gene-specific LAMP primers for detection of TDH-producing V. parahaemolyticus. The specificity of this assay was evaluated with 32 strains of TDH-producing V. parahaemolyticus, one strain of TDH-producing Grimontia hollisae, 10 strains of TDH-nonproducing V. parahaemolyticus, and 94 strains of TDH-nonproducing bacteria, and the sensitivity was high enough to detect one cell per test. Moreover, to investigate the detection of TDH-producing V. parahaemolyticus in oysters, the LAMP assay was performed with enrichment culture in alkaline peptone water of oyster samples inoculated with TDH-producing V. parahaemolyticus and TDH-nonproducing V. parahaemolyticus and V. alginolyticus after enrichment in alkaline peptone water. These results suggest that the LAMP assay targeting tdh gene has high sensitivity and specificity and is useful to detect TDH-producing V. parahaemolyticus in oyster after enrichment.

  14. Detection of shrimp Taura syndrome virus by loop-mediated isothermal amplification using a designed portable multi-channel turbidimeter.

    Science.gov (United States)

    Sappat, Assawapong; Jaroenram, Wansadaj; Puthawibool, Teeranart; Lomas, Tanom; Tuantranont, Adisorn; Kiatpathomchai, Wansika

    2011-08-01

    In this study, a portable turbidimetric end-point detection method was devised and tested for the detection of Taura syndrome virus (TSV) using spectroscopic measurement of a loop-mediated isothermal amplification (LAMP) by-product: magnesium pyrophosphate (Mg(2)P(2)O(7)). The device incorporated a heating block that maintained an optimal temperature of 63°C for the duration of the RT-LAMP reaction. Turbidity of the RT-LAMP by-product was measured when light from a light-emitting diode (LED) passed through the tube to reach a light dependent resistance (LDR) detector. Results revealed that turbidity measurement of the RT-LAMP reactions using this device provided the same detection sensitivity as the agarose gel electrophoresis detection of RT-LAMP and nested RT-PCR (IQ2000™) products. Cross reactions with other shrimp viruses were not found, indicating that the RT-LAMP-turbidity measurement was highly specific to TSV. The combination of 10 min for rapid RNA preparation with 30 min for RT-LAMP amplification followed by turbidity measurement resulted in a total assay time of less than 1h compared to 4-8h for the nested RT-PCR method. RT-LAMP plus turbidity measurement constitutes a platform for the development of more rapid and user-friendly detection of TSV in the field.

  15. Activation of proto-oncogenes by disruption of chromosome neighborhoods.

    Science.gov (United States)

    Hnisz, Denes; Weintraub, Abraham S; Day, Daniel S; Valton, Anne-Laure; Bak, Rasmus O; Li, Charles H; Goldmann, Johanna; Lajoie, Bryan R; Fan, Zi Peng; Sigova, Alla A; Reddy, Jessica; Borges-Rivera, Diego; Lee, Tong Ihn; Jaenisch, Rudolf; Porteus, Matthew H; Dekker, Job; Young, Richard A

    2016-03-25

    Oncogenes are activated through well-known chromosomal alterations such as gene fusion, translocation, and focal amplification. In light of recent evidence that the control of key genes depends on chromosome structures called insulated neighborhoods, we investigated whether proto-oncogenes occur within these structures and whether oncogene activation can occur via disruption of insulated neighborhood boundaries in cancer cells. We mapped insulated neighborhoods in T cell acute lymphoblastic leukemia (T-ALL) and found that tumor cell genomes contain recurrent microdeletions that eliminate the boundary sites of insulated neighborhoods containing prominent T-ALL proto-oncogenes. Perturbation of such boundaries in nonmalignant cells was sufficient to activate proto-oncogenes. Mutations affecting chromosome neighborhood boundaries were found in many types of cancer. Thus, oncogene activation can occur via genetic alterations that disrupt insulated neighborhoods in malignant cells.

  16. A one-step reverse transcription loop-mediated isothermal amplification for detection and discrimination of infectious bursal disease virus

    Directory of Open Access Journals (Sweden)

    Qi Xiaole

    2011-03-01

    Full Text Available Abstract Background Infectious bursal disease (IBD is a highly contagious immunosuppressive disease in young chickens caused by infectious bursal disease virus (IBDV. It causes huge economic losses to the poultry industry. The objective of this study is to develop a loop-mediated isothermal amplification (LAMP method for the detection and discrimination of IBDV. Results In this study, we applied reverse transcription loop-mediated isothermal amplification (RT-LAMP to detect IBDV in one simple step and further identified the very virulent strain from non-vvIBDVs with a simply post-amplification restriction enzyme analysis. Based on sequence analysis, a set of two inner, two outer and two loop primers were designed to target the VP5 gene and they showed great specificity with no cross reaction to the other common avian pathogens. The detection limit determined by both color change inspection and agarose gel electrophoresis was 28 copies viral RNA, which was almost as sensitive as a real-time RT-PCR previous developed in our laboratory. We also identified a unique Tfi I restriction site located exclusively in non-vvIBDVs, so very virulent strain could be distinguished from current vaccine strains. By screening a panel of clinical specimens, results showed that this method is high feasible in clinical settings, and it obtained results 100% correlated with real-time RT-PCR. Conclusion RT-LAMP is a rapid, simple and sensitive assay. In combination with the Tfi I restriction analysis, this method holds great promises not only in laboratory detection and discrimination of IBDV but also in large scale field and clinical studies.

  17. GMO detection using a bioluminescent real time reporter (BART of loop mediated isothermal amplification (LAMP suitable for field use

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    Kiddle Guy

    2012-04-01

    Full Text Available Abstract Background There is an increasing need for quantitative technologies suitable for molecular detection in a variety of settings for applications including food traceability and monitoring of genetically modified (GM crops and their products through the food processing chain. Conventional molecular diagnostics utilising real-time polymerase chain reaction (RT-PCR and fluorescence-based determination of amplification require temperature cycling and relatively complex optics. In contrast, isothermal amplification coupled to a bioluminescent output produced in real-time (BART occurs at a constant temperature and only requires a simple light detection and integration device. Results Loop mediated isothermal amplification (LAMP shows robustness to sample-derived inhibitors. Here we show the applicability of coupled LAMP and BART reactions (LAMP-BART for determination of genetically modified (GM maize target DNA at low levels of contamination (0.1-5.0% GM using certified reference material, and compare this to RT-PCR. Results show that conventional DNA extraction methods developed for PCR may not be optimal for LAMP-BART quantification. Additionally, we demonstrate that LAMP is more tolerant to plant sample-derived inhibitors, and show this can be exploited to develop rapid extraction techniques suitable for simple field-based qualitative tests for GM status determination. We also assess the effect of total DNA assay load on LAMP-BART quantitation. Conclusions LAMP-BART is an effective and sensitive technique for GM detection with significant potential for quantification even at low levels of contamination and in samples derived from crops such as maize with a large genome size. The resilience of LAMP-BART to acidic polysaccharides makes it well suited to rapid sample preparation techniques and hence to both high throughput laboratory settings and to portable GM detection applications. The impact of the plant sample matrix and genome loading

  18. GMO detection using a bioluminescent real time reporter (BART) of loop mediated isothermal amplification (LAMP) suitable for field use.

    Science.gov (United States)

    Kiddle, Guy; Hardinge, Patrick; Buttigieg, Neil; Gandelman, Olga; Pereira, Clint; McElgunn, Cathal J; Rizzoli, Manuela; Jackson, Rebecca; Appleton, Nigel; Moore, Cathy; Tisi, Laurence C; Murray, James A H

    2012-04-30

    There is an increasing need for quantitative technologies suitable for molecular detection in a variety of settings for applications including food traceability and monitoring of genetically modified (GM) crops and their products through the food processing chain. Conventional molecular diagnostics utilising real-time polymerase chain reaction (RT-PCR) and fluorescence-based determination of amplification require temperature cycling and relatively complex optics. In contrast, isothermal amplification coupled to a bioluminescent output produced in real-time (BART) occurs at a constant temperature and only requires a simple light detection and integration device. Loop mediated isothermal amplification (LAMP) shows robustness to sample-derived inhibitors. Here we show the applicability of coupled LAMP and BART reactions (LAMP-BART) for determination of genetically modified (GM) maize target DNA at low levels of contamination (0.1-5.0% GM) using certified reference material, and compare this to RT-PCR. Results show that conventional DNA extraction methods developed for PCR may not be optimal for LAMP-BART quantification. Additionally, we demonstrate that LAMP is more tolerant to plant sample-derived inhibitors, and show this can be exploited to develop rapid extraction techniques suitable for simple field-based qualitative tests for GM status determination. We also assess the effect of total DNA assay load on LAMP-BART quantitation. LAMP-BART is an effective and sensitive technique for GM detection with significant potential for quantification even at low levels of contamination and in samples derived from crops such as maize with a large genome size. The resilience of LAMP-BART to acidic polysaccharides makes it well suited to rapid sample preparation techniques and hence to both high throughput laboratory settings and to portable GM detection applications. The impact of the plant sample matrix and genome loading within a reaction must be controlled to ensure

  19. Rapid and sensitive detection of Yersinia pestis using amplification of plague diagnostic bacteriophages monitored by real-time PCR.

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    Kirill V Sergueev

    Full Text Available BACKGROUND: Yersinia pestis, the agent of plague, has caused many millions of human deaths and still poses a serious threat to global public health. Timely and reliable detection of such a dangerous pathogen is of critical importance. Lysis by specific bacteriophages remains an essential method of Y. pestis detection and plague diagnostics. METHODOLOGY/PRINCIPAL FINDINGS: The objective of this work was to develop an alternative to conventional phage lysis tests--a rapid and highly sensitive method of indirect detection of live Y. pestis cells based on quantitative real-time PCR (qPCR monitoring of amplification of reporter Y. pestis-specific bacteriophages. Plague diagnostic phages phiA1122 and L-413C were shown to be highly effective diagnostic tools for the detection and identification of Y. pestis by using qPCR with primers specific for phage DNA. The template DNA extraction step that usually precedes qPCR was omitted. phiA1122-specific qPCR enabled the detection of an initial bacterial concentration of 10(3 CFU/ml (equivalent to as few as one Y. pestis cell per 1-microl sample in four hours. L-413C-mediated detection of Y. pestis was less sensitive (up to 100 bacteria per sample but more specific, and thus we propose parallel qPCR for the two phages as a rapid and reliable method of Y. pestis identification. Importantly, phiA1122 propagated in simulated clinical blood specimens containing EDTA and its titer rise was detected by both a standard plating test and qPCR. CONCLUSIONS/SIGNIFICANCE: Thus, we developed a novel assay for detection and identification of Y. pestis using amplification of specific phages monitored by qPCR. The method is simple, rapid, highly sensitive, and specific and allows the detection of only live bacteria.

  20. Loop-mediated isothermal amplification assay for the detection of Ehrlichia canis DNA in blood samples from dogs

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    SA Faggion

    2013-01-01

    Full Text Available The rickettsial bacterium Ehrlichia canis is the etiological agent of canine monocytic ehrlichiosis, one of the most important canine tick-borne diseases in the world. In this study, a loop-mediated isothermal amplification (LAMP assay was developed for detection of E. canis DNA using LAMP primers targeting the groESL operon. Reactions were performed at 60°C for 60 min and the results were visualized by gel electrophoresis. Successful amplification was obtained using plasmid DNA containing a fragment of the groESL operon and DNA extracted from blood samples that tested positive for E. canis by real-time PCR. The specificity of amplification was confirmed by EcoRI restriction of internal sites in the LAMP primers and no cross-reactivity with blood samples positive for Babesia spp., another common tick-borne pathogen, was observed. The high cost of nucleic acid tests (NAT is one of the disadvantages for their large-scale use as routine diagnostic tests. The E. canis LAMP assay developed here is an interesting alternative to PCR since it does not require a thermocycler, thus reducing costs for the veterinary clinical laboratory.

  1. Development of multiple cross displacement amplification label-based gold nanoparticles lateral flow biosensor for detection of Listeria monocytogenes

    Science.gov (United States)

    Wang, Yi; Li, Hui; Wang, Yan; Li, Hua; Luo, Lijuan; Xu, Jianguo; Ye, Changyun

    2017-01-01

    Listeria monocytogenes, one of most problematic foodborne pathogens, is responsible for listeriosis in both humans and animals and mainly transmitted through the food chain. In this report, we propose a simple, rapid, and nearly instrument-free molecular technique using multiple cross displacement amplification (MCDA) label-based gold nanoparticles lateral flow biosensor (LFB) for specific, sensitive, and visual detection of L. monocytogenes. The MCDA-LFB method was carried out at a constant temperature (61°C) for only 20 min during the reaction stage, and then the amplification mixtures were directly detected by using LFB, eliminating the use of an electrophoresis instrument, special reagents, or amplicon analysis equipment. The whole procedure, from sample processing to result indicating, was finished within 1 h. The analytical specificity of MCDA-LFB method was successfully determined by distinguishing the target bacterium from other pathogens. The analytical sensitivity of the MCDA-LFB assay was 10 fg of genomic templates per reaction in pure culture, which was in complete accordance with MCDA by gel electrophoresis, real-time turbidity, and colorimetric indicator. The assay was also successfully applied to detecting L. monocytogenes in pork samples. Therefore, the rapidity, simplicity, and nearly equipment-free platform of the MCDA-LFB technique make it possible for food control, clinical diagnosis, and more. The proof-of-concept assay can be reconfigured to detect various target sequences by redesigning the specific MCDA primers. PMID:28138243

  2. Optimization of loop-mediated isothermal amplification (LAMP) assays for the detection of Leishmania DNA in human blood samples.

    Science.gov (United States)

    Abbasi, Ibrahim; Kirstein, Oscar D; Hailu, Asrat; Warburg, Alon

    2016-10-01

    Visceral leishmaniasis (VL), one of the most important neglected tropical diseases, is caused by Leishmania donovani eukaryotic protozoan parasite of the genus Leishmania, the disease is prevalent mainly in the Indian sub-continent, East Africa and Brazil. VL can be diagnosed by PCR amplifying ITS1 and/or kDNA genes. The current study involved the optimization of Loop-mediated isothermal amplification (LAMP) for the detection of Leishmania DNA in human blood or tissue samples. Three LAMP systems were developed; in two of those the primers were designed based on shared regions of the ITS1 gene among different Leishmania species, while the primers for the third LAMP system were derived from a newly identified repeated region in the Leishmania genome. The LAMP tests were shown to be sufficiently sensitive to detect 0.1pg of DNA from most Leishmania species. The green nucleic acid stain SYTO16, was used here for the first time to allow real-time monitoring of LAMP amplification. The advantage of real time-LAMP using SYTO 16 over end-point LAMP product detection is discussed. The efficacy of the real time-LAMP tests for detecting Leishmania DNA in dried blood samples from volunteers living in endemic areas, was compared with that of qRT-kDNA PCR.

  3. One-step reverse transcription loop-mediated isothermal amplification for the rapid detection of cucumber green mottle mosaic virus.

    Science.gov (United States)

    Li, Jin-yu; Wei, Qi-wei; Liu, Yong; Tan, Xin-qiu; Zhang, Wen-na; Wu, Jian-yan; Charimbu, Miriam Karwitha; Hu, Bai-shi; Cheng, Zhao-bang; Yu, Cui; Tao, Xiao-rong

    2013-11-01

    Cucumber green mottle mosaic virus (CGMMV) has caused serious damage to Cucurbitaceae crops worldwide. The virus is considered one of the most serious Cucurbitaceae quarantine causes in many countries. In this study, a highly efficient and practical one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) was developed for the detection of CGMMV. The total RNA or crude RNA extracted from watermelon plants or seeds could be detected easily by this RT-LAMP assay. The RT-LAMP assay was conducted in isothermal (63°C) conditions within 1h. The amplified products of CGMMV could be detected as ladder-like bands using agarose gel electrophoresis or visualized in-tube under UV light with the addition of a fluorescent dye. The RT-LAMP amplification was specific to CGMMV, as no cross-reaction was observed with other viruses. The RT-LAMP assay was 100-fold more sensitive than that of reverse-transcription polymerase chain reaction (RT-PCR). This is the first report of the application of the RT-LAMP assay to detect CGMMV. The sensitive, specific and rapid RT-LAMP assay developed in this study can be applied widely in laboratories, the field and quarantine surveillance of CGMMV.

  4. Evaluation of Loop-Mediated Isothermal Amplification Assay for the Detection of Pneumocystis jirovecii in Immunocompromised Patients

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    Preeti Singh

    2015-01-01

    Full Text Available Pneumocystis pneumonia (PCP is one of the common opportunistic infection among HIV and non-HIV immunocompromised patients. The lack of a rapid and specific diagnostic test necessitates a more reliable laboratory diagnostic test for PCP. In the present study, the loop-mediated isothermal amplification (LAMP assay was evaluated for the detection of Pneumocystis jirovecii. 185 clinical respiratory samples, including both BALF and IS, were subjected to GMS staining, nested PCR, and LAMP assay. Of 185 respiratory samples, 12/185 (6.5%, 41/185 (22.2%, and 49/185 (26.5% samples were positive by GMS staining, nested PCR, and LAMP assay, respectively. As compared to nested PCR, additional 8 samples were positive by LAMP assay and found to be statistically significant (p<0.05 with the detection limit of 1 pg. Thus, the LAMP assay may serve as a better diagnostic tool for the detection of P. jirovecii with high sensitivity and specificity, less turn-around time, operational simplicity, single-step amplification, and immediate visual detection.

  5. Development and evaluation of a loop-mediated isothermal amplification assay for rapid detection of lymphocystis disease virus.

    Science.gov (United States)

    Li, Qiong; Yue, Zhiqin; Liu, Hong; Liang, Chengzhu; Zheng, Xiaolong; Zhao, Yuran; Chen, Xiao; Xiao, Xizhi; Chen, Changfu

    2010-02-01

    A loop-mediated isothermal amplification (LAMP) assay was developed for the detection of lymphocystis disease virus (LCDV). A set of five specific primers, two inner and two outer primers and a loop primer, were designed on the basis of the major capsid protein gene of LCDV. The reaction time and temperatures were optimized for 60 min at 63 degrees C, respectively. LAMP amplification products were detected by a ladder-like appearance on agarose gel electrophoresis or a naked-eye inspection of a color change in the reaction tube by addition of SYBR Green I. The assay was specific for LCDV, and there was no cross-reactivity with white spot syndrome virus (WSSV) or six other Iridoviridae viruses (epizootic hematopoietic necrosis virus, EHNV; tiger frog virus, TFV; Bohle iridovirus, BIV; soft-shelled turtle iridovirus, STIV; infectious spleen and kidney necrosis virus, ISKNV; red sea bream iridovirus, RSIV). The detection limit of the LAMP assay was 15 fg, which was similar to that of real-time quantitative polymerase chain reaction (PCR) and 10-fold higher than the conventional PCR. The LAMP assay was evaluated using 109 clinical samples, and the results indicated the suitability and simplicity of the test as a rapid, field diagnostic tool for detection of LCDV. The LCDV LAMP assay has potential for early diagnosis of LCDV infection.

  6. Development and evaluation of a novel and rapid detection assay for Botrytis cinerea based on loop-mediated isothermal amplification.

    Science.gov (United States)

    Duan, Ya-Bing; Ge, Chang-Yan; Zhang, Xiao-Ke; Wang, Jian-Xin; Zhou, Ming-Guo

    2014-01-01

    Botrytis cinerea is a devastating plant pathogen that causes grey mould disease. In this study, we developed a visual detection method of B. cinerea based on the Bcos5 sequence using loop-mediated isothermal amplification (LAMP) with hydroxynaphthol blue dye (HNB). The LAMP reaction was optimal at 63 °C for 45 min. When HNB was added prior to amplification, samples with B. cinerea DNA developed a characteristic sky blue color after the reaction but those without DNA or with DNA of other plant pathogenic fungi did not. Results of HNB staining method were reconfirmed when LAMP products were subjected to gel electrophoresis. The detection limit of this LAMP assay for B. cinerea was 10(-3) ng µL(-1) of genomic DNA per reaction, which was 10-fold more sensitive than conventional PCR (10(-2) ng µL(-1)). Detection of the LAMP assay for inoculum of B. cinerea was possible in the inoculated tomato and strawberry petals. In the 191 diseased samples, 180 (94.2%) were confirmed as positive by LAMP, 172 (90.1%) positive by the tissue separation, while 147 (77.0%) positive by PCR. Because the LAMP assay performed well in aspects of sensitivity, specificity, repeatability, reliability, and visibility, it is suitable for rapid detection of B. cinerea in infected plant materials prior to storage and during transportation, such as cut flowers, fruits and vegetables.

  7. A microfluidic platform for transcription- and amplification-free detection of zepto-mole amounts of nucleic acid molecules.

    Science.gov (United States)

    Mayr, Reinhard; Haider, Michaela; Thünauer, Roland; Haselgrübler, Thomas; Schütz, Gerhard J; Sonnleitner, Alois; Hesse, Jan

    2016-04-15

    Here we report the development of a device for the transcription- and amplification-free detection of DNA and RNA molecules down to the zepto-mole range. A microfluidic chip with a built-in microarray was used for manipulation of nano-liter sample volumes. Specific staining and immobilization of the target molecules was achieved via a double hybridization approach thereby avoiding bias due to enzymatic processes like reverse transcription and PCR amplification. Therefore, target molecules were indirectly labeled by pre-hybridization to complementary Cy5-labeled probes. The remaining single-stranded portion of each target molecule could subsequently hybridize to complementary capture probes of a microarray. Thus a target-mediated immobilization of labeled DNA took place. By means of an ultra-sensitive fluorescence readout, all molecules hybridized to the microarray could be detected. The combination of minimized sample volume and single molecule detection yielded a detection limit of 39 fM (831 molecules in 35.4 nl assay volume) for target DNA and 16 fM (338 molecules) for target RNA after 1h on-chip hybridization.

  8. Development and evaluation of a novel and rapid detection assay for Botrytis cinerea based on loop-mediated isothermal amplification.

    Directory of Open Access Journals (Sweden)

    Ya-Bing Duan

    Full Text Available Botrytis cinerea is a devastating plant pathogen that causes grey mould disease. In this study, we developed a visual detection method of B. cinerea based on the Bcos5 sequence using loop-mediated isothermal amplification (LAMP with hydroxynaphthol blue dye (HNB. The LAMP reaction was optimal at 63 °C for 45 min. When HNB was added prior to amplification, samples with B. cinerea DNA developed a characteristic sky blue color after the reaction but those without DNA or with DNA of other plant pathogenic fungi did not. Results of HNB staining method were reconfirmed when LAMP products were subjected to gel electrophoresis. The detection limit of this LAMP assay for B. cinerea was 10(-3 ng µL(-1 of genomic DNA per reaction, which was 10-fold more sensitive than conventional PCR (10(-2 ng µL(-1. Detection of the LAMP assay for inoculum of B. cinerea was possible in the inoculated tomato and strawberry petals. In the 191 diseased samples, 180 (94.2% were confirmed as positive by LAMP, 172 (90.1% positive by the tissue separation, while 147 (77.0% positive by PCR. Because the LAMP assay performed well in aspects of sensitivity, specificity, repeatability, reliability, and visibility, it is suitable for rapid detection of B. cinerea in infected plant materials prior to storage and during transportation, such as cut flowers, fruits and vegetables.

  9. Signal amplification in electrochemical detection of buckwheat allergenic protein using field effect transistor biosensor by introduction of anionic surfactant

    Directory of Open Access Journals (Sweden)

    Sho Hideshima

    2016-03-01

    Full Text Available Food allergens, especially buckwheat proteins, sometimes induce anaphylactic shock in patients after ingestion. Development of a simple and rapid screening method based on a field effect transistor (FET biosensor for food allergens in food facilities or products is in demand. In this study, we achieved the FET detection of a buckwheat allergenic protein (BWp16, which is not charged enough to be electrically detected by FET biosensors, by introducing additional negative charges from anionic surfactants to the target proteins. A change in the FET characteristics reflecting surface potential caused by the adsorption of target charged proteins was observed when the target sample was coupled with the anionic surfactant (sodium dodecyl sulfate; SDS, while no significant response was detected without any surfactant treatment. It was suggested that the surfactant conjugated with the protein could be useful for the charge amplification of the target proteins. The surface plasmon resonance analysis revealed that the SDS-coupled proteins were successfully captured by the receptors immobilized on the sensing surface. Additionally, we obtained the FET responses at various concentrations of BWp16 ranging from 1 ng/mL to 10 μg/mL. These results suggest that a signal amplification method for FET biosensing is useful for allergen detection in the food industry.

  10. Rapid Molecular Detection of Zika Virus in Acute-Phase Urine Samples Using the Recombinase Polymerase Amplification Assay

    Science.gov (United States)

    Abd El Wahed, Ahmed; Sanabani, Sabri Saeed; Faye, Oumar; Pessôa, Rodrigo; Patriota, João Veras; Giorgi, Ricardo Rodrigues; Patel, Pranav; Böhlken-Fascher, Susanne; Landt, Olfert; Niedrig, Matthias; Zanotto, Paolo Marinho de Andrade; Czerny, Claus Peter; Sall, Amadou A.; Weidmann, Manfred

    2017-01-01

    Background: Currently the detection of Zika virus (ZIKV) in patient samples is done by real-time RT-PCR. Samples collected from rural area are sent to highly equipped laboratories for screening. A rapid point-of-care test is needed to detect the virus, especially at low resource settings. Methodology/Principal Findings: In this report, we describe the development of a reverse transcription isothermal recombinase polymerase amplification (RT-RPA) assay for the identification of ZIKV. RT-RPA assay was portable, sensitive (21 RNA molecules), and rapid (3-15 minutes). No cross-reactivity was detected to other flaviviruses, alphaviruses and arboviruses. Compared to real-time RT-PCR, the diagnostic sensitivity was 92%, while the specificity was 100%. Conclusions/Significance: The developed assay is a promising platform for rapid point of need detection of ZIKV in low resource settings and elsewhere (e.g. during mass gathering). PMID:28239513

  11. Development of a Rapid and Sensitive Method for Detection of African Swine Fever Virus Using Loop-Mediated Isothermal Amplification

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    Xulong Wu

    Full Text Available ABSTRACT A loop-mediated isothermal amplification (LAMP assay was developed for rapid, sensitive and specific detection of African swine fever virus (ASFV. A set of LAMP primers was designed based on the sequence of the ASFV gene K205R. Reaction temperature and time were optimized to 64 oC and 60 min, respectively. LAMP products were detected by agarose gel electrophoresis or visually with the addition of fluorescent dye. The detection limit of the LAMP assay was approximately 6 copies of the target gene per microliter, 100 times more sensitive than conventional PCR. LAMP is a simple and inexpensive molecular assay format for ASFV detection. To date, African swine fever has not been reported in China. LAMP can be used to monitor ASFV spread into China, thereby reducing the threat of ASF.

  12. Comparison of conventional PCR, multiplex PCR, and loop-mediated isothermal amplification assays for rapid detection of Arcobacter species.

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    Wang, Xiaoyu; Seo, Dong Joo; Lee, Min Hwa; Choi, Changsun

    2014-02-01

    This study aimed to develop a loop-mediated isothermal amplification (LAMP) method for the rapid detection of Arcobacter species. Specific primers targeting the 23S ribosomal RNA gene were used to detect Arcobacter butzleri, Arcobacter cryaerophilus, and Arcobacter skirrowii. The specificity of the LAMP primer set was assessed using DNA samples from a panel of Arcobacter and Campylobacter species, and the sensitivity was determined using serial dilutions of Arcobacter species cultures. LAMP showed a 10- to 1,000-fold-higher sensitivity than multiplex PCR, with a detection limit of 2 to 20 CFU per reaction in vitro. Whereas multiplex PCR showed cross-reactivity with Campylobacter species, the LAMP method developed in this study was more sensitive and reliable than conventional PCR or multiplex PCR for the detection of Arcobacter species.

  13. Novel real-time simultaneous amplification and testing method to accurately and rapidly detect Mycobacterium tuberculosis complex.

    Science.gov (United States)

    Cui, Zhenling; Wang, Yongzhong; Fang, Liang; Zheng, Ruijuan; Huang, Xiaochen; Liu, Xiaoqin; Zhang, Gang; Rui, Dongmei; Ju, Jinliang; Hu, Zhongyi

    2012-03-01

    The aim of this study was to establish and evaluate a simultaneous amplification and testing method for detection of the Mycobacterium tuberculosis complex (SAT-TB assay) in clinical specimens by using isothermal RNA amplification and real-time fluorescence detection. In the SAT-TB assay, a 170-bp M. tuberculosis 16S rRNA fragment is reverse transcribed to DNA by use of Moloney murine leukemia virus (M-MLV) reverse transcriptase, using specific primers incorporating the T7 promoter sequence, and undergoes successive cycles of amplification using T7 RNA polymerase. Using a real-time PCR instrument, hybridization of an internal 6-carboxyfluorescein-4-[4-(dimethylamino)phenylazo] benzoic acid N-succinimidyl ester (FAM-DABCYL)-labeled fluorescent probe can be used to detect RNA amplification. The SAT-TB assay takes less than 1.5 h to perform, and the sensitivity of the assay for detection of M. tuberculosis H37Rv is 100 CFU/ml. The TB probe has no cross-reactivity with nontuberculous mycobacteria or other common respiratory tract pathogens. For 253 pulmonary tuberculosis (PTB) specimens and 134 non-TB specimens, the SAT-TB results correlated with 95.6% (370/387 specimens) of the Bactec MGIT 960 culture assay results. The sensitivity, specificity, and positive and negative predictive values of the SAT-TB test for the diagnosis of PTB were 67.6%, 100%, 100%, and 62.0%, respectively, compared to 61.7%, 100%, 100%, and 58.0% for Bactec MGIT 960 culture. For PTB diagnosis, the sensitivities of the SAT-TB and Bactec MGIT 960 culture methods were 97.6% and 95.9%, respectively, for smear-positive specimens and 39.2% and 30.2%, respectively, for smear-negative specimens. In conclusion, the SAT-TB assay is a novel, simple test with a high specificity which may enhance the detection rate of TB. It is therefore a promising tool for rapid diagnosis of M. tuberculosis infection in clinical microbiology laboratories.

  14. Genome-wide copy number profiling to detect gene amplifications in neural progenitor cells

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    U. Fischer

    2014-12-01

    Full Text Available DNA sequence amplification occurs at defined stages during normal development in amphibians and flies and seems to be restricted in humans to drug-resistant and tumor cells only. We used array-CGH to discover copy number changes including gene amplifications and deletions during differentiation of human neural progenitor cells. Here, we describe cell culture features, DNA extraction, and comparative genomic hybridization (CGH analysis tailored towards the identification of genomic copy number changes. Further detailed analysis of amplified chromosome regions associated with this experiment, was published by Fischer and colleagues in PLOS One in 2012 (Fischer et al., 2012. We provide detailed information on deleted chromosome regions during differentiation and give an overview on copy number changes during differentiation induction for two representative chromosome regions.

  15. Recombinase polymerase and enzyme-linked immunosorbent assay as a DNA amplification-detection strategy for food analysis

    Energy Technology Data Exchange (ETDEWEB)

    Santiago-Felipe, S.; Tortajada-Genaro, L.A.; Puchades, R.; Maquieira, A., E-mail: amaquieira@qim.upv.es

    2014-02-06

    Graphical abstract: -- Highlights: •Recombinase polymerase amplification is a powerful DNA method operating at 40 °C. •The combination RPA–ELISA gives excellent performances for high-throughput analysis. •Screening of food safety threats has been done using standard laboratory equipment. •Allergens, GMOs, bacteria, and fungi have been successfully determined. -- Abstract: Polymerase chain reaction in conjunction with enzyme-linked immunosorbent assay (PCR–ELISA) is a well-established technique that provides a suitable rapid, sensitive, and selective method for a broad range of applications. However, the need for precise rapid temperature cycling of PCR is an important drawback that can be overcome by employing isothermal amplification reactions such as recombinase polymerase amplification (RPA). The RPA–ELISA combination is proposed for amplification at a low, constant temperature (40 °C) in a short time (40 min), for the hybridisation of labelled products to specific 5′-biotinylated probes/streptavidin in coated microtiter plates at room temperature, and for detection by colorimetric immunoassay. RPA–ELISA was applied to screen common safety threats in foodstuffs, such as allergens (hazelnut, peanut, soybean, tomato, and maize), genetically modified organisms (P35S and TNOS), pathogenic bacteria (Salmonella sp. and Cronobacter sp.), and fungi (Fusarium sp.). Satisfactory sensitivity and reproducibility results were achieved for all the targets. The RPA–ELISA technique does away with thermocycling and provides a suitable sensitive, specific, and cost-effective method for routine applications, and proves particularly useful for resource-limited settings.

  16. Rapid detection of microbial DNA by a novel isothermal genome exponential amplification reaction (GEAR) assay.

    Science.gov (United States)

    Prithiviraj, Jothikumar; Hill, Vincent; Jothikumar, Narayanan

    2012-04-20

    In this study we report the development of a simple target-specific isothermal nucleic acid amplification technique, termed genome exponential amplification reaction (GEAR). Escherichia coli was selected as the microbial target to demonstrate the GEAR technique as a proof of concept. The GEAR technique uses a set of four primers; in the present study these primers targeted 5 regions on the 16S rRNA gene of E. coli. The outer forward and reverse Tab primer sequences are complementary to each other at their 5' end, whereas their 3' end sequences are complementary to their respective target nucleic acid sequences. The GEAR assay was performed at a constant temperature 60 °C and monitored continuously in a real-time PCR instrument in the presence of an intercalating dye (SYTO 9). The GEAR assay enabled amplification of as few as one colony forming units of E. coli per reaction within 30 min. We also evaluated the GEAR assay for rapid identification of bacterial colonies cultured on agar media directly in the reaction without DNA extraction. Cells from E. coli colonies were picked and added directly to GEAR assay mastermix without prior DNA extraction. DNA in the cells could be amplified, yielding positive results within 15 min.

  17. G-quadruplex based Exo III-assisted signal amplification aptasensor for the colorimetric detection of adenosine.

    Science.gov (United States)

    Xu, Lei; Shen, Xin; Li, Bingzhi; Zhu, Chunhong; Zhou, Xuemin

    2017-08-08

    Adenosine is an endogenous nucleotide pivotally involved in nucleic acid and energy metabolism. Its excessive existence may indicate tumorigenesis, typically lung cancer. Encouraged by its significance as the clinical biomarker, sensitive assay methods towards adenosine have been popularized, with high cost and tedious procedures as the inevitable defects. Herein, we report a label-free aptamer-based exonuclease III (Exo III) amplification colorimetric aptasensor for the highly sensitive and cost-effective detection of adenosine. The strategy employed two unlabeled hairpin DNA oligonucleotides (HP1 and HP2), where HP1 contained the aptamer towards adenosine and HP2 embedded the guanine-rich sequence (GRS). In the presence of adenosine, hairpin HP1 could form specific binding with adenosine and trigger the unfolding of HP1's hairpin structure. The resulting adenosine-HP1 complex could hybridize with HP2, generating the Exo III recognition site. After Exo III-assisted degradation, the GRS was released from HP2, and the adenosine-HP1 was released back to the solution to combine another HP2, inducing the cycling amplification. After multiple circulations, the released ample GRSs were induced to form G-quadruplex, further catalyzing the oxidation of TMB, yielding a color change which was finally mirrored in the absorbance change. On the contrary, the absence of adenosine failed to unfold HP1, remaining color unchanged eventually. Thanks to the amplification strategy, the limit of detection was lowered to 17 nM with a broad linear range from 50 nM to 6 μM. The proposed method was successfully applied to the detection of adenosine in biological samples and satisfying recoveries were acquired. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Rapid detection of newly isolated Tembusu-related Flavivirus by reverse-transcription loop-mediated isothermal amplification assay

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    Wang Youling

    2011-12-01

    Full Text Available Abstract Background From April 2010 to January 2011, a severe new viral disease had devastated most duck-farming regions in China. This disease affected not only laying ducks but also meat ducks, causing huge economic losses for the poultry industry. The objective of this study is to develop a one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP assay for the detection of the new virus related to Tembusu-related Flavivirus. Results The RT-LAMP assay is very simple and rapid, and the amplification can be completed within 50 min under isothermal conditions at 63°C by a set of 6 primers targeting the E gene based on the sequences analysis of the newly isolated viruses and other closely related Flavivirus.The monitoring of gene amplification can also be visualized by using SYBR green I fluorescent dye. In addition, the RT-LAMP assay for newly isolated Tembusu-related Flavivirus showed higher sensitivity with an RNA detection-limit of 2 copies/μL compared with 190 copies/μL of the conventional RT-PCR method. The specificity was identified without cross reaction to other common avian pathogens. By screening a panel of clinical samples this method was more feasible in clinical settings and there was higher positive coincidence rate than conventional RT-PCR and virus isolation. Conclusion The RT-LAMP assay for newly isolated Tembusu-related Flavivirus is a valuable tool for the rapid and real-time detection not only in well-equipped laboratories but also in general conditions.

  19. Method to detect the end-point for PCR DNA amplification using an ionically labeled probe and measuring impedance change

    Science.gov (United States)

    Miles, Robin R.; Belgrader, Phillip; Fuller, Christopher D.

    2007-01-02

    Impedance measurements are used to detect the end-point for PCR DNA amplification. A pair of spaced electrodes are located on a surface of a microfluidic channel and an AC or DC voltage is applied across the electrodes to produce an electric field. An ionically labeled probe will attach to a complementary DNA segment, and a polymerase enzyme will release the ionic label. This causes the conductivity of the solution in the area of the electrode to change. This change in conductivity is measured as a change in the impedance been the two electrodes.

  20. Development of a loop-mediated Isothermal amplification assay for sensitive and rapid detection of Vibrio parahaemolyticus

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    Kawahara Ryuji

    2008-09-01

    Full Text Available Abstract Background Vibrio parahaemolyticus is a marine seafood-borne pathogen causing gastrointestinal disorders in humans. Thermostable direct hemolysin (TDH and TDH-related hemolysin (TRH are known as major virulence determinants of V. parahaemolyticus. Most V. parahaemolyticus isolates from the environment do not produce TDH or TRH. Total V. parahaemolyticus has been used as an indicator for control of seafood contamination toward prevention of infection. Detection of total V. parahaemolyticus using conventional culture- and biochemical-based assays is time-consuming and laborious, requiring more than three days. Thus, we developed a novel and highly specific loop-mediated isothermal amplification (LAMP assay for the sensitive and rapid detection of Vibrio parahaemolyticus. Results The assay provided markedly more sensitive and rapid detection of V. parahaemolyticus strains than conventional biochemical and PCR assays. The assay correctly identified 143 V. parahaemolyticus strains, but did not detect 33 non-parahaemolyticus Vibrio and 56 non-Vibrio strains. Sensitivity of the LAMP assay for direct detection of V. parahaemolyticus in pure cultures and in spiked shrimp samples was 5.3 × 102 CFU per ml/g (2.0 CFU per reaction. The sensitivity of the LAMP assay was 10-fold more sensitive than that of the conventional PCR assay. The LAMP assay was markedly faster, requiring for amplification 13–22 min in a single colony on TCBS agar from each of 143 V. parahaemolyticus strains and less than 35 min in spiked shrimp samples. The LAMP assay for detection of V. parahaemolyticus required less than 40 min in a single colony on thiosulfate citrate bile salt sucrose (TCBS agar and 60 min in spiked shrimp samples from the beginning of DNA extraction to final determination. Conclusion The LAMP assay is a sensitive, rapid and simple tool for the detection of V. parahaemolyticus and will facilitate the surveillance for control of contamination of V

  1. Specific, sensitive and rapid detection of human plasmodium knowlesi infection by loop-mediated isothermal amplification (LAMP in blood samples

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    Anthony Claudia N

    2011-07-01

    Full Text Available Abstract Background The emergence of Plasmodium knowlesi in humans, which is in many cases misdiagnosed by microscopy as Plasmodium malariae due to the morphological similarity has contributed to the needs of detection and differentiation of malaria parasites. At present, nested PCR targeted on Plasmodium ssrRNA genes has been described as the most sensitive and specific method for Plasmodium detection. However, this method is costly and requires trained personnel for its implementation. Loop-mediated isothermal amplification (LAMP, a novel nucleic acid amplification method was developed for the clinical detection of P. knowlesi. The sensitivity and specificity of LAMP was evaluated in comparison to the results obtained via microscopic examination and nested PCR. Methods LAMP assay was developed based on P. knowlesi genetic material targeting the apical membrane antigen-1 (AMA-1 gene. The method uses six primers that recognize eight regions of the target DNA and it amplifies DNA within an hour under isothermal conditions (65°C in a water-bath. Results LAMP is highly sensitive with the detection limit as low as ten copies for AMA-1. LAMP detected malaria parasites in all confirm cases (n = 13 of P. knowlesi infection (sensitivity, 100% and none of the negative samples (specificity, 100% within an hour. LAMP demonstrated higher sensitivity compared to nested PCR by successfully detecting a sample with very low parasitaemia ( Conclusion With continuous efforts in the optimization of this assay, LAMP may provide a simple and reliable test for detecting P. knowlesi malaria parasites in areas where malaria is prevalent.

  2. Shewanella putrefaciens in cultured tilapia detected by a new calcein-loop-mediated isothermal amplification (Ca-LAMP) method.

    Science.gov (United States)

    Suebsing, Rungkarn; Kampeera, Jantana; Sirithammajak, Sarawut; Pradeep, Padmaja Jayaprasad; Jitrakorn, Sarocha; Arunrut, Narong; Sangsuriya, Pakkakul; Saksmerprome, Vanvimon; Senapin, Saengchan; Withyachumnarnkul, Boonsirm; Kiatpathomchai, Wansika

    2015-12-09

    Shewanella putrefaciens is being increasingly isolated from a wide variety of sources and is pathogenic to many marine and freshwater fish. For better control of this pathogen, there is a need for the development of simple and inexpensive but highly specific, sensitive, and rapid detection methods suitable for application in field laboratories. Our colorogenic loop-mediated isothermal amplification (LAMP) assay combined with calcein (Ca-LAMP) for unaided visual confirmation of LAMP amplicons is a simple method for fish pathogen detection in cultured tilapia. Here, we describe the detection of S. putrefaciens using the same platform. As before, the method gave positive results (orange to green color change) in 45 min at 63°C with sensitivity 100 times higher than that of a conventional PCR assay, with no cross-amplification of other known fish bacterial pathogens tested. Using the assay with 389 samples of gonads, fertilized eggs, and fry of farmed Nile and red tilapia Oreochromis spp., 35% of samples were positive for S. putrefaciens. The highest prevalence was found in samples of gonads (55%) and fertilized eggs (55%) from adult breeding stocks, indicating that S. putrefaciens could be passed on easily to fry used for stocking production ponds. Tissue tropism assays revealed that the spleen showed the highest colonization by S. putrefaciens in naturally infected tilapia and that it would be the most suitable organ for screening and monitoring fish stocks for presence of the bacteria.

  3. An electrochemical biosensor for sensitive detection of microRNA-155: combining target recycling with cascade catalysis for signal amplification.

    Science.gov (United States)

    Wu, Xiaoyan; Chai, Yaqin; Zhang, Pu; Yuan, Ruo

    2015-01-14

    In this work, a new electrochemical biosensor based on catalyzed hairpin assembly target recycling and cascade electrocatalysis (cytochrome c (Cyt c) and alcohol oxidase (AOx)) for signal amplification was constructed for highly sensitive detection of microRNA (miRNA). It is worth pointing out that target recycling was achieved only based on strand displacement process without the help of nuclease. Moreover, porous TiO2 nanosphere was synthesized, which could offer more surface area for Pt nanoparticles (PtNPs) enwrapping and enhance the amount of immobilized DNA strand 1 (S1) and Cyt c accordingly. With the mimicking sandwich-type reaction, the cascade catalysis amplification strategy was carried out by AOx catalyzing ethanol to acetaldehyde with the concomitant formation of high concentration of H2O2, which was further electrocatalyzed by PtNPs and Cyt c. This newly designed biosensor provided a sensitive detection of miRNA-155 from 0.8 fM to 1 nM with a relatively low detection limit of 0.35 fM.

  4. Highly sensitive detection of small ruminant bovine spongiform encephalopathy within transmissible spongiform encephalopathy mixes by serial protein misfolding cyclic amplification.

    Science.gov (United States)

    Gough, Kevin C; Bishop, Keith; Maddison, Ben C

    2014-11-01

    It is assumed that sheep and goats consumed the same bovine spongiform encephalopathy (BSE)-contaminated meat and bone meal that was fed to cattle and precipitated the BSE epidemic in the United Kingdom that peaked more than 20 years ago. Despite intensive surveillance for cases of BSE within the small ruminant populations of the United Kingdom and European Union, no instances of BSE have been detected in sheep, and in only two instances has BSE been discovered in goats. If BSE is present within the small ruminant populations, it may be at subclinical levels, may manifest as scrapie, or may be masked by coinfection with scrapie. To determine whether BSE is potentially circulating at low levels within the European small ruminant populations, highly sensitive assays that can specifically detect BSE, even within the presence of scrapie prion protein, are required. Here, we present a novel assay based on the specific amplification of BSE PrP(Sc) using the serial protein misfolding cyclic amplification assay (sPMCA), which specifically amplified small amounts of ovine and caprine BSE agent which had been mixed into a range of scrapie-positive brain homogenates. We detected the BSE prion protein within a large excess of classical, atypical, and CH1641 scrapie isolates. In a blind trial, this sPMCA-based assay specifically amplified BSE PrP(Sc) within brain mixes with 100% specificity and 97% sensitivity when BSE agent was diluted into scrapie-infected brain homogenates at 1% (vol/vol).

  5. A Portable Automatic Endpoint Detection System for Amplicons of Loop Mediated Isothermal Amplification on Microfluidic Compact Disk Platform

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    Shah Mukim Uddin

    2015-03-01

    Full Text Available In recent years, many improvements have been made in foodborne pathogen detection methods to reduce the impact of food contamination. Several rapid methods have been developed with biosensor devices to improve the way of performing pathogen detection. This paper presents an automated endpoint detection system for amplicons generated by loop mediated isothermal amplification (LAMP on a microfluidic compact disk platform. The developed detection system utilizes a monochromatic ultraviolet (UV emitter for excitation of fluorescent labeled LAMP amplicons and a color sensor to detect the emitted florescence from target. Then it processes the sensor output and displays the detection results on liquid crystal display (LCD. The sensitivity test has been performed with detection limit up to 2.5 × 10−3 ng/µL with different DNA concentrations of Salmonella bacteria. This system allows a rapid and automatic endpoint detection which could lead to the development of a point-of-care diagnosis device for foodborne pathogens detection in a resource-limited environment.

  6. Sensitive and rapid detection of the insect pathogenic fungus Metarhizium anisopliae var. anisopliae by loop-mediated isothermal amplification.

    Science.gov (United States)

    Li, Ya; Cai, Shuang-Hu

    2011-05-01

    A set of six specific primers was designed by targeting intergenic spacer region (IGS) sequences. With Bst DNA polymerase, the products could be clearly amplified for 60 min at 62 °C in a simple water bath. The sensitivity of the loop-mediated isothermal amplification (LAMP) for detecting Metarhizium anisopliae var. anisopliae was about 0.01 pg fungal DNA per reaction (equivalent to 27 conidia). LAMP products could be judged with agar gel or naked eye after addition of SYBR Green I. There were no cross reactions with other fungal isolates indicating high specificity of the LAMP. The LAMP could detect the presence of M. anisopliae var. anisopliae from soil. The detection limits for M. anisopliae var. anisopliae of LAMP reaction was 50 conidia per reaction in soil.

  7. Antibody-Conjugated Rubpy Dye-Doped Silica Nanoparticles as Signal Amplification for Microscopic Detection of Vibrio cholerae O1

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    Nualrahong Thepwiwatjit

    2013-01-01

    Full Text Available This study demonstrated the potential application of antibody-conjugated Rubpy dye-doped silica nanoparticles for immunofluorescence microscopic detection of Vibrio cholerae O1. The particle synthesis of 20X of the original ratio was accomplished yielding spherical nanoparticles with an average size of 45±3 nm. The nanoparticles were carboxyl functionalized and then conjugated with either monoclonal antibody or polyclonal antibody against V. cholerae O1. The antibody-conjugated nanoparticles were tested with two target bacteria and three challenge strains. The result showed that monoclonal antibody-conjugated Rubpy dye-doped silica nanoparticles could be effectively used as signal amplification to detect V. cholerae O1 under a fluorescence microscope. Their extremely strong fluorescence signal also enables the detection of a single cell bacterium.

  8. Loop-Mediated Isothermal Amplification Assay Targeting the MOMP Gene for Rapid Detection of Chlamydia psittaci Abortus Strain

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    Guo-Zhen Lin, Fu-Ying Zheng, Ji-Zhang Zhou, Guang-Hua Wang, Xiao-An Cao, Xiao-Wei Gong and Chang-Qing Qiu*

    2012-05-01

    Full Text Available For rapid detection of the Chlamydia psittaci abortus strain, a loop-mediated isothermal amplification (LAMP assay was developed and evaluated in this study. The primers for the LAMP assay were designed on the basis of the main outer membrane protein (MOMP gene sequence of C. psittaci. Analysis showed that the assay could detect the abortus strain of C. psittaci with adequate specificity. The sensitivity of the test was the same as that of the nested-conventional PCR and higher than that of chick embryo isolation. Testing of 153 samples indicated that the LAMP assay could detect the genome of the C. psittaci abortus strain effectively in clinical samples. This assay is a useful tool for rapid diagnosis of C. psittaci infection in sheep, swine and cattle.

  9. Development of a loop-mediated isothermal amplification method for rapid detection of streptococcal pyrogenic exotoxin B.

    Science.gov (United States)

    Cao, Cuiming; Zhang, Fang; Ji, Mingyu; Pei, Fengyan; Fan, Xiujie; Shen, Hong; Wang, Qingxi; Yang, Weihua; Wang, Yunshan

    2016-07-01

    We developed a visual loop-mediated isothermal amplification (LAMP) technique to detect the streptococcal pyrogenic exotoxin B (speB) gene. Fifteen strains (from American Type Culture Collection or clinical isolates) were used to determine the specificity and sensitivity of the LAMP assay. Clinical samples were collected from 132 patients with suspected Streptococcus pyogenes (S. pyogenes) infection to verify the feasibility of the LAMP assay for detection of the speB gene. By using a set of five primers (a pair of outer primers, a pair of inner primers and one loop primer) targeting the speB gene, the amplification reaction was rapidly performed in a regular water bath under isothermal conditions at 63 °C for approximately 60 min. Only the two S. pyogenes strains showed positive results which were easily observed with the naked eye, and the other strains showed negative results. The detection limit of the LAMP assay was 0.01 ng/μl of template, showing higher sensitivity than conventional PCR (with a detection limit of 1.0 ng/μl). The detection rate of the speB gene in clinical samples was 71.21% and was consistent with the PCR results. The rapid detection of the speB gene by the LAMP assay is highly specific and sensitive, is simple to perform and cost-effective, and is expected to be a new reliable method for the rapid diagnosis of S. pyogenes infection, that is particularly suitable for rural or community hospitals in developing countries.

  10. Genomic amplification patterns of human telomerase RNA gene and C-MYC in liquid-based cytological specimens used for the detection of high-grade cervical intraepithelial neoplasia

    Science.gov (United States)

    2012-01-01

    Background The amplification of oncogenes initiated by high-risk human papillomavirus (HPV) infection is an early event in cervical carcinogenesis and can be used for cervical lesion diagnosis. We measured the genomic amplification rates and the patterns of human telomerase RNA gene (TERC) and C-MYC in the liquid-based cytological specimens to evaluate the diagnostic characteristics for the detection of high-grade cervical lesions. Methods Two hundred and forty-three residual cytological specimens were obtained from outpatients aged 25 to 64 years at Qilu Hospital, Shandong University. The specimens were evaluated by fluorescence in situ hybridization (FISH) using chromosome probes to TERC (3q26) and C-MYC (8q24). All of the patients underwent colposcopic examination and histological evaluation. A Chi-square test was used for categorical data analysis. Results In the normal, cervical intraepithelial neoplasia grade 1 (CIN1), grade 2 (CIN2), grade 3 (CIN3) and squamous cervical cancer (SCC) cases, the TERC positive rates were 9.2%, 17.2%, 76.2%, 100.0% and 100.0%, respectively; the C-MYC positive rates were 20.7%, 31.0%, 71.4%, 81.8% and 100.0%, respectively. The TERC and C-MYC positive rates were higher in the CIN2+ (CIN2, CIN3 and SCC) cases than in the normal and CIN1 cases (p C-MYC test showed lower sensitivity (80.0% vs. 84.0%) and higher specificity (77.7% vs. 64.3%). Using a cut-off value of 5% or more aberrant cells, the TERC test showed the highest combination of sensitivity and specificity. The CIN2+ group showed more high-level TERC gene copy number (GCN) cells than did the normal/CIN1 group (p C-MYC, no significant difference between the two histological categories was detected (p > 0.05). Conclusions The TERC test is highly sensitive and is therefore suitable for cervical cancer screening. The C-MYC test is not suitable for cancer screening because of its lower sensitivity. The amplification patterns of TERC become more diverse and complex as the

  11. Gene amplification in carcinogenesis

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    Lucimari Bizari

    2006-01-01

    Full Text Available Gene amplification increases the number of genes in a genome and can give rise to karyotype abnormalities called double minutes (DM and homogeneously staining regions (HSR, both of which have been widely observed in human tumors but are also known to play a major role during embryonic development due to the fact that they are responsible for the programmed increase of gene expression. The etiology of gene amplification during carcinogenesis is not yet completely understood but can be considered a result of genetic instability. Gene amplification leads to an increase in protein expression and provides a selective advantage during cell growth. Oncogenes such as CCND1, c-MET, c-MYC, ERBB2, EGFR and MDM2 are amplified in human tumors and can be associated with increased expression of their respective proteins or not. In general, gene amplification is associated with more aggressive tumors, metastases, resistance to chemotherapy and a decrease in the period during which the patient stays free of the disease. This review discusses the major role of gene amplification in the progression of carcinomas, formation of genetic markers and as possible therapeutic targets for the development of drugs for the treatment of some types of tumors.

  12. Adaptation of a visualized loop-mediated isothermal amplification technique for field detection of Plasmodium vivax infection

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    Wang Wei-Ming

    2011-06-01

    Full Text Available Abstract Background Loop-mediated isothermal amplification (LAMP is a high performance method for detecting DNA and holds promise for use in the molecular detection of infectious pathogens, including Plasmodium spp. However, in most malaria-endemic areas, which are often resource-limited, current LAMP methods are not feasible for diagnosis due to difficulties in accurately interpreting results with problems of sensitive visualization of amplified products, and the risk of contamination resulting from the high quantity of amplified DNA produced. In this study, we establish a novel visualized LAMP method in a closed-tube system, and validate it for the diagnosis of malaria under simulated field conditions. Methods A visualized LAMP method was established by the addition of a microcrystalline wax-dye capsule containing the highly sensitive DNA fluorescence dye SYBR Green I to a normal LAMP reaction prior to the initiation of the reaction. A total of 89 blood samples were collected on filter paper and processed using a simple boiling method for DNA extraction, and then tested by the visualized LAMP method for Plasmodium vivax infection. Results The wax capsule remained intact during isothermal amplification, and released the DNA dye to the reaction mixture only when the temperature was raised to the melting point following amplification. Soon after cooling down, the solidified wax sealed the reaction mix at the bottom of the tube, thus minimizing the risk of aerosol contamination. Compared to microscopy, the sensitivity and specificity of LAMP were 98.3% (95% confidence interval (CI: 91.1-99.7% and 100% (95% CI: 88.3-100%, and were in close agreement with a nested polymerase chain reaction method. Conclusions This novel, cheap and quick visualized LAMP method is feasible for malaria diagnosis in resource-limited field settings.

  13. Loop-mediated Isothermal Amplification Assay to Rapidly Detect Wheat Streak Mosaic Virus in Quarantined Plants

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    Siwon Lee

    2015-12-01

    Full Text Available We developed a loop-mediated isothermal amplification (LAMP method to rapidly diagnose Wheat streak mosaic virus (WSMV during quarantine inspections of imported wheat, corn, oats, and millet. The LAMP method was developed as a plant quarantine inspection method for the first time, and its simplicity, quickness, specificity and sensitivity were verified compared to current reverse transcription-polymerase chain reaction (RT-PCR and nested PCR quarantine methods. We were able to quickly screen for WSMV at quarantine sites with many test samples; thus, this method is expected to contribute to plant quarantine inspections.

  14. Information Limited Oligonucleotide Amplification Assay for Affinity-Based, Parallel Detection Studies.

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    Harish Bokkasam

    Full Text Available Molecular communication systems encounter similar constraints as telecommunications. In either case, channel crosstalk at the receiver end will result in information loss that statistical analysis cannot compensate. This is because in any communication channel there is a physical limit to the amount of information that can be transmitted. We present a novel and simple modified end amplification (MEA technique to generate reduced and defined amounts of specific information in form of short fragments from an oligonucleotide source that also contains unrelated and redundant information. Our method can be a valuable tool to investigate information overflow and channel capacity in biomolecular recognition systems.

  15. Rapid detection of nusG and fadA in Fusobacterium nucleatum by loop-mediated isothermal amplification.

    Science.gov (United States)

    Huang, Simo; Yang, Zhan; Zou, Dayang; Dong, Derong; Liu, Anheng; Liu, Wei; Huang, Liuyu

    2016-08-01

    Fusobacterium nucleatum is associated with various human diseases such as periodontal disease and colorectal cancer (CRC); thus, F. nucleatum detection might serve as a novel diagnostic tool. Here, we describe the development of a sensitive and rapid molecular method for detecting two F. nucleatum genes: the highly conserved nusG and fadA, which encode a critical host colonization factor. Loop-mediated isothermal amplification (LAMP) primer sets for the rapid detection of nusG and fadA were designed and optimized. The nusG primers yielded consistent negative results for 20 non-F. nucleatum bacterial strains, confirming the high specificity of the primers. LAMP reaction primer sensitivity was determined, and its detection rate in comparison to conventional PCR was assessed using 57 clinical stool samples. The LAMP detection limit for nusG and fadA was 22.5 and 0.225 pg µl-1, respectively, indicating that the sensitivity of this method was 10-fold higher than that of conventional PCR. These results suggest that the LAMP technique is able to effectively identify F. nucleatum via nusG as well as detect its virulence factor. To the best of our knowledge, this study is the first to report the application of LAMP for the detection of nusG and fadA in F. nucleatum. The LAMP method constitutes a sensitive and specific visual assay for the rapid detection of the pathogen F. nucleatum.

  16. Multiple displacement amplification as an adjunct to PCR-based detection of Staphylococcus aureus in synovial fluid

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    Johnson Sandra

    2010-10-01

    Full Text Available Abstract Background Detection of bacterial nucleic acids in synovial fluid following total joint arthroplasty with suspected infection can be difficult; among other technical challenges, inhibitors in the specimens require extensive sample preparation and can diminish assay sensitivity even using polymerase chain reaction (PCR-based methods. To address this problem a simple protocol for prior use of multiple displacement amplification (MDA as an adjunct to PCR was established and tested on both purified S. aureus DNA as well as on clinical samples known to contain S. aureus nucleic acids. Findings A single round of MDA on purified nucleic acids resulted in a > 300 thousand-fold increase in template DNA on subsequent quantitative PCR (qPCR analysis. MDA use on clinical samples resulted in at least a 100-fold increase in sensitivity on subsequent qPCR and required no sample preparation other than a simple alkali/heat lysis step. Mixed samples of S. aureus DNA with a 103 - 104-fold excess of human genomic DNA still allowed for MDA amplification of the minor bacterial component to the threshold of detectability. Conclusion MDA is a promising technique that may serve to significantly enhance the sensitivity of molecular assays in cases of suspected joint infection while simultaneously reducing the specimen handling required.

  17. Development of a loop-mediated isothermal amplification assay for the detection of Streptococcus agalactiae in bovine milk.

    Science.gov (United States)

    Bosward, Katrina L; House, John K; Deveridge, Amber; Mathews, Karen; Sheehy, Paul A

    2016-03-01

    Streptococcus agalactiae is a well-characterized bovine mastitis pathogen that is known to be highly contagious and capable of spreading rapidly in affected dairy herds. Loop-mediated isothermal amplification (LAMP) is a novel molecular diagnostic method that has the capability to provide rapid, cost-effective screening for pathogens to support on-farm disease control and eradication programs. In the current study, a LAMP test was developed to detect S. agalactiae in milk. The assay was validated on a bank of existing clinical mastitis milk samples that had previously been identified as S. agalactiae positive via traditional microbiological culture techniques and PCR. The LAMP assay was conducted on bacterial colonies and DNA extracted from milk in tube- and plate-based formats using multiple detection platforms. The 1-h assay conducted at 64 °C exhibited repeatability (coefficient of variation) of 2.07% (tube) and 8.3% (plate), sensitivity to ~20 pg of extracted DNA/reaction, and specificity against a panel of known bacterial mastitis pathogens. Of the 109 known S. agalactiae isolates assessed by LAMP directly from bacterial cells in culture, 108 were identified as positive, in accordance with PCR analysis. The LAMP analysis from the corresponding milk samples indicated that 104 of these milks exhibited a positive amplification curve. Although exhibiting some limitations, this assay provides an opportunity for rapid screening of milk samples to facilitate on-farm management of this pathogen.

  18. Clinical significance of fluorescence in situ hybridization for detection of hTERC gene amplification in cervical cancer and precancerous tissues cases

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    Shuang LIU

    2012-06-01

    Full Text Available Objective  To detect the human telomerase RNA gene (hTERC amplification in cervical lesions, and explore its clinical significance. Methods  The tissues of the cervical lesions were collected from 195 patients, including 33 of chronic cervicitis, 34 of CINⅠ, 37 of CIN Ⅱ-Ⅲ, 30 of cervical squamous cell carcinoma, and 61 of cervica1 adenocarcinoma, and abnormal hTERC was detected with amplification of fluorescence in situhybridization (FISH. The relationship between hTERC gene amplification and clinicopathological parameters was analyzed. Results  Among the 195 patients, the positive rate of hTERC gene amplification was 3.03% (1/33, 29.41% (10/34, 72.97% (27/37, 100% (30/30, 91.8% (56/61 in chronic cervicitis, CINⅠ, CIN Ⅱ-Ⅲ, cervical squamous cell carcinoma and cervica1 adenocarcinoma respectively, and the results showed that hTERC amplification rate was significantly higher in group CIN Ⅱ-Ⅲthan in group CINⅠ(P 0.05. Conclusion  Detection of gene amplification by FISH technology can be used as a means for accurate diagnosis and prediction of the histologically difficult-to-diagnose lesion and for risk assessment after treatment of cervical precancerous lesions.

  19. Development of a loop-mediated isothermal amplification assay for rapid, sensitive and specific detection of a Campylobacter jejuni clone.

    Science.gov (United States)

    Luo, Yan; Sahin, Orhan; Dai, Lei; Sippy, Rachel; Wu, Zuowei; Zhang, Qijing

    2012-05-01

    Loop-mediated isothermal amplification (LAMP) assay is a simple, rapid and specific detection method and has been used for detection and identification of different Campylobacter species. In this study, we develop a LAMP assay specific for detection of a particular clone (clone SA) of Campylobacter jejuni, associated with the vast majority of recent sheep abortions in the U.S. Using a set of specific primers for C. jejuni IA3902 (a clone SA isolate) and genomic DNA or boiled cell extract as template, the target DNA was amplified at 63 °C for 50 min in a water bath. A positive reaction was identified visually as white precipitate or fluorescence under UV, and confirmed by gel electrophoresis. Detection limit of the assay was comparable to that of conventional PCR. The LAMP was shown to be specific for detection of clone SA when tested on a number of C. jejuni strains of different genetic backgrounds. Applicability of the LAMP assay for specific detection of clone SA was demonstrated in animal tissues experimentally infected with IA3902 or genetically diverse C. jejuni strains. Since clone SA is the predominant cause of sheep abortions in the U.S. and is a zoonotic pathogen, the LAMP assay may be a valuable detection tool in future epidemiological studies.

  20. Application of a molecular beacon based real-time isothermal amplification (MBRTIA) technology for simultaneous detection of Bacillus cereus and Staphylococcus aureus.

    Science.gov (United States)

    Mandappa, I M; Joglekar, Prasanna; Manonmani, H K

    2015-07-01

    A multiplex real-time isothermal amplification assay was developed using molecular beacons for the detection of Bacillus cereus and Staphylococcus aureus by targeting four important virulence genes. A correlation between targeting highly accessible DNA sequences and isothermal amplification based molecular beacon efficiency and sensitivity was demonstrated using phi(Φ)29 DNA polymerase at a constant isothermal temperature of 30 °C. It was very selective and consistently detected down to 10(1) copies of DNA. The specificity and sensitivity of this assay, when tested with pure culture were high, surpassing those of currently used PCR assays for the detection of these organisms. The molecular beacon based real-time isothermal amplification (MBRTIA) assay could be carried out entirely in 96 well plates or well strips, enabling a rapid and high-throughput detection of food borne pathogens.

  1. An isothermal, label-free, and rapid one-step RNA amplification/detection assay for diagnosis of respiratory viral infections.

    Science.gov (United States)

    Koo, Bonhan; Jin, Choong Eun; Lee, Tae Yoon; Lee, Jeong Hoon; Park, Mi Kyoung; Sung, Heungsup; Park, Se Yoon; Lee, Hyun Jung; Kim, Sun Mi; Kim, Ji Yeun; Kim, Sung-Han; Shin, Yong

    2017-04-15

    Recently, RNA viral infections caused by respiratory viruses, such as influenza, parainfluenza, respiratory syncytial virus, coronavirus, and Middle East respiratory syndrome-coronavirus (MERS-CoV), and Zika virus, are a major public health threats in the world. Although myriads of diagnostic methods based on RNA amplification have been developed in the last decades, they continue to lack speed, sensitivity, and specificity for clinical use. A rapid and accurate diagnostic method is needed for appropriate control, including isolation and treatment of the patients. Here, we report an isothermal, label-free, one-step RNA amplification and detection system, termed as iROAD, for the diagnosis of respiratory diseases. It couples a one-step isothermal RNA amplification method and a bio-optical sensor for simultaneous viral RNA amplification/detection in a label-free and real-time manner. The iROAD assay offers a one-step viral RNA amplification/detection example to rapid analysis (<20min). The detection limit of iROAD assay was found to be 10-times more sensitive than that of real-time reverse transcription-PCR method. We confirmed the clinical utility of the iROAD assay by detecting viral RNAs obtained from 63 human respiratory samples. We envision that the iROAD assay will be useful and potentially adaptable for better diagnosis of emerging infectious diseases including respiratory diseases.

  2. Development of multiple cross displacement amplification label-based gold nanoparticles lateral flow biosensor for detection of Listeria monocytogenes

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    Wang Y

    2017-01-01

    Full Text Available Yi Wang,1 Hui Li,1,2 Yan Wang,1 Hua Li,1 Lijuan Luo,1 Jianguo Xu,1 Changyun Ye1 1State Key Laboratory of Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Chinese Center for Disease Control and Prevention, Changping, Beijing, 2Department of Microbiology, GuiZhou Medical University, Guiyang, Guizhou, People’s Republic of China Abstract: Listeria monocytogenes, one of most problematic foodborne pathogens, is responsible for listeriosis in both humans and animals and mainly transmitted through the food chain. In this report, we propose a simple, rapid, and nearly instrument-free molecular technique using multiple cross displacement amplification (MCDA label-based gold nanoparticles lateral flow biosensor (LFB for specific, sensitive, and visual detection of L. monocytogenes. The MCDA-LFB method was carried out at a constant temperature (61°C for only 20 min during the reaction stage, and then the amplification mixtures were directly detected by using LFB, eliminating the use of an electrophoresis instrument, special reagents, or amplicon analysis equipment. The whole procedure, from sample processing to result indicating, was finished within 1 h. The analytical specificity of MCDA-LFB method was successfully determined by distinguishing the target bacterium from other pathogens. The analytical sensitivity of the MCDA-LFB assay was 10 fg of genomic templates per reaction in pure culture, which was in complete accordance with MCDA by gel electrophoresis, real-time turbidity, and colorimetric indicator. The assay was also successfully applied to detecting L. monocytogenes in pork samples. Therefore, the rapidity, simplicity, and nearly equipment-free platform of the MCDA-LFB technique make it possible for food control, clinical diagnosis, and more. The proof-of-concept assay can be reconfigured to detect

  3. Hairpin DNA Switch for Ultrasensitive Spectrophotometric Detection of DNA Hybridization Based on Gold Nanoparticles and Enzyme Signal Amplification

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Youyu; Tang, Zhiwen; Wang, Jun; Wu, Hong; Maham, Aihui; Lin, Yuehe

    2010-08-01

    A novel DNA detection platform based on a hairpin-DNA switch, nanoparticles, and enzyme signal amplification for ultrasensitive detection of DNA hybridization has been developed in this work. In this DNA assay, a “stem-loop” DNA probe dually labeled with a thiol at its 5’ end and a biotin at its 3’ end, respectively, was used. This probe was immobilized on the gold nanoparticles (AuNPs) anchored by a protein, globulin, on a 96-well microplate. In the absence of target DNA, the immobilized probe with the stem-loop structure shields the biotin from being approached by a bulky horseradish peroxidase linked-avidin (avidin-HRP) conjugate due to the steric hindrance. However, in the presence of target DNA, the hybridization between the hairpin DNA probe and the target DNA causes significant conformational change of the probe, which forces biotin away from the surface of AuNPs. As a result, the biotin becomes accessible by the avidin-HRP, and the target hybridization event can be sensitively detected via the HRP catalyzed substrate 3, 3', 5, 5'-tetramethylbenzidine using spectrophometric method. Some experimental parameters governing the performance of the assay have been optimized. At optimal conditions, this DNA assay can detect DNA at the concentration of femtomolar level by means of a signal amplification strategy based on the combination of enzymes and nanoparticles. This approach also has shown excellent specificity to distinguish single-base mismatches of DNA targets because of the intrinsic high selectivity of the hairpin DNA probe.

  4. Multiple oncogenic mutations related to targeted therapy in nasopharyngeal carcinoma

    Institute of Scientific and Technical Information of China (English)

    Jian-Wei Zhang; Hong-Yuan Zhao; Yu-Xiang Ma; Zhi-Huang Hu; Pei-Yu Huang; Li Zhang; Tao Qin; Shao-Dong Hong; Jing Zhang; Wen-Feng Fang; Yuan-Yuan Zhao; Yun-Peng Yang; Cong Xue; Yan Huang

    2015-01-01

    Introduction:An increasing number of targeted drugs have been tested for the treatment of nasopharyngeal carcinoma (NPC). However, targeted therapy-related oncogenic mutations have not been fully evaluated. This study aimed to detect targeted therapy-related oncogenic mutations in NPC and to determine which targeted therapy might be potentially effective in treating NPC. Methods:By using the SNaPshot assay, a rapid detection method, 19 mutation hotspots in 6 targeted therapy-related oncogenes were examined in 70 NPC patients. The associations between oncogenic mutations and clinicopathologic factors were analyzed. Results:Among 70 patients, 12 (17.1%) had mutations in 5 oncogenes:7 (10.0%) had v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (KIT) mutation, 2 (2.8%) had epidermal growth factor receptor (EGFR) mutation, 1 (1.4%) had phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) mutation, 1 (1.4%) had Kirsten rat sarcoma viral oncogene homolog (KRAS) mutation, and 1 (1.4%) had simultaneous EGFR and v-Raf murine sarcoma viral oncogene homolog B1 (BRAF) mutations. No significant differences were observed between oncogenic mutations and clinicopathologic characteristics. Additionally, these oncogenic mutations were not associated with tumor recurrence and metastasis. Conclusions:Oncogenic mutations are present in NPC patients. The efficacy of targeted drugs on patients with the related oncogenic mutations requires further validation.

  5. Loop Mediated Isothermal Amplification for Detection of Trypanosoma brucei gambiense in Urine and Saliva Samples in Nonhuman Primate Model

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    Maina Ngotho

    2015-01-01

    Full Text Available Human African trypanosomiasis (HAT is a vector-borne parasitic zoonotic disease. The disease caused by Trypanosoma brucei gambiense is the most prevalent in Africa. Early diagnosis is hampered by lack of sensitive diagnostic techniques. This study explored the potential of loop mediated isothermal amplification (LAMP and polymerase chain reaction (PCR in the detection of T. b. gambiense infection in a vervet monkey HAT model. Six vervet monkeys were experimentally infected with T. b. gambiense IL3253 and monitored for 180 days after infection. Parasitaemia was scored daily. Blood, cerebrospinal fluid (CSF, saliva, and urine samples were collected weekly. PCR and LAMP were performed on serum, CSF, saliva, and urine samples. The detection by LAMP was significantly higher than that of parasitological methods and PCR in all the samples. The performance of LAMP varied between the samples and was better in serum followed by saliva and then urine samples. In the saliva samples, LAMP had 100% detection between 21 and 77 dpi, whereas in urine the detection it was slightly lower, but there was over 80% detection between 28 and 91 dpi. However, LAMP could not detect trypanosomes in either saliva or urine after 140 and 126 dpi, respectively. The findings of this study emphasize the importance of LAMP in diagnosis of HAT using saliva and urine samples.

  6. Loop Mediated Isothermal Amplification for Detection of Trypanosoma brucei gambiense in Urine and Saliva Samples in Nonhuman Primate Model.

    Science.gov (United States)

    Ngotho, Maina; Kagira, John Maina; Gachie, Beatrice Muthoni; Karanja, Simon Muturi; Waema, Maxwell Wambua; Maranga, Dawn Nyawira; Maina, Naomi Wangari

    2015-01-01

    Human African trypanosomiasis (HAT) is a vector-borne parasitic zoonotic disease. The disease caused by Trypanosoma brucei gambiense is the most prevalent in Africa. Early diagnosis is hampered by lack of sensitive diagnostic techniques. This study explored the potential of loop mediated isothermal amplification (LAMP) and polymerase chain reaction (PCR) in the detection of T. b. gambiense infection in a vervet monkey HAT model. Six vervet monkeys were experimentally infected with T. b. gambiense IL3253 and monitored for 180 days after infection. Parasitaemia was scored daily. Blood, cerebrospinal fluid (CSF), saliva, and urine samples were collected weekly. PCR and LAMP were performed on serum, CSF, saliva, and urine samples. The detection by LAMP was significantly higher than that of parasitological methods and PCR in all the samples. The performance of LAMP varied between the samples and was better in serum followed by saliva and then urine samples. In the saliva samples, LAMP had 100% detection between 21 and 77 dpi, whereas in urine the detection it was slightly lower, but there was over 80% detection between 28 and 91 dpi. However, LAMP could not detect trypanosomes in either saliva or urine after 140 and 126 dpi, respectively. The findings of this study emphasize the importance of LAMP in diagnosis of HAT using saliva and urine samples.

  7. Novel multifunction-integrated molecular beacon for the amplification detection of DNA hybridization based on primer/template-free isothermal polymerization.

    Science.gov (United States)

    Dong, Haiyan; Wu, Zai-Sheng; Xu, Jianguo; Ma, Ji; Zhang, Huijuan; Wang, Jie; Shen, Weiyu; Xie, Jingjing; Jia, Lee

    2015-10-15

    Molecular beacon (MB) is widely explored as a signaling probe in powerful biosensing systems, for example, enzyme-assisted strand displacement amplification (SDA)-based system. The existing polymerization-based amplification system is often composed of recognition element, primer, template and fluorescence reporter. To develop a new MB sensing system and simply the signal amplification design, we herein attempted to propose a multifunctional integrated MB (MI-MB) for the polymerization amplification detection of target DNA via introducing a G-rich fragment into the loop of MB without using any exogenous auxiliary oligonucleotide probe. Utilizing only one MI-MB probe, the p53 target gene could trigger the cycles of hybridization/polymerization/displacement, resulting in amplification of the target hybridization event. Thus, the p53 gene can be detected down to 5 × 10(-10)M with the linear response range from 5 × 10(-10)M to 4 × 10(-7)M. Using the MI-MB, we could readily discriminate the point mutation-contained p53 from the wild-type one. As a proof-of-concept study, owing to its simplicity and multifunction, including recognition, replication, amplification and signaling, the MI-MB exhibits the great potential for the development of different biosensors for various biomedical applications, especially, for early cancer diagnosis.

  8. Rapid detection of Salmonella in raw chicken breast using real-time PCR combined with immunomagnetic separation and whole genome amplification.

    Science.gov (United States)

    Hyeon, Ji-Yeon; Deng, Xiangyu

    2017-05-01

    We presented the first attempt to combine immunomagnetic separation (IMS), whole genome amplification by multiple displacement amplification (MDA) and real-time PCR for detecting a bacterial pathogen in a food sample. This method was effective in enabling real-time PCR detection of low levels of Salmonella enterica Serotype Enteritidis (SE) (∼10 CFU/g) in raw chicken breast without culture enrichment. In addition, it was able to detect refrigeration-stressed SE cells at lower concentrations (∼0.1 CFU/g) in raw chicken breast after a 4-h culture enrichment, shortening the detection process from days to hours and displaying no statistical difference in detection rate in comparison with a culture-based detection method. By substantially improving performance in SE detection over conventional real-time PCR, we demonstrated the potential of IMS-MDA real-time PCR as a rapid, sensitive and affordable method for detecting Salmonella in food.

  9. Development of a rapid assay to detect the jellyfish Cyanea nozakii using a loop-mediated isothermal amplification method.

    Science.gov (United States)

    Liu, Zhongyuan; Dong, Zhijun; Liu, Dongyan

    2016-07-01

    Blooms of the harmful jellyfish Cyanea nozakii, which are a severe nuisance to fisheries and tourisms, frequently occur in the northern East China Sea, Yellow Sea, and Bohai Sea. To provide early warning of this species, a simple and effective molecular method for identifying C. nozakii was developed using the loop-mediated isothermal amplification method (LAMP). The LAMP assay is highly specific and uses a set of four primers that target six different regions on the mitochondrial cytochrome c oxidase subunit I (COI) gene of C. nozakii. The amplification conditions, including the dNTP and betaine concentrations, the inner primer to outer primer concentration ratio, reaction time and temperature, were optimized. The LAMP assay amplified DNA extracted from tissue samples of C. nozakii but did not amplify DNA from other common scyphozoans and hydrozoans collected in the same region. In addition, the LAMP assay was more sensitive than conventional PCR. Therefore, the established LAMP assay is a sensitive, specific, fast, and easily performed method for detection of C. nozakii at different stages in their life cycle.

  10. Tuning backbones and side-chains of cationic conjugated polymers for optical signal amplification of fluorescent DNA detection.

    Science.gov (United States)

    Huang, Yan-Qin; Liu, Xing-Fen; Fan, Qu-Li; Wang, Lihua; Song, Shiping; Wang, Lian-Hui; Fan, Chunhai; Huang, Wei

    2009-06-15

    Three cationic conjugated polymers (CCPs) exhibiting different backbone geometries and charge densities were used to investigate how their conjugated backbone and side chain properties, together with the transitions of DNA amphiphilic properties, interplay in the CCP/DNA-C* (DNA-C*: fluorophore-labeled DNA) complexes to influence the optical signal amplification of fluorescent DNA detection based on Förster resonance energy transfer (FRET). By examining the FRET efficiencies to dsDNA-C* (dsDNA: double-stranded DNA) and ssDNA-C* (ssDNA: single-stranded DNA) for each CCP, twisted conjugated backbones and higher charge densities were proved to facilitate electrostatic attraction in CCP/dsDNA-C* complexes, and induced improved sensitivity to DNA hybridization. Especially, by using the CCP with twisted conjugated backbone and the highest charge density, a more than 7-fold higher efficiency of FRET to dsDNA-C* was found than to ssDNA-C*, indicating a high signal amplification for discriminating between dsDNA and ssDNA. By contrast, linear conjugated backbones and lower charge density were demonstrated to favor hydrophobic interactions in CCP/ssDNA-C* complexes. These findings provided guidelines for the design of novel sensitive CCP, which can be useful to recognize many other important DNA activities involving transitions of DNA amphiphilic properties like DNA hybridization, such as specific DNA binding with ions, some secondary or tertiary structural changes of DNA, and so forth.

  11. Integrated biochip for label-free and real-time detection of DNA amplification by contactless impedance measurements based on interdigitated electrodes.

    Science.gov (United States)

    Fang, Xinxin; Jin, Qinghui; Jing, Fengxiang; Zhang, Huanqian; Zhang, Feng; Mao, Hongju; Xu, Baojian; Zhao, Jianlong

    2013-06-15

    Here, we introduce an integrated biochip which offers accurate thermal control and sensitive electrochemical detection of DNA amplification in real-time. The biochip includes a 10-μl microchamber, a temperature sensor, a heater, and a contactless impedance biosensor. A pair of interdigitated electrodes is employed as the impedance biosensor and the products of the amplification are determined directly through tracing the impedance change, without using any labels, redox indicators, or probes. Real-time monitoring of strand-displacement amplification and rolling circle amplification was successfully performed on the biochip and a detection limit of 1 nM was achieved. Amplification starting at an initial concentration of 10 nM could be discriminated from that starting at 1 nM started concentration as well as from the negative control. Since an insulation layer covers the electrodes, the electrodes are spared from erosion, hydrolysis and bubble formation on the surface, thus, ensuring a long lifetime and a high reusability of the sensor. In comparison to bench-top apparatus, our chip shows good efficiency, sensitivity, accuracy, and versatility. Our system requires only simple equipments and simple skills, and can easily be miniaturized into a micro-scale system. The system will then be suitable for a handheld portable device, which can be applied in remote areas. It covers merits such as low cost, low-power consumption, rapid response, real-time monitoring, label-free detection, and high-throughput detection.

  12. Design and application of a loop-mediated isothermal amplification assay for the rapid detection of Staphylococcus pseudintermedius.

    Science.gov (United States)

    Diribe, Onyinye; North, Sarah; Sawyer, Jason; Roberts, Lisa; Fitzpatrick, Noel; La Ragione, Roberto

    2014-01-01

    Staphylococcus pseudintermedius is a commensal and opportunistic pathogen of dogs. It is mainly implicated in canine pyoderma, as well as other suppurative conditions of dogs. Although bacterial culture is routinely used for clinical diagnosis, molecular methods are required to accurately identify and differentiate S. pseudintermedius from other members of the Staphylococcus intermedius group. These methods, owing largely to their cost, are not easy to implement in nonspecialized laboratories or veterinary practices. In the current study, loop-mediated isothermal amplification (LAMP), a novel isothermal nucleic acid amplification procedure, was employed to develop a rapid, specific, and sensitive S. pseudintermedius assay. Different detection strategies, including the use of a lateral flow device, were evaluated. The assay was evaluated for cross-reactivity against 30 different bacterial species and validated on a panel of 108 S. pseudintermedius isolates, originating from different dog breeds and locations within the United Kingdom. The assay was specific, showing no cross-reactivity during in silico and in vitro testing. When tested using DNA extracts prepared directly from 35 clinical surgical site swabs, the assay could detect S. pseudintermedius in less than 15 min, with a diagnostic sensitivity of 94.6%, superior to that of a polymerase chain reaction method. The LAMP assay also had an analytical sensitivity in the order of 10(1) gene copies, and the amplified products were readily detected using a lateral flow device. The LAMP assay described in the present study is simple and rapid, opening up the possibility of its use as a diagnostic tool within veterinary practices.

  13. Loop mediated isothermal amplification (LAMP) assay for detection of coconut root wilt disease and arecanut yellow leaf disease phytoplasma.

    Science.gov (United States)

    Nair, Smita; Manimekalai, Ramaswamy; Ganga Raj, Palliyath; Hegde, Vinayaka

    2016-07-01

    The coconut root wilt disease (RWD) and the arecanut yellow leaf disease (YLD) are two major phytoplasma associated diseases affecting palms in South India. Greatly debilitating the palm health, these diseases cause substantial yield reduction and economic loss to farmers. A rapid and robust diagnostic technique is crucial in efficient disease management. We established phytoplasma 16S rDNA targeted loop mediated isothermal amplification (LAMP) and real time LAMP based diagnostics for coconut RWD and arecanut YLD. The LAMP reaction was set at 65 °C and end point detection made using hydroxynaphthol blue (HNB) and agarose gel electrophoresis. Molecular typing of LAMP products were made with restriction enzyme HpyCH4 V. Conventional PCR with LAMP external primers and sequencing of amplicons was carried out. Real time LAMP was performed on the Genei II platform (Optigene Ltd., UK). An annealing curve analysis was programmed at the end of the incubation to check the fidelity of the amplicons. The phytoplasma positive samples produced typical ladder like bands on agarose gel, showed colour change from violet to blue with HNB and produced unique annealing peak at 85 ± 0.5 °C in the real time detection. Restriction digestion produced predicted size fragments. Sequencing and BLASTN analysis confirmed that the amplification corresponded to phytoplasma 16S rRNA gene. LAMP method devised here was found to be more robust compared to conventional nested PCR and hence has potential applications in detection of phytoplasma from symptomatic palm samples and in rapid screening of healthy seedlings.

  14. Development of mitochondrial loop-mediated isothermal amplification for detection of the small liver fluke Opisthorchis viverrini (Opisthorchiidae; Trematoda; Platyhelminthes).

    Science.gov (United States)

    Le, Thanh Hoa; Nguyen, Nga Thi Bich; Truong, Nam Hai; De, Nguyen Van

    2012-04-01

    Mitochondrial DNA sequences offer major advantages over the more usual nuclear targets for loop-mediated isothermal amplification approaches (mito-LAMP) because multiple copies occur in every cell. Four LAMP primers [F3, FIP(F1c+F2), BIP(B1c+B2), and B3] were designed based on the mitochondrial nad1 sequence of Opisthorchis viverrini and used for a highly specific assay (mito-OvLAMP) to distinguish DNA of O. viverrini from that of another opisthorchiid (Clonorchis sinensis) and other trematodes (Haplorchis pumilio, Haplorchis taichui, Fasciola hepatica, and Fasciola gigantica). Conventional PCR was applied using F3/B3 primer pairs to verify the specificity of the primers for O. viverrini DNA templates. All LAMP-positive samples could be detected with the naked eye in sunlight, by gel electrophoresis (stained with ethidium bromide), and by addition of SYBR green I to the product in sunlight or under UV light. Only DNA from O. viverrini yielded amplification products by LAMP (and by PCR verification), and the LAMP limit of detection was as little as 100 fg (10(-4) ng DNA), indicating that this assay is 10 to 100 times more sensitive than PCR. Field testing was done using representative egg and metacercarial samples collected from localities where the fluke is endemic. With the advantages of simplicity, rapidity, sensitivity, and cost effectiveness, mito-OvLAMP is a good tool for molecular detection and epidemiology studies in regions or countries where O. viverrini is endemic, which can lead to more effective control of opisthorchiasis and trematodiasis.

  15. Highly Sensitive Detection of Low-Abundance White Spot Syndrome Virus by a Pre-Amplification PCR Method.

    Science.gov (United States)

    Pan, Xiaoming; Zhang, Yanfang; Sha, Xuejiao; Wang, Jing; Li, Jing; Dong, Ping; Liang, Xingguo

    2017-03-28

    White spot syndrome virus (WSSV) is a major threat to the shrimp farming industry and so far there is no effective therapy for it, and thus early diagnostic of WSSV is of great importance. However, at the early stage of infection, the extremely low-abundance of WSSV DNA challenges the detection sensitivity and accuracy of PCR. To effectively detect low-abundance WSSV, here we developed a pre-amplification PCR (pre-amp PCR) method to amplify trace amounts of WSSV DNA from massive background genomic DNA. Combining with normal specific PCR, 10 copies of target WSSV genes were detected from ~10(10) magnitude of backgrounds. In particular, multiple target genes were able to be balanced amplified with similar efficiency due to the usage of the universal primer. The efficiency of the pre-amp PCR was validated by nested-PCR and quantitative PCR, and pre-amp PCR showed higher efficiency than nested-PCR when multiple targets were detected. The developed method is particularly suitable for the super early diagnosis of WSSV, and has potential to be applied in other low-abundance sample detection cases.

  16. Rapid and sensitive detection of human astrovirus in water samples by loop-mediated isothermal amplification with hydroxynaphthol blue dye

    Science.gov (United States)

    2014-01-01

    Background The aim of this paper was to develop a reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for rapid, sensitive and inexpensive detection of astrovirus. Results The detection limit of LAMP using in vitro RNA transcripts was 3.6×10 copies·μL-1, which is as sensitive as the presently used PCR assays. However, the LAMP products could be identified as different colors with the naked eye following staining with hydroxynaphthol blue dye (HNB). No cross-reactivity with other gastroenteric viruses (rotavirus and norovirus) was observed, indicating the relatively high specificity of LAMP. The RT-LAMP method with HNB was used to effectively detect astrovirus in reclaimed water samples. Conclusions The LAMP technique described in this study is a cheap, sensitive, specific and rapid method for the detection of astrovirus. The RT-LAMP method can be simply applied for the specific detection of astrovirus and has the potential to be utilized in the field as a screening test. PMID:24524254

  17. Recombinase Polymerase Amplification (RPA of CaMV-35S Promoter and nos Terminator for Rapid Detection of Genetically Modified Crops

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    Chao Xu

    2014-10-01

    Full Text Available Recombinase polymerase amplification (RPA is a novel isothermal DNA amplification and detection technology that enables the amplification of DNA within 30 min at a constant temperature of 37–42 °C by simulating in vivo DNA recombination. In this study, based on the regulatory sequence of the cauliflower mosaic virus 35S (CaMV-35S promoter and the Agrobacterium tumefaciens nopaline synthase gene (nos terminator, which are widely incorporated in genetically modified (GM crops, we designed two sets of RPA primers and established a real-time RPA detection method for GM crop screening and detection. This method could reliably detect as few as 100 copies of the target molecule in a sample within 15–25 min. Furthermore, the real-time RPA detection method was successfully used to amplify and detect DNA from samples of four major GM crops (maize, rice, cotton, and soybean. With this novel amplification method, the test time was significantly shortened and the reaction process was simplified; thus, this method represents an effective approach to the rapid detection of GM crops.

  18. Recombinase polymerase amplification (RPA) of CaMV-35S promoter and nos terminator for rapid detection of genetically modified crops.

    Science.gov (United States)

    Xu, Chao; Li, Liang; Jin, Wujun; Wan, Yusong

    2014-10-10

    Recombinase polymerase amplification (RPA) is a novel isothermal DNA amplification and detection technology that enables the amplification of DNA within 30 min at a constant temperature of 37-42 °C by simulating in vivo DNA recombination. In this study, based on the regulatory sequence of the cauliflower mosaic virus 35S (CaMV-35S) promoter and the Agrobacterium tumefaciens nopaline synthase gene (nos) terminator, which are widely incorporated in genetically modified (GM) crops, we designed two sets of RPA primers and established a real-time RPA detection method for GM crop screening and detection. This method could reliably detect as few as 100 copies of the target molecule in a sample within 15-25 min. Furthermore, the real-time RPA detection method was successfully used to amplify and detect DNA from samples of four major GM crops (maize, rice, cotton, and soybean). With this novel amplification method, the test time was significantly shortened and the reaction process was simplified; thus, this method represents an effective approach to the rapid detection of GM crops.

  19. A signal amplification assay for HSV type 1 viral DNA detection using nanoparticles and direct acoustic profiling

    Directory of Open Access Journals (Sweden)

    Hammond Richard

    2010-02-01

    Full Text Available Abstract Background Nucleic acid based recognition of viral sequences can be used together with label-free biosensors to provide rapid, accurate confirmation of viral infection. To enhance detection sensitivity, gold nanoparticles can be employed with mass-sensitive acoustic biosensors (such as a quartz crystal microbalance by either hybridising nanoparticle-oligonucleotide conjugates to complimentary surface-immobilised ssDNA probes on the sensor, or by using biotin-tagged target oligonucleotides bound to avidin-modified nanoparticles on the sensor. We have evaluated and refined these signal amplification assays for the detection from specific DNA sequences of Herpes Simplex Virus (HSV type 1 and defined detection limits with a 16.5 MHz fundamental frequency thickness shear mode acoustic biosensor. Results In the study the performance of semi-homogeneous and homogeneous assay formats (suited to rapid, single step tests were evaluated utilising different diameter gold nanoparticles at varying DNA concentrations. Mathematical models were built to understand the effects of mass transport in the flow cell, the binding kinetics of targets to nanoparticles in solution, the packing geometries of targets on the nanoparticle, the packing of nanoparticles on the sensor surface and the effect of surface shear stiffness on the response of the acoustic sensor. This lead to the selection of optimised 15 nm nanoparticles that could be used with a 6 minute total assay time to achieve a limit of detection sensitivity of 5.2 × 10-12 M. Larger diameter nanoparticles gave poorer limits of detection than smaller particles. The limit of detection was three orders of magnitude lower than that observed using a hybridisation assay without nanoparticle signal amplification. Conclusions An analytical model was developed to determine optimal nanoparticle diameter, concentration and probe density, which allowed efficient and rapid optimisation of assay parameters

  20. Dissection of the Oncogenic MYCN Transcriptional Network Reveals a Large Set of Clinically Relevant Cell Cycle Genes as Drivers of Neuroblastoma Tumorigenesis

    NARCIS (Netherlands)

    D.M. Murphy; P.G. Buckley; K. Bryan; K.M. Watters; J. Koster; P. van Sluis; J. Molenaar; R. Versteeg; R.L. Stallings

    2011-01-01

    Amplification of the oncogenic transcription factor MYCN plays a major role in the pathogenesis of several pediatric cancers, including neuroblastoma, medulloblastoma, and rhabodomyosarcoma. For neuroblastoma, MYCN amplification is the most powerful genetic predictor of poor patient survival, yet th

  1. Impact of isolated hepatitis C virus (HCV) core-specific antibody detection and viral RNA amplification among HCV-seronegative dialysis patients at risk for infection.

    Science.gov (United States)

    Barril, Guillermina; Quiroga, Juan A; Arenas, María Dolores; Espinosa, Mario; García-Fernández, Nuria; Cigarrán, Secundino; Herrero, José A; del Peso, Gloria; Caro, Pilar; García-Agudo, Rebeca; Amézquita, Yésica; Blanco, Ana; Martínez-Rubio, Pilar; Alcázar, José M; González-Parra, Emilio; Martín-Gómez, Adoración; Castillo, Inmaculada; Bartolomé, Javier; Carreño, Vicente

    2014-08-01

    Amplification of hepatitis C virus (HCV) RNA from blood detected occult HCV infections in 30.9% of 210 HCV-seronegative dialysis patients with abnormal liver enzyme levels that had evaded standard HCV testing practices. Isolated HCV core-specific antibody detection identified three additional anti-HCV screening-negative patients lacking HCV RNA amplification in blood who were considered potentially infectious. Together, these findings may affect management of the dialysis setting. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  2. Detection and identification of Brettanomyces/Dekkera sp. yeasts with a loop-mediated isothermal amplification method.

    Science.gov (United States)

    Hayashi, Nobuyuki; Arai, Ritsuko; Tada, Setsuzo; Taguchi, Hiroshi; Ogawa, Yutaka

    2007-01-01

    Primer sets for a loop-mediated isothermal amplification (LAMP) method were developed to specifically identify each of the four Brettanomyces/Dekkera species, Dekkera anomala, Dekkera bruxellensis, Dekkera custersiana and Brettanomyces naardenensis. Each primer set was designed with target sequences in the ITS region of the four species and could specifically amplify the target DNA of isolates from beer, wine and soft drinks. Furthermore, the primer sets differentiated strains of the target species from strains belonging to other species, even within the genus Brettanomyces/Dekkera. Moreover, the LAMP method with these primer sets could detect about 1 x 10(1) cfu/ml of Brettanomyces/Dekkera yeasts from suspensions in distilled water, wine and beer. This LAMP method with primer sets for the identification of Brettanomyces/Dekkera yeasts is advantageous in terms of specificity, sensitivity and ease of operation compared with standard PCR methods.

  3. Gold Nanoparticles Based Colorimetric Detection of Target DNA After Loop-mediated Isothermal Amplification

    Institute of Scientific and Technical Information of China (English)

    ZHOU Chao; MU Ying; YANG Meng-chao; WU Qing-qing; XU Wei; ZHANG Ying; JIN Wei

    2013-01-01

    We have developed a rapid,simple and label-free colorimetric method for the identification of target DNA.It is based on loop-mediated isothermal amplification(LAMP).Plain gold nanoparticles(AuNPs) are used to indicate the occurrence of LAMP.The amplified product is mixed with AuNPs in an optimized ratio,at which the deoxyribonucleotides(dNTPs) bind to the AuNPs via ligand-metal interactions and thus enhance AuNPs stability.If a target DNA is amplified,the dramatic reduction of the dNTPs leads to the aggregation of AuNPs and a color change from red to blue.The success of the method strongly depends on the ionic strength of the solution and the initial concentration of dNTPs.Unlike other methods for the identification of isothermal products,this method is simple and can be readily applied on site where instrumentation is inadequate or even lacking.

  4. Rapid, Sensitive Detection of Bartonella quintana by Loop-Mediated Isothermal Amplification of the groEL Gene

    Science.gov (United States)

    Hu, Shoukui; Niu, Lina; Luo, Lijuan; Song, Xiuping; Sun, Jimin; Liu, Qiyong

    2016-01-01

    Trench fever, caused by Bartonella quintana, is recognized as a re-emerging and neglected disease. Rapid and sensitive detection approaches are urgently required to monitor and help control B. quintana infections. Here, loop-mediated isothermal amplification (LAMP), which amplifies target DNA at a fixed temperature with high sensitivity, specificity and rapidity, was employed to detect B. quintana. Thirty-six strains, including 10 B. quintana, 13 other Bartonella spp., and 13 other common pathogens, were applied to verify and evaluate the LAMP assay. The specificity of the LAMP assay was 100%, and the limit of detection was 125 fg/reaction. The LAMP assay was compared with qPCR in the examination of 100 rhesus and 20 rhesus-feeder blood samples; the diagnostic accuracy was found to be 100% when LAMP was compared to qPCR, but the LAMP assay was significantly more sensitive (p < 0.05). Thus, LAMP methodology is a useful for diagnosis of trench fever in humans and primates, especially in low-resource settings, because of its rapid, sensitive detection that does not require sophisticated equipment. PMID:27916953

  5. Rapid, Sensitive Detection of Bartonella quintana by Loop-Mediated Isothermal Amplification of the groEL Gene

    Directory of Open Access Journals (Sweden)

    Shoukui Hu

    2016-12-01

    Full Text Available Trench fever, caused by Bartonella quintana, is recognized as a re-emerging and neglected disease. Rapid and sensitive detection approaches are urgently required to monitor and help control B. quintana infections. Here, loop-mediated isothermal amplification (LAMP, which amplifies target DNA at a fixed temperature with high sensitivity, specificity and rapidity, was employed to detect B. quintana. Thirty-six strains, including 10 B. quintana, 13 other Bartonella spp., and 13 other common pathogens, were applied to verify and evaluate the LAMP assay. The specificity of the LAMP assay was 100%, and the limit of detection was 125 fg/reaction. The LAMP assay was compared with qPCR in the examination of 100 rhesus and 20 rhesus-feeder blood samples; the diagnostic accuracy was found to be 100% when LAMP was compared to qPCR, but the LAMP assay was significantly more sensitive (p < 0.05. Thus, LAMP methodology is a useful for diagnosis of trench fever in humans and primates, especially in low-resource settings, because of its rapid, sensitive detection that does not require sophisticated equipment.

  6. Rapid and sensitive detection of Plesiomonas shigelloides by loop-mediated isothermal amplification of the hugA gene.

    Directory of Open Access Journals (Sweden)

    Shuang Meng

    Full Text Available Plesiomonas shigelloides is one of the causative agents of human gastroenteritis, with increasing number of reports describing such infections in recent years. In this study, the hugA gene was chosen as the target to design loop-mediated isothermal amplification (LAMP assays for the rapid, specific, and sensitive detection of P. shigelloides. The performance of the assay with reference plasmids and spiked human stools as samples was evaluated and compared with those of quantitative PCR (qPCR. No false-positive results were observed for the 32 non-P. shigelloides strains used to evaluate assay specificity. The limit of detection for P. shigelloides was approximately 20 copies per reaction in reference plasmids and 5×10(3 CFU per gram in spiked human stool, which were more sensitive than the results of qPCR. When applied in human stool samples spiked with 2 low levels of P. shigelloides, the LAMP assays achieved accurate detection after 6-h enrichment. In conclusion, the LAMP assay developed in this study is a valuable method for rapid, cost-effective, and simple detection of P. shigelloides in basic clinical and field laboratories in the rural areas of China.

  7. Loop-mediated isothermal amplification applied to filarial parasites detection in the mosquito vectors: Dirofilaria immitis as a study model

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    Nelson Bryce

    2009-03-01

    Full Text Available Abstract Background Despite recent advances in our understanding of the basic biology behind transmission of zoonotic infectious diseases harbored by arthropod vectors these diseases remain threatening public health concerns. For effective control of vector and treatment, precise sampling indicating the prevalence of such diseases is essential. With an aim to develop a quick and simple method to survey zoonotic pathogen-transmitting vectors, LAMP (loop-mediated isothermal amplification was applied to the detection of filarial parasites using a filarial parasite-transmitting experimental model that included one of the mosquito vectors, Aedes aegypti, and the canine heartworm, Dirofilaria immitis. Results LAMP reactions amplifying the cytochrome oxidase subunit I gene demonstrated high sensitivity when a single purified D. immitis microfilaria was detected. Importantly, the robustness of the LAMP reaction was revealed upon identification of an infected mosquito carrying just a single parasite, a level easily overlooked using conventional microscopic analysis. Furthermore, successful detection of D. immitis in wild-caught mosquitoes demonstrated its applicability to field surveys. Conclusion Due to its simplicity, sensitivity, and reliability, LAMP is suggested as an appropriate diagnostic method for routine diagnosis of mosquito vectors carrying filarial parasites. This method can be applied to the survey of not only canine filariasis but also lymphatic filariasis, another major public health problem. Therefore, this method offers great promise as a useful diagnostic method for filarial parasite detection in endemic filariasis regions.

  8. Development and application of reverse transcription loop-mediated isothermal amplification for detecting live Shewanella putrefaciens in preserved fish sample.

    Science.gov (United States)

    Li, Chenghua; Ying, Qi; Su, Xiurong; Li, Taiwu

    2012-04-01

    Given that live Shewanella putrefaciens is one of the major causes of spoilage for aquatic products even in chill storage, the rapid and accurate detection process is the first priority. In the present study, a novel reverse transcription loop-mediated isothermal amplification (RT-LAMP) detecting assay was developed by targeting internal transcribed spacer (ITS) sequence between 16S and 23S rRNA. At the same time, a new procaryotic mRNA isolation strategy was also established by introducing a polyA tail to RNA during cDNA synthesis step. Under the optimal reaction time (60 min) and temperature (64.1 °C), S. putrefaciens could be specially identified from a variety of other tested bacteria by RT-LAMP. The sensitivity analysis showed that RT-LAMP could be identified as lower as 5.4 copies per reaction, which is over 200-fold higher than that of standard PCR (1.08 × 10³ copies per reaction). The method could be effectively identified S. putrefaciens in artificially contaminated or spoilaged fish samples with dose-dependent manners. To our knowledge, this is the first report using RT-LAMP assay to detect live S. putrefaciens in fish. The study provided a rapid and accurate detection method for live bacteria in aquatic food and established a new procaryotic mRNA isolation strategy at the same time, which will be useful for food preservation. © 2012 Institute of Food Technologists®

  9. Loop-mediated isothermal amplification (LAMP) assay for the rapid detection of the sexually-transmitted parasite, Trichomonas vaginalis.

    Science.gov (United States)

    Adao, Davin Edric V; Rivera, Windell L

    2016-01-01

    A loop-mediated isothermal amplification (LAMP) assay was developed to detect the sexually-transmitted parasite, Trichomonas vaginalis in vaginal swabs. The presence of T. vaginalis was detected from 121 female sex workers attending a social hygiene clinic in Balibago, Angeles City, Pampanga, Philippines using culture, polymerase chain reaction (PCR), and the developed LAMP assay. The high analytical sensitivity of LAMP detected a higher prevalence of T. vaginalis (42.06%) compared to culture (8.26%) and PCR (7.44%). Additionally, this assay did not cross-react with DNAs of other trichomonads that can infect humans such as Trichomonas tenax and Pentatrichomonas hominis as well as the pathogens, Candida albicans and Staphylococcus aureus. The LAMP assay developed had a limit of detection (0.036 ng/μl) lower than that of PCR using the primers TvK3 and TvK7 (0.36 ng/μl). Prevalence of T. vaginalis in female sex workers in this area of the Philippines may be higher than previously estimated. Discordant results of PCR and LAMP may be due to different reactions to different kinds of inhibitors in the vaginal swabs.

  10. A pH Indicator-linked Immunosorbent assay following direct amplification strategy for colorimetric detection of protein biomarkers.

    Science.gov (United States)

    Shao, Fengying; Jiao, Lei; Miao, Luyang; Wei, Qin; Li, He

    2017-04-15

    A new pH indicator-linked immunosorbent assay (PILISA) reached pg/mL sensitivity based on pH indicator molecules loaded carbon nitride nanosheets as signal enhancer has been developed for colorimetric detection of protein biomarkers. As the secondary antibody binds to the carbon nitride nanosheets, the carbon nitride nanosheets and pH indicator complex as the signal amplification platform for colour change by detecting absorbance of pH indicator. The colour change was resulted from the releasing of pH indicator molecules from carbon nitride nanosheets triggered by alkali solution (AS). In this novel PILISA, the intensity absorbance of pH indicator is proportional to the concentration of the disease marker. The outstanding detection performance of the PILISA can be attributed to the following reasons: (1) ultrathin carbon nitride nanosheets with a larger surface area could adsorb abundant phenolphthalein (PP) molecules through hydrophobic interactions as well as the resulted PP anions can be free easily released into aqueous solution, leading to an obvious allochroic response; (2) the signal intensity is precisely determined by the amount of PP molecules loading onto the carbon nitride nanosheets surface, which ensures simple, low-cost and stable colorimetric detection. As expected, this new PILISA method offered an enzyme-free approach followed enzyme-linked immunosorbent assay format, which showed great promising potential as an innovative robust assay method for practical clinical applications. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. Efficient detection of pathogen virus in sand dabs,Paralichthys olivaceus using loop-mediated isothermal amplification (LAMP)

    Institute of Scientific and Technical Information of China (English)

    HWANG Jinik; PARK So Yun; SUH Sung-Suk; PARK Mirye; LEE Sukchan; LEE Taek-Kyun

    2016-01-01

    Viral hemorrhagic septicemia virus (VHSV) and marine birnavirus (MABV) are the causative pathogens for some of the most explosive epidemics of emerging viral diseases in many Asian countries, leading to huge economic losses in aquaculture. Rapid molecular detection for surveillance or diagnosis has been a critical component in reducing the prevalence of pathogen infection. The loop-mediated isothermal amplification (LAMP) of DNA is currently one of the most commonly used molecular diagnostic tools, as it is simple, quick, and easy to amplify target DNA under isothermal conditions. In the present study, a novel and highly specific LAMP assay for the sensitive and rapid detection of VHSV and MABV infection in fish was developed. Using a set of synthesized primers matching a specific region of the genome, the efficiency and specificity of the LAMP assay were optimized in terms of the reaction temperature and DNA polymerase concentration, as they are the main determinants of the sensitivity and specificity of the LAMP assay. In particular, we demonstrated that our assay could be applied to efficient detection of VHSV and MABV infection in the wild fish,Paralichthys olivaceus. Our results demonstrate the simplicity and convenience of this method for the detection of viral infection in aquatic organisms.

  12. Establishment and Evaluation of a Loop-Mediated Isothermal Amplification Assay for Detection of Raccoon Dog in Meat Mixtures

    Directory of Open Access Journals (Sweden)

    Jinhua Liu

    2017-01-01

    Full Text Available Raccoon dog (Nyctereutes procyonoides is an economically important animal used for fur production, but consuming its meat is injurious to human health. Currently, no rapid and sensitive method for detecting raccoon dog meat in meat mixtures is available. In this study, we developed an easily applicable, rapid, and economically feasible method for identifying the presence of raccoon dog in meat mixtures based on loop-mediated isothermal amplification (LAMP. Four sets of LAMP primers were tested at different temperatures, and the primers that worked best at 62°C (set 2 were determined. In the LAMP assay, there was no cross-reactivity with the meat procured from other species of animals and the detection limit of DNA concentration was 0.1 pg·μL−1, slightly higher than TaqMan real-time PCR (0.01 pg·μL−1, but sensitivity of 0.1 pg·μL−1 complies with most requirements of routine analysis. Moreover, by the LAMP method, the meat mixtures containing more than 0.5% of the raccoon dog component were directly detected (without DNA extraction in the supernatant isolated from the meat mixtures after performing repeated cycles of thawing and freezing of minced meat mixtures. Our results show that LAMP assay is a valuable, straightforward, and sensitive detection tool for identification of raccoon dog meat in mixtures.

  13. Electrochemical DNA probe for Hg(2+) detection based on a triple-helix DNA and Multistage Signal Amplification Strategy.

    Science.gov (United States)

    Wang, Huan; Zhang, Yihe; Ma, Hongmin; Ren, Xiang; Wang, Yaoguang; Zhang, Yong; Wei, Qin

    2016-12-15

    In this work, an ultrasensitive electrochemical sensor was developed for detection of Hg(2+). Gold nanoparticles decorated bovine serum albumin reduction of graphene oxide (AuNP-BSA-rGO) were used as subsurface material for the immobilization of triple-helix DNA. The triple-helix DNA containing a thiol labelled single-stranded DNA (sDNA) and a thymine-rich DNA (T-rich DNA), which could be unwinded in the present of Hg(2+) to form more stable thymine-Hg(2+)-thymine (T-Hg(2+)-T) complex. T-Hg(2+)-T complex was then removed and the sDNA was left on the electrode. At this time, gold nanoparticle carrying thiol labelled cytosine-rich complementary DNA (cDNA-AuNP) could bind with the free sDNA. Meanwhile, the other free cDNA on AuNP could bind with each other in the present of Ag(+) to form the stable cytosine-Ag(+)-cytosine (C-Ag(+)-C) complex and circle amplification. Plenty of C-Ag(+)-C could form silver nanoclusters by electrochemical reduction and the striping signal of Ag could be measured for purpose of the final electrochemical detection of Hg(2+). This sensor could detect Hg(2+) over a wide concentration range from 0.1 to 130nM with a detection limit of 0.03nM.

  14. Development of a Rapid Detection Method for Potato virus X by Reverse Transcription Loop-Mediated Isothermal Amplification

    Directory of Open Access Journals (Sweden)

    Joojin Jeong

    2015-09-01

    Full Text Available The primary step for efficient control of viral diseases is the development of simple, rapid, and sensitive virus detection. Reverse transcription loop-mediated isothermal amplification (RT-LAMP has been used to detect viral RNA molecules because of its simplicity and high sensitivity for a number of viruses. RT-LAMP for the detection of Potato virus X (PVX was developed and compared with conventional reverse transcription polymerase chain reaction (RT-PCR to demonstrate its advantages over RT-PCR. RT-LAMP reactions were conducted with or without a set of loop primers since one out of six primers showed PVX specificity. Based on real-time monitoring, RT-LAMP detected PVX around 30 min, compared to 120 min for RT-PCR. By adding a fluorescent reagent during the reaction, the extra step of visualization by gel electrophoresis was not necessary. RT-LAMP was conducted using simple inexpensive instruments and a regular incubator to evaluate whether RNA could be amplified at a constant temperature instead of using an expensive thermal cycler. This study shows the potential of RT-LAMP for the diagnosis of viral diseases and PVX epidemiology because of its simplicity and rapidness compared to RT-PCR.

  15. Graphene oxide based fluorescence resonance energy transfer and loop-mediated isothermal amplification for white spot syndrome virus detection.

    Science.gov (United States)

    Waiwijit, U; Phokaratkul, D; Kampeera, J; Lomas, T; Wisitsoraat, A; Kiatpathomchai, W; Tuantranont, A

    2015-10-20

    Graphene oxide (GO) is attractived for biological or medical applications due to its unique electrical, physical, optical and biological properties. In particular, GO can adsorb DNA via π-π stacking or non-covalent interactions, leading to fluorescence quenching phenomenon applicable for bio-molecular detection. In this work, a new method for white spot syndrome virus (WSSV)-DNA detection is developed based on loop-mediated isothermal amplification (LAMP) combined with fluorescence resonance energy transfer (FRET) between GO and fluorescein isothiocyanate-labeled probe (FITC-probe). The fluorescence quenching efficiency of FITC-probe was found to increase with increasing GO concentration and reached 98.7% at a GO concentration of 50 μg/ml. The fluorescence intensity of FITC-probe was recovered after hybridization with WSSV LAMP product with an optimal hybridization time of 10 min and increased accordingly with increasing amount of LAMP products. The detection limit was estimated to be as low as 10 copies of WSSV plasmid DNA or 0.6 fg of the total DNA extracted from shrimp infected with WSSV. In addition, no cross reaction was observed with other common shrimp viral pathogens. Therefore, the GO-FRET-LAMP technique is promising for fast, sensitive and specific detection of DNAs.

  16. Characterization of Leukemia-Inducing Genes Using a Proto-Oncogene/Homeobox Gene Retroviral Human cDNA Library in a Mouse In Vivo Model.

    Directory of Open Access Journals (Sweden)

    Su Hwa Jang

    Full Text Available The purpose of this research is to develop a method to screen a large number of potential driver mutations of acute myeloid leukemia (AML using a retroviral cDNA library and murine bone marrow transduction-transplantation system. As a proof-of-concept, murine bone marrow (BM cells were transduced with a retroviral cDNA library encoding well-characterized oncogenes and homeobox genes, and the virus-transduced cells were transplanted into lethally irradiated mice. The proto-oncogenes responsible for leukemia initiation were identified by PCR amplification of cDNA inserts from genomic DNA isolated from leukemic cells. In an initial screen of ten leukemic mice, the MYC proto-oncogene was detected in all the leukemic mice. Of ten leukemic mice, 3 (30% had MYC as the only transgene, and seven mice (70% had additional proto-oncogene inserts. We repeated the same experiment after removing MYC-related genes from the library to characterize additional leukemia-inducing gene combinations. Our second screen using the MYC-deleted proto-oncogene library confirmed MEIS1and the HOX family as cooperating oncogenes in leukemia pathogenesis. The model system we introduced in this study will be valuable in functionally screening novel combinations of genes for leukemogenic potential in vivo, and the system will help in the discovery of new targets for leukemia therapy.

  17. Characterization of Leukemia-Inducing Genes Using a Proto-Oncogene/Homeobox Gene Retroviral Human cDNA Library in a Mouse In Vivo Model.

    Science.gov (United States)

    Jang, Su Hwa; Lee, Sohyun; Chung, Hee Yong

    2015-01-01

    The purpose of this research is to develop a method to screen a large number of potential driver mutations of acute myeloid leukemia (AML) using a retroviral cDNA library and murine bone marrow transduction-transplantation system. As a proof-of-concept, murine bone marrow (BM) cells were transduced with a retroviral cDNA library encoding well-characterized oncogenes and homeobox genes, and the virus-transduced cells were transplanted into lethally irradiated mice. The proto-oncogenes responsible for leukemia initiation were identified by PCR amplification of cDNA inserts from genomic DNA isolated from leukemic cells. In an initial screen of ten leukemic mice, the MYC proto-oncogene was detected in all the leukemic mice. Of ten leukemic mice, 3 (30%) had MYC as the only transgene, and seven mice (70%) had additional proto-oncogene inserts. We repeated the same experiment after removing MYC-related genes from the library to characterize additional leukemia-inducing gene combinations. Our second screen using the MYC-deleted proto-oncogene library confirmed MEIS1and the HOX family as cooperating oncogenes in leukemia pathogenesis. The model system we introduced in this study will be valuable in functionally screening novel combinations of genes for leukemogenic potential in vivo, and the system will help in the discovery of new targets for leukemia therapy.

  18. Determination of tertiary amines based on pH junctions and field amplification in capillary electrophoresis with electrochemiluminescence detection.

    Science.gov (United States)

    Sreedhar, Mallipattu; Lin, Yang-Wei; Tseng, Wei-Lung; Chang, Huan-Tsung

    2005-08-01

    A stacking approach based on pH junction and field amplification has been developed for determining amines by capillary electrophoresis (CE) with electrochemiluminescence (ECL) detection. A two-electrode configuration was employed with an indium/tin oxide-coated glass as a working electrode and a platinum wire as a pseudoreference electrode. The ECL system also contains a flow cell (poly(dimethylsiloxane)-aluminum oxide) that was made from a mixture of Sylgard 184 silicone elastomer, a curing agent, and aluminum oxide. In order to improve the sensitivity of the present CE-ECL system using tris(2,2'-bipyridyl) ruthenium(II) (Ru(bpy)(3) (2+)), a stacking approach based on pH junctions and field amplification has been tested for the analysis of triethylamine (TEA), tripropylamine (TPA), and tributylamine (TBA). Once amines (cations) prepared in citric acid solution (pH < 4.0) migrate towards the background electrolyte (15 mM sodium borate at pH 8.0), they slow down and are stacked at the boundary as a result of deprotonation and decreases in the electric field. By applying hydrodynamic injection of the sample for 60 s, this method provides the concentration limits of detection (signal-to-noise ratio = 3) of 24, 20, and 32 nM for TEA, TPA, and TBA, respectively. The results indicate that the stacking CE-ECL system is better than CE-ECL systems using a two-electrode configuration and comparable to those using a three-electrode configuration. The potential applicability of the new and low-cost CE-ECL system has been demonstrated by the determination of 1.0 microM lidocaine, a local anesthetic drug, in urine without any tedious sample preparation.

  19. Values of mutations of K-ras oncogene at codon 12 in detection of pancreatic cancer:15-year experience

    Institute of Scientific and Technical Information of China (English)

    De-Qing Mu; You-Shu Peng; Qiao-Jian Xu

    2004-01-01

    AIM: To summarize progress in the study of K-ras gene studies in pancreatic cancer and its potential clinical significance in screening test for early detection of pancreatic cancer, and to differentiate pancreatic cancer from chronic pancreatitis in recent decade.METHODS: Literature search (MEDLINE 1986-2003) was performed using the key words K-ras gene, pancreatic cancer, chronic pancreatitis, and diagnosis. Two kind of opposite points of view on the significance of K-ras gene in detection early pancreatic cancer and differentiation pancreatic cancer from chronic pancreatitis were investigated.The presence of a K-ras gene mutation at codon 12 has been seen in 75-100% of pancreatic cancers, and is not rare in patients with chronic pancreatitis, and represents an increased risk of developing pancreatic cancer. However, the significance of the detection of this mutation in specimens obtained by needle aspiration from pure pancreatic juice and from stools for its utilization for the detection of early pancreatic cancer, and differentiation pancreatic cancer from chronic pancreatitis remains controversial. CONCLUSION: The value of K-ras gene mutation for the detection of early pancreatic cancer and differentiation pancreatic cancer from chronic pancreatitis remains uncertains in clinical pratice. Nevertheless, K-ras mutation screening may increase the sensitivity of FNA and ERP cytology and may be useful in identifying pancreatitis patients at high risk for developing cancer, and as a adjunct with cytology to differentiate pancreatic cancer from chronic pancreatitis.

  20. Signal Amplification by Glyco-qPCR for Ultrasensitive Detection of Carbohydrates: Applications in Glycobiology**

    OpenAIRE

    Kwon, Seok Joon; Lee, Kyung Bok; Solakyildirim, Kemal; Masuko, Sayaka; Ly, Mellisa; Zhang, Fuming; Li, Lingyun; Dordick, Jonathan S.; Robert J Linhardt

    2012-01-01

    Tiny amounts of carbohydrates (ca. 1 zmol) can be detected quantitatively by a real-time method based on the conjugation of carbohydrates with DNA markers (see picture). The proposed method (glyco-qPCR) provides uniform, ultrasensitive detection of carbohydrates, which can be applied to glycobiology, as well as carbohydrate-based drug discovery.

  1. Application of Single—labelled Probe—primer in PCR Amplification to the Detection of Hepatitis B Virus DNA

    Institute of Scientific and Technical Information of China (English)

    KONG,De-Ming; SHEN,Han-Xi

    2003-01-01

    A new method based on the incorporation of a single-lablled probe-primer into polymerase chain reaction(PCR) for the detection of PCR-amplified DNA in a closed system is reported.The probeprimerc consists of a specific probe sequence on the 5''''''''-end and a primer sequence on the 3''''''''-end.A flurophore is located at the 5''''''''end.The primeR-quencher is an oligonucleotide,which is complementary to the probe sequence of probe-primer and labelled with a quencher at the 3''''''''-end.In the duplex formed by probe-primer and primer-quencher.the fluorophore and quencher are kept in close proximity to each other.Therefore the fluorescence is quenched.During PCR amplificatio,the specific probe sequence of probeprimer binds to its complement within the same strand of DNA,and is cleaved by Taq DNA polymerase,resulting in the restoration of fluorescence.This system has the same energy transfer mechanism as molecular beacons,and a good quenching effciency can be ensured.Following optimization of PCR conditions,this method was used to detect hepatitis b virus(HBV) dna in patient sera.This technology eliminates the risk of carry-over contamination,simplifies the amplification assay and opens up new possibilities for the real-time detection of the amplified DNA.

  2. Loop-Mediated Isothermal Amplification Method for the Rapid Detection of Ralstonia solanacearum Phylotype I Mulberry Strains in China

    Science.gov (United States)

    Huang, Wen; Zhang, Hao; Xu, Jingsheng; Wang, Shuai; Kong, Xiangjiu; Ding, Wei; Xu, Jin; Feng, Jie

    2017-01-01

    Ralstonia solanacearum phylotype I mulberry strains are causative agent of bacterial wilt of mulberry. Current diagnostic methods are not adopted to the mulberry wilt disease. In this study, we developed a rapid method, loop-mediated isothermal amplification (LAMP), to detect R. solanacearum phylotype I mulberry strains. A set of six primers was designed to target the clone MG67 sequence in this LAMP detection which can be completed in 20 min at 64°C. The results of the LAMP reaction could be observed with the naked eye due to magnesium pyrophosphate precipitate produced during the reaction or the color change after adding SYBR Green I. The specificity of the LAMP was confirmed using DNA from 46 representative strains of R. solanacearum and 7 other soil-borne bacteria strains. This method was also of high sensitivity and could be used to detect the presence of less than 160 fg genomic DNA or 2.2 × 102 CFU/ml of bacterial cells per 25 μl reaction volume, moreover, the presence of plant tissue fluid did not affect the sensitivity. Since it does not require expensive equipment or specialized techniques, this LAMP-based diagnostic method has the potential to be used under field conditions to make disease forecasting more accurate and efficient.

  3. Protocols for the in situ PCR-amplification and detection of mRNA and DNA sequences.

    Science.gov (United States)

    Bagasra, Omar

    2007-01-01

    In this protocol we describe the in situ PCR method for the amplification of both DNA and mRNA targets [in situ reverse transcriptase-PCR (RT-PCR)], from frozen or paraffin-fixed tissue sections, cell culture or other single-cell suspensions. Detection of amplicons can be achieved by the hybridization and detection of labeled probes. The protocol includes the following steps: (i) tissue preparation, (ii) in situ PCR (or in situ RT-PCR), (iii) probe hybridization, (iv) signal detection. The technique has high sensitivity (geometrically PCR-amplifying 150-350 bp fragments of a gene of interest in situ) and specificity (derived from in situ hybridization with specific fluorescent or biotinylated probes for the target genes). The ability to identify individual cells, expressing or carrying specific genes of interest in a latent form in a tissue section under the microscope provides a visual account of silent genes, and allows the determination of various aspects of normal versus pathological conditions, or latent versus active viral replication. An average of 48 h is required to carry out the technique.

  4. Detection of Staphylococcus epidermidis by a Quartz Crystal Microbalance Nucleic Acid Biosensor Array Using Au Nanoparticle Signal Amplification

    Directory of Open Access Journals (Sweden)

    Weiling Fu

    2008-10-01

    Full Text Available Staphylococcus epidermidis is a critical pathogen of nosocomial blood infections, resulting in significant morbidity and mortality. A piezoelectric quartz crystal microbalance (QCM nucleic acid biosensor array using Au nanoparticle signal amplification was developed to rapidly detect S. epidermidis in clinical samples. The synthesized thiolated probes specific targeting S. epidermidis 16S rRNA gene were immobilized on the surface of QCM nucleic acid biosensor arrays. Hybridization was induced by exposing the immobilized probes to the PCR amplified fragments of S. epidermidis, resulting in a mass change and a consequent frequency shift of the QCM biosensor. To further enhance frequency shift results from above described hybridizations, streptavidin coated Au nanoparticles were conjugated to the PCR amplified fragments. The results showed that the lowest detection limit of current QCM system was 1.3×103 CFU/mL. A linear correlation was found when the concentration of S. epidermidis varied from 1.3×103 to 1.3×107 CFU/mL. In addition, 55 clinical samples were detected with both current QCM biosensor system and conventional clinical microbiological method, and the sensitivity and specificity of current QCM biosensor system were 97.14% and 100%, respectively. In conclusion, the current QCM system is a rapid, low-cost and sensitive method that can be used to identify infection of S. epidermidis in clinical samples.

  5. Rapid detection of peste des petits ruminants virus by a reverse transcription loop-mediated isothermal amplification assay.

    Science.gov (United States)

    Li, Lin; Bao, Jingyue; Wu, Xiaodong; Wang, Zhiliang; Wang, Junwei; Gong, Mingxia; Liu, Chunju; Li, Jinming

    2010-12-01

    Peste des petits ruminants virus (PPRV) is the causative agent of peste des petits ruminants (PPR), an economically important viral disease of small ruminants. In this report, a one-step, single-tube, reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the detection of PPRV. A set of six LAMP primers were designed based on the matrix gene sequence of PPRV to amplify the target RNA by incubation at 63°C for 60min with Bst DNA polymerase and reverse transcriptase. The amplified products could be observed by the naked eye. The specificity of the RT-LAMP assay was validated by amplifying eight strains of PPRV isolated in different geographical areas. No cross-reactivity with other related viruses, including rinderpest virus, canine distemper virus and measles virus, was detected. The sensitivity of the assay was similar to that of real-time reverse transcription polymerase chain reaction (RT-PCR) and 10-fold higher than that of conventional RT-PCR. Twenty clinical samples were evaluated by the RT-LAMP assay, and the results were consistent with those of real-time RT-PCR. As a simple, rapid and accurate detection method, this RT-LAMP assay has important potential applications in the clinical diagnosis of PPR and the surveillance of PPRV. Copyright © 2010 Elsevier B.V. All rights reserved.

  6. Loop-mediated isothermal amplification: rapid visual and real-time methods for detection of genetically modified crops.

    Science.gov (United States)

    Randhawa, Gurinder Jit; Singh, Monika; Morisset, Dany; Sood, Payal; Zel, Jana

    2013-11-27

    A rapid, reliable, and sensitive loop-mediated isothermal amplification (LAMP) system was developed for screening of genetically modified organisms (GMOs). The optimized LAMP assays using designed primers target commonly employed promoters, i.e., Cauliflower Mosaic Virus 35S (P-35S) and Figwort Mosaic Virus promoter (P-FMV), and marker genes, i.e., aminoglycoside 3'-adenyltransferase (aadA), neomycin phosphotransferase II (nptII), and β-glucuronidase (uidA). The specificity and performance of the end-point and real-time LAMP assays were confirmed using eight genetically modified (GM) cotton events on four detection systems, employing two chemistries. LAMP assays on the isothermal real-time system were found to be most sensitive, detecting up to four target copies, within 35 min. The LAMP assays herein presented using alternate detection systems can be effectively utilized for rapid and cost-effective screening of the GM status of a sample, irrespective of the crop species or GM trait. These assays coupled with a fast and simple DNA extraction method may further facilitate on-site GMO screening.

  7. Development of Au-Nanoprobes Combined with Loop-Mediated Isothermal Amplification for Detection of Isoniazid Resistance in Mycobacterium tuberculosis

    Directory of Open Access Journals (Sweden)

    Jutturong Ckumdee

    2016-01-01

    Full Text Available Multidrug resistant tuberculosis (MDR-TB is Mycobacterium tuberculosis that does not respond to isoniazid and rifampicin, so the condition worsens continuously and creates difficulties for treatment by public health control programmes, especially in developing countries. The real time polymerase chain reaction (PCR combined with agarose gel electrophoresis or strip tests is useful molecular tools for diagnosis of MDR-TB. Novel loop-mediated isothermal amplification (LAMP can also detect drug resistance, which is a one-point mutation, by designing inner primers of 5′ end specific with the mutant. Au-nanoprobes on hybridisation with LAMP products containing target-specific sequences remain red, whereas test samples without specific sequences in the probe turn purple due to salt-induced aggregation of the Au-nanoprobes. In this study, a strategy was designed based on the LAMP of a DNA sample coupled to specific Au-nanoprobes, which showed the potential to provide a rapid and sensitive method for detecting isoniazid resistance at katG gene position 315 (G→C. 46 clinical samples were tested and showed 100% specificity and sensitivity compared with Genotype® MDR-TB Plus. This method was advantageous because it is rapid, cheap, specific, and sensitive. Further, it does not require thermal cycles for MDR-TB detection.

  8. Field Evaluation of a High Throughput Loop Mediated Isothermal Amplification Test for the Detection of Asymptomatic Plasmodium Infections in Zanzibar.

    Science.gov (United States)

    Aydin-Schmidt, Berit; Morris, Ulrika; Ding, Xavier C; Jovel, Irina; Msellem, Mwinyi I; Bergman, Daniel; Islam, Atiqul; Ali, Abdullah S; Polley, Spencer; Gonzalez, Iveth J; Mårtensson, Andreas; Björkman, Anders

    2017-01-01

    New field applicable diagnostic tools are needed for highly sensitive detection of residual malaria infections in pre-elimination settings. Field performance of a high throughput DNA extraction system for loop mediated isothermal amplification (HTP-LAMP) was therefore evaluated for detecting malaria parasites among asymptomatic individuals in Zanzibar. HTP-LAMP performance was evaluated against real-time PCR on 3008 paired blood samples collected on filter papers in a community-based survey in 2015. The PCR and HTP-LAMP determined malaria prevalences were 1.6% (95%CI 1.3-2.4) and 0.7% (95%CI 0.4-1.1), respectively. The sensitivity of HTP-LAMP compared to PCR was 40.8% (CI95% 27.0-55.8) and the specificity was 99.9% (CI95% 99.8-100). For the PCR positive samples, there was no statistically significant difference between the geometric mean parasite densities among the HTP-LAMP positive (2.5 p/μL, range 0.2-770) and HTP-LAMP negative (1.4 p/μL, range 0.1-7) samples (p = 0.088). Two lab technicians analysed up to 282 samples per day and the HTP-LAMP method was experienced as user friendly. Although field applicable, this high throughput format of LAMP as used here was not sensitive enough to be recommended for detection of asymptomatic low-density infections in areas like Zanzibar, approaching malaria elimination.

  9. Rapid genome detection of Schmallenberg virus and bovine viral diarrhea virus by use of isothermal amplification methods and high-speed real-time reverse transcriptase PCR.

    Science.gov (United States)

    Aebischer, Andrea; Wernike, Kerstin; Hoffmann, Bernd; Beer, Martin

    2014-06-01

    Over the past few years, there has been an increasing demand for rapid and simple diagnostic tools that can be applied outside centralized laboratories by using transportable devices. In veterinary medicine, such mobile test systems would circumvent barriers associated with the transportation of samples and significantly reduce the time to diagnose important infectious animal diseases. Among a wide range of available technologies, high-speed real-time reverse transcriptase quantitative PCR (RT-qPCR) and the two isothermal amplification techniques loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA) represent three promising candidates for integration into mobile pen-side tests. The aim of this study was to investigate the performance of these amplification strategies and to evaluate their suitability for field application. In order to enable a valid comparison, novel pathogen-specific assays have been developed for the detection of Schmallenberg virus and bovine viral diarrhea virus. The newly developed assays were evaluated in comparison with established standard RT-qPCR using samples from experimentally or field-infected animals. Even though all assays allowed detection of the target virus in less than 30 min, major differences were revealed concerning sensitivity, specificity, robustness, testing time, and complexity of assay design. These findings indicated that the success of an assay will depend on the integrated amplification technology. Therefore, the application-specific pros and cons of each method that were identified during this study provide very valuable insights for future development and optimization of pen-side tests.

  10. Study of DNA extraction methods for use in loop-mediated isothermal amplification detection of single resting cysts in the toxic dinoflagellates Alexandrium tamarense and A. catenella.

    Science.gov (United States)

    Nagai, Satoshi; Yamamoto, Keigo; Hata, Naotugu; Itakura, Shigeru

    2012-09-01

    In a previous study, we experienced instable amplification and a low amplification success in loop-mediated isothermal amplification (LAMP) reactions from naturally occurring vegetative cells or resting cysts of the toxic dinoflagellates Alexandrium tamarense and Alexandrium catenella. In this study, we examined 4 methods for extracting DNA from single resting cysts of A. tamarense and A. catenella to obtain more stable and better amplification success and to facilitate unambiguous detection using the LAMP method. Apart from comparing the 4 different DNA extraction methods, namely, (1) boiling in Tris-EDTA (TE) buffer, (2) heating at 65 °C in hexadecyltrimethylammonium bromide buffer, (3) boiling in 0.5% Chelex buffer, and (4) boiling in 5% Chelex buffer, we also examined the need for homogenization to crush the resting cysts before DNA extraction in each method. Homogenization of resting cysts was found to be essential for DNA extraction in all 4 methods. The detection time was significantly shorter in 5% Chelex buffer than in the other buffers and the amplification success was 100% (65/65), indicating the importance of DNA extraction and the effectiveness of 5% Chelex buffer in the Alexandrium LAMP.

  11. Development of a loop-mediated isothermal amplification assay for rapid, sensitive detection of Campylobacter jejuni in cattle farm samples.

    Science.gov (United States)

    Dong, Hee-Jin; Cho, Ae-Ri; Hahn, Tae-Wook; Cho, Seongbeom

    2014-09-01

    Campylobacter jejuni is a leading cause of bacterial foodborne disease worldwide. The detection of this organism in cattle and their environment is important for the control of C. jejuni transmission and the prevention of campylobacteriosis. Here, we describe the development of a rapid and sensitive method for the detection of C. jejuni in naturally contaminated cattle farm samples, based on real-time loop-mediated isothermal amplification (LAMP) of the hipO gene. The LAMP assay was specific (100% inclusivity and exclusivity for 84 C. jejuni and 41 non-C. jejuni strains, respectively), sensitive (detection limit of 100 fg/μl), and quantifiable (R(2) = 0.9133). The sensitivity of the LAMP assay was then evaluated for its application to the naturally contaminated cattle farm samples. C. jejuni strains were isolated from 51 (20.7%) of 246 cattle farm samples, and the presence of the hipO gene was tested using the LAMP assay. Amplification of the hipO gene by LAMP within 30 min (mean ~10.8 min) in all C. jejuni isolates (n = 51) demonstrated its rapidity and accuracy. Next, template DNA was prepared from a total of 186 enrichment broth cultures of cattle farm samples either by boiling or using a commercial kit, and the sensitivity of detection of C. jejuni was compared between the LAMP and PCR assays. In DNA samples prepared by boiling, the higher sensitivity of the LAMP assay (84.4%) compared with the PCR assay (35.5%) indicates that it is less susceptible to the existence of inhibitors in sample material. In DNA samples prepared using a commercial kit, both the LAMP and PCR assays showed 100% sensitivity. We anticipate that the use of this rapid, sensitive, and simple LAMP assay, which is the first of its kind for the identification and screening of C. jejuni in cattle farm samples, may play an important role in the prevention of C. jejuni contamination in the food chain, thereby reducing the risk of human campylobacteriosis.

  12. Enhanced charge detection: Amplification factor, phase reversal and measurement time dependence

    Energy Technology Data Exchange (ETDEWEB)

    Thorgrimson, J.; Sachrajda, A. S. [National Research Council Canada, Ottawa, ON Canada K1A 0R6 and Department of Physics, McGill University, 3600 rue University, Montreal, QC (Canada); Studenikin, S. A.; Bogan, A. [National Research Council Canada, Ottawa, ON Canada K1A 0R6 and Department of Physics and Astronomy, University of Waterloo, 200 University Avenue West, Waterloo, ON (Canada); Aers, G. C.; Kam, A.; Zawadzki, P.; Wasilewski, Z. R. [National Research Council Canada, Ottawa, ON (Canada)

    2013-12-04

    Studenikin et al. recently demonstrated a significant enhancement of the fringe contrast of coherent Landau-Zener-Stückelberg (LZS) oscillations between singlet S and triplet T+ two-spin states using a modified charge detection technique called enhanced charge detection (ECD). In this paper we explain the amplitude phase reversal and confirm the magnitude of the effect is consistent with our calibrations. We also show that the enhancement cannot be explained by a T{sub 1} effect.

  13. A correction to the research article titled: "Amplification of the driving oncogene, KRAS or BRAF, underpins acquired resistance to MEK1/2 inhibitors in colorectal cancer cells" by A. S. Little, K. Balmanno, M. J. Sale, S. Newman, J. R. Dry, M. Hampson, P. A. W. Edwards, P. D. Smith, S. J. Cook.

    Science.gov (United States)

    Little, Annette S; Balmanno, Kathryn; Sale, Matthew J; Newman, Scott; Dry, Jonathan R; Hampson, Mark; Edwards, Paul A W; Smith, Paul D; Cook, Simon J

    2011-01-01

    The acquisition of resistance to protein kinase inhibitors is a growing problem in cancer treatment. We modeled acquired resistance to the MEK1/2 (mitogen-activated or extracellular signal–regulated protein kinase kinases 1 and 2) inhibitor selumetinib (AZD6244) in colorectal cancer cell lines harboring mutations in BRAF (COLO205 and HT29 lines) or KRAS (HCT116 and LoVo lines). AZD6244-resistant derivatives were refractory to AZD6244-induced cell cycle arrest and death and exhibited a marked increase in ERK1/2 (extracellular signal–regulated kinases 1 and 2) pathway signaling and cyclin D1 abundance when assessed in the absence of inhibitor. Genomic sequencing revealed no acquired mutations in MEK1 or MEK2, the primary target of AZD6244. Rather, resistant lines showed a marked up-regulation of their respective driving oncogenes, BRAF600E or KRAS13D, due to intrachromosomal amplification. Inhibition of BRAF reversed resistance to AZD6244 in COLO205 cells, which suggested that combined inhibition of MEK1/2 and BRAF may reduce the likelihood of acquired resistance in tumors with BRAF600E. Knockdown of KRAS reversed AZD6244 resistance in HCT116 cells as well as reduced the activation of ERK1/2 and protein kinase B; however, the combined inhibition of ERK1/2 and phosphatidylinositol 3-kinase signaling had little effect on AZD6244 resistance, suggesting that additional KRAS effector pathways contribute to this process. Microarray analysis identified increased expression of an 18-gene signature previously identified as reflecting MEK1/2 pathway output in resistant cells. Thus, amplification of the driving oncogene (BRAF600E or KRAS13D) can drive acquired resistance to MEK1/2 inhibitors by increasing signaling through the ERK1/2 pathway. However, up-regulation of KRAS13D leads to activation of multiple KRAS effector pathways, underlining the therapeutic challenge posed by KRAS mutations. These results may have implications for the use of combination therapies.

  14. Development of reverse transcription loop-mediated isothermal amplification assay as a simple detection method of Chrysanthemum stem necrosis virus in chrysanthemum and tomato.

    Science.gov (United States)

    Suzuki, Ryoji; Fukuta, Shiro; Matsumoto, Yuho; Hasegawa, Toru; Kojima, Hiroko; Hotta, Makiko; Miyake, Noriyuki

    2016-10-01

    For a simple and rapid detection of Chrysanthemum stem necrosis virus (CSNV) from chrysanthemum and tomato, a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed. A primer set designed to the genome sequences of CSNV worked most efficiently at 63°C and could detect CSNV RNA within 12min by fluorescence monitoring using an isothermal DNA amplification and fluorescence detection device. The result of a specificity test using seven other viruses and one viroid-infectable chrysanthemum or tomato showed that the assay could amplify CSNV specifically, and a sensitivity comparison showed that the RT-LAMP assay was as sensitive as the reverse transcriptase polymerase chain reaction. The RT-LAMP assay using crude RNA, extracted simply, could detect CSNV. Overall, the RT-LAMP assay was found to be a simple, specific, convenient, and time-saving method for CSNV detection.

  15. Clinical evaluation of a loop-mediated isothermal amplification (LAMP assay for rapid detection of Neisseria meningitidis in cerebrospinal fluid.

    Directory of Open Access Journals (Sweden)

    DoKyung Lee

    Full Text Available Neisseria meningitidis (Nm is a leading causative agent of bacterial meningitis in humans. Traditionally, meningococcal meningitis has been diagnosed by bacterial culture. However, isolation of bacteria from patients' cerebrospinal fluid (CSF is time consuming and sometimes yields negative results. Recently, polymerase chain reaction (PCR-based diagnostic methods of detecting Nm have been considered the gold standard because of their superior sensitivity and specificity compared with culture. In this study, we developed a loop-mediated isothermal amplification (LAMP method and evaluated its ability to detect Nm in cerebrospinal fluid (CSF.We developed a meningococcal LAMP assay (Nm LAMP that targets the ctrA gene. The primer specificity was validated using 16 strains of N. meningitidis (serogroup A, B, C, D, 29-E, W-135, X, Y, and Z and 19 non-N. meningitidis species. Within 60 min, the Nm LAMP detected down to ten copies per reaction with sensitivity 1000-fold more than that of conventional PCR. The LAMP assays were evaluated using a set of 1574 randomly selected CSF specimens from children with suspected meningitis collected between 1998 and 2002 in Vietnam, China, and Korea. The LAMP method was shown to be more sensitive than PCR methods for CSF samples (31 CSF samples were positive by LAMP vs. 25 by PCR. The detection rate of the LAMP method was substantially higher than that of the PCR method. In a comparative analysis of the PCR and LAMP assays, the clinical sensitivity, specificity, positive predictive value, and negative predictive value of the LAMP assay were 100%, 99.6%, 80.6%, and 100%, respectively.Compared to PCR, LAMP detected Nm with higher analytical and clinical sensitivity. This sensitive and specific LAMP method offers significant advantages for screening patients on a population basis and for diagnosis in clinical settings.

  16. Rapid detection of all known ebolavirus species by reverse transcription-loop-mediated isothermal amplification (RT-LAMP).

    Science.gov (United States)

    Oloniniyi, Olamide K; Kurosaki, Yohei; Miyamoto, Hiroko; Takada, Ayato; Yasuda, Jiro

    2017-03-26

    Ebola virus disease (EVD), a highly virulent infectious disease caused by ebolaviruses, has a fatality rate of 25-90%. Without a licensed chemotherapeutic agent or vaccine for the treatment and prevention of EVD, control of outbreaks requires accurate and rapid diagnosis of cases. In this study, five sets of six oligonucleotide primers targeting the nucleoprotein gene were designed for specific identification of each of the five ebolavirus species using reverse transcription-loop mediated isothermal amplification (RT-LAMP) assay. The detection limits of the ebolavirus species-specific primer sets were evaluated using in vitro transcribed RNAs. The detection limit of species-specific RT-LAMP assays for Zaire ebolavirus, Sudan ebolavirus, Taï Forest ebolavirus, and Bundibugyo ebolavirus was 256 copies/reaction, while the detection limit for Reston ebolavirus was 64 copies/reaction, and the detection time for each of the RT-LAMP assays was 13.3±3.0, 19.8±4.6, 14.3±0.6, 16.1±4.7, and 19.8±2.4min (mean±SD), respectively. The sensitivity of the species-specific RT-LAMP assays were similar to that of the established RT-PCR and quantitative RT-PCR assays for diagnosis of EVD and are suitable for field or point-of-care diagnosis. The RT-LAMP assays were specific for the detection of the respective species of ebolavirus with no cross reaction with other species of ebolavirus and other viral hemorrhagic fever viruses such as Marburg virus, Lassa fever virus, and Dengue virus. The species-specific RT-LAMP assays developed in this study are rapid, sensitive, and specific and could be useful in case of an EVD outbreak.

  17. Evaluation of loop-mediated isothermal amplification for the rapid, reliable, and robust detection of Salmonella in produce.

    Science.gov (United States)

    Yang, Qianru; Wang, Fei; Jones, Kelly L; Meng, Jianghong; Prinyawiwatkul, Witoon; Ge, Beilei

    2015-04-01

    Rapid, reliable, and robust detection of Salmonella in produce remains a challenge. In this study, loop-mediated isothermal amplification (LAMP) was comprehensively evaluated against real-time quantitative PCR (qPCR) for detecting diverse Salmonella serovars in various produce items (cantaloupe, pepper, and several varieties of lettuce, sprouts, and tomato). To mimic real-world contamination events, produce samples were surface-inoculated with low concentrations (1.1-2.9 CFU/25 g) of individual Salmonella strains representing ten serovars and tested after aging at 4 °C for 48 h. Four DNA extraction methods were also compared using produce enrichment broths. False-positive or false-negative results were not observed among 178 strains (151 Salmonella and 27 non-Salmonella) used to evaluate assay specificity. The detection limits for LAMP were 1.8-4 CFU per reaction in pure culture and 10(4)-10(6) CFU per 25 g (i.e., 10(2)-10(4) CFU per g) in produce without enrichment, comparable to those obtained by qPCR. After 6-8 h of enrichment, both LAMP and qPCR consistently detected these low concentrations of Salmonella of diverse serovars in all produce items except sprouts. The PrepMan Ultra sample preparation reagent yielded the best results among the four DNA extraction methods. Upon further validation, LAMP may be a valuable tool for routine Salmonella testing in produce. The difficulty of detecting Salmonella in sprouts, whether using LAMP or qPCR, warrants further study.

  18. Development of loop-mediated isothermal amplification (LAMP) assay for the rapid detection of Penicillium nordicum in dry-cured meat products.

    Science.gov (United States)

    Ferrara, M; Perrone, G; Gallo, A; Epifani, F; Visconti, A; Susca, A

    2015-06-02

    The need of powerful diagnostic tools for rapid, simple, and cost-effective detection of food-borne fungi has become very important in the area of food safety. Currently, several isothermal nucleic acid amplification methods have been developed as an alternative to PCR-based analyses. Loop-mediated isothermal amplification (LAMP) is one of these innovative methods; it requires neither gel electrophoresis to separate and visualize the products nor expensive laboratory equipment and it has been applied already for detection of pathogenic organisms. In the current study, we developed a LAMP assay for the specific detection of Penicillium nordicum, the major causative agent of ochratoxin A contamination in protein-rich food, especially dry-cured meat products. The assay was based on targeting otapksPN gene, a key gene in the biosynthesis of ochratoxin A (OTA) in P. nordicum. Amplification of DNA during the reaction was detected directly in-tube by color transition of hydroxynaphthol blue from violet to sky blue, visible to the naked eye, avoiding further post amplification analyses. Only DNAs isolated from several P. nordicum strains led to positive results and no amplification was observed from non-target OTA and non OTA-producing strains. The assay was able to detect down to 100 fg of purified targeted genomic DNA or 10(2) conidia/reaction within 60 min. The LAMP assay for detection and identification of P. nordicum was combined with a rapid DNA extraction method set up on serially diluted conidia, providing an alternative rapid, specific and sensitive DNA-based method suitable for application directly "on-site", notably in key steps of dry-cured meat production.

  19. Evaluation of a real-time PCR and a loop-mediated isothermal amplification for detection of Xanthomonas arboricola pv. pruni in plant tissue samples

    NARCIS (Netherlands)

    Palacio-Bielsa, Ana; López-Soriano, Pablo; Bühlmann, Andreas; Doorn, van Joop; Pham, Khanh; Cambra, Miguel A.; Berruete, Isabel M.; Pothier, Joël F.; Duffy, Brion; Olmos, Antonio; López, María M.

    2015-01-01

    Operational capacity of real-time PCR and loop-mediated isothermal amplification (LAMP) diagnostic assays for detection of Xanthomonas arboricola pv. pruni was established in a ring-test involving four laboratories. Symptomatic and healthy almond leaf samples with two methods of sample

  20. Evaluation of a real-time PCR and a loop-mediated isothermal amplification for detection of Xanthomonas arboricola pv. pruni in plant tissue samples

    NARCIS (Netherlands)

    Palacio-Bielsa, Ana; López-Soriano, Pablo; Bühlmann, Andreas; Doorn, van Joop; Pham, Khanh; Cambra, Miguel A.; Berruete, Isabel M.; Pothier, Joël F.; Duffy, Brion; Olmos, Antonio; López, María M.

    2015-01-01

    Operational capacity of real-time PCR and loop-mediated isothermal amplification (LAMP) diagnostic assays for detection of Xanthomonas arboricola pv. pruni was established in a ring-test involving four laboratories. Symptomatic and healthy almond leaf samples with two methods of sample preparatio

  1. Development and implementation of real-time nucleic acid amplification for the detection of enterovirus infections in comparison to rapid culture of various clinical specimens

    NARCIS (Netherlands)

    van Doornum, G J J; Schutten, Martin; Voermans, J; Guldemeester, G J J; Niesters, H G M

    2007-01-01

    Several real-time PCR and nucleic acid sequence-based amplification (NASBA) primer pairs and a modified real-time PCR primer pair for the detection of enteroviruses were compared. The modified real-time PCR primer pair was evaluated on clinical samples in comparison with cell culture using the Magna

  2. Development of reverse transcription loop-mediated isothermal amplification assay for rapid detection of an emerging potyvirus: tomato necrotic stunt virus

    Science.gov (United States)

    Tomato necrotic stunt virus (ToNStV) is an emerging potyvirus that causes severe stunting to the infected tomato plants. A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for a sensitive detection of ToNStV. The sensitivity of RT-LAMP was comparable to th...

  3. Development of a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of Sugarcane mosaic virus and Sorghum mosaic virus in sugarcane

    Science.gov (United States)

    A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for detecting Sugarcane mosaic virus (SCMV) and Sorghum mosaic virus (SrMV) in sugarcane. Six sets of four primers corresponding to the conserved coat protein gene were designed for each virus and their succ...

  4. Development of a rapid diagnostic assay for the detection of tomato chlorotic dwarf viroid based on isothermal reverse-transcription-recombinase polymerase amplification

    Science.gov (United States)

    A molecular diagnostic assay utilizing reverse transcription-recombinase polymerase amplification (RT-RPA) at an isothermal constant temperature of 39 °C and target-specific primers and probe were developed for the rapid, sensitive, and specific detection of tomato chlorotic dwarf viroid (TCDVd) in ...

  5. Rapid detection of TEM, SHV and CTX-M extended-spectrum ß-lactamases in Enterobacteriaceae using ligation-mediated amplification with microarray analysis

    NARCIS (Netherlands)

    Cohen Stuart, J.; Dierikx, C.M.; Naiemi, Al N.; Karczmarek, A.; Hoek, A.; Vos, P.; Fluit, A.C.; Scharringa, J.; Duim, B.; Mevius, D.J.; Leverstein-van Hall, M.A.

    2010-01-01

    Objectives Fast and adequate detection of extended-spectrum ß-lactamases (ESBLs) is crucial for infection control measures and the choice of antimicrobial therapy. The aim of this study was to develop and evaluate a novel ESBL assay using ligation-mediated amplification combined with microarray

  6. Hetero-enzyme-based two-round signal amplification strategy for trace detection of aflatoxin B1 using an electrochemical aptasensor.

    Science.gov (United States)

    Zheng, Wanli; Teng, Jun; Cheng, Lin; Ye, Yingwang; Pan, Daodong; Wu, Jingjing; Xue, Feng; Liu, Guodong; Chen, Wei

    2016-06-15

    An electrochemical aptasensor for trace detection of aflatoxin B1 (AFB1) was developed by using an aptamer as the recognition unit while adopting the telomerase and EXO III based two-round signal amplification strategy as the signal enhancement units. The telomerase amplification was used to elongate the ssDNA probes on the surface of gold nanoparticles, by which the signal response range of the signal-off model electrochemical aptasensor could be correspondingly enlarged. Then, the EXO III amplification was used to hydrolyze the 3'-end of the dsDNA after the recognition of target AFB1, which caused the release of bounded AFB1 into the sensing system, where it participated in the next recognition-sensing cycle. With this two-round signal amplified electrochemical aptasensor, target AFB1 was successfully measured at trace concentrations with excellent detection limit of 0.6*10(-4)ppt and satisfied specificity due to the excellent affinity of the aptamer against AFB1. Based on this designed two-round signal amplification strategy, both the sensing range and detection limit were greatly improved. This proposed ultrasensitive electrochemical aptasensor method was also validated by comparison with the classic instrumental methods. Importantly, this hetero-enzyme based two-round signal amplified electrochemical aptasensor offers a great promising protocol for ultrasensitive detection of AFB1 and other mycotoxins by replacing the core recognition sequence of the aptamer.

  7. Reliability of nucleic acid amplification methods for detection of Chlamydia trachomatis in urine: results of the first international collaborative quality control study among 96 laboratories

    NARCIS (Netherlands)

    R.P.A.J. Verkooyen (Roel); G.T. Noordhoek; P.E. Klapper; J. Reid; J. Schirm; G.M. Cleator; M. Ieven; G. Hoddevik

    2003-01-01

    textabstractThe first European Quality Control Concerted Action study was organized to assess the ability of laboratories to detect Chlamydia trachomatis in a panel of urine samples by nucleic acid amplification tests (NATs). The panel consisted of lyophilized urine samples, includ

  8. Amplification of interference color by using liquid crystal for protein detection

    Energy Technology Data Exchange (ETDEWEB)

    Zhu, Qingdi; Yang, Kun-Lin, E-mail: cheyk@nus.edu.sg [Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, 117576 Singapore (Singapore)

    2013-12-09

    Micrometer-sized, periodic protein lines printed on a solid surface cause interference color which is invisible to the naked eye. However, the interference color can be amplified by using a thin layer of liquid crystal (LC) covered on the surface to form a phase diffraction grating. Strong interference color can thus be observed under ambient light. By using the LC-amplified interference color, we demonstrate naked-eye detection of a model protein—immunoglobulin G (IgG). Limit of detection can reach 20 μg/ml of IgG without using any instrumentation. This detection method is potentially useful for the development of low-cost and portable biosensors.

  9. Detection of Flavobacterium psychrophilum from fish tissue and water samples by PCR amplification

    DEFF Research Database (Denmark)

    Wiklund, T.; Madsen, Lone; Bruun, Morten Sichlau

    2000-01-01

    Rainbow trout fry syndrome and cold-water disease, caused by Flavobacterium psychrophilum, are important diseases in farmed salmonids. Some of the presently available techniques for the detection of Fl. psychrophilum are either time consuming or lack sufficient sensitivity. In the present...... investigation, the possible detection of Fl. psychrophilum from fish tissue and water samples was examined using nested PCR with DNA probes against a sequence of the 16S rRNA genes. The DNA was extracted using Chelex(R) 100 chelating resin. The primers, which were tested against strains isolated from diseased...... to be more sensitive than agar cultivation of tissue samples from the brain of rainbow trout injected with Fl. psychrophilum. In non-sterile fresh water seeded with Fl. psychrophilum the detection limit of the PCR- assay was 1.7 cfu in the PCR tube, corresponding to 110 cfu ml(-1) water. The PCR...

  10. The use of a two-tiered testing strategy for the simultaneous detection of small EGFR mutations and EGFR amplification in lung cancer.

    Directory of Open Access Journals (Sweden)

    Marzena Anna Lewandowska

    Full Text Available Lung cancer is the leading cause of cancer-related death worldwide. Recent progress in lung cancer diagnosis and treatment has been achieved due to a better understanding the molecular mechanisms of the disease and the identification of biomarkers that allow more specific cancer treatments. One of the best known examples of personalized therapy is the use of tyrosine kinase inhibitors, such as gefitinib and erlotinib, for the successful treatment of non-small-cell lung cancer patients selected based on the specific EGFR mutations. Therefore, the reliable detection of mutations is critical for the application of appropriate therapy. In this study, we tested a two-tiered mutation detection strategy using real-time PCR assays as a well-validated high-sensitivity method and multiplex ligation-dependent probe amplification (MLPA-based EGFRmut+ assay as a second-tier standard-sensitivity method. One additional advantage of the applied MLPA method is that it allows the simultaneous detection of EGFR mutations and copy-number alterations (i.e., amplifications in EGFR, MET and ERBB2. Our analysis showed high concordance between these two methods. With the use of this two-tier strategy, we reliably determined the frequency of EGFR mutations and EGFR, MET and ERBB2 amplifications in over 200 lung cancer samples. Additionally, taking advantage of simultaneous copy number and small mutation analyses, we showed a very strong correlation between EGFR mutations and EGFR amplifications and a mutual exclusiveness of EGFR mutations/amplifications with MET and ERBB2 amplifications. Our results proved the reliability and usefulness of the two-tiered EGFR testing strategy.

  11. The use of a two-tiered testing strategy for the simultaneous detection of small EGFR mutations and EGFR amplification in lung cancer.

    Science.gov (United States)

    Lewandowska, Marzena Anna; Czubak, Karol; Klonowska, Katarzyna; Jozwicki, Wojciech; Kowalewski, Janusz; Kozlowski, Piotr

    2015-01-01

    Lung cancer is the leading cause of cancer-related death worldwide. Recent progress in lung cancer diagnosis and treatment has been achieved due to a better understanding the molecular mechanisms of the disease and the identification of biomarkers that allow more specific cancer treatments. One of the best known examples of personalized therapy is the use of tyrosine kinase inhibitors, such as gefitinib and erlotinib, for the successful treatment of non-small-cell lung cancer patients selected based on the specific EGFR mutations. Therefore, the reliable detection of mutations is critical for the application of appropriate therapy. In this study, we tested a two-tiered mutation detection strategy using real-time PCR assays as a well-validated high-sensitivity method and multiplex ligation-dependent probe amplification (MLPA)-based EGFRmut+ assay as a second-tier standard-sensitivity method. One additional advantage of the applied MLPA method is that it allows the simultaneous detection of EGFR mutations and copy-number alterations (i.e., amplifications) in EGFR, MET and ERBB2. Our analysis showed high concordance between these two methods. With the use of this two-tier strategy, we reliably determined the frequency of EGFR mutations and EGFR, MET and ERBB2 amplifications in over 200 lung cancer samples. Additionally, taking advantage of simultaneous copy number and small mutation analyses, we showed a very strong correlation between EGFR mutations and EGFR amplifications and a mutual exclusiveness of EGFR mutations/amplifications with MET and ERBB2 amplifications. Our results proved the reliability and usefulness of the two-tiered EGFR testing strategy.

  12. Rapid and Sensitive Salmonella Typhi Detection in Blood and Fecal Samples Using Reverse Transcription Loop-Mediated Isothermal Amplification.

    Science.gov (United States)

    Fan, Fenxia; Yan, Meiying; Du, Pengcheng; Chen, Chen; Kan, Biao

    2015-09-01

    Typhoid fever caused by Salmonella enterica serovar Typhi remains a significant public health problem in developing countries. Although the main method for diagnosing typhoid fever is blood culture, the test is time consuming and not always able to detect infections. Thus, it is very difficult to distinguish typhoid from other infections in patients with nonspecific symptoms. A simple and sensitive laboratory detection method remains necessary. The purpose of this study is to establish and evaluate a rapid and sensitive reverse transcription-based loop-mediated isothermal amplification (RT-LAMP) method to detect Salmonella Typhi infection. In this study, a new specific gene marker, STY1607, was selected to develop a STY1607-RT-LAMP assay; this is the first report of specific RT-LAMP detection assay for typhoid. Human-simulated and clinical blood/stool samples were used to evaluate the performance of STY1607-RT-LAMP for RNA detection; this method was compared with STY1607-LAMP, reverse transcription real-time polymerase chain reaction (rRT-PCR), and bacterial culture methods for Salmonella Typhi detection. Using mRNA as the template, STY1607-RT-LAMP exhibited 50-fold greater sensitivity than STY1607-LAMP for DNA detection. The STY1607-RT-LAMP detection limit is 3 colony-forming units (CFU)/mL for both the pure Salmonella Typhi samples and Salmonella Typhi-simulated blood samples and was 30 CFU/g for the simulated stool samples, all of which were 10-fold more sensitive than the rRT-PCR method. RT-LAMP exhibited improved Salmonella Typhi detection sensitivity compared to culture methods and to rRT-PCR of clinical blood and stool specimens from suspected typhoid fever patients. Because it can be performed without sophisticated equipment or skilled personnel, RT-LAMP is a valuable tool for clinical laboratories in developing countries. This method can be applied in the clinical diagnosis and care of typhoid fever patients as well as for a quick public health response.

  13. A reverse transcription loop-mediated isothermal amplification (LAMP) assay for the detection of feline Coronavirus

    National Research Council Canada - National Science Library

    Angelica Stranieri; Stefania Lauzi; Alessia Giordano; Saverio Paltrinieri

    2016-01-01

    ...). The addition of two loop primers allows the reaction time to be of one hour only (Nagamine et al., 2002). The aim of this study was to develop a reverse transcription LAMP assay for an easy and inexpensive detection of feline Coronavirus...

  14. On-chip detection of a single nucleotide polymorphism without polymerase amplification.

    Science.gov (United States)

    Han, Jinhee; Tan, Matthew; Sudheendra, Lakshmana; Weiss, Robert H; Kennedy, Ian M

    2014-09-01

    A nanoparticle-assembled photonic crystal (PC) array was used to detect single nucleotide polymorphism (SNP). The assay platform with PC nanostructure enhanced the fluorescent signal from nanoparticle-hybridized DNA complexes due to phase matching of excitation and emission. Nanoparticles coupled with probe DNA were trapped into nanowells in an array by using an electrophoretic particle entrapment system. The PC/DNA assay platform was able to identify a 1 base pair (bp) difference in synthesized nucleotide sequences that mimicked the mutation seen in a feline model of human autosomal dominant polycystic kidney disease (PKD) with a sensitivity of 0.9 fg/mL (50 aM)-sensitivity, which corresponds to 30 oligos/array. The reliability of the PC/DNA assay platform to detect SNP in a real sample was demonstrated by using genomic DNA (gDNA) extracted from the urine and blood of two PKD(-) wild type and three PKD positive cats. The standard curves for PKD positive (PKD(+)) and negative (PKD(-)) DNA were created using two feline-urine samples. An additional three urine samples were analyzed in a similar fashion and showed satisfactory agreement with the standard curve, confirming the presence of the mutation in affected urine. The limit of detection (LOD) was 0.005 ng/mL which corresponds to 6 fg per array for gDNA in urine and blood. The PC system demonstrated the ability to detect a number of genome equivalents for the PKD SNP that was very similar to the results reported with real time polymerase chain reaction (PCR). The favorable comparison with quantitative PCR suggests that the PC technology may find application well beyond the detection of the PKD SNP, into areas where a simple, cheap and portable nucleic acid analysis is desirable.

  15. Double-hairpin molecular-beacon-based amplification detection for gene diagnosis linked to cancer.

    Science.gov (United States)

    Xu, Huo; Zhang, Rongbo; Li, Feng; Zhou, Yingying; Peng, Ting; Wang, Xuedong; Shen, Zhifa

    2016-09-01

    A powerful double-hairpin molecular beacon (DHMB) was developed for cancer-related KRAS gene detection based on the one-to-two stoichiometry. During target DNA detection, DHMB can execute signal transduction even if no any exogenous element is involved. Unlike the conventional molecular beacon based on the one-to-one interaction, one target DNA not only hybridizes with one DHMB and opens its hairpin but also promotes the interaction between two DHMBs, causing the separation of two fluorophores from quenchers. This leads to an enhanced fluorescence signal. As a result, the target KRAS gene is able to be detected within a wide dynamic range from 0.05 to 200 nM with the detection limit of 50 pM, indicating a dramatic improvement compared with traditional molecular beacons. Moreover, the point mutations existing in target DNAs can be easily screened. The potential application for target species in real samples was indicated by the analysis of PCR amplicons of DNAs from the DNA extracted from SW620 cell. Besides becoming a promising candidate probe for molecular biology research and clinical diagnosis of genetic diseases, the DHMB is expected to provide a significant insight into the design of DNA probe-based homogenous sensing systems. Graphical Abstract A powerful double-hairpin molecular beacon (DHMB) was developed for cancer-related gene KRAS detection based on the one-to-two stoichiometry. Without the help of any exogenous probe, the point mutation is easily screened, and the target DNA can be quantified down to 50 pM, indicating a dramatic improvement compared with traditional molecular beacons.

  16. Loop-mediated isothermal amplification (LAMP) assays for rapid detection and differentiation of Nosema apis and N. ceranae in honeybees.

    Science.gov (United States)

    Ptaszyńska, Aneta A; Borsuk, Grzegorz; Woźniakowski, Grzegorz; Gnat, Sebastian; Małek, Wanda

    2014-08-01

    Nosemosis is a contagious disease of honeybees (Apis mellifera) manifested by increased winter mortality, poor spring build-up and even the total extinction of infected bee colonies. In this paper, loop-mediated isothermal amplifications (LAMP) were used for the first time to identify and differentiate N. apis and N. ceranae, the causative agents of nosemosis. LAMP assays were performed at a constant temperature of 60 °C using two sets of six species-specific primers, recognising eight distinct fragments of 16S rDNA gene and GspSSD polymerase with strand displacement activity. The optimal time for LAMP and its Nosema species sensitivity and specificity were assessed. LAMP only required 30 min for robust identification of the amplicons. Ten-fold serial dilutions of total DNA isolated from bees infected with microsporidia were used to determine the detection limit of N. apis and N. ceranae DNAs by LAMP and standard PCR assays. LAMP appeared to be 10(3) -fold more sensitive than a standard PCR in detecting N. apis and N. ceranae. LAMP methods developed by us are highly Nosema species specific and allow to identify and differentiate N. apis and N. ceranae.

  17. Multiplex ligation-dependent probe amplification for rapid detection of deletions and duplications in the dystrophin gene

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Objective:Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are X-linked disorders caused by mutations in the dystrophin gene. The majority of recognized mutations are copy number changes of individual exons. The objective of the present study was to assess the multiplex ligation-dependent probe amplification (MLPA) effects of detection of gene mutations. Methods: Samples of 20 control males and 80 males and their mothers referred to our diagnostic facility on the clinical suspicion of DMD or BMD were tested by MLPA and multiplex PCR. Results: The mean DQs for all peak of 20 control male samples was 1.02 (range from 0.83 to 1.21) by MLPA. Deletions or duplications were identified in 6 out of 31 families that had been previously tested as negative by multiplex PCR. One case of complex rearrangement involving a duplication of two regions: dupEX3-9 and dupEX 17-41 were found by MLPA. Conclusions: MLPA is a highly sensitive method and rapid alternative to multiplex PCR for detection of DMD and BMD.

  18. Specific Detection of Clavibacter michiganensis subsp. sepedonicus by Amplification of Three Unique DNA Sequences Isolated by Subtraction Hybridization.

    Science.gov (United States)

    Mills, D; Russell, B W; Hanus, J W

    1997-08-01

    ABSTRACT Three single-copy, unique DNA fragments, designated Cms50, Cms72, and Cms85, were isolated from strain CS3 of Clavibacter michiganensis subsp. sepedonicus by subtraction hybridization using driver DNA from C. michiganensis subsp. insidiosus, C. michiganensis subsp. michiganensis, and Rhodococcus facians. Radio-labeled probes made of these fragments and used in Southern blot analysis revealed each to be absolutely specific to all North American C. michiganensis subsp. sepedonicus strains tested, including plasmidless and nonmucoid strains. The probes have no homology with genomic DNA from related C. michiganensis subspecies insidiosus, michiganensis, and tessellarius, nor with DNA from 11 additional bacterial species and three unidentified strains, some of which have been previously reported to display cross-reactivity with C. michiganensis subsp. sepedonicus-specific antisera. The three fragments shared no homology, and they appeared to be separated from each other by at least 20 kbp in the CS3 genome. Internal primer sets permitted amplification of each fragment by the polymerase chain reaction (PCR) only from C. michiganensis subsp. sepedonicus DNA. In a PCR-based sensitivity assay using a primer set that amplifies Cms85, the lowest level of detection of C. michiganensis subsp. sepedonicus was 100 CFU per milliliter when cells were added to potato core fluid. Erroneous results that may arise from PCR artifacts and mutational events are, therefore, minimized by the redundancy of the primer sets, and the products should be verifiable with unique capture probes in sequence-based detection systems.

  19. Mtp-40 and alpha antigen gene fragment amplification for the detection of Mycobacterium tuberculosis in Colombian clinical specimens

    Directory of Open Access Journals (Sweden)

    Rosalba Alfonso

    2002-12-01

    Full Text Available In this study, the use of Mtp-40 and alpha antigen polymerase chain reaction (PCR amplification fragments for the precise tuberculosis (TB diagnosis was evaluated. One hundred and ninety two different samples were obtained from 113 patients with suspected TB. Mtp-40 and alpha antigen protein genes were amplified by the PCR technique and compared to both the "gold standard" (culture test, as well as the clinical parameters (including a clinical record and X-ray film exam in 113 patients. Thirty-eight of the 113 patients had a presumptive clinical diagnosis of TB; 74% being detected by PCR technique, 58% by culture and 44% by direct microscopic visualization. Weconclude that it is possible to use PCR as a suitable technique for the detection of any mycobacteria by means of the alpha antigen product, or the specific infection of Mycobacterium tuberculosis by means of the mtp-40 gene. This might be a good supporting tool in difficult clinical TB diagnosis and pauci-bacillary cases.

  20. Loop-mediated isothermal amplification (LAMP) assays for detection and identification of aquaculture pathogens: current state and perspectives.

    Science.gov (United States)

    Biswas, Gouranga; Sakai, Masahiro

    2014-04-01

    Since its invention in 2000, loop-mediated isothermal amplification (LAMP) assay has been one of the most extensively used molecular diagnostic tools in bio-medical fields due to the rapidity, accuracy, and cost-effectiveness of the technique. This technique has also earned popularity in aquaculture disease diagnosis. Aquaculture, as a result of its rapid intensification and expansion, experiences increased infectious disease occurrences. For maintenance of economic viability, rapid, sensitive and efficient diagnosis of disease causing agents is an important step prior to undertaking effective prevention and control measures in aquaculture. Constraints on time and expertise required for conventional biochemical, serological and polymerase chain reaction (PCR)-based techniques offer avenues in adoption of the LAMP by the aquaculturists at field conditions. This assay has been successfully applied in detection of several bacterial, viral and parasitic pathogens causing serious diseases in aquaculture. In this review, we endeavored to accommodate the LAMP methodology with its different recent improvements and an overview of its application for the detection of aquaculture-associated pathogens.

  1. Detection and identification of Trichophyton tonsurans from clinical isolates and hairbrush samples by loop-mediated isothermal amplification system.

    Science.gov (United States)

    Yo, Ayaka; Yamamoto, Mikachi; Nakayama, Takako; Ishikawa, Jun; Makimura, Koichi

    2016-09-01

    Since the 1990s, there have been reports of the spread of dermatophytosis caused by Trichophyton tonsurans among contact sports athletes in several countries, including Japan. This study was performed to develop a loop-mediated isothermal amplification (LAMP) system for rapid and accurate detection and identification of T. tonsurans from clinical isolates or hairbrush samples for diagnosis and to prevent the spread of infection. A specific primer set was prepared by comparing the whole genome sequence of T. tonsurans with those of six other closely related dermatophytes. After confirming the sensitivity and specificity of this system, LAMP assay was performed using 37 clinical samples obtained from three healthy volunteers and 24 judo athletes. A total of 155 fungal isolates (56 strains of various standard fungi, 96 identified T. tonsurans isolates, three hairbrush-cultured isolates from judo athletes) and 37 hairbrush samples (34 samples from 24 judo athletes, and three samples from three healthy volunteers) were used for culture and LAMP assay, respectively. The assay showed no cross-reactivity to standard strains other than T. tonsurans. The detection limit was 100 copies of DNA template per tube. All of the 96 T. tonsurans isolates were amplified, and all samples from healthy volunteers showed negative results. Four of the 34 hairbrush samples obtained from judo athletes showed positive results in LAMP assay, and two of the four were positive in both culture and LAMP assay. We developed a rapid LAMP system with high specificity and sensitivity for diagnosis of T. tonsurans infection.

  2. Loop-mediated isothermal amplification (LAMP) test for specific and rapid detection of Brucella abortus in cattle.

    Science.gov (United States)

    Karthik, K; Rathore, Rajesh; Thomas, Prasad; Arun, T R; Viswas, K N; Agarwal, R K; Manjunathachar, H V; Dhama, Kuldeep

    2014-01-01

    Brucella abortus, the major causative agent of abortion in cattle and a zoonotic pathogen, needs to be diagnosed at an early stage. Loop-mediated isothermal amplification (LAMP) test is easy to perform and also promising to be adapted at field level. To develop a LAMP assay for specific and rapid detection of B. abortus from clinical samples of cattle. LAMP primers were designed targeting BruAb2_0168 region using specific software tool and LAMP was optimized. The developed LAMP was tested for its specificity with 3 Brucella spp. and 11 other non-Brucella spp. Sensitivity of the developed LAMP was also carried out with known quantity of DNA. Cattle whole blood samples and aborted fetal stomach contents were collected and used for testing with developed LAMP assay and results were compared with polymerase chain reaction (PCR). The developed LAMP assay works at 61 °C for 60 min and the detection limit was observed to be 100-fold more than the conventional PCR that is commonly used for diagnosis of B. abortus. Clinical sensitivity and specificity of the developed LAMP assay was 100% when compared with Rose Bengal plate test and standard tube agglutination test. SYB® green dye I was used to visualize the result with naked eye. The novelty of the developed LAMP assay for specifically detecting B. abortus infection in cattle along with its inherent rapidness and high sensitivity can be employed for detecting this economically important pathogen of cattle at field level as well be exploited for screening of human infections.

  3. Visual detection of the human metapneumovirus using reverse transcription loop-mediated isothermal amplification with hydroxynaphthol blue dye

    Directory of Open Access Journals (Sweden)

    Wang Xiang

    2012-07-01

    Full Text Available Abstract Background Human metapneumovirus (hMPV is a major cause of acute respiratory infections ranging from wheezing to bronchiolitis and pneumonia in children worldwide. The objective of this study is to develop a visual reverse transcription loop-mediated isothermal amplification (RT-LAMP assay for the detection of hMPV and applied to the clinical samples. Results In this study, visual RT-LAMP assay for hMPV was performed in one step with the addition of hydroxynaphthol blue (HNB, and were used to detect respiratory samples. Six primers, including two outer primers (F3 and B3, two inner primers (FIP, BIP and two loop primers (LF and LB, were designed for hMPV N gene by the online software. Moreover, the RT-LAMP assay showed good specificity and no cross-reactivity was observed with human rhinovirus (HRV, human respiratory syncytial Virus (RSV, or influenza virus A/PR/8/34 (H1N1. The detection limit of the RT-LAMP assay was approximately ten viral RNA copies, lower than that of traditional reverse transcriptase polymerase chain reaction (RT-PCR 100 RNA copies. In the 176 nasopharyngeal samples, 23 (13.1% were conformed as hMPV positive by RT-LAMP, but 18 (10.2% positive by RT-PCR. Conclusion Compared with conventional RT-PCR, the visual hMPV RT-LAMP assay performed well in the aspect of detect time, sensitivity, specificity and visibility. It is anticipated that the RT-LAMP will be used for clinical tests in hospital or field testing during outbreaks and in emergency.

  4. Oncogenic viruses and cancer

    Institute of Scientific and Technical Information of China (English)

    Guangxiang; George; Luo; Jing-hsiung; James; Ou

    2015-01-01

    <正>This special issue of the journal is dedicated to the important topic of oncogenic viruses and cancer.It contains seven review articles covering all known oncogenic viruses except for human T-lymphotropic virus type1(HTLV-1).These review articles are contributed by experts on specific viruses and their associated human cancers.Viruses account for about 20%of total human cancer cases.Although many viruses can cause various tumors in animals,only seven of them

  5. Imaging oncogene expression

    Energy Technology Data Exchange (ETDEWEB)

    Mukherjee, Archana [Department of Radiology, Thomas Jefferson University, Philadelphia, PA 19107 (United States)], E-mail: Archana.Mukherjee@jefferson.edu; Wickstrom, Eric [Department of Biochemistry and Molecular Biology, Thomas Jefferson University, 233S, 10th street, Philadelphia, PA 19107 (United States)], E-mail: eric@tesla.jci.tju.edu; Thakur, Mathew L. [Department of Radiology, Thomas Jefferson University, Philadelphia, PA 19107 (United States)], E-mail: Mathew.Thakur@jefferson.edu

    2009-05-15

    This review briefly outlines the importance of molecular imaging, particularly imaging of endogenous gene expression for noninvasive genetic analysis of radiographic masses. The concept of antisense imaging agents and the advantages and challenges in the development of hybridization probes for in vivo imaging are described. An overview of the investigations on oncogene expression imaging is given. Finally, the need for further improvement in antisense-based imaging agents and directions to improve oncogene mRNA targeting is stated.

  6. Development and clinical performance of high throughput loop-mediated isothermal amplification for detection of malaria

    OpenAIRE

    Perera, Rushini S.; Ding, Xavier C; Tully, Frank; Oliver, James; Bright, Nigel; Bell, David; Chiodini, Peter L; Gonzalez, Iveth J.; Spencer D Polley

    2017-01-01

    Background Accurate and efficient detection of sub-microscopic malaria infections is crucial for enabling rapid treatment and interruption of transmission. Commercially available malaria LAMP kits have excellent diagnostic performance, though throughput is limited by the need to prepare samples individually. Here, we evaluate the clinical performance of a newly developed high throughput (HTP) sample processing system for use in conjunction with the Eiken malaria LAMP kit. Methods The HTP syst...

  7. Development and clinical performance of high throughput loop-mediated isothermal amplification for detection of malaria.

    OpenAIRE

    Perera, RS; Ding, XC; Tully, F.; Oliver, J.; Bright, N; Bell, D.; Chiodini, PL; Gonzalez, IJ; Polley, SD

    2017-01-01

    Background Accurate and efficient detection of sub-microscopic malaria infections is crucial for enabling rapid treatment and interruption of transmission. Commercially available malaria LAMP kits have excellent diagnostic performance, though throughput is limited by the need to prepare samples individually. Here, we evaluate the clinical performance of a newly developed high throughput (HTP) sample processing system for use in conjunction with the Eiken malaria LAMP kit. Methods The HTP syst...

  8. Surface plasmon resonance detection of silver ions and cysteine using DNA intercalator-based amplification.

    Science.gov (United States)

    Chang, Chia-Chen; Lin, Shenhsiung; Wei, Shih-Chung; Chu-Su, Yu; Lin, Chii-Wann

    2012-03-01

    We report the development of a surface plasmon resonance sensor based on the silver ion (Ag(+))-induced conformational change of a cytosine-rich, single-stranded DNA for the detection of Ag(+) and cysteine (Cys) in aqueous solutions. In the free state, single-stranded oligonucleotides fold into double-helical structures through the addition of Ag(+) to cytosine–cytosine (C–C) mismatches. However, in the presence of Cys, which competitively binds to Ag(+), the formation of the C–Ag(+)–C assembly is inhibited, resulting in free-state, single-stranded oligonucleotides. To enhance sensitivity, the DNA intercalator, daunorubicin, was employed to achieve signal enhancement. The detection limit for Ag(+) was 10 nM with a measurement range of 50–2,000 nM, and the detection limit for Cys was 50 nM with a measurement range of 50–2,000 nM. This simple assay was also used to individually determine the spiked Ag(+) concentration in water samples and Cys concentrations in biological fluid samples.

  9. Two sequential PCR amplifications for detection of Schistosoma mansoni in stool samples with low parasite load

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    Maria Cristina Carvalho do Espírito-Santo

    2012-10-01

    Full Text Available Schistosomiasis constitutes a major public health problem, with an estimated 200 million individuals infected worldwide and 700 million people living in risk areas. In Brazil there are areas of high, medium and low endemicity. Studies have shown that in endemic areas with a low prevalence of Schistosoma infection the sensitivity of parasitological methods is clearly reduced. Consequently diagnosis is often impeded due to the presence of false-negative results. The aim of this study is to present the PCR reamplification (Re-PCR protocol for the detection of Schistosoma mansoni in samples with low parasite load (with less than 100 eggs per gram (epg of feces. Three methods were used for the lysis of the envelopes of the S. mansoni eggs and two techniques of DNA extraction were carried out. Extracted DNA was quantified, and the results suggested that the extraction technique, which mixed glass beads with a guanidine isothiocyanate/phenol/chloroform (GT solution, produced good results. PCR reamplification was conducted and detection sensitivity was found to be five eggs per 500 mg of artificially marked feces. The results achieved using these methods suggest that they are potentially viable for the detection of Schistosoma infection with low parasite load.

  10. Non-instrumented DNA isolation, amplification and microarray-based hybridization for a rapid on-site detection of devastating Phytophthora kernoviae.

    Science.gov (United States)

    Schwenkbier, Lydia; Pollok, Sibyll; Rudloff, Anne; Sailer, Sebastian; Cialla-May, Dana; Weber, Karina; Popp, Jürgen

    2015-10-07

    A rapid and simple instrument-free detection system was developed for the identification of the plant pathogen Phytophthora kernoviae (P. kernoviae). The on-site operable analysis steps include magnetic particle based DNA isolation, helicase-dependent amplification (HDA) and chip-based DNA hybridization. The isothermal approach enabled the convenient amplification of the yeast GTP-binding protein (Ypt1) target gene in a miniaturized HDA-zeolite-heater (HZH) by an exothermic reaction. The amplicon detection on the chip was performed under room temperature conditions – either by successive hybridization and enzyme binding or by a combined step. A positive signal is displayed by enzymatically generated silver nanoparticle deposits, which serve as robust endpoint signals allowing an immediate visual readout. The hybridization assay enabled the reliable detection of 10 pg μL(-1) target DNA. This is the first report of an entirely electricity-free, field applicable detection approach for devastating Phytophthora species, exemplarily shown for P. kernoviae.

  11. Multiplex isothermal solid-phase recombinase polymerase amplification for the specific and fast DNA-based detection of three bacterial pathogens.

    Science.gov (United States)

    Kersting, Sebastian; Rausch, Valentina; Bier, Frank F; von Nickisch-Rosenegk, Markus

    2014-01-01

    We report on the development of an on-chip RPA (recombinase polymerase amplification) with simultaneous multiplex isothermal amplification and detection on a solid surface. The isothermal RPA was applied to amplify specific target sequences from the pathogens Neisseria gonorrhoeae, Salmonella enterica and methicillin-resistant Staphylococcus aureus (MRSA) using genomic DNA. Additionally, a positive plasmid control was established as an internal control. The four targets were amplified simultaneously in a quadruplex reaction. The amplicon is labeled during on-chip RPA by reverse oligonucleotide primers coupled to a fluorophore. Both amplification and spatially resolved signal generation take place on immobilized forward primers bount to expoxy-silanized glass surfaces in a pump-driven hybridization chamber. The combination of microarray technology and sensitive isothermal nucleic acid amplification at 38 °C allows for a multiparameter analysis on a rather small area. The on-chip RPA was characterized in terms of reaction time, sensitivity and inhibitory conditions. A successful enzymatic reaction is completed in isothermal nucleic acid amplification with RPA and spatially-resolved signal generation on specific immobilized oligonucleotides.

  12. Amplification of an invA gene sequence of Salmonella typhimurium by polymerase chain reaction as a specific method of detection of Salmonella.

    Science.gov (United States)

    Rahn, K; De Grandis, S A; Clarke, R C; McEwen, S A; Galán, J E; Ginocchio, C; Curtiss, R; Gyles, C L

    1992-08-01

    Amplification of nucleotide sequences within the invA gene of Salmonella typhimurium was evaluated as a means of detecting Salmonella. A collection of 630 strains of Salmonella comprising over 100 serovars, including the 20 most prevalent serovars isolated from animals and humans in Canada, was examined. Controls consisted of 142 non-Salmonella strains comprising 21 genera of bacteria. Cultures were screened by inoculating a single colony of bacteria directly into a polymerase chain reaction (PCR) mixture which contained a pair of primers specific for the invA gene. The specific PCR product was a 284 bp DNA fragment which was visualized in 2% agarose gels. With the exception of two S. litchfield and two S. senftenberg strains, all Salmonella strains were detected. In contrast, none of the non-Salmonella strains yielded the specific amplification product. Non-specific amplification of a few non-Salmonella strains resulted in a product that was distinctly different in size from the specific 284 bp product. Specificity of amplification was further confirmed by demonstration of hybridization of a 32P-labelled invA gene fragment only to the specific 284 bp product. The detection of 99.4% of Salmonella strains tested and the failure to specifically amplify DNA from non-Salmonella strains confirm that the invA gene contains sequences unique to Salmonella and demonstrate that this gene is a suitable PCR target, with potential diagnostic applications.

  13. How to detect gold, silver and mercury in human brain and other tissues by autometallographic silver amplification.

    Science.gov (United States)

    Danscher, G; Stoltenberg, M; Juhl, S

    1994-10-01

    Gold, silver, mercury and zinc bind chemically to sulphide or selenide ions and create crystal lattices that can be detected in histological sections by a silver amplification technique called autometallography (AMG). The technique specifically magnifies such nanometer-sized catalytic crystals. For each metal, a detailed protocol has been worked out. If several different AMG metals/metal molecules are present in the same tissue, it is possible to distinguish one from another. The AMG technique is based on the capability of small crystal lattices of the aforementioned metals and metal molecules to initiate AMG silver amplification. Electrons released from adhering hydroquinone molecules reduce silver ions that are integrally connected with the crystal lattices. In this manner, particles consisting of only a few atoms of, say, gold, or molecules of mercury selenide (Figure 1), can be silver amplified to a size at which they can be detected in the electron microscope, or even further to dimensions that can be observed in the light microscope. Thus the AMG technique opens up the possibility of visualizing gold, e.g. in the nervous system of rheumatic patients who have been treated with aurothiomalate. Mercury can similarly be visualized in tissues from individuals who have been exposed to mercury, either through leaching from amalgam dental fillings, through eating fish, or by occupational exposure, and silver in the central (CNS) and peripheral nervous systems (PNS) and other tissues from individuals exposed to silver in one form or another. In the future, the possibility of demonstrating vesicular zinc, a particular pool of endogenous zinc that is found in terminals of zinc-enriched neurons (ZEN neurons), might prove valuable for pathological interpretation of diseases such as Alzheimer's disease. The vesicular zinc, present in some of the synaptic vesicles of ZEN neuron terminals, is most impressively demonstrated by AMG in telencephalic structures. It is becoming

  14. Detection of Q Fever Specific Antibodies Using Recombinant Antigen in ELISA with Peroxidase Based Signal Amplification

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    Hua-Wei Chen

    2014-01-01

    Full Text Available Currently, the accepted method for Q fever serodiagnosis is indirect immunofluorescent antibody assay (IFA using the whole cell antigen. In this study, we prepared the recombinant antigen of the 27-kDa outer membrane protein (Com1 which has been shown to be recognized by Q fever patient sera. The performance of recombinant Com1 was evaluated in ELISA by IFA confirmed serum samples. Due to the low titers of IgG and IgM in Q fever patients, the standard ELISA signals were further amplified by using biotinylated anti-human IgG or IgM plus streptavidin-HRP polymer. The modified ELISA can detect 88% (29 out of 33 of Q fever patient sera collected from Marines deployed to Iraq. Less than 5% (5 out of 156 of the sera from patients with other febrile diseases reacted with the Com1. These results suggest that the modified ELISA using Com1 may have the potential to improve the detection of Q fever specific antibodies.

  15. Detection of Staphylococcus aureus using 15N-labeled bacteriophage amplification coupled with matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry.

    Science.gov (United States)

    Pierce, Carrie L; Rees, Jon C; Fernández, Facundo M; Barr, John R

    2011-03-15

    A novel approach to rapid bacterial detection using an isotopically labeled (15)N bacteriophage and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) is introduced. Current phage amplification detection (PAD) via mass spectrometric analysis is limited because host bacteria must be inoculated with low phage titers in such a way that initial infecting phage concentrations must be below the detection limit of the instrument, thus lengthening incubation times. Additionally, PAD techniques cannot distinguish inoculate input phage from output phage which can increase the possibility of false positive results. Here, we report a rapid and accurate PAD approach for identification of Staphylococcus aureus via detection of bacteriophage capsid proteins. This approach uses both a wild-type (14)N and a (15)N-isotopically labeled S. aureus-specific bacteriophage. High (15)N phage titers, above our instrument's detection limits, were used to inoculate S. aureus. MALDI-TOF MS detection of the (14)N progeny capsid proteins in the phage-amplified culture indicated the presence of the host bacteria. Successful phage amplification was observed after 90 min of incubation. The amplification was observed by both MALDI-TOF MS analysis and by standard plaque assay measurements. This method overcomes current limitations by improving analysis times while increasing selectivity when compared to previously reported PAD methodologies.

  16. Modeling bacteriophage amplification as a predictive tool for optimized MALDI-TOF MS-based bacterial detection.

    Science.gov (United States)

    Cox, Christopher R; Rees, Jon C; Voorhees, Kent J

    2012-11-01

    Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a valuable tool for rapid bacterial detection and identification but is limited by the need for relatively high cell count samples, which have been grown under strictly controlled conditions. These requirements can be eliminated by the natural infection of a viable bacterial species of interest with a host-specific phage. This produces a rapid increase in phage protein concentrations in comparison to bacterial concentrations, which can in turn be exploited as a method for signal amplification during MALDI-TOF MS. One drawback to this approach is the requirement for repetitive, time-consuming sample preparation and analysis applied over the course of a phage infection to monitor phage concentrations as a function of time to determine the MALDI-TOF MS detection limit. To reduce the requirement for repeated preparation and analysis, a modified phage therapy model was investigated as a means for predicting the time during a given phage infection when a detectable signal would occur. The modified model used a series of three differential equations composed of predetermined experimental parameters including phage burst size and burst time to predict progeny phage concentrations as a function of time. Using Yersinia pestis with plague diagnostic phage φA1122 and Escherichia coli with phage MS2 as two separate, well-characterized model phage-host pairs, we conducted in silico modeling of the infection process and compared it with experimental infections monitored in real time by MALDI-TOF MS. Significant agreement between mathematically calculated phage growth curves and those experimentally obtained by MALDI-TOF MS was observed, thus verifying this method's utility for significant time and labor reduction.

  17. An enhanced chemiluminescence resonance energy transfer aptasensor based on rolling circle amplification and WS2 nanosheet for Staphylococcus aureus detection.

    Science.gov (United States)

    Hao, Liling; Gu, Huajie; Duan, Nuo; Wu, Shijia; Ma, Xiaoyuan; Xia, Yu; Tao, Zui; Wang, Zhouping

    2017-03-22

    A chemiluminescence resonance energy transfer aptasensor was fabricated for the detection of Staphylococcus aureus (S. aureus) with Co(2+) enhanced N-(aminobutyl)-N-(ethylisoluminol) (ABEI) functional flowerlike gold nanoparticles (Co(2+)/ABEI-AuNFs) as donor and WS2 nanosheet as acceptor. In the presence of S. aureus, rolling circle amplification (RCA) can be started. Partially complementary sequence of RCA product functional ABEI-AuNFs (cDNA-ABEI-AuNFs) were then annealed to multiple sites of the RCA product to form duplex complex. This complex is less adsorbed onto the WS2 nanosheet, thus attenuating the quenching of ABEI-AuNFs chemiluminescence by WS2 nanosheet. In the absence of target S. aureus (and hence the absence of RCA and duplex formation), the free cDNA-ABEI-AuNFs is completely adsorbed onto the WS2 nanosheet and chemiluminescence quenching ensues. Under optimal conditions, the logarithmic correlation between the concentration of S. aureus and the CL signal was found to be linear within the range of 50 cfu/mL to 1.5 × 10(5) cfu/mL (R(2) = 0.9913). The limits of detection of the developed method were found to be 15 cfu/mL for S. aureus. The selectivity and the capability of the biosensor in meat samples were also studied. Therefore, this simple and easy operation method can be used to detect S. aureus with high sensitivity and specificity.

  18. DNA Amplification Techniques for the Detection of Toxoplasma gondii Tissue Cysts in Meat Producing Animals: A Narrative Review Article

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    Farooq RIAZ

    2016-12-01

    Full Text Available Background: Toxoplasma gondii is an intracellular parasite, which infects one-third population of world. Humans and animals acquire infection by ingesting oocytes from feces of cats or by meat of other animals having cysts that may lead to congenital, ocular or cephalic toxoplasmosis. Either it is important to detect T. gondii from meat of food animals from retail shops or directly at slaughterhouses, which is meant for export.Methods: The current research was done without time limitation using such terms as follows: “Toxoplasma gondii”, “Meat”, “Tissue cyst”, “PCR”, “LAMP”, “Screening” and “Immunological assay” alone or in combination, in English language. The used electronic databases for searching included as follows: PubMed, Scopus, Google Scholar, Web of Science and Science Direct. The searches were limited to the published papers to English language.Results: Sensitivity of different molecular techniques for diagnosis of Toxoplasma is real-time PCR > LAMP > conventional PCR. In addition to these DNA analysis tools, bioassay in mice and cats is considered as “gold standard” to detect T. gondii. Conclusion: This review article will help the readers for grasping advantages and limitations of different diagnostic tools for screening meat samples for T. gondii. This review also makes bibliography about the type of meat sample to be processed for diagnosis and different primers or sequences to be targeted for T. gondii by number of researches for its detection from meat or tissue sample using DNA amplification techniques.

  19. Field Evaluation of a High Throughput Loop Mediated Isothermal Amplification Test for the Detection of Asymptomatic Plasmodium Infections in Zanzibar

    Science.gov (United States)

    Morris, Ulrika; Ding, Xavier C.; Jovel, Irina; Msellem, Mwinyi I.; Bergman, Daniel; Islam, Atiqul; Ali, Abdullah S.; Polley, Spencer; Gonzalez, Iveth J.; Mårtensson, Andreas; Björkman, Anders

    2017-01-01

    Background New field applicable diagnostic tools are needed for highly sensitive detection of residual malaria infections in pre-elimination settings. Field performance of a high throughput DNA extraction system for loop mediated isothermal amplification (HTP-LAMP) was therefore evaluated for detecting malaria parasites among asymptomatic individuals in Zanzibar. Methods HTP-LAMP performance was evaluated against real-time PCR on 3008 paired blood samples collected on filter papers in a community-based survey in 2015. Results The PCR and HTP-LAMP determined malaria prevalences were 1.6% (95%CI 1.3–2.4) and 0.7% (95%CI 0.4–1.1), respectively. The sensitivity of HTP-LAMP compared to PCR was 40.8% (CI95% 27.0–55.8) and the specificity was 99.9% (CI95% 99.8–100). For the PCR positive samples, there was no statistically significant difference between the geometric mean parasite densities among the HTP-LAMP positive (2.5 p/μL, range 0.2–770) and HTP-LAMP negative (1.4 p/μL, range 0.1–7) samples (p = 0.088). Two lab technicians analysed up to 282 samples per day and the HTP-LAMP method was experienced as user friendly. Conclusions Although field applicable, this high throughput format of LAMP as used here was not sensitive enough to be recommended for detection of asymptomatic low-density infections in areas like Zanzibar, approaching malaria elimination. PMID:28095434

  20. Detection of influenza A and B with the Alere™ i Influenza A & B: a novel isothermal nucleic acid amplification assay

    Science.gov (United States)

    Hazelton, Briony; Gray, Timothy; Ho, Jennifer; Ratnamohan, V Mala; Dwyer, Dominic E; Kok, Jen

    2015-01-01

    Background Rapid influenza diagnostic tests (RIDTs) have an important role in clinical decision-making; however, the performances of currently available assays vary widely. Objectives We evaluated the performance of the Alere™ i Influenza A&B (Alere™ iNAT), a rapid isothermal nucleic acid amplification assay that has recently received FDA clearance, for the detection of influenza A and B viruses during the Australian influenza season of 2013. Results were compared to two other RIDTs tested in parallel; Quidel Sofia® Influenza A+B fluorescent immunoassay (FIA) and Alere™ BinaxNOW® Influenza A & B immunochromatographic (ICT) assay. Methods A total of 202 paired nasopharyngeal swabs collected from patients ≥16 years old with an influenza-like illness (ILI) were eluted in 2 ml of universal transport medium (UTM) that was used to perform all three RIDTs in parallel. Reverse-transcription polymerase chain reaction (RT-PCR) was used as the reference standard. Results Compared to RT-PCR, Alere™ iNAT detected 77·8% influenza A positive samples versus 71·4% and 44·4% for the Quidel Sofia® Influenza A+B FIA and BinaxNOW® Influenza A & B ICT assay, respectively. For influenza B, Alere™ iNAT detected 75% of those positive by RT-PCR, versus 33·3% and 25·0% for Sofia® and BinaxNOW®, respectively. The specificity of Alere™ iNAT was 100% for influenza A and 99% for influenza B. Conclusions Alere™ i Influenza A&B is a promising new rapid influenza diagnostic assay with potential point-of-care applications. PMID:25728758

  1. Development of loop-mediated isothermal amplification to detect Streptococcus suis and its application to retail pork meat in Japan.

    Science.gov (United States)

    Arai, Sakura; Tohya, Mari; Yamada, Ryoko; Osawa, Ro; Nomoto, Ryohei; Kawamura, Yoshiaki; Sekizaki, Tsutomu

    2015-09-02

    We here developed a novel loop-mediated isothermal amplification (LAMP) method to detect Streptococcus suis in raw pork meat. This method, designated LAMPSS, targeted the recombination/repair protein (recN) gene of S. suis and detected all serotypes of S. suis, except those taxonomically removed from authentic S. suis, i.e., serotypes 20, 22, 26, 32, 33, and 34. The specificity of LAMPSS was confirmed and its detection limit was 5.4cfu/reaction. Among the 966 raw pork meat samples examined, including sliced pork, minced pork, and the liver, tongue, heart, and small intestine, 255 samples tested positive with LAMPSS. The rate of contamination was higher in the organs than in pork. No significant difference was observed in the total bacterial count between LAMPSS-positive and -negative samples. The number of shops that provided LAMPSS-positive pork was slightly higher in those that sold swine organs and pork than in those that sold only pork, suggesting that cross contamination occurred from the organs to pork. Among the 255 which tested positive for LAMPSS, only 47 samples tested positive for the previously described LAMP specific for S. suis serotype 2. Two isolates of S. suis serotype 2, belonging to sequence type 28, which is potentially hazardous to humans, as well as those of some other serotypes were obtained from 19 out of 47 samples by combining LAMP with a replica plating method. These results suggest that LAMPSS will be a useful tool for the surveillance of raw pork meat in the retail market. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. Point-Counterpoint: A Nucleic Acid Amplification Test for Streptococcus pyogenes Should Replace Antigen Detection and Culture for Detection of Bacterial Pharyngitis.

    Science.gov (United States)

    Pritt, Bobbi S; Patel, Robin; Kirn, Thomas J; Thomson, Richard B

    2016-10-01

    Nucleic acid amplification tests (NAATs) have frequently been the standard diagnostic approach when specific infectious agents are sought in a clinic specimen. They can be applied for specific agents such as S. pyogenes, or commercial multiplex NAATs for detection of a variety of pathogens in gastrointestinal, bloodstream, and respiratory infections may be used. NAATs are both rapid and sensitive. For many years, S. pyogenes testing algorithms used a rapid and specific group A streptococcal antigen test to screen throat specimens, followed, in some clinical settings, by a throat culture for S. pyogenes to increase the sensitivity of its detection. Now S. pyogenes NAATs are being used with increasing frequency. Given their accuracy, rapidity, and ease of use, should they replace antigen detection and culture for the detection of bacterial pharyngitis? Bobbi Pritt and Robin Patel of the Mayo Clinic, where S. pyogenes NAATs have been used for well over a decade with great success, will explain the advantages of this approach, while Richard (Tom) Thomson and Tom Kirn of the NorthShore University HealthSystem will discuss their concerns about this approach to diagnosing bacterial pharyngitis. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  3. Two types of nanoparticle-based bio-barcode amplification assays to detect HIV-1 p24 antigen

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    Dong Huahuang

    2012-08-01

    Full Text Available Abstract Background HIV-1 p24 antigen is a major viral component of human immunodeficiency virus type 1 (HIV-1 which can be used to identify persons in the early stage of infection and transmission of HIV-1 from infected mothers to infants. The detection of p24 is usually accomplished by using an enzyme-linked immunosorbent assay (ELISA with low detection sensitivity. Here we report the use of two bio-barcode amplification (BCA assays combined with polymerase chain reaction (PCR and gel electrophoresis to quantify HIV-1 p24 antigen. Method A pair of anti-p24 monoclonal antibodies (mAbs were used in BCA assays to capture HIV-1 p24 antigen in a sandwich format and allowed for the quantitative measurement of captured p24 using PCR and gel electrophoresis. The first 1 G12 mAb was coated on microplate wells or magnetic microparticles (MMPs to capture free p24 antigens. Captured p24 in turn captured 1D4 mAb coated gold nanoparticle probes (GNPs containing double-stranded DNA oligonucleotides. One strand of the oligonucleotides was covalently immobilized whereas the unbound complimentary bio-barcode DNA strand could be released upon heating. The released bio-barcode DNA was amplified by PCR, electrophoresed in agarose gel and quantified. Results The in-house ELISA assay was found to quantify p24 antigen with a limit of detection (LOD of 1,000 pg/ml and a linear range between 3,000 and 100,000 pg/ml. In contrast, the BCA-based microplate method yielded an LOD of 1 pg/ml and a linear detection range from 1 to 10,000 pg/ml. The BCA-based MMP method yielded an LOD of 0.1 pg/ml and a linear detection range from 0.1 to 1,000 pg/ml. Conclusions When combined with PCR and simple gel electrophoresis, BCA-based microplate and MMPs assays can be used to quantify HIV-1 p24 antigen. These methods are 3–4 orders of magnitude more sensitive than our in-house ELISA-based assay and may provide a useful approach to detect p24 in patients newly infected

  4. Loop-mediated isothermal amplification of specific endoglucanase gene sequence for detection of the bacterial wilt pathogen Ralstonia solanacearum.

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    Rok Lenarčič

    Full Text Available The increased globalization of crops production and processing industries also promotes the side-effects of more rapid and efficient spread of plant pathogens. To prevent the associated economic losses, and particularly those related to bacterial diseases where their management relies on removal of the infected material from production, simple, easy-to-perform, rapid and cost-effective tests are needed. Loop-mediated isothermal amplification (LAMP assays that target 16S rRNA, fliC and egl genes were compared and evaluated as on-site applications. The assay with the best performance was that targeted to the egl gene, which shows high analytical specificity for diverse strains of the betaproteobacterium Ralstonia solanacearum, including its non-European and non-race 3 biovar 2 strains. The additional melting curve analysis provides confirmation of the test results. According to our extensive assessment, the egl LAMP assay requires minimum sample preparation (a few minutes of boiling for the identification of pure cultures and ooze from symptomatic material, and it can also be used in a high-throughput format in the laboratory. This provides sensitive and reliable detection of R. solanacearum strains of different phylotypes.

  5. Rapid and ultrasensitive detection of microRNA by target-assisted isothermal exponential amplification coupled with poly (thymine)-templated fluorescent copper nanoparticles

    Science.gov (United States)

    Park, Kwan Woo; Batule, Bhagwan S.; Kang, Kyoung Suk; Park, Ki Soo; Park, Hyun Gyu

    2016-10-01

    We devised a novel method for rapid and ultrasensitive detection of target microRNA (miRNA) by employing target-assisted isothermal exponential amplification (TAIEA) combined with poly (thymine)-templated fluorescent copper nanoparticles (CuNPs) as signaling probes. The target miRNA hybridizes to the unimolecular template DNA and works as a primer for the extension reaction to form double-stranded product, which consequently generates two nicking endonuclease recognition sites. By simultaneous nicking and displacement reactions, exponential amplification generates many poly (thymine) strands as final products, which are employed for the synthesis of fluorescent CuNPs. Based on the fluorescent signal from CuNPs, target miRNA is detected as low as 0.27 fM around 1 h of total analysis time. The diagnostic capability of this system has been successfully demonstrated by reliably detecting target miRNA from different cell lysates, showing its great potential towards real clinical applications.

  6. G-quadruplex fluorescent probe-mediated real-time rolling circle amplification strategy for highly sensitive microRNA detection.

    Science.gov (United States)

    Jiang, Hong-Xin; Liang, Zhen-Zhen; Ma, Yan-Hong; Kong, De-Ming; Hong, Zhang-Yong

    2016-11-02

    Real-time PCR has revolutionized PCR from qualitative to quantitative. As an isothermal DNA amplification technique, rolling circular amplification (RCA) has been demonstrated to be a versatile tool in many fields. Development of a simple, highly sensitive, and specific strategy for real-time monitoring of RCA will increase its usefulness in many fields. The strategy reported here utilized the specific fluorescence response of thioflavin T (ThT) to G-quadruplexes formed by RCA products. Such a real-time monitoring strategy works well in both traditional RCA with linear amplification efficiency and modified RCA proceeded in an exponential manner, and can be readily performed in commercially available real-time PCR instruments, thereby achieving high-throughput detection and making the proposed technique more suitable for biosensing applications. As examples, real-time RCA-based sensing platforms were designed and successfully used for quantitation of microRNA over broad linear ranges (8 orders of magnitude) with a detection limit of 4 aM (or 0.12 zmol). The feasibility of microRNA analysis in human lung cancer cells was also demonstrated. This work provides a new method for real-time monitoring of RCA by using unique nucleic acid secondary structures and their specific fluorescent probes. It has the potential to be extended to other isothermal single-stranded DNA amplification techniques.

  7. Electrochemical Detection of Sequence-Specific DNA with the Amplification of Gold Nanoparticles

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    Yuzhong Zhang

    2011-01-01

    Full Text Available A sensitive electrochemical DNA biosensor was prepared based on mercaptoacetic acid (MAA/gold nanoparticles (AuNPs modified electrode. Probe DNA (NH2-DNA was covalently linked to the carboxyl group of MAA in the presence of 1-ethyl-3-(3-dimethylaminopropyl carbodiimide hydrochloride (EDC and N-hydroxyl-succinimide (NHS. Scanning electron microscopy (SEM and electrochemical impedance spectra (EIS were used to investigate the film assembly process. The DNA hybridization events were monitored by differential pulse voltammetry (DPV, and adriamycin was used as the electrochemical indicator. Also the factors influencing the performance of the DNA hybridization were investigated in detail. Under the optimal conditions, the signal was linearly changed with target DNA concentration increased from 5.0 × 10−13 to 1.0 × 10−9 M and had a detection limit of 1.7 × 10−13 M (signal/noise ratio of 3. In addition, the DNA biosensor showed good reproducibility and stability during DNA assay.

  8. Ultrasensitive electrical detection of protein using nanogap electrodes and nanoparticle-based DNA amplification.

    Science.gov (United States)

    Chang, Tien-Li; Tsai, Chien-Ying; Sun, Chih-Chen; Chen, Chun-Chi; Kuo, Long-Sheng; Chen, Ping-Hei

    2007-06-15

    The present study describes an ultrasensitive protein biochip that employs nanogap electrodes and self-assembled nanoparticles to electrically detect protein. A bio-barcode DNA technique amplifies the concentration of target antigen at least 100-fold. This technique requires the establishment of conjugate magnetic nanoparticles (MNPs) and gold nanoparticles (AuNPs) through binding between monoclonal antibodies (2B2), the target antigen, and polyclonal antibodies (GP). Both GP and capture ssDNA (single-strand DNA) bonds to bio-barcode ssDNA are immobilized on the surface of AuNPs. A denature process releases the bio-barcode ssDNAs into the solution, and a hybridization process establishes multilayer AuNPs over the gap surface between electrodes. Electric current through double-layer self-assembled AuNPs is much greater than that through self-assembled monolayer AuNPs. This significant increase in electric current provides evidence that the solution contains the target antigen. Results show that the protein biochip attains a sensitivity of up to 1 pg/ microL.

  9. Development of loop-mediated isothermal amplification (LAMP) for detection of Theileria equi.

    Science.gov (United States)

    Xie, Junren; Liu, Guangyuan; Tian, Zhancheng; Luo, Jin

    2013-09-01

    Several approaches have been developed for diagnosis of Theileria equi infection in horses and donkeys but all of them have limitations in practice. Due to numerous strengths including easy operation, cheapness and high sensitivity and specificity, LAMP has been already extensively used for surveillance of a number of diseases. We here set up a LAMP assay based on 18S rRNA gene for T. equi diagnosis. The approach was specific enough to differentiate T. equi from other evolutionary-related protozoa. Moreover, it was sensitive enough that LAMP was capable of detecting as much low as 10 copy target gene and 1 pg/μl blood genomic DNA. It was further demonstrated that LAMP was much more sensitive than canonical blood smear and comparable to PCR using test and field samples. The present results support an idea that LAMP developed in this study is reliable, reproducible and highly sensitive and specific, being a potential to be globally used for surveillance of T. equi infection in the field.

  10. Ranque-Hilsch vortex tube thermocycler for fast DNA amplification and real-time optical detection

    Science.gov (United States)

    Ebmeier, Ryan J.; Whitney, Scott E.; Sarkar, Amitabha; Nelson, Michael; Padhye, Nisha V.; Gogos, George; Viljoen, Hendrik J.

    2004-12-01

    An innovative polymerase chain reaction (PCR) thermocycler capable of performing real-time optical detection is described below. This device utilizes the Ranque-Hilsch vortex tube in a system to efficiently and rapidly cycle three 20 μL samples between the denaturation, annealing, and elongation temperatures. The reaction progress is displayed real-time by measuring the size of a fluorescent signal emitted by SYBR green/double-stranded DNA complexes. This device can produce significant reaction yields with very small amounts of initial DNA, for example, it can amplify 0.25 fg (˜5 copies) of a 96 bp bacteriophage λ-DNA fragment 2.7×1011-fold by performing 45 cycles in less than 12 min. The optical threshold (150% of the baseline intensity) was passed 8 min into the reaction at cycle 34. Besides direct applications, the speed and sensitivity of this device enables it to be used as a scientific instrument for basic studies such as PCR assembly and polymerase kinetics.

  11. Amplification of rabbit hepatocyte growth factor and detection of its expression in COS-7 cell line.

    Science.gov (United States)

    Yao, H; Han, J; Wang, J; Wang, L; Gong, C; Li, L; Liang, Z; Tian, Y

    2015-11-25

    We used RT-PCR, nested PCR to acquire the partial 5'- race fragment of rabbit HGF cDNA and the partial 3'- race fragment of rabbit HGF cDNA. Then, we used recombination PCR to acquire rabbit HGF successfully. Homology analysis was conducted among the sequence of RABHGF and known human and rat HGF by DNAStar. It was proved that high level of homology existed among the sequences of those three HGF genes. We used the acquired gene of RABHGF to construct its recombinant eukaryotic expression vector pcDNA3.1(+)-RABHGF (pRABHGF). The identification of the eukaryotic expression vector pRABHGF by PCR, restriction enzyme and sequencing analysis showed that rabbit HGF gene was correctly inserted into the vector. pRABHGF and pcDNA3.1(+) as controls were transfected into COS-7 cells by lipofectamine. It takes 24h-36h after transfection to detect the expression of RABHGF protein by indirect immunofluorescence assay (IFA). The proliferation of cos-7 cells were evaluated by MTT assay. The result displayed positive effect of RABHGF protein on the proliferation of COS-7 cells. This study lays the foundation for a new gene therapy method for ischemic heart disease.

  12. Detection of virus mRNA within infected host cells using an isothermal nucleic acid amplification assay: marine cyanophage gene expression within Synechococcus sp

    Directory of Open Access Journals (Sweden)

    Hall Matthew J

    2007-06-01

    Full Text Available Abstract Background Signal-Mediated Amplification of RNA Technology (SMART is an isothermal nucleic acid amplification technology, developed for the detection of specific target sequences, either RNA (for expression or DNA. Cyanophages are viruses that infect cyanobacteria. Marine cyanophages are ubiquitous in the surface layers of the ocean where they infect members of the globally important genus Synechococcus. Results Here we report that the SMART assay allowed us to differentiate between infected and non-infected host cultures. Expression of the cyanophage strain S-PM2 portal vertex gene (g20 was detected from infected host Synechococcus sp. WH7803 cells. Using the SMART assay, we demonstrated that g20 mRNA peaked 240 – 360 minutes post-infection, allowing us to characterise this as a mid to late transcript. g20 DNA was also detected, peaking 10 hours post-infection, coinciding with the onset of host lysis. Conclusion The SMART assay is based on isothermal nucleic acid amplification, allowing the detection of specific sequences of DNA or RNA. It was shown to be suitable for differentiating between virus-infected and non-infected host cultures and for the detection of virus gene expression: the first reported use of this technology for such applications.

  13. Development and clinical performance of high throughput loop-mediated isothermal amplification for detection of malaria

    Science.gov (United States)

    Perera, Rushini S.; Ding, Xavier C.; Tully, Frank; Oliver, James; Bright, Nigel; Bell, David; Chiodini, Peter L.; Gonzalez, Iveth J.; Polley, Spencer D.

    2017-01-01

    Background Accurate and efficient detection of sub-microscopic malaria infections is crucial for enabling rapid treatment and interruption of transmission. Commercially available malaria LAMP kits have excellent diagnostic performance, though throughput is limited by the need to prepare samples individually. Here, we evaluate the clinical performance of a newly developed high throughput (HTP) sample processing system for use in conjunction with the Eiken malaria LAMP kit. Methods The HTP system utilised dried blood spots (DBS) and liquid whole blood (WB), with parallel sample processing of 94 samples per run. The system was evaluated using 699 samples of known infection status pre-determined by gold standard nested PCR. Results The sensitivity and specificity of WB-HTP-LAMP was 98.6% (95% CI, 95.7–100), and 99.7% (95% CI, 99.2–100); sensitivity of DBS-HTP-LAMP was 97.1% (95% CI, 93.1–100), and specificity 100% against PCR. At parasite densities greater or equal to 2 parasites/μL, WB and DBS HTP-LAMP showed 100% sensitivity and specificity against PCR. At densities less than 2 p/μL, WB-HTP-LAMP sensitivity was 88.9% (95% CI, 77.1–100) and specificity was 99.7% (95% CI, 99.2–100); sensitivity and specificity of DBS-HTP-LAMP was 77.8% (95% CI, 54.3–99.5) and 100% respectively. Conclusions The HTP-LAMP system is a highly sensitive diagnostic test, with the potential to allow large scale population screening in malaria elimination campaigns. PMID:28166235

  14. Development and clinical performance of high throughput loop-mediated isothermal amplification for detection of malaria.

    Science.gov (United States)

    Perera, Rushini S; Ding, Xavier C; Tully, Frank; Oliver, James; Bright, Nigel; Bell, David; Chiodini, Peter L; Gonzalez, Iveth J; Polley, Spencer D

    2017-01-01

    Accurate and efficient detection of sub-microscopic malaria infections is crucial for enabling rapid treatment and interruption of transmission. Commercially available malaria LAMP kits have excellent diagnostic performance, though throughput is limited by the need to prepare samples individually. Here, we evaluate the clinical performance of a newly developed high throughput (HTP) sample processing system for use in conjunction with the Eiken malaria LAMP kit. The HTP system utilised dried blood spots (DBS) and liquid whole blood (WB), with parallel sample processing of 94 samples per run. The system was evaluated using 699 samples of known infection status pre-determined by gold standard nested PCR. The sensitivity and specificity of WB-HTP-LAMP was 98.6% (95% CI, 95.7-100), and 99.7% (95% CI, 99.2-100); sensitivity of DBS-HTP-LAMP was 97.1% (95% CI, 93.1-100), and specificity 100% against PCR. At parasite densities greater or equal to 2 parasites/μL, WB and DBS HTP-LAMP showed 100% sensitivity and specificity against PCR. At densities less than 2 p/μL, WB-HTP-LAMP sensitivity was 88.9% (95% CI, 77.1-100) and specificity was 99.7% (95% CI, 99.2-100); sensitivity and specificity of DBS-HTP-LAMP was 77.8% (95% CI, 54.3-99.5) and 100% respectively. The HTP-LAMP system is a highly sensitive diagnostic test, with the potential to allow large scale population screening in malaria elimination campaigns.

  15. One-Pot Reverse Transcriptional Loop-Mediated Isothermal Amplification (RT-LAMP) for Detecting MERS-CoV.

    Science.gov (United States)

    Lee, Se Hee; Baek, Yun Hee; Kim, Yang-Hoon; Choi, Young-Ki; Song, Min-Suk; Ahn, Ji-Young

    2016-01-01

    Due to the limitation of rapid development of specific antiviral drug or vaccine for novel emerging viruses, an accurate and rapid diagnosis is a key to manage the virus spread. We developed an efficient and rapid method with high specificity for the Middle East Respiratory Syndrome coronavirus (MERS-CoV), based on one-pot reverse transcription loop-mediated isothermal amplification (one-pot RT-LAMP). A set of six LAMP primers [F3, B3, FIP, BIP, LF (Loop-F), and LB (Loop-B)] were designed using the sequence of nucleocapsid (N) gene with optimized RT-LAMP enzyme conditions: 100 U M-MLV RTase and 4 U Bst polymerase, implying that the reaction was able to detect four infectious viral genome copies of MERS-CoV within a 60 min reaction time period. Significantly, EvaGreen dye has better signal read-out properties in one-pot RT-LAMP reaction and is more compatible with DNA polymerase than SYBR green I. Isothermally amplified specific N genes were further evaluated using field-deployable microchamber devices, leading to the specific identification of as few as 0.4 infectious viral genome copies, with no cross-reaction to the other acute respiratory disease viruses, including influenza type A (H1N1 and H3N2), type B, human coronavirus 229E, and human metapneumovirus. This sensitive, specific and feasible method provides a large-scale technical support in emergencies, and is also applied as a sample-to-detection module in Point of Care Testing devices.

  16. HER2 missense mutations have distinct effects on oncogenic signaling and migration

    Science.gov (United States)

    Zabransky, Daniel J.; Yankaskas, Christopher L.; Cochran, Rory L.; Wong, Hong Yuen; Croessmann, Sarah; Chu, David; Kavuri, Shyam M.; Red Brewer, Monica; Rosen, D. Marc; Dalton, W. Brian; Cimino-Mathews, Ashley; Cravero, Karen; Button, Berry; Kyker-Snowman, Kelly; Cidado, Justin; Erlanger, Bracha; Parsons, Heather A.; Manto, Kristen M.; Bose, Ron; Lauring, Josh; Arteaga, Carlos L.; Konstantopoulos, Konstantinos; Park, Ben Ho

    2015-01-01

    Recurrent human epidermal growth factor receptor 2 (HER2) missense mutations have been reported in human cancers. These mutations occur primarily in the absence of HER2 gene amplification such that most HER2-mutant tumors are classified as “negative” by FISH or immunohistochemistry assays. It remains unclear whether nonamplified HER2 missense mutations are oncogenic and whether they are targets for HER2-directed therapies that are currently approved for the treatment of HER2 gene-amplified breast cancers. Here we functionally characterize HER2 kinase and extracellular domain mutations through gene editing of the endogenous loci in HER2 nonamplified human breast epithelial cells. In in vitro and in vivo assays, the majority of HER2 missense mutations do not impart detectable oncogenic changes. However, the HER2 V777L mutation increased biochemical pathway activation and, in the context of a PIK3CA mutation, enhanced migratory features in vitro. However, the V777L mutation did not alter in vivo tumorigenicity or sensitivity to HER2-directed therapies in proliferation assays. Our results suggest the oncogenicity and potential targeting of HER2 missense mutations should be considered in the context of cooperating genetic alterations and provide previously unidentified insights into functional analysis of HER2 mutations and strategies to target them. PMID:26508629

  17. HER2 missense mutations have distinct effects on oncogenic signaling and migration.

    Science.gov (United States)

    Zabransky, Daniel J; Yankaskas, Christopher L; Cochran, Rory L; Wong, Hong Yuen; Croessmann, Sarah; Chu, David; Kavuri, Shyam M; Red Brewer, Monica; Rosen, D Marc; Dalton, W Brian; Cimino-Mathews, Ashley; Cravero, Karen; Button, Berry; Kyker-Snowman, Kelly; Cidado, Justin; Erlanger, Bracha; Parsons, Heather A; Manto, Kristen M; Bose, Ron; Lauring, Josh; Arteaga, Carlos L; Konstantopoulos, Konstantinos; Park, Ben Ho

    2015-11-10

    Recurrent human epidermal growth factor receptor 2 (HER2) missense mutations have been reported in human cancers. These mutations occur primarily in the absence of HER2 gene amplification such that most HER2-mutant tumors are classified as "negative" by FISH or immunohistochemistry assays. It remains unclear whether nonamplified HER2 missense mutations are oncogenic and whether they are targets for HER2-directed therapies that are currently approved for the treatment of HER2 gene-amplified breast cancers. Here we functionally characterize HER2 kinase and extracellular domain mutations through gene editing of the endogenous loci in HER2 nonamplified human breast epithelial cells. In in vitro and in vivo assays, the majority of HER2 missense mutations do not impart detectable oncogenic changes. However, the HER2 V777L mutation increased biochemical pathway activation and, in the context of a PIK3CA mutation, enhanced migratory features in vitro. However, the V777L mutation did not alter in vivo tumorigenicity or sensitivity to HER2-directed therapies in proliferation assays. Our results suggest the oncogenicity and potential targeting of HER2 missense mutations should be considered in the context of cooperating genetic alterations and provide previously unidentified insights into functional analysis of HER2 mutations and strategies to target them.

  18. Detection and identification of human Plasmodium species with real-time quantitative nucleic acid sequence-based amplification

    Directory of Open Access Journals (Sweden)

    Kager Piet A

    2006-10-01

    Full Text Available Abstract Background Decisions concerning malaria treatment depend on species identification causing disease. Microscopy is most frequently used, but at low parasitaemia (Plasmodium antigen detection do often not allow for species discrimination as microscopy does, but also become insensitive at Methods This paper reports the development of a sensitive and specific real-time Quantitative Nucleic Acid Sequence Based Amplification (real-time QT-NASBA assays, based on the small-subunit 18S rRNA gene, to identify the four human Plasmodium species. Results The lower detection limit of the assay is 100 – 1000 molecules in vitro RNA for all species, which corresponds to 0.01 – 0.1 parasite per diagnostic sample (i.e. 50 μl of processed blood. The real-time QT-NASBA was further evaluated using 79 clinical samples from malaria patients: i.e. 11 Plasmodium. falciparum, 37 Plasmodium vivax, seven Plasmodium malariae, four Plasmodium ovale and 20 mixed infections. The initial diagnosis of 69 out of the 79 samples was confirmed with the developed real-time QT-NASBA. Re-analysis of seven available original slides resolved five mismatches. Three of those were initially identified as P. malariae mono-infection, but after re-reading the slides P. falciparum was found, confirming the real-time QT-NASBA result. The other two slides were of poor quality not allowing true species identification. The remaining five discordant results could not be explained by microscopy, but may be due to extreme low numbers of parasites present in the samples. In addition, 12 Plasmodium berghei isolates from mice and 20 blood samples from healthy donors did not show any reaction in the assay. Conclusion Real-time QT-NASBA is a very sensitive and specific technique with a detection limit of 0.1 Plasmodium parasite per diagnostic sample (50 μl of blood and can be used for the detection, identification and quantitative measurement of low parasitaemia of Plasmodium species, thus

  19. Pesticides and oncogenic modulation.

    Science.gov (United States)

    Vakonaki, Elena; Androutsopoulos, Vasilis P; Liesivuori, Jyrki; Tsatsakis, Aristidis M; Spandidos, Demetrios A

    2013-05-10

    Pesticides constitute a diverse class of chemicals used for the protection of agricultural products. Several lines of evidence demonstrate that organochlorine and organophosphate pesticides can cause malignant transformation of cells in in vitro and in vivo models. In the current minireview a comprehensive summary of recent in vitro findings is presented along with data reported from human population studies, regarding the impact of pesticide exposure on activation or dysregulation of oncogenes and tumor suppressor genes. Substantial mechanistic work suggests that pesticides are capable of inducing mutations in oncogenes and increase their transcriptional expression in vitro, whereas human population studies indicate associations between pesticide exposure levels and mutation occurrence in cancer-related genes. Further work is required to fully explore the exact mechanisms by which pesticide exposure affects the integrity and normal function of oncogenes and tumor suppressor genes in human populations.

  20. A lab-on-a-chip system with integrated sample preparation and loop-mediated isothermal amplification for rapid and quantitative detection of Salmonella spp. in food samples.

    Science.gov (United States)

    Sun, Yi; Quyen, Than Linh; Hung, Tran Quang; Chin, Wai Hoe; Wolff, Anders; Bang, Dang Duong

    2015-04-21

    Foodborne disease is a major public health threat worldwide. Salmonellosis, an infectious disease caused by Salmonella spp., is one of the most common foodborne diseases. Isolation and identification of Salmonella by conventional bacterial culture or molecular-based methods are time consuming and usually take a few hours to days to complete. In response to the demand for rapid on line or on site detection of pathogens, in this study, we describe for the first time an eight-chamber lab-on-a-chip (LOC) system with integrated magnetic bead-based sample preparation and loop-mediated isothermal amplification (LAMP) for rapid and quantitative detection of Salmonella spp. in food samples. The whole diagnostic procedures including DNA isolation, isothermal amplification, and real-time detection were accomplished in a single chamber. Up to eight samples could be handled simultaneously and the system was capable to detect Salmonella at concentration of 50 cells per test within 40 min. The simple design, together with high level of integration, isothermal amplification, and quantitative analysis of multiple samples in short time, will greatly enhance the practical applicability of the LOC system for rapid on-site screening of Salmonella for applications in food safety control, environmental surveillance, and clinical diagnostics.

  1. Paper-based electrochemiluminescence origami device for protein detection using assembled cascade DNA-carbon dots nanotags based on rolling circle amplification.

    Science.gov (United States)

    Wu, Ludan; Ma, Chao; Zheng, Xiaoxiao; Liu, Haiyun; Yu, Jinghua

    2015-06-15

    In this work, we developed a cascade signal amplification strategy for detection of IgG antigen by combining the rolling circle amplification (RCA) technique with oligonucleotide functionalized carbon dots (CDs), based on a paper-based electrochemiluminescence (ECL) origami device (PECLOD) for the first time. In this PECLOD, three-dimensional (3D) macroporous Au-paper electrode was fabricated and employed as the working electrode for specific and efficient antibodies capture. The RCA product containing tandem-repeat sequences could serve as an excellent template for periodic assembly of CDs, which presented per protein recognition event to numerous CDs tags for ECL readout. Under the optimal conditions, the proposed strategy showed remarkable amplification efficiency, very little nonspecific adsorption with good stability, reproducibility, and accuracy. Using human IgG (H-IgG) as a model protein, the designed strategy was successfully demonstrated for the ultrasensitive detection of protein target. The results revealed that the strategy exhibited a dynamic response to H-IgG range from 1.0 fM to 25 pM with a limit of detection as low as 0.15 fM. Importantly, the methodology can be further extended to the detection of other proteins or biomarkers.

  2. A signal-on electrochemical aptasensor for ultrasensitive detection of endotoxin using three-way DNA junction-aided enzymatic recycling and graphene nanohybrid for amplification

    Science.gov (United States)

    Bai, Lijuan; Chai, Yaqin; Pu, Xiaoyun; Yuan, Ruo

    2014-02-01

    Endotoxin, also known as lipopolysaccharide (LPS), is able to induce a strong immune response on its internalization into mammalian cells. To date, aptamer-based biosensors for LPS detection have been rarely reported. This work describes a new signal-on electrochemical aptasensor for the ultrasensitive detection of LPS by combining the three-way DNA hybridization process and nanotechnology-based amplification. With the help of DNA1 (associated with the concentration of target LPS), the capture probe hybridizes with DNA1 and the assistant probe to open its hairpin structure and form a ternary ``Y'' junction structure. The DNA1 can be released from the structure in the presence of nicking endonuclease to initiate the next hybridization process. Then a great deal of cleaved capture probe produced in the cyclic process can bind with DNA2-nanocomposite, which contains the electroactive toluidine blue (Tb) with the amplification materials graphene (Gra) and gold nanoparticles (AuNPs). Thus, an enhanced electrochemical signal can be easily read out. With the cascade signal amplification, this newly designed protocol provides an ultrasensitive electrochemical detection of LPS down to the femtogram level (8.7 fg mL-1) with a linear range of 6 orders of magnitude (from 10 fg mL-1 to 50 ng mL-1). Moreover, the high sensitivity and specificity make this method versatile for the detection of other biomolecules by changing the corresponding sequences of the capture probe and the assistant probe.

  3. DNA aneuploidy and integration of human papillomavirus type 16 e6/e7 oncogenes in intraepithelial neoplasia and invasive squamous cell carcinoma of the cervix uteri.

    Science.gov (United States)

    Melsheimer, Peter; Vinokurova, Svetlana; Wentzensen, Nicolas; Bastert, Gunther; von Knebel Doeberitz, Magnus

    2004-05-01

    Increasingly deregulated expression of the E6-E7 oncogenes of high-risk human papillomaviruses (HR-HPVs) has been identified as the major transforming factor in the pathogenesis of cervical dysplasia and derived cancers. The expression of these genes in epithelial stem cells first results in chromosomal instability and induces chromosomal aneuploidy. It is speculated that this subsequently favors integration of HR-HPV genomes into cellular chromosomes. This in turn leads to expression of viral cellular fusion transcripts and further enhanced expression of the E6-E7 oncoproteins. Chromosomal instability and aneuploidization thus seems to precede and favor integration of HR-HPV genomes. To prove this sequential concept, we analyzed here the sequence of events of DNA aneuploidization and integration in a series of HPV-16-positive cervical dysplastic lesions and carcinomas. Eighty-five punch biopsies of HPV-16-positive cervical lesions (20 CIN1/2, 50 CIN3, and 15 CxCa) were analyzed for DNA ploidy by DNA flow cytometry and for integration of HPV E6/E7 oncogenes using the amplification of papillomavirus oncogene transcripts assay, a reverse transcription-PCR method to detect integrate-derived human papillomavirus oncogene transcripts. DNA aneuploidy and viral genome integration were both associated with increasing dysplasia (P oncogene expression appears to result first in chromosomal instability and aneuploidization and is subsequently followed by integration of HR-HPV genomes in the affected cell clones.

  4. A novel electrochemical sensing strategy for rapid and ultrasensitive detection of Salmonella by rolling circle amplification and DNA–AuNPs probe

    Energy Technology Data Exchange (ETDEWEB)

    Zhu, Dan; Yan, Yurong; Lei, Pinhua; Shen, Bo [Key Laboratory of Clinical Laboratory Diagnostics (Ministry of Education), College of Laboratory Medicine, Chongqing Medical University, Chongqing 400016 (China); Cheng, Wei [Key Laboratory of Clinical Laboratory Diagnostics (Ministry of Education), College of Laboratory Medicine, Chongqing Medical University, Chongqing 400016 (China); The Center for Clinical Molecular Medical detection, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016 (China); Ju, Huangxian [Key Laboratory of Clinical Laboratory Diagnostics (Ministry of Education), College of Laboratory Medicine, Chongqing Medical University, Chongqing 400016 (China); State Key Laboratory of Analytical Chemistry for Life Science, Department of Chemistry, Nanjing University, Nanjing 210093 (China); Ding, Shijia, E-mail: dingshijia@163.com [Key Laboratory of Clinical Laboratory Diagnostics (Ministry of Education), College of Laboratory Medicine, Chongqing Medical University, Chongqing 400016 (China)

    2014-10-10

    A novel electrochemical sensing strategy was developed for ultrasensitive and rapid detection of Salmonella by combining the rolling circle amplification with DNA–AuNPs probe. - Highlights: • This paper presented a novel sensing strategy for the rapid and ultrasensitive detection for Salmonella. • Combination of rolling circle amplification and DNA–AuNPs probe is the first time for Salmonella electrochemical detection. • The method displayed excellent sensitivity and specificity for detection of Salmonella. • The fabricated biosensor was successfully applied to detect Salmonella in milk samples. - Abstract: A novel electrochemical sensing strategy was developed for ultrasensitive and rapid detection of Salmonella by combining the rolling circle amplification with DNA–AuNPs probe. The target DNA could be specifically captured by probe 1 on the sensing interface. Then the circularization mixture was added to form a typical sandwich structure. In the presence of dNTPs and phi29 DNA polymerase, the RCA was initiated to produce micrometer-long single-strand DNA. Finally, the detection probe (DNA–AuNPs) could recognize RCA product to produce enzymatic electrochemical signal. Under optimal conditions, the calibration curve of synthetic target DNA had good linearity from 10 aM to 10 pM with a detection limit of 6.76 aM (S/N = 3). The developed method had been successfully applied to detect Salmonella as low as 6 CFU mL{sup −1} in real milk sample. This proposed strategy showed great potential for clinical diagnosis, food safety and environmental monitoring.

  5. Evaluation of a field-portable DNA microarray platform and nucleic acid amplification strategies for the detection of arboviruses, arthropods, and bloodmeals.

    Science.gov (United States)

    Grubaugh, Nathan D; Petz, Lawrence N; Melanson, Vanessa R; McMenamy, Scott S; Turell, Michael J; Long, Lewis S; Pisarcik, Sarah E; Kengluecha, Ampornpan; Jaichapor, Boonsong; O'Guinn, Monica L; Lee, John S

    2013-02-01

    Highly multiplexed assays, such as microarrays, can benefit arbovirus surveillance by allowing researchers to screen for hundreds of targets at once. We evaluated amplification strategies and the practicality of a portable DNA microarray platform to analyze virus-infected mosquitoes. The prototype microarray design used here targeted the non-structural protein 5, ribosomal RNA, and cytochrome b genes for the detection of flaviviruses, mosquitoes, and bloodmeals, respectively. We identified 13 of 14 flaviviruses from virus inoculated mosquitoes and cultured cells. Additionally, we differentiated between four mosquito genera and eight whole blood samples. The microarray platform was field evaluated in Thailand and successfully identified flaviviruses (Culex flavivirus, dengue-3, and Japanese encephalitis viruses), differentiated between mosquito genera (Aedes, Armigeres, Culex, and Mansonia), and detected mammalian bloodmeals (human and dog). We showed that the microarray platform and amplification strategies described here can be used to discern specific information on a wide variety of viruses and their vectors.

  6. Detection of denitrification genes by in situ rolling circle amplification - fluorescence in situ hybridization (in situ RCA-FISH) to link metabolic potential with identity inside bacterial cells

    DEFF Research Database (Denmark)

    Hoshino, Tatsuhiko; Schramm, Andreas

    2010-01-01

    A target-primed in situ rolling circle amplification (in situ RCA) protocol was developed for detection of single-copy genes inside bacterial cells and optimized with Pseudomonas stutzeri, targeting nitrite and nitrous oxide reductase genes (nirS and nosZ). Two padlock probes were designed per gene......). Nevertheless, multiple genes (nirS and nosZ; nirS and the 16S rRNA gene) could be detected simultaneously in P. stutzeri. Environmental application of in situ RCA-FISH was demonstrated on activated sludge by the differential detection of two types of nirS-defined denitrifiers; one of them was identified...

  7. Rapid, simple and sensitive detection of Q fever by loop-mediated isothermal amplification of the htpAB gene.

    Directory of Open Access Journals (Sweden)

    Lei Pan

    Full Text Available BACKGROUND: Q fever is the most widespread zoonosis, and domestic animals are the most common sources of transmission. It is not only difficult to distinguish from other febrile diseases because of the lack of specific clinical manifestations in humans, but it is also difficult to identify the disease in C. burnetii-carrying animals because of the lack of identifiable features. Conventional serodiagnosis requires sera from the acute and convalescent stages of infection, which are unavailable at early diagnosis. Nested PCR and real-time PCR require equipment. In this study, we developed a Loop-Mediated Isothermal Amplification (LAMP assay to identify C. burnetii rapidly and sensitively. METHODS: A universal LAMP primer set was designed to detect the repeated sequence IS1111a of the htpAB gene of C. burnetii using PrimerExplorer V4 software. The sensitivity of the LAMP assay was evaluated using known quantities of recombined reference plasmids containing the targeted genes. The specificity of the developed LAMP assay was determined using 26 members of order Rickettsiae and 18 other common pathogens. The utility of the LAMP assay was further compared with real time PCR by the examination 24 blood samples including 6 confirmed and 18 probable Q fever cases, which diagnosed by IFA serological assessment and real time PCR. In addition, 126 animal samples from 4 provinces including 97 goats, 7 cattle, 18 horses, 3 marmots and 1 deer were compared by these two methods. RESULTS: The limits of detection of the LAMP assay for the htpAB gene were 1 copy per reaction. The specificity of the LAMP assay was 100%, and no cross-reaction was observed among the bacteria used in the study. The positive rate of unknown febrile patients was 33.3%(95%CI 30.2%-36.4% for the LAMP assay and 8.3%(95%CI 7.4%-9.2% for the real time PCR(P<0.05. Similarly, the total positive rate of animals was 7.9%(95%CI 7.1%-8.7% for the LAMP assay and 0.8%(95%CI 0.7%-0.9%for the real time

  8. Multiplex, rapid and sensitive isothermal detection of nucleic-acid sequence by endonuclease restriction-mediated real-time multiple cross displacement amplification

    Directory of Open Access Journals (Sweden)

    Yi eWang

    2016-05-01

    Full Text Available We have devised a novel isothermal amplification technology, termed endonuclease restriction-mediated real-time multiple cross displacement amplification (ET-MCDA, which facilitated multiplex, rapid, specific and sensitive detection of nucleic-acid sequences at a constant temperature. The ET-MCDA integrated multiple cross displacement amplification strategy, restriction endonuclease cleavage and real-time fluorescence detection technique. In the ET-MCDA system, the functional cross primer E-CP1 or E-CP2 was constructed by adding a short sequence at the 5’ end of CP1 or CP2, respectively, and the new E-CP1 or E-CP2 primer was labelled at the 5’ end with a fluorophore and in the middle with a dark quencher. The restriction endonuclease Nb.BsrDI specifically recognized the short sequence and digested the newly synthesized double-stranded terminal sequences (5’ end short sequences and their complementary sequences, which released the quenching, resulting on a gain of fluorescence signal. Thus, the ET-MCDA allowed real-time detection of single or multiple targets in only a single reaction, and the positive results were observed in as short as 12 minutes, detecting down to 3.125 fg of genomic DNA per tube. Moreover, the analytical specificity and the practical application of the ET-MCDA were also successfully evaluated in this study. Here we provided the details on the novel ET-MCDA technique and expounded the basic ET-MCDA amplification mechanism.

  9. Label-free and sensitive fluorescence detection of nucleic acid, based on combination of a graphene oxid /SYBR green I dye platform and polymerase assisted signal amplification

    Science.gov (United States)

    Zhu, Xiao; Xing, Da

    2012-12-01

    A new label-free isothermal fluorescence amplification detection for nucleic acid has been developed. In this paper, we first developed a novel sensitive and specific detection platform with an unmodified hairpin probe (HP) combination of the graphene oxid (GO)/ SYBR green I dye (SG), which was relied on the selective principle of adsorption and the high quenching efficiency of GO. Then for the application of this new strategy, we used Mirco RNA-21 (Mir-21) as the target to evaluate this working principle of our design. When the target was hybridizing with the HP and inducing its conformation of change, an efficient isothermal circular strand-displacement polymerization reaction was activating to assist the first signal amplification. In this format, the formed complex conformation of DNA would interact with its high affinity dye, then detached from the surface of GO after incubating with the platform of GO/intercalating dye. This reaction would accompany with obvious fluorescence recovery, and accomplish farther signal enhancement by a mass of intercalating dye inserting into the minor groove of the long duplex replication product. By taking advantage of the multiple amplification of signal, this method exerted substantial enhancement in sensitivity and could be used for rapid and selective detection of Mir-21 with attomole range. It is expected that this cost-effective GO based sensor might hold considerable potential to apply in bioanalysis studies.

  10. Multiplexed real-time PCR amplification of tlh, tdh and trh genes in Vibrio parahaemolyticus and its rapid detection in shellfish and Gulf of Mexico water.

    Science.gov (United States)

    Rizvi, Amy V; Bej, Asim K

    2010-10-01

    In this study, we have developed a SYBR Green I-based real-time multiplexed PCR assay for the detection of Vibrio parahaemolyticus in Gulf of Mexico water (gulf water), artificially seeded and natural oysters targeting three hemolysin genes, tlh, tdh and trh in a single reaction. Post-amplification melt-temperature analysis confirmed the amplification of all three targeted genes with high specificity. The detection sensitivity was 10 cfu (initial inoculum) in 1 ml of gulf water or oyster tissue homogenate, following 5 h enrichment. The results showed 58% of the oysters to be positive for tlh, indicating the presence of V. parahaemolyticus; of which 21% were positive for tdh; and 0.7% for trh, signifying the presence of pathogenic strains. The C(t) values showed that oyster tissue matrix had some level of inhibition, whereas the gulf water had negligible effect on PCR amplification. The assay was rapid (approximately 8 h), specific and sensitive, meeting the ISSC guidelines. Rapid detection using real-time multiplexed PCR will help reduce V. parahaemolyticus-related disease outbreaks, thereby increasing consumer confidence and economic success of the seafood industry.

  11. Visual detection of West Nile virus using reverse transcription loop-mediated isothermal amplification combined with a vertical flow visualization strip

    Directory of Open Access Journals (Sweden)

    Zengguo eCao

    2016-04-01

    Full Text Available West Nile virus (WNV causes a severe zoonosis, which can lead to a large number of casualties and considerable economic losses. A rapid and accurate identification methodfor WNV for use in field laboratories is urgently needed. Here, a method utilizing reverse transcription loop-mediated isothermal amplification combined with a vertical flow visualization strip (RT-LAMP-VF was developed to detect the envelope (E gene of WNV. The RT-LAMP-VF assay could detect 102 copies/μl ofan WNV RNA standard using a 40 min amplification reaction followed by a 2 min incubationof the amplification product on the visualization strip, and no cross-reaction with other closely related members of theFlavivirus genus was observed. The assay was further evaluated using cells and mouse brain tissues infected with a recombinant rabies virus expressing the E protein of WNV.The assay produced sensitivities of 101.5TCID50/ml and 101.33 TCID50/ml for detection of the recombinant virus in the cells and brain tissues, respectively. Overall, the RT-LAMP-VF assay developed in this study is rapid, simple and effective, and it is therefore suitable for clinical application in the field.

  12. An integrated direct loop-mediated isothermal amplification microdevice incorporated with an immunochromatographic strip for bacteria detection in human whole blood and milk without a sample preparation step.

    Science.gov (United States)

    Lee, Dohwan; Kim, Yong Tae; Lee, Jee Won; Kim, Do Hyun; Seo, Tae Seok

    2016-05-15

    We have developed an integrated direct loop-mediated isothermal amplification (Direct LAMP) microdevice incorporated with an immunochromatographic strip (ICS) to identify bacteria contaminated in real samples. The Direct LAMP is a novel isothermal DNA amplification technique which does not require thermal cycling steps as well as any sample preparation steps such as cell lysis and DNA extraction for amplifying specific target genes. In addition, the resultant amplicons were colorimetrically detected on the ICS, thereby enabling the entire genetic analysis process to be simplified. The two functional units (Direct LAMP and ICS) were integrated on a single device without use of the tedious and complicated microvalve and tubing systems. The utilization of a slidable plate allows us to manipulate the fluidic control in the microchannels manually and the sequential operation of the Direct LAMP and ICS detection could be performed by switching the slidable plate to each functional unit. Thus, the combination of the direct isothermal amplification without any sample preparation and thermal cycling steps, the ICS based amplicon detection by naked eyes, and the slidable plate to eliminate the microvalves in the integrated microdevice would be an ideal platform for point-of-care DNA diaganotics. On the integrated Direct LAMP-ICS microdevice, we could analyze Staphylococcus aureus (S. aureus) and Escherichia coli O157:H7 (E. coli O157:H7) contaminated in human whole blood or milk at a single-cell level within 1h.

  13. Sensitive electrochemical detection of telomerase activity using spherical nucleic acids gold nanoparticles triggered mimic-hybridization chain reaction enzyme-free dual signal amplification.

    Science.gov (United States)

    Wang, Wen-Jing; Li, Jing-Jing; Rui, Kai; Gai, Pan-Pan; Zhang, Jian-Rong; Zhu, Jun-Jie

    2015-03-03

    We report an electrochemical sensor for telomerase activity detection based on spherical nucleic acids gold nanoparticles (SNAs AuNPs) triggered mimic-hybridization chain reaction (mimic-HCR) enzyme-free dual signal amplification. In the detection strategy, SNAs AuNPs and two hairpin probes were employed. SNAs AuNPs as the primary amplification element, not only hybridized with the telomeric repeats on the electrode to amplify signal but also initiated the subsequent secondary amplification, mimic-hybridization chain reaction of two hairpin probes. If the cells' extracts were positive for telomerase activity, SNAs AuNPs could be captured on the electrode. The carried initiators could trigger an alternative hybridization reaction of two hairpin probes that yielded nicked double helices. The signal was further amplified enzyme-free by numerous hexaammineruthenium(III) chloride ([Ru(NH3)6](3+), RuHex) inserting into double-helix DNA long chain by electrostatic interaction, each of which could generate an electrochemical signal at appropriate potential. With this method, a detection limit of down to 2 HeLa cells and a dynamic range of 10-10,000 cells were achieved. Telomerase activities of different cell lines were also successfully evaluated.

  14. Detection of methicillin-resistant Staphylococcus aureus using phage amplification combined with matrix-assisted laser desorption/ionization mass spectrometry.

    Science.gov (United States)

    Rees, Jon C; Barr, John R

    2017-02-01

    Antibiotic resistance continues to contribute significantly to morbidity and mortality across the world. Developing new tests for antibiotic-resistant bacteria is a core action to combat resistant infections. We describe a method that uses phage amplification detection (PAD) combined with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) to rapidly identify Staphylococcus aureus and determine phenotypic susceptibility to cefoxitin. Samples tested for S. aureus are incubated together with bacteriophage in the presence and absence of cefoxitin and subjected to rapid trypsin digestion followed by MALDI-MS analysis. Tryptic peptides derived from amplified phage proteins can be detected by MALDI-MS, as validated by time-of-flight (TOF)/TOF analysis of each peptide combined with database searching. Methicillin-resistant S. aureus show significant phage amplification in the presence of cefoxitin, while methicillin-sensitive S. aureus show no phage amplification relative to a no-antibiotic control. We also show that PAD methodology can be implemented on an FDA-approved commercial MALDI-MS bacterial identification system to identify S. aureus and determine antibiotic susceptibility. The novelty of this assay includes the use of phage-derived tryptic peptides as detected by MALDI-MS to monitor the results of PAD on an instrument common to many modern microbiology laboratories.

  15. Visual detection and differentiation of Classic Swine Fever Virus strains using nucleic acid sequence-based amplification (NASBA) and G-quadruplex DNAzyme assay

    Science.gov (United States)

    Lu, Xiaolu; Shi, Xueyao; Wu, Gege; Wu, Tiantian; Qin, Rui; Wang, Yi

    2017-01-01

    The split G-quadruplex DNAzyme has emerged as a valuable tool for visual DNA detection. Here, we successfully integrated colorimetric split G-quadruplex DNAzyme assay with nucleic acid sequence-based amplification to generate a novel detection approach, allowing visual and rapid detection for the RNA of Shimen and HCLV strains of Classic Swine Fever Virus (CSFV). CSFV is a RNA virus that causes a highly contagious disease in domestic pigs and wild boar. With this method, we were able to detect as little as 10 copies/ml of CSF viral RNA within 3 h in serum samples taken from the field. No interference was encountered in the amplification and detection of Classic Swine Fever Virus in the presence of non-target RNA or DNA. Moreover, Shimen and HCLV strains of Classic Swine Fever Virus could be easily differentiated using the NASBA-DNAzyme system. These findings indicate the NASBA-DNAzyme system is a rapid and practical technique for detecting and discriminating CSFV strains and may be applied to the detection of other RNA viruses. PMID:28287135

  16. Development and Application of a Loop-Mediated Isothermal Amplification (LAMP Approach for the Rapid Detection of Dirofilaria repens from Biological Samples.

    Directory of Open Access Journals (Sweden)

    Donato Antonio Raele

    2016-06-01

    Full Text Available Dirofilariasis by Dirofilaria repens is an important mosquito vector borne parasitosis, and the dog represents the natural host and reservoir of the parasite. This filarial nematode can also induce disease in humans, and in the last decades an increasing number of cases have been being reported. The present study describes the first loop mediated isothermal amplification (LAMP assay to detect D. repens DNA in blood and mosquitoes. Two versions of the technique have been developed and described: in the first, the amplification is followed point by point through a real time PCR instrument (ReT-LAMP; in the second, the amplification is visualized by checking UV fluorescence of the reaction mixture after addition of propidium iodide (PI-LAMP. The two variants use the same set of 4 primers targeting the D. repens cytochrome oxidase subunit I (COI gene. To assess the specificity of the method, reactions were carried out by using DNA from the major zoonotic parasites of the family of Onchocercidae, and no amplification was observed. The lower limit of detection of the ReT-LAMP assay was 0.15 fg/μl (corresponding to about 50 copy of COI gene per μl. Results suggest that the described assay is specific, and its sensitivity is higher than the conventional PCR based on the same gene. It is also provide a rapid and cost-effective molecular detection of D. repens, mainly when PI-LAMP is applied, and it should be performed in areas where this emerging parasitosis is endemic.

  17. Effects of c-myc oncogene modulation on differentiation of human small cell lung carcinoma cell lines

    NARCIS (Netherlands)

    Van Waardenburg, RCAM; Meijer, C; Pinto-Sietsma, SJ; De Vries, EGE; Timens, W; Mulder, NM

    1998-01-01

    Amplification and over-expression of oncogenes of the myc family are related to the prognosis of certain solid tumors such as small cell lung cancer (SCLC). For SCLC, c-myc is the oncogene most consistently found to correlate with the end stage behaviour of the tumour, in particular with survival af

  18. Loop-mediated isothermal amplification as an emerging technology for detection of Yersinia ruckeri the causative agent of enteric red mouth disease in fish

    Directory of Open Access Journals (Sweden)

    Soliman Hatem

    2008-08-01

    Full Text Available Abstract Background Enteric Redmouth (ERM disease also known as Yersiniosis is a contagious disease affecting salmonids, mainly rainbow trout. The causative agent is the gram-negative bacterium Yersinia ruckeri. The disease can be diagnosed by isolation and identification of the causative agent, or detection of the Pathogen using fluorescent antibody tests, ELISA and PCR assays. These diagnostic methods are laborious, time consuming and need well trained personnel. Results A loop-mediated isothermal amplification (LAMP assay was developed and evaluated for detection of Y. ruckeri the etiological agent of enteric red mouth (ERM disease in salmonids. The assay was optimised to amplify the yruI/yruR gene, which encodes Y. ruckeri quorum sensing system, in the presence of a specific primer set and Bst DNA polymerase at an isothermal temperature of 63°C for one hour. Amplification products were detected by visual inspection, agarose gel electrophoresis and by real-time monitoring of turbidity resulted by formation of LAMP amplicons. Digestion with HphI restriction enzyme demonstrated that the amplified product was unique. The specificity of the assay was verified by the absence of amplification products when tested against related bacteria. The assay had 10-fold higher sensitivity compared with conventional PCR and successfully detected Y. ruckeri not only in pure bacterial culture but also in tissue homogenates of infected fish. Conclusion The ERM-LAMP assay represents a practical alternative to the microbiological approach for rapid, sensitive and specific detection of Y. ruckeri in fish farms. The assay is carried out in one hour and needs only a heating block or water bath as laboratory furniture. The advantages of the ERM-LAMP assay make it a promising tool for molecular detection of enteric red mouth disease in fish farms.

  19. Detection and differentiation of Fusarium oxysporum f. sp. lycopersici race 1 using loop-mediated isothermal amplification with three primer sets.

    Science.gov (United States)

    Ayukawa, Y; Komatsu, K; Kashiwa, T; Akai, K; Yamada, M; Teraoka, T; Arie, T

    2016-09-01

    Fusarium oxysporum f. sp. lycopersici (Fol) causes tomato wilt. Based on the difference in pathogenicity towards tomato cultivars, Fol is classified into three races. In this study, a rapid method is developed for the detection and discrimination of Fol race 1 using a loop-mediated isothermal amplification (LAMP) assay with two primer sets targeting a region of the nucleotide sequence of the SIX4 gene specific for race 1 and a primer set targeting the SIX5 gene, conserved in all known Fol isolates. Upon LAMP reaction, amplification using all three primer sets was observed only when DNA of Fol race 1 was used as a template, and not when DNA of other Fol races or other fungal species was used. This method could detect 300 fg of Fol race 1 DNA, a 100-fold higher sensitivity than that obtained by conventional PCR. The method can also detect DNA extracted from soil artificially infested with Fol race 1. It is now possible to detect Fol race 1 in colonies and infected tomato stems without DNA isolation. This method is a rapid and simple tool for discrimination of Fol race 1. This study developed a loop-mediated isothermal amplification (LAMP) assay for detection and differentiation of Fusarium oxysporum f. sp. lycopersici (Fol) race 1 by using three primer sets targeting for the SIX4 and SIX5 genes. These genes are present together only in Fol race 1. This method can detect Fol race 1 in infected tomato stems without DNA extraction, affording an efficient diagnosis of Fusarium wilt on tomatoes in the field. © 2016 The Society for Applied Microbiology.

  20. Development of a Reverse Transcription Loop-Mediated Isothermal Amplification Method for the Rapid Detection of Subtype H7N9 Avian Influenza Virus

    Directory of Open Access Journals (Sweden)

    Hongmei Bao

    2014-01-01

    Full Text Available A novel influenza A (H7N9 virus has emerged in China. To rapidly detect this virus from clinical samples, we developed a reverse transcription loop-mediated isothermal amplification (RT-LAMP method for the detection of the H7N9 virus. The minimum detection limit of the RT-LAMP assay was 0.01 PFU H7N9 virus, making this method 100-fold more sensitive to the detection of the H7N9 virus than conventional RT-PCR. The H7N9 virus RT-LAMP assays can efficiently detect different sources of H7N9 influenza virus RNA (from chickens, pigeons, the environment, and humans. No cross-reactive amplification with the RNA of other subtype influenza viruses or of other avian respiratory viruses was observed. The assays can effectively detect H7N9 influenza virus RNA in drinking water, soil, cloacal swab, and tracheal swab samples that were collected from live poultry markets, as well as human H7N9 virus, in less than 30 min. These results suggest that the H7N9 virus RT-LAMP assays were efficient, practical, and rapid diagnostic methods for the epidemiological surveillance and diagnosis of influenza A (H7N9 virus from different resource samples.

  1. Rapid detection of Staphylococcus aureus in dairy and meat foods by combination of capture with silica-coated magnetic nanoparticles and thermophilic helicase-dependent isothermal amplification.

    Science.gov (United States)

    Chen, Xingxing; Wu, Xiaoli; Gan, Min; Xu, Feng; He, Lihua; Yang, Dong; Xu, Hengyi; Shah, Nagendra P; Wei, Hua

    2015-03-01

    Staphylococcus aureus is one of the main pathogens in dairy and meat products; therefore, developing a highly sensitive and rapid method for its detection is necessary. In this study, a quantitative detection method for Staph. aureus was developed using silica-coated magnetic nanoparticles and thermophilic helicase-dependent isothermal amplification. First, genomic DNA was extracted from lysed bacteria using silica-coated magnetic nanoparticles and amplified using thermophilic helicase-dependent isothermal amplification. After adding the nucleic-acid dye SYBR Green I to the amplicons, the fluorescence intensity was observed using a UV lamp or recorded using a fluorescence spectrophotometer. This detection system had a detection limit of 5×10(0) cfu/mL in pure culture and milk-powder samples and 5×10(1) cfu/mL in pork samples using a UV light in less than 2h. In addition, a good linear relationship was obtained between fluorescence intensity and bacterial concentrations ranging from 10(2) to 10(4) cfu/mL under optimal conditions. Furthermore, the results from contaminated milk powder and pork samples suggested that the detection system could be used for the quantitative analysis of Staph. aureus and applied potentially to the food industry for the detection of this pathogen.

  2. Real-time sequence-validated loop-mediated isothermal amplification assays for detection of Middle East respiratory syndrome coronavirus (MERS-CoV.

    Directory of Open Access Journals (Sweden)

    Sanchita Bhadra

    Full Text Available The Middle East respiratory syndrome coronavirus (MERS-CoV, an emerging human coronavirus, causes severe acute respiratory illness with a 35% mortality rate. In light of the recent surge in reported infections we have developed asymmetric five-primer reverse transcription loop-mediated isothermal amplification (RT-LAMP assays for detection of MERS-CoV. Isothermal amplification assays will facilitate the development of portable point-of-care diagnostics that are crucial for management of emerging infections. The RT-LAMP assays are designed to amplify MERS-CoV genomic loci located within the open reading frame (ORF1a and ORF1b genes and upstream of the E gene. Additionally we applied one-step strand displacement probes (OSD for real-time sequence-specific verification of LAMP amplicons. Asymmetric amplification effected by incorporating a single loop primer in each assay accelerated the time-to-result of the OSD-RT-LAMP assays. The resulting assays could detect 0.02 to 0.2 plaque forming units (PFU (5 to 50 PFU/ml of MERS-CoV in infected cell culture supernatants within 30 to 50 min and did not cross-react with common human respiratory pathogens.

  3. Development of PCR primer systems for amplification of nitrite reductase genes (nirK and nirS) to detect denitrifying bacteria in environmental samples

    Energy Technology Data Exchange (ETDEWEB)

    Braker, G.; Witzel, K.P. [Max-Planck-Inst. fuer Limnologie, Ploen (Germany); Fesefeldt, A. [Univ. Kiel (Germany). Inst. fuer Allgemeine Mikrobiologie

    1998-10-01

    A system was developed for the detection of denitrifying bacteria by the application of specific nitrite reductase gene fragments with PCR. Primer sequences were found for the amplification of fragments from both nitrite reductase genes (nirK and nirS) after comparative sequence analysis. Whenever amplification was tried with these primers, the known nir type of denitrifying laboratory cultures could be confirmed. Likewise, the method allowed a determination of the nir type of five laboratory strains. The nirK gene could be amplified from Blastobacter denitrificans, Alcaligenes xylosoxidans, and Alcaligenes sp. (DSM 30128); the nirS gene was amplified from Alcaligenes eutrophus DSM 530 and from the denitrifying isolate IFAM 3698. For each of the two genes, at least one primer combination amplified successfully for all of the test strains. Specific amplification products were not obtained wit h nondenitrifying bacteria or with strains of the other nir type. The specificity of the amplified products was confirmed by subsequent sequencing. These results suggest the suitability of the method for the qualitative detection of denitrifying bacteria in environmental samples. This was shown by applying the generally amplifying primer combination for each nir gene developed in this study to total DNA preparations from aquatic habitats.

  4. [Rapid detection of Macrobrachium rosenbergii nodavirus isolated in China by a reverse-transcription loop-mediated isothermal amplification assay combined with a lateral flow dipstick method].

    Science.gov (United States)

    Lin, Feng; Liu, Li; Hao, Gui-Jie; Cao, Zheng; Sheng, Peng-Cheng; Wu, Ying-Lei; Shen, Jin-Yu

    2014-09-01

    White coloration of the muscle of the giant river prawn (Macrobrachium rosenbergii) is a serious problem in China. The Macrobrachium rosenbergii Nodavirus (MrNV) has been confirmed to be the pathogen that causes this disorder. To develop a rapid, sensitive and specific technology for the detection of Macrobrachium rosenbergii Nodavirus isolated from China (MrNV-China), a reverse-transcription loop- mediated isothermal amplification assay combined with a lateral flow dipstick (RT-LAMP-LFD) assay method is described. A set of four primers and a labeled probe were designed specifically to recognize six distinct regions of the MrNV RNA2 gene. Results showed the sensitivity of the RT-LAMP-LFD assay was ten-times higher than the reverse-transcription loop-mediated isothermal amplification assay (RT-LAMP) with agarose gel electrophoresis. The assay was conducted with one-step amplification at 61°C in a single tube within 45 min. No product was generated from shrimps infected with other viruses, including DNA viruses (infectious hypodermal and hematopoietic necrosis virus (IHHNV); white spot syndrome virus (WSSV)) and RNA viruses (Taura syndrome virus (TSV); infectious myonecrosis virus (IMNV); yellow head virus (YHV)). Results were visualized by the LFD method. Therefore, the described rapid and sensitive assay is potentially useful for MrNV detection.

  5. A label-free colorimetric isothermal cascade amplification for the detection of disease-related nucleic acids based on double-hairpin molecular beacon.

    Science.gov (United States)

    Wu, Dong; Xu, Huo; Shi, Haimei; Li, Weihong; Sun, Mengze; Wu, Zai-Sheng

    2017-03-08

    K-Ras mutations at codon 12 play an important role in an early step of carcinogenesis. Here, a label-free colorimetric isothermal cascade amplification for ultrasensitive and specific detection of K-Ras point mutation is developed based on a double-hairpin molecular beacon (DHMB). The biosensor consists of DHMB probe and a primer-incorporated polymerization template (PPT) designed partly complementary to DHMB. In the presence of polymerase, target DNA is designed to trigger strand displacement amplification (SDA) via promote the hybridization of PPT with DHMB and subsequently initiates cascade amplification process with the help of the nicking endonuclease. During the hybridization and enzymatic reaction, G-quadruplex/hemin DNAzymes are generated, catalyzing the oxidation of ABTS(2-) by H2O2 in the presence of hemin. Utilizing the proposed facile colorimetric scheme, the target DNA can be quantified down to 4 pM with the dynamic response range of 5 orders of magnitude, indicating the substantially improved detection capability. Even more strikingly, point mutation in K-ras gene can be readily observed by the naked eye without the need for the labeling or expensive equipment. Given the high-performance for K-Ras analysis, the enhanced signal transduction capability associated with double-hairpin structure of DHMB provides a novel rout to screen biomarkers, and the descripted colorimetric biosensor seems to hold great promise for diagnostic applications of genetic diseases. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. Visual Detection of Canine Parvovirus Based on Loop-Mediated Isothermal Amplification Combined with Enzyme-Linked Immunosorbent Assay and with Lateral Flow Dipstick

    OpenAIRE

    Sun, Yu-Ling; Yen, Chon-Ho; Tu, Ching-Fu

    2013-01-01

    ABSTRACT Loop-mediated isothermal amplification (LAMP) combined with enzyme-linked immunosorbent assay (LAMP–ELISA) and with lateral flow dipstick (LAMP–LFD) are rapid, sensitive and specific methods for the visual detection of clinical pathogens. In this study, LAMP–ELISA and LAMP–LFD were developed for the visual detection of canine parvovirus (CPV). For LAMP, a set of four primers (biotin-labeled forward inner primers) was designed to specifically amplify a region of the VP2 gene of CPV. T...

  7. Application of a Real-time Reverse Transcription Loop Mediated Amplification Method to the Detection of Rabies Virus in Arctic Foxes in Greenland

    DEFF Research Database (Denmark)

    Wakeley, Philip; Johnson, Nicholas; Rasmussen, Thomas Bruun

    Reverse transcription loop mediated amplification (RT-LAMP) offers a rapid, isothermal method for amplification of virus RNA. In this study a panel of positive rabies virus samples originally prepared from arctic fox brain tissue was assessed for the presence of rabies viral RNA using a real time...... RT-LAMP. The method had previously been shown to work with samples from Ghana which clustered with cosmopolitan lineage rabies viruses but the assay had not been assessed using samples from animals infected with rabies from the arctic region. The assay is designed to amplify both cosmopolitan strains...... virus of arctic origin virus can be detected using RT-LAMP and the method reported is more rapid than the real-time RT-PCR. Further arctic fox samples are under analysis in order to confirm these findings....

  8. Biochip for Real-Time Monitoring of Hepatitis B Virus (HBV) by Combined Loop-Mediated Isothermal Amplification and Solution-Phase Electrochemical Detection

    Science.gov (United States)

    Tien, Bui Quang; Ngoc, Nguyen Thy; Loc, Nguyen Thai; Thu, Vu Thi; Lam, Tran Dai

    2017-06-01

    Accurate in situ diagnostic tests play a key role in patient management and control of most infectious diseases. To achieve this, use of handheld biochips that implement sample handling, sample analysis, and result readout together is an ideal approach. We present herein a fluid-handling biochip for real-time electrochemical monitoring of nucleic acid amplification based on loop-mediated isothermal amplification and real-time electrochemical detection on a microfluidic platform. Intercalation between amplifying DNA and free redox probe in solution phase was used to monitor the number of DNA copies. The whole diagnostic process is completed within 70 min. Our platform offers a fast and easy tool for quantification of viral pathogens in shorter time and with limited risk of all potential forms of cross-contamination. Such diagnostic tools have potential to make a huge difference to the lives of millions of people worldwide.

  9. A loop-mediated isothermal amplification assay and sample preparation procedure for sensitive detection of Xanthomonas fragariae in strawberry