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Sample records for rapid multiplex detection

  1. Multiplexed plasmon sensor for rapid label-free analyte detection.

    Science.gov (United States)

    Rosman, Christina; Prasad, Janak; Neiser, Andreas; Henkel, Andreas; Edgar, Jonathan; Sönnichsen, Carsten

    2013-07-10

    Efficient and cost-effective multiplexed detection schemes for proteins in small liquid samples would bring drastic advances to fields like disease detection or water quality monitoring. We present a novel multiplexed sensor with randomly deposited aptamer functionalized gold nanorods. The spectral position of plasmon resonances of individual nanorods, monitored by dark-field spectroscopy, respond specifically to different proteins. We demonstrate nanomolar sensitivity, sensor recycling, and the potential to upscale to hundreds or thousands of targets.

  2. Rapid detection of methicillin-resistant staphylococci by multiplex PCR

    African Journals Online (AJOL)

    Administrator

    2010-11-08

    Nov 8, 2010 ... Deletion screening of the Duchenne muscular dystrophy locus via multiplex DNA amplification. Nucleic Acids Res. 16: 11141-11156. Fang H, Hedin G (2003). Rapid screening and identification of methicillin-resistant Staphylococcus aureus from clinical samples by selective-broth and real-time PCR assay.

  3. Rapid detection of methicillin-resistant staphylococci by multiplex PCR

    African Journals Online (AJOL)

    A rapid and sensitive method for excluding the presence of methicillin-resistant Staphylococcus aureus (MRSA) in clinical samples was developed. The combination of MRSA detection by mecA coaA PCR with prior enrichment in selective broth was tested for 300 swabs. PCR identified 26 MRSApositive samples, ...

  4. Development of multiplex-PCR assay for rapid detection of Candida spp.

    Directory of Open Access Journals (Sweden)

    Ni Made A. Tarini

    2010-05-01

    Full Text Available Aim Candida spp. infection commonly occur in immunocompromised patients. Biochemical assay for identification of Candida spp. is time-consuming and shows many undetermined results. Specific detection for antibody, antigen and metabolites of Candida spp. had low sensitivity and specificity. In this study, we developed a rapid diagnostic method, Multiplex-PCR, to identify Candida spp.Methods Five Candida spp. isolates were cultured, identifi ed with germ tube and API® 20 C AUX (BioMerieux® SA kit. Furthermore, DNA was purified by QIAamp DNA mini (Qiagen® kit for Multiplex-PCR assay.Results DNA detection limit by Multiplex-PCR assays for C. albicans, C. tropicalis, C. parapsilosis, C. krusei and C. glabrata were 4 pg, 0.98 pg, 0.98 pg, 0.5 pg and 16 pg respectively. This assay was also more sensitive than culture in that Multiplex-PCR could detect 2.6-2.9 x 100 CFU/ml, whereas culture 2.6-2.9 x 102 CFU/ml.Conclusion Multiplex-PCR is much more sensitive than culture and thus, can be recommended as a sensitive and specific assay for identification of Candida spp. (Med J Indones 2010; 19:83-7Keywords: Candida spp., multiplex-PCR

  5. Development of a novel multiplex PCR assay for rapid detection of virulence associated genes of Pasteurella multocida from pigs

    National Research Council Canada - National Science Library

    Rajkhowa, S

    2015-01-01

    Significance and Impact of the Study: The study reports the development and evaluation of a novel multiplex PCR assay for the rapid detection of 11 important VAGs of Pasteurella multocida isolates from pigs...

  6. Direct PCR - A rapid method for multiplexed detection of different serotypes of Salmonella in enriched pork meat samples

    DEFF Research Database (Denmark)

    Chin, Wai Hoe; Sun, Yi; Høgberg, Jonas

    2017-01-01

    , in this study, we developed a multiplex Direct PCR method for rapid detection of different Salmonella serotypes directly from pork meat samples without any DNA purification steps. An inhibitor-resistant Phusion Pfu DNA polymerase was used to overcome PCR inhibition. Four pairs of primers including a pair...... of newly designed primers targeting Salmonella spp. at subtype level were incorporated in the multiplex Direct PCR. To maximize the efficiency of the Direct PCR, the ratio between sample and dilution buffer was optimized. The sensitivity and specificity of the multiplex Direct PCR were tested using...... and integration into a point-of-need Lab-on-a-chip system for rapid online pathogen detection....

  7. Rapid detection of Actinobacillus actinomycetemcomitans, Prevotella intermedia and Porphyromona gingivalis by multiplex PCR.

    Science.gov (United States)

    García, L; Tercero, J C; Legido, B; Ramos, J A; Alemany, J; Sanz, M

    1998-01-01

    The identification of specific periodontal pathogens by conventional methods, mainly anaerobic cultivation, is difficult, time consuming and even sometimes unreliable. Therefore, a multiplex PCR method for simultaneous detection of Actinobacillus actinomycetemcomitans (A.a.), Porphyromona gingivalis (P.g.) and Prevotella intermedia (P.i.) was developed for rapid and easy identification of these specific bacterial pathogens in subgingival plaque samples. In this paper, there is a detailed description of the oligonucleotide primer selection, DNA extraction and PCR conditions and the sequencing of the amplified products. The locus chosen to be amplified is a highly variable region in the 16S ribosomal DNA. For the development of this technique ATCC cultures and pure cultures from subgingival plaque samples taken from periodontitis patients were used. As an internal positive control a recombinant plasmid was developed. This simple DNA extraction procedure and the DNA amplification and visualization of the amplified product permits the detection of the bacteria in a working day. Thus, this multiplex PCR method is a rapid and effective detection method for specific periodontal pathogens.

  8. Development a rapid and accurate multiplex real time PCR method for the detection Chlamydia trachomatis and Mycoplasma hominis.

    Science.gov (United States)

    Safarkar, Roya; Mehrabadi, Jalil Fallah; Noormohammadi, Zahra; Mirnejad, Reza

    2017-11-01

    Sexually transmitted diseases easily spread among sexually active people and often have no symptoms. Rapid and accurate method for detecting these infections are necessary in early stages. The traditional detection methods of them are difficult and time-consuming. In this study, multiplex real time PCR was optimized for rapid identification of Chlamydia trachomatis and Mycoplasma hominis in a single tube and was performed with our designed primers. The sensitivity test was carried out to designed primers with diluted genomic DNA. To defined the specificity, non STD bacteria were used as DNA template. This study indicated that the developed multiplex real time PCR can be an effective alternative procedure to the conventional methods for rapid and accurate identification of C Chlamydia trachomatis and Mycoplasma hominis. Multiplex real-time PCR Results of them were checked with melting curves. The sensitivity of our designed primer by multiplex real time PCR for Chlamydia trachomatis and Mycoplasma hominis were 4.78×1010 and 8.35×1010 , respectively, Which the primers did not amplify any product from a non-STD species. Multiplex real time PCR by our new primers and analysis of melting curves were successfully usable for rapid and accurate detection of Chlamydia trachomatis and Mycoplasma hominis. This assay instead of traditional culture method, has considerable potential to be rapid, accurate and highly sensitive molecular diagnostic tool for simultaneous and direct detection. © 2017 Wiley Periodicals, Inc.

  9. A multiplex RT-PCR for rapid and simultaneous detection of viruses and viroids in chrysanthemum.

    Science.gov (United States)

    Song, A; You, Y; Chen, F; Li, P; Jiang, J; Chen, S

    2013-01-01

    Chrysanthemum plants are subject to serious virus diseases, so detection and identification of virus pathogens is important to prevent the virus spread. A reliable one-step multiplex RT-PCR was developed to simultaneously detect two viruses and two viriods: chrysanthemum virus B, tomato Aspermy virus, chrysanthemum stunt viroid and chrysanthemum chlorotic mottle viroid. In addition, we investigated the detection limit and the efficiency of single and multiplex RT-PCR assays. The results showed that the multiplex RT-PCR assay proved to be as sensitive as the single one. In conclusion, this technique is potentially useful in routine diagnosis of chrysanthemum viruses and viroids. The multiplex RT-PCR assay described in this study is the first report of simultaneous detection of virus and viroid in chrysanthemum, which provides a fast, convenient, cost-saving way to detect the virus and viroid mixed infections in plants. © 2012 The Society for Applied Microbiology.

  10. Rapid DNA haplotyping using a multiplex heteroduplex approach: Application to Duchenne muscula dystrophy carrier detection

    Energy Technology Data Exchange (ETDEWEB)

    Prior, T.W.; Wenger, G.D.; Moore, J. [Ohio State Univ., Columbus, OH (United States)] [and others

    1994-09-01

    A new strategy has been developed for rapid haplotype analysis. It is based on an initial multiplex amplification of several polymorphic sites, followed by heteroduplex detection. Heteroduplexes formed between two different alleles are detected because they migrate differently than the corresponding homoduplexes in Hydrolink-MDE gel. The method is simple, rapid, does not depend on specific sequences such as restriction enzyme sites or CA boxes and does not require the use of isotope. This approach has been tested using 12 commonly occurring polymorphisms spanning the dystrophin gene as a model. We describe the use of the method to assign the carrier status of females in Duchenne muscular dystrophy (DMD) pedigrees. As a result of expanding the number of detectable polymorphisms throughout the dystrophin gene, we show how the method can easily be combined with dinucleotide analysis to improve the accuracy of carrier detection in the nondeletion cases. The technique is also shown to be used as an effective screen for improving carrier detection in several families with deletions. The finding of heterozygosity within the deletion identifies the at-risk female as a noncarrier. Using this method, we have identified and incorporated 3 new dystrophin polymorphisms (one of which in exon 16 is unique to African Americans). The method may be used other genetic diseases when mutations are unknown, or there are few dinucleotide markers in the gene proximity, or for the identification of haplotype backgrounds of mutant alleles.

  11. Clinical usefulness of multiplex PCR lateral flow in MRSA detection: a novel, rapid genetic testing method.

    Science.gov (United States)

    Nihonyanagi, Shin; Kanoh, Yuhsaku; Okada, Kiyomi; Uozumi, Toshiki; Kazuyama, Yukumasa; Yamaguchi, Tokiko; Nakazaki, Nobuhiko; Sakurai, Keizou; Hirata, Yasuyoshi; Munekata, Shinichi; Ohtani, Shinichi; Takemoto, Tsuyoshi; Bandoh, Yuki; Akahoshi, Tohru

    2012-06-01

    Methicillin-resistant Staphylococcus aureus (MRSA) with exogenous cassette DNA containing the methicillin-resistant gene mecA (SCCmec) poses a problem as a drug-resistant bacterium responsible for hospital- and community-acquired infections. The frequency of MRSA detection has recently been increasing rapidly in Japan, and SCCmec has also been classified more diversely into types I-V. A rapid test is essential for early diagnosis and treatment of MRSA infections, but detection by conventional methods requires at least two days. The newly developed multiplex PCR lateral flow method allows specific amplification of femA to detect S. aureus, mecA to detect SCCmec, and kdpC to detect SCCmec type II; moreover, PCR products can be evaluated visually in about 3 h. In the present study, we developed a PCR lateral flow method for MRSA using this method and investigated its clinical usefulness in the detection of MRSA. The results showed a diagnostic concordance rate of 91.7% for MRSA and methicillin-susceptible S. aureus between bacteriological examination and PCR lateral flow, and a high level of specificity in PCR lateral flow. In addition, a higher detection rate for S. aureus using the same sample was observed for PCR lateral flow (70.2%) than for bacteriological tests (48.6%). The above results show that PCR lateral flow for MRSA detection has high sensitivity, specificity, and speed, and its clinical application as a method for early diagnosis of MRSA infections appears to be feasible.

  12. Multiplex real-time PCR assay for rapid detection of methicillin-resistant staphylococci directly from positive blood cultures.

    Science.gov (United States)

    Wang, Hye-Young; Kim, Sunghyun; Kim, Jungho; Park, Soon-Deok; Uh, Young; Lee, Hyeyoung

    2014-06-01

    Methicillin-resistant Staphylococcus aureus (MRSA) is the most prevalent cause of bloodstream infections (BSIs) and is recognized as a major nosocomial pathogen. This study aimed to evaluate a newly designed multiplex real-time PCR assay capable of the simultaneous detection of mecA, S. aureus, and coagulase-negative staphylococci (CoNS) in blood culture specimens. The Real-MRSA and Real-MRCoNS multiplex real-time PCR assays (M&D, Republic of Korea) use the TaqMan probes 16S rRNA for Staphylococcus spp., the nuc gene for S. aureus, and the mecA gene for methicillin resistance. The detection limit of the multiplex real-time PCR assay was 10(3) CFU/ml per PCR for each gene target. The multiplex real-time PCR assay was evaluated using 118 clinical isolates from various specimen types and a total of 350 positive blood cultures from a continuous monitoring blood culture system. The results obtained with the multiplex real-time PCR assay for the three targets were in agreement with those of conventional identification and susceptibility testing methods except for one organism. Of 350 positive bottle cultures, the sensitivities of the multiplex real-time PCR kit were 100% (166/166 cultures), 97.2% (35/36 cultures), and 99.2% (117/118 cultures) for the 16S rRNA, nuc, and mecA genes, respectively, and the specificities for all three targets were 100%. The Real-MRSA and Real-MRCoNS multiplex real-time PCR assays are very useful for the rapid accurate diagnosis of staphylococcal BSIs. In addition, the Real-MRSA and Real-MRCoNS multiplex real-time PCR assays could have an important impact on the choice of appropriate antimicrobial therapy, based on detection of the mecA gene. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  13. Multiplex Real-Time PCR Assay for Rapid Detection of Methicillin-Resistant Staphylococci Directly from Positive Blood Cultures

    Science.gov (United States)

    Wang, Hye-young; Kim, Sunghyun; Kim, Jungho; Park, Soon-Deok

    2014-01-01

    Methicillin-resistant Staphylococcus aureus (MRSA) is the most prevalent cause of bloodstream infections (BSIs) and is recognized as a major nosocomial pathogen. This study aimed to evaluate a newly designed multiplex real-time PCR assay capable of the simultaneous detection of mecA, S. aureus, and coagulase-negative staphylococci (CoNS) in blood culture specimens. The Real-MRSA and Real-MRCoNS multiplex real-time PCR assays (M&D, Republic of Korea) use the TaqMan probes 16S rRNA for Staphylococcus spp., the nuc gene for S. aureus, and the mecA gene for methicillin resistance. The detection limit of the multiplex real-time PCR assay was 103 CFU/ml per PCR for each gene target. The multiplex real-time PCR assay was evaluated using 118 clinical isolates from various specimen types and a total of 350 positive blood cultures from a continuous monitoring blood culture system. The results obtained with the multiplex real-time PCR assay for the three targets were in agreement with those of conventional identification and susceptibility testing methods except for one organism. Of 350 positive bottle cultures, the sensitivities of the multiplex real-time PCR kit were 100% (166/166 cultures), 97.2% (35/36 cultures), and 99.2% (117/118 cultures) for the 16S rRNA, nuc, and mecA genes, respectively, and the specificities for all three targets were 100%. The Real-MRSA and Real-MRCoNS multiplex real-time PCR assays are very useful for the rapid accurate diagnosis of staphylococcal BSIs. In addition, the Real-MRSA and Real-MRCoNS multiplex real-time PCR assays could have an important impact on the choice of appropriate antimicrobial therapy, based on detection of the mecA gene. PMID:24648566

  14. Direct PCR - A rapid method for multiplexed detection of different serotypes of Salmonella in enriched pork meat samples.

    Science.gov (United States)

    Chin, Wai Hoe; Sun, Yi; Høgberg, Jonas; Quyen, Than Linh; Engelsmann, Pia; Wolff, Anders; Bang, Dang Duong

    2017-04-01

    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 method is time consuming. In response to the demand for rapid on line or at site detection of pathogens, in this study, we developed a multiplex Direct PCR method for rapid detection of different Salmonella serotypes directly from pork meat samples without any DNA purification steps. An inhibitor-resistant Phusion Pfu DNA polymerase was used to overcome PCR inhibition. Four pairs of primers including a pair of newly designed primers targeting Salmonella spp. at subtype level were incorporated in the multiplex Direct PCR. To maximize the efficiency of the Direct PCR, the ratio between sample and dilution buffer was optimized. The sensitivity and specificity of the multiplex Direct PCR were tested using naturally contaminated pork meat samples for detecting and subtyping of Salmonella spp. Conventional bacterial culture methods were used as reference to evaluate the performance of the multiplex Direct PCR. Relative accuracy, sensitivity and specificity of 98.8%; 97.6% and 100%, respectively, were achieved by the method. Application of the multiplex Direct PCR to detect Salmonella in pork meat at slaughter reduces the time of detection from 5 to 6 days by conventional bacterial culture and serotyping methods to 14 h (including 12 h enrichment time). Furthermore, the method poses a possibility of miniaturization and integration into a point-of-need Lab-on-a-chip system for rapid online pathogen detection. Copyright © 2016 Elsevier Ltd. All rights reserved.

  15. Rapid detection of chromosomal aneuploidies in uncultured amniocytes by multiplex ligation-dependent probe amplification (MLPA)

    NARCIS (Netherlands)

    Hochstenbach, R; Meijer, J; van de Brug, J; Vossebeld-Hoff, I; Jansen, R; van der Luijt, R B; Sinke, R J; Page-Christiaens, G C M L; Ploos van Amstel, J-K; de Pater, J M

    2005-01-01

    OBJECTIVE: To test whether multiplex ligation-dependent probe amplification (MLPA) can be used for the detection of aneuploidy of chromosomes 13, 18, 21, X, and Y in uncultured amniocytes. METHODS: We performed a prospective study based on 527 amniotic fluid samples. Chromosome copy numbers were

  16. Rapid detection and differentiation of mycobacterial species using a multiplex PCR system

    Directory of Open Access Journals (Sweden)

    Andrea Santos Lima

    2013-07-01

    Full Text Available Introduction The early diagnosis of mycobacterial infections is a critical step for initiating treatment and curing the patient. Molecular analytical methods have led to considerable improvements in the speed and accuracy of mycobacteria detection. Methods The purpose of this study was to evaluate a multiplex polymerase chain reaction system using mycobacterial strains as an auxiliary tool in the differential diagnosis of tuberculosis and diseases caused by nontuberculous mycobacteria (NTM Results Forty mycobacterial strains isolated from pulmonary and extrapulmonary origin specimens from 37 patients diagnosed with tuberculosis were processed. Using phenotypic and biochemical characteristics of the 40 mycobacteria isolated in LJ medium, 57.5% (n=23 were characterized as the Mycobacterium tuberculosis complex (MTBC and 20% (n=8 as nontuberculous mycobacteria (NTM, with 22.5% (n=9 of the results being inconclusive. When the results of the phenotypic and biochemical tests in 30 strains of mycobacteria were compared with the results of the multiplex PCR, there was 100% concordance in the identification of the MTBC and NTM species, respectively. A total of 32.5% (n=13 of the samples in multiplex PCR exhibited a molecular pattern consistent with NTM, thus disagreeing with the final diagnosis from the attending physician. Conclusions Multiplex PCR can be used as a differential method for determining TB infections caused by NTM a valuable tool in reducing the time necessary to make clinical diagnoses and begin treatment. It is also useful for identifying species that were previously not identifiable using conventional biochemical and phenotypic techniques.

  17. Rapid detection and identification of 12 respiratory viruses using a dual priming oligonucleotide system-based multiplex PCR assay.

    Science.gov (United States)

    Kim, Suk Ran; Ki, Chang-Seok; Lee, Nam Yong

    2009-03-01

    Acute viral respiratory infections are among the most common causes of human disease. Rapid and accurate diagnosis of viral respiratory infections is important for providing timely therapeutic interventions. This study evaluated a new multiplex PCR assay (Seegene Inc., Seoul, Korea) for simultaneous detection and identification of 12 respiratory viruses using two primer mixes. The viruses included parainfluenza viruses 1, 2, and 3, human metapneumovirus, human coronavirus 229E/NL63 and OC43, adenovirus, influenza viruses A and B, human respiratory syncytial viruses A and B, and human rhinovirus A. The analytical sensitivity of the assay was 10-100 copies per reaction for each type of virus. There was no cross-reactivity with common bacterial or viral pathogens. A comparison with conventional viral culture and immunofluorescence was carried out using 101 respiratory specimens from 92 patients. Using viral culture, 57 specimens (56.4%) were positive without co-infection. The same viruses were identified in all 57 specimens using the multiplex PCR. Seven of the 57 specimens (12.3%) were found to be co-infected with other respiratory viruses, and 19 of 44 (43.2%) specimens which were negative by culture were positive by the multiplex PCR. The Seeplex Respiratory Virus Detection assay represents a significant improvement over the conventional methods for the detection of a broad spectrum of respiratory viruses.

  18. Development of a novel multiplex PCR assay for rapid detection of virulence associated genes of Pasteurella multocida from pigs.

    Science.gov (United States)

    Rajkhowa, S

    2015-09-01

    As the pathogenicity of Pasteurella multocida is associated with various virulence factors (VFs), the aim of the study was to develop a novel multiplex PCR (m-PCR) assay for the rapid detection of important virulence associated genes (VAGs) of P. multocida isolates from pigs. The target recognized VFs used in the study were diverse adhesins (ptfA and pfhA), toxins (toxA), siderophores (tonB and hgbA), sialidases (nanB, nanH) and outer membrane proteins (ompA, ompH, oma87 and plpB). The primers for the genes encoding these VFs were designed by primer3 software (http://bioinfo.ut.ee/primer3-0.4.0/) using gene sequences available in Genbank. The detection limit of the developed assay was 10(2)  CFU ml(-1) . The m-PCR did not produce any nonspecific amplification products when tested against Bordetella bronchiseptica which also commonly infects pigs. We applied m-PCR to the field samples, and the results obtained were the same as the single PCR results. The developed assay would be very useful for veterinary diagnostic laboratories and for others interested in the rapid virulence profiling of porcine P. multocida isolates circulating in the piggeries. The study reports the development and evaluation of a novel multiplex PCR assay for the rapid detection of 11 important VAGs of Pasteurella multocida isolates from pigs. Rapid and simultaneous detection of recognized VFs of the organism are essential to know the virulo-types of P. multocida isolates circulating in the piggeries. The developed novel assay will be very useful for the rapid detection of VAGs of P. multocida isolates from pigs. © 2015 The Society for Applied Microbiology.

  19. Rapid detection of Shigella and enteroinvasive Escherichia coli in produce enrichments by a conventional multiplex PCR assay.

    Science.gov (United States)

    Binet, Rachel; Deer, Deanne M; Uhlfelder, Samantha J

    2014-06-01

    Faster detection of contaminated foods can prevent adulterated foods from being consumed and minimize the risk of an outbreak of foodborne illness. A sensitive molecular detection method is especially important for Shigella because ingestion of as few as 10 of these bacterial pathogens can cause disease. The objectives of this study were to compare the ability of four DNA extraction methods to detect Shigella in six types of produce, post-enrichment, and to evaluate a new and rapid conventional multiplex assay that targets the Shigella ipaH, virB and mxiC virulence genes. This assay can detect less than two Shigella cells in pure culture, even when the pathogen is mixed with background microflora, and it can also differentiate natural Shigella strains from a control strain and eliminate false positive results due to accidental laboratory contamination. The four DNA extraction methods (boiling, PrepMan Ultra [Applied Biosystems], InstaGene Matrix [Bio-Rad], DNeasy Tissue kit [Qiagen]) detected 1.6 × 10(3)Shigella CFU/ml post-enrichment, requiring ∼18 doublings to one cell in 25 g of produce pre-enrichment. Lower sensitivity was obtained, depending on produce type and extraction method. The InstaGene Matrix was the most consistent and sensitive and the multiplex assay accurately detected Shigella in less than 90 min, outperforming, to the best of our knowledge, molecular assays currently in place for this pathogen. Published by Elsevier Ltd.

  20. Rapid detection and typing of pathogenic nonpneumophila Legionella spp. isolates using a multiplex real-time PCR assay.

    Science.gov (United States)

    Benitez, Alvaro J; Winchell, Jonas M

    2016-04-01

    We developed a single tube multiplex real-time PCR assay that allows for the rapid detection and typing of 9 nonpneumophila Legionella spp. isolates that are clinically relevant. The multiplex assay is capable of simultaneously detecting and discriminating L. micdadei, L. bozemanii, L. dumoffii, L. longbeachae, L. feeleii, L. anisa, L. parisiensis, L. tucsonensis serogroup (sg) 1 and 3, and L. sainthelensis sg 1 and 2 isolates. Evaluation of the assay with nucleic acid from each of these species derived from both clinical and environmental isolates and typing strains demonstrated 100% sensitivity and 100% specificity when tested against 43 other Legionella spp. Typing of L. anisa, L. parisiensis, and L. tucsonensis sg 1 and 3 isolates was accomplished by developing a real-time PCR assay followed by high-resolution melt (HRM) analysis targeting the ssrA gene. Further typing of L. bozemanii, L. longbeachae, and L. feeleii isolates to the serogroup level was accomplished by developing a real-time PCR assay followed by HRM analysis targeting the mip gene. When used in conjunction with other currently available diagnostic tests, these assays may aid in rapidly identifying specific etiologies associated with Legionella outbreaks, clusters, sporadic cases, and potential environmental sources. Published by Elsevier Inc.

  1. Rapid detection of carbapenemase genes by multiplex real-time PCR.

    Science.gov (United States)

    Monteiro, Jussimara; Widen, Raymond H; Pignatari, Antonio C C; Kubasek, Carly; Silbert, Suzane

    2012-04-01

    To develop a single multiplex real-time PCR assay to detect six different genetic types of carbapenemases already identified in Enterobacteriaceae (KPC, GES, NDM, IMP, VIM and OXA-48). A total of 58 bacterial isolates were tested. Thirty were previously characterized as resistant to carbapenems and documented by PCR and sequencing analysis to carry the following genes: bla(KPC) type, bla(GES) type, bla(IMP) type, bla(VIM) type, bla(OXA-48) and bla(NDM-1). These positive strains included 21 Enterobacteriaceae, 1 Acinetobacter baumannii and 8 Pseudomonas aeruginosa isolates. The remaining 28 isolates previously tested susceptible to carbapenems and were negative for these genes. Bacterial DNA was extracted using the easyMag extractor (bioMérieux, France). The real-time PCR was performed using the Rotor-Gene 6000 instrument (Corbett Life Science, Australia) and specific primers for each carbapenemase target were designed using the DNAStar software (Madison, WI, USA). Each one of the six carbapenemase genes tested presented a different melting curve after PCR amplification. The melting temperature (T(m)) analysis of the amplicons identified was as follows: bla(IMP) type (T(m) 80.1°C), bla(OXA-48) (T(m) 81.6°C), bla(NDM-1) (T(m) 84°C), bla(GES) type (T(m) 88.6°C), bla(VIM) type (T(m) 90.3°C) and bla(KPC) type (T(m) 91.6°C). No amplification was detected among the negative samples. The results showed 100% concordance with the genotypes previously identified. The new assay was able to detect the presence of six different carbapenemase gene types in a single 3 h PCR.

  2. [Application of multiplex ligation-dependent probe amplification for rapid detection of aneuploidies and structural chromosomal abnormalities in prenatal diagnosis].

    Science.gov (United States)

    Zhang, Jingjing; Hu, Ping; Luo, Chunyu; Ji, Qiuqing; Zhou, Jing; Liu, An; Ma, Dingyuan; Xu, Zhengfeng

    2014-02-01

    To explore the value of multiplex ligation-dependent probe amplification (MLPA) for rapid detection of aneuploidies and structural chromosomal abnormalities during prenatal diagnosis. Two hundred and eight six amniotic fluid samples were analyzed with both MLPA and conventional karyotyping. Structural abnormalities were verified with array comparative genomic hybridization. Ten cases of trisomy 21, 2 cases of trisomy 18, 1 case of trisomy 13, 1 case of mosaic trisomy 21, 1 case of 45,X, 1 case of large deletion of Xp, 1 case of trisomy 18p and 1 case of large deletion of 18p and 18q were identified. The same results were derived by both MLPA and conventional karyotyping. Structural abnormalities were verified by array comparative genomic hybridization (aCGH) with 100% accuracy. In addition to aneuploidies, MLPA can rapidly identify large deletions and duplications of chromosomes 21, 18, 13, X and Y. MLPA is supplementary to conventional karyotyping for identification of such chromosomal abnormalities prenatal diagnosis.

  3. Rapid detection and identification of viral and bacterial fish pathogens using a DNA array‐based multiplex assay

    DEFF Research Database (Denmark)

    Lievens, B.; Frans, I.; Heusdens, C.

    2011-01-01

    for the simultaneous detection and identification of all cyprinid herpesviruses (CyHV‐1, CyHV‐2 and CyHV‐3) and some of the most important fish pathogenic Flavobacterium species, including F. branchiophilum, F. columnare and F. psychrophilum. For virus identification, the DNA polymerase and helicase genes were......Fish diseases can be caused by a variety of diverse organisms, including bacteria, fungi, viruses and protozoa, and pose a universal threat to the ornamental fish industry and aquaculture. The lack of rapid, accurate and reliable means by which fish pathogens can be detected and identified has been...... one of the main limitations in fish pathogen diagnosis and fish disease management and has consequently stimulated the search for alternative diagnostic techniques. Here, we describe a method based on multiplex and broad‐range PCR amplification combined with DNA array hybridization...

  4. Rapid focused sequencing: a multiplexed assay for simultaneous detection and strain typing of Bacillus anthracis, Francisella tularensis, and Yersinia pestis.

    Directory of Open Access Journals (Sweden)

    Rosemary S Turingan

    Full Text Available BACKGROUND: The intentional release of Bacillus anthracis in the United States in 2001 has heightened concern about the use of pathogenic microorganisms in bioterrorism attacks. Many of the deadliest bacteria, including the Class A Select Agents Bacillus anthracis, Francisella tularensis, and Yersinia pestis, are highly infectious via the pulmonary route when released in aerosolized form. Hence, rapid, sensitive, and reliable methods for detection of these biothreats and characterization of their potential impact on the exposed population are of critical importance to initiate and support rapid military, public health, and clinical responses. METHODOLOGY/PRINCIPAL FINDINGS: We have developed microfluidic multiplexed PCR and sequencing assays based on the simultaneous interrogation of three pathogens per assay and ten loci per pathogen. Microfluidic separation of amplified fluorescently labeled fragments generated characteristic electrophoretic signatures for identification of each agent. The three sets of primers allowed significant strain typing and discrimination from non-pathogenic closely-related species and environmental background strains based on amplicon sizes alone. Furthermore, sequencing of the 10 amplicons per pathogen, termed "Rapid Focused Sequencing," allowed an even greater degree of strain discrimination and, in some cases, can be used to determine virulence. Both amplification and sequencing assays were performed in microfluidic biochips developed for fast thermal cycling and requiring 7 µL per reaction. The 30-plex sequencing assay resulted in genotypic resolution of 84 representative strains belonging to each of the three biothreat species. CONCLUSIONS/SIGNIFICANCE: The microfluidic multiplexed assays allowed identification and strain differentiation of the biothreat agents Bacillus anthracis, Francisella tularensis, and Yersinia pestis and clear discrimination from closely-related species and several environmental

  5. Development and application of a multiplex PCR assay for rapid detection of 4 major bacterial pathogens in ducks.

    Science.gov (United States)

    Wei, B; Cha, S-Y; Kang, M; Park, I-J; Moon, O-K; Park, C-K; Jang, H-K

    2013-05-01

    Infections with Pasteurella multocida, Salmonella enterica, Riemerella anatipestifer, and Escherichia coli result in high morbidity and mortality, which cause significant economic loss in the poultry industry. It can be difficult to distinguish these pathogens based on clinical signs because these pathogens can cause similar clinical signs and coinfections can occur. Thus, rapid and sensitive detection of these 4 major bacterial pathogens are important in ducks. The aim of this study was to develop a multiplex PCR (mPCR) assay for simultaneously detecting and identifying these 4 pathogenic bacteria in a single tube reaction. The target genes used were KMT1 of P. multocida, the invasion protein gene of S. enterica, 16S rDNA of R. anatipestifer, and the alkaline phosphatase gene of E. coli. The detection limit of the assay for all bacterial DNA was 10 pg. The mPCR did not produce any nonspecific amplification products when tested against other related pathogens, including Staphylococcus aureus, Streptococcus pyogenes, Clostridium perfringens, Mycoplasma gallinarum, Mycoplasma synoviae, and Mycoplasma gallisepticum, which can also infect ducks. We applied mPCR to field samples, and the results were the same as the single PCR results. These results suggest that mPCR for the 4 bacteria is a useful and rapid technique to apply to field samples.

  6. [Evaluation of the efficacies of rapid antigen test, multiplex PCR, and real-time PCR for the detection of a novel influenza A (H1N1) virus].

    Science.gov (United States)

    Hwang, Yusun; Kim, Kyounghee; Lee, Miae

    2010-04-01

    In April 2009, a novel influenza A (H1N1) virus was detected in the US, and at the time of conducting this study, H1N1 infection had reached pandemic proportions. In Korea, rapid antigen tests and PCR assays have been developed to detect the H1N1 virus. We evaluated the efficacies of rapid antigen test, multiplex PCR, and real-time PCR for detecting the H1N1 virus. From August to September 2009, we tested 734 samples obtained from nasopharyngeal swab or nasal swab using rapid antigen test (SD Influenza Antigen, Standard Diagnostics, Inc., Korea) and multiplex PCR (Seeplex FluA ACE Subtyping, Seegene, Korea). We also tested 224 samples using the AdvanSure real-time PCR (LG Life Sciences, Korea) to compare the results obtained using real-time PCR with those obtained using multiplex PCR. Furthermore, 99 samples were tested using the AdvanSure real-time PCR and the AccuPower real-time PCR (Bioneer, Korea). In comparison with the results of multiplex PCR, the sensitivity and specificity of the rapid antigen test were 48.0% and 99.8%, respectively. The concordance rate for multiplex PCR and the AdvanSure real-time PCR was 99.6% (kappa=0.991, P=0.000), and that for the AdvanSure real-time PCR and the AccuPower real-time PCR was 97.0% (kappa=0.936, P=0.000). The rapid antigen test is significantly less sensitive than PCR assay; therefore, it is not useful for H1N1 detection; however multiplex PCR, the AdvanSure real-time PCR, and the Accu-Power real-time PCR can be useful for H1N1 detection.

  7. Improved Multiplex Polymerase Chain Reaction for Rapid Staphylococcus Aureus Detection in Meat and Milk Matrices

    Directory of Open Access Journals (Sweden)

    Šramková Zuzana

    2016-06-01

    Full Text Available Staphylococcal food poisoning represents one of the most frequently occurring intoxications, caused by staphylococcal enterotoxins (SE-s and staphylococcal enterotoxin-like proteins (SEl-s. Therefore, there is a need for rapid, sensitive and specific detection method for this human pathogen and its toxin genes in food matrices. The present work is focused on Staphylococcus aureus detection by a nonaplex polymerase chain reaction, which targets the 23S rRNA gene for identification of S. aureus at the species level, genes for classical SE-s (SEA, SEC, SED, new SE-s (SEH, SEI, SEl-s (SEK, SEL and tsst-1 gene (toxic shock syndrome toxin. Primers were properly designed to avoid undesirable interactions and to create a reliably identifiable profile of amplicons when visualized in agarose gel. According to obtained results, this approach is able to reach the detection sensitivity of 12 colony forming units from milk and meat matrices without prior culturing and DNA extraction.

  8. A Rapid Screening Analysis of Antioxidant Compounds in Native Australian Food Plants Using Multiplexed Detection with Active Flow Technology Columns

    Directory of Open Access Journals (Sweden)

    Emmanuel Janaka Rochana Rupesinghe

    2016-01-01

    Full Text Available Conventional techniques for identifying antioxidant and phenolic compounds in native Australian food plants are laborious and time-consuming. Here, we present a multiplexed detection technique that reduces analysis time without compromising separation performance. This technique is achieved using Active Flow Technology-Parallel Segmented Flow (AFT-PSF columns. Extracts from cinnamon myrtle (Backhousia myrtifolia and lemon myrtle (Backhousia citriodora leaves were analysed via multiplexed detection using an AFT-PSF column with underivatised UV-VIS, mass spectroscopy (MS, and the 2,2-diphenyl-1-picrylhydrazyl (DPPH• derivatisation for antioxidants as detection methods. A number of antioxidant compounds were detected in the extracts of each leaf extract.

  9. Rapid detection of coliforms in drinking water of Arak city using multiplex PCR method in comparison with the standard method of culture (Most Probably Number)

    OpenAIRE

    fatemeh, Dehghan; Reza, Zolfaghari Mohammad; Mohammad, Arjomandzadegan; Salomeh, Kalantari; Reza, Ahmari Gholam; Hossein, Sarmadian; Maryam, Sadrnia; Azam, Ahmadi; Mana, Shojapoor; Negin, Najarian; Reza, Kasravi Alii; Saeed, Falahat

    2014-01-01

    Objective: To analyse molecular detection of coliforms and shorten the time of PCR. Methods: Rapid detection of coliforms by amplification of lacZ and uidA genes in a multiplex PCR reaction was designed and performed in comparison with most probably number (MPN) method for 16 artificial and 101 field samples. The molecular method was also conducted on isolated coliforms from positive MPN samples; standard sample for verification of microbial method certificated reference material; isolated...

  10. Multiplexed chromosome conformation capture sequencing for rapid genome-scale high-resolution detection of long-range chromatin interactions.

    Science.gov (United States)

    Stadhouders, Ralph; Kolovos, Petros; Brouwer, Rutger; Zuin, Jessica; van den Heuvel, Anita; Kockx, Christel; Palstra, Robert-Jan; Wendt, Kerstin S; Grosveld, Frank; van Ijcken, Wilfred; Soler, Eric

    2013-03-01

    Chromosome conformation capture (3C) technology is a powerful and increasingly popular tool for analyzing the spatial organization of genomes. Several 3C variants have been developed (e.g., 4C, 5C, ChIA-PET, Hi-C), allowing large-scale mapping of long-range genomic interactions. Here we describe multiplexed 3C sequencing (3C-seq), a 4C variant coupled to next-generation sequencing, allowing genome-scale detection of long-range interactions with candidate regions. Compared with several other available techniques, 3C-seq offers a superior resolution (typically single restriction fragment resolution; approximately 1-8 kb on average) and can be applied in a semi-high-throughput fashion. It allows the assessment of long-range interactions of up to 192 genes or regions of interest in parallel by multiplexing library sequencing. This renders multiplexed 3C-seq an inexpensive, quick (total hands-on time of 2 weeks) and efficient method that is ideal for the in-depth analysis of complex genetic loci. The preparation of multiplexed 3C-seq libraries can be performed by any investigator with basic skills in molecular biology techniques. Data analysis requires basic expertise in bioinformatics and in Linux and Python environments. The protocol describes all materials, critical steps and bioinformatics tools required for successful application of 3C-seq technology.

  11. Rapid detection method for Bacillus anthracis using a combination of multiplexed real-time PCR and pyrosequencing and its application for food biodefense.

    Science.gov (United States)

    Janzen, Timothy W; Thomas, Matthew C; Goji, Noriko; Shields, Michael J; Hahn, Kristen R; Amoako, Kingsley K

    2015-02-01

    Bacillus anthracis, the causative agent of anthrax, has the capacity to form highly resilient spores as part of its life cycle. The potential for the dissemination of these spores using food as a vehicle is a huge public health concern and, hence, requires the development of a foodborne bioterrorism response approach. In this work, we address a critical gap in food biodefense by presenting a novel, combined, sequential method involving the use of real-time PCR and pyrosequencing for the rapid, specific detection of B. anthracis spores in three food matrices: milk, apple juice, and bottled water. The food samples were experimentally inoculated with 40 CFU ml(-1), and DNA was extracted from the spores and analyzed after immunomagnetic separation. Applying the combination of multiplex real-time PCR and pyrosequencing, we successfully detected the presence of targets on both of the virulence plasmids and the chromosome. The results showed that DNA amplicons generated from a five-target multiplexed real-time PCR detection using biotin-labeled primers can be used for single-plex pyrosequencing detection. The combined use of multiplexed real-time PCR and pyrosequencing is a novel, rapid detection method for B. anthracis from food and provides a tool for accurate, quantitative identification with potential biodefense applications.

  12. A multiplex PCR assay for the rapid and sensitive detection of methicillin-resistant Staphylococcus aureus and simultaneous discrimination of Staphylococcus aureus from coagulase-negative staphylococci.

    Science.gov (United States)

    Xu, Benjin; Liu, Ling; Liu, Li; Li, Xinping; Li, Xiaofang; Wang, Xin

    2012-11-01

    Methicillin-resistant Staphylococcus aureus (MRSA) is a global health concern, which had been detected in food and food production animals. Conventional testing for detection of MRSA takes 3 to 5 d to yield complete information of the organism and its antibiotic sensitivity pattern. So, a rapid method is needed to diagnose and treat the MRSA infections. The present study focused on the development of a multiplex PCR assay for the rapid and sensitive detection of MRSA. The assay simultaneously detected 4 genes, namely, 16S rRNA of the Staphylococcus genus, femA of S. aureus, mecA that encodes methicillin resistance, and one internal control. It was rapid and yielded results within 4 h. The analytical sensitivity and specificity of the multiplex PCR assay was evaluated by comparing it with the conventional method. The analytical sensitivity of the multiplex PCR assay at the DNA level was 10 ng DNA. The analytical specificity was evaluated with 10 reference staphylococci strains and was 100%. The diagnostic evaluation of MRSA was carried out using 360 foodborne staphylococci isolates, and showed 99.1% of specificity, 96.4% of sensitivity, 97.5% of positive predictive value, and 97.3% of negative predictive value compared to the conventional method. The inclusion of an internal control in the multiplex PCR assay is important to exclude false-negative cases. This test can be used as an effective diagnostic and surveillance tool to investigate the spread and emergence of MRSA. © 2012 Institute of Food Technologists®

  13. Rapid Detection of Mycobacterium tuberculosis Strains Resistant to Isoniazid and/or Rifampicin: Standardization of Multiplex Polymerase Chain Reaction Analysis.

    Science.gov (United States)

    Collantes, Jimena; Solari, Francesca Barletta; Rigouts, Leen

    2016-12-07

    Drug susceptibility testing using molecular techniques can enhance the identification of drug-resistant Mycobacterium tuberculosis Two multiplex real-time polymerase chain reaction (qPCR) assays were developed to detect the most common resistance-associated mutations to isoniazid (katGS315T, inhA-15C → T), and rifampicin (rpoBH526Y and rpoBS531L). To assess the species specificity of the qPCR, we selected 31 nontuberculous mycobacteria (NTM) reference strains belonging to 17 species from the public collection of mycobacterial cultures (BCCM/ITM). Additionally, we tested 17 isoniazid and/or rifampicin-resistant strains with other mutations in the target genes to assess mutation specificity. The limit of detection for all the targeted mutations was 20 bacilli/reaction. Multiplex 1 showed 90%, 95%, and 100% efficiency for wild type (WT), Mut katGS315T, and Mut rpoBS531L, respectively; whereas Multiplex 2 showed 97%, 94%, and 90% efficiency for WT, Mut inhA-15, and Mut rpoBH526Y, respectively. Three of 17 strains that presented other mutations in the target genes were identified as rifampicin resistant and only 3/31 NTM showed a similar melting temperature to rpoBL531 and/or katGT315 mutants. Thus, our proposed cascade of specific tuberculosis detection followed by drug resistance testing showed sensitivities for katGS315T, rpoBS531L, rpoBH526Y, and inhA-15 detection of 100%, 100%, 100%, and 96%, respectively; and specificities of 98%, 95%, 100%, and 100, respectively. © The American Society of Tropical Medicine and Hygiene.

  14. Clinical impact of a commercially available multiplex PCR system for rapid detection of pathogens in patients with presumed sepsis

    Directory of Open Access Journals (Sweden)

    Linde Hans-Jörg

    2009-08-01

    Full Text Available Abstract Background Timely identification of pathogens is crucial to minimize mortality in patients with severe infections. Detection of bacterial and fungal pathogens in blood by nucleic acid amplification promises to yield results faster than blood cultures (BC. We analyzed the clinical impact of a commercially available multiplex PCR system in patients with suspected sepsis. Methods Blood samples from patients with presumed sepsis were cultured with the Bactec 9240™ system (Becton Dickinson, Heidelberg, Germany and aliquots subjected to analysis with the LightCycler® SeptiFast® (SF Test (Roche Diagnostics, Mannheim, Germany at a tertiary care centre. For samples with PCR-detected pathogens, the actual impact on clinical management was determined by chart review. Furthermore a comparison between the time to a positive blood culture result and the SF result, based on a fictive assumption that it was done either on a once or twice daily basis, was made. Results Of 101 blood samples from 77 patients, 63 (62% yielded concordant negative results, 14 (13% concordant positive and 9 (9% were BC positive only. In 14 (13% samples pathogens were detected by SF only, resulting in adjustment of antibiotic therapy in 5 patients (7,7% of patients. In 3 samples a treatment adjustment would have been made earlier resulting in a total of 8 adjustments in all 101 samples (8%. Conclusion The addition of multiplex PCR to conventional blood cultures had a relevant impact on clinical management for a subset of patients with presumed sepsis.

  15. Rapid detection of coliforms in drinking water of Arak city using multiplex PCR method in comparison with the standard method of culture (Most Probably Number)

    Science.gov (United States)

    Fatemeh, Dehghan; Reza, Zolfaghari Mohammad; Mohammad, Arjomandzadegan; Salomeh, Kalantari; Reza, Ahmari Gholam; Hossein, Sarmadian; Maryam, Sadrnia; Azam, Ahmadi; Mana, Shojapoor; Negin, Najarian; Reza, Kasravi Alii; Saeed, Falahat

    2014-01-01

    Objective To analyse molecular detection of coliforms and shorten the time of PCR. Methods Rapid detection of coliforms by amplification of lacZ and uidA genes in a multiplex PCR reaction was designed and performed in comparison with most probably number (MPN) method for 16 artificial and 101 field samples. The molecular method was also conducted on isolated coliforms from positive MPN samples; standard sample for verification of microbial method certificated reference material; isolated strains from certificated reference material and standard bacteria. The PCR and electrophoresis parameters were changed for reducing the operation time. Results Results of PCR for lacZ and uidA genes were similar in all of standard, operational and artificial samples and showed the 876 bp and 147 bp bands of lacZ and uidA genes by multiplex PCR. PCR results were confirmed by MPN culture method by sensitivity 86% (95% CI: 0.71-0.93). Also the total execution time, with a successful change of factors, was reduced to less than two and a half hour. Conclusions Multiplex PCR method with shortened operation time was used for the simultaneous detection of total coliforms and Escherichia coli in distribution system of Arak city. It's recommended to be used at least as an initial screening test, and then the positive samples could be randomly tested by MPN. PMID:25182727

  16. Rapid detection of coliforms in drinking water of Arak city using multiplex PCR method in comparison with the standard method of culture (Most Probably Number).

    Science.gov (United States)

    Fatemeh, Dehghan; Reza, Zolfaghari Mohammad; Mohammad, Arjomandzadegan; Salomeh, Kalantari; Reza, Ahmari Gholam; Hossein, Sarmadian; Maryam, Sadrnia; Azam, Ahmadi; Mana, Shojapoor; Negin, Najarian; Reza, Kasravi Alii; Saeed, Falahat

    2014-05-01

    To analyse molecular detection of coliforms and shorten the time of PCR. Rapid detection of coliforms by amplification of lacZ and uidA genes in a multiplex PCR reaction was designed and performed in comparison with most probably number (MPN) method for 16 artificial and 101 field samples. The molecular method was also conducted on isolated coliforms from positive MPN samples; standard sample for verification of microbial method certificated reference material; isolated strains from certificated reference material and standard bacteria. The PCR and electrophoresis parameters were changed for reducing the operation time. Results of PCR for lacZ and uidA genes were similar in all of standard, operational and artificial samples and showed the 876 bp and 147 bp bands of lacZ and uidA genes by multiplex PCR. PCR results were confirmed by MPN culture method by sensitivity 86% (95% CI: 0.71-0.93). Also the total execution time, with a successful change of factors, was reduced to less than two and a half hour. Multiplex PCR method with shortened operation time was used for the simultaneous detection of total coliforms and Escherichia coli in distribution system of Arak city. It's recommended to be used at least as an initial screening test, and then the positive samples could be randomly tested by MPN.

  17. An experimental study for rapid detection and quantification of endodontic microbiota following photo-activated disinfection via new multiplex real-time PCR assay.

    Science.gov (United States)

    Pourhajibagher, Maryam; Raoofian, Reza; Ghorbanzadeh, Roghayeh; Bahador, Abbas

    2018-01-11

    The infected root canal system harbors one of the highest accumulations of polymicrobial infections. Since the eradication of endopathogenic microbiota is a major goal in endodontic infection therapy, photo-activated disinfection (PAD) can be used as an alternative therapeutic method in endodontic treatment. Compared to cultivation-based approaches, molecular techniques are more reliable for identifying microbial agents associated with endodontic infections. The purpose of this study was to evaluate the ability of designed multiplex real-time PCR protocol for the rapid detection and quantification of six common microorganisms involved in endodontic infection before and after the PAD. Samples were taken from the root canals of 50 patients with primary and secondary/persistent endodontic infections using sterile paper points. PAD with toluidine blue O (TBO) plus diode laser was performed on root canals. Resampling was then performed, and the samples were transferred to transport medium. Then, six target microorganisms were detected using multiplex real-time PCR before and after the PAD. Veillonella parvula was found using multiplex real-time PCR to have the highest frequency among samples collected before the PAD (29.4%), followed by Porphyromonas. gingivalis (23.1%), Aggregatibacter actinomycetemcomitans (13.6%), Actinomyces naeslundii (13.0%), Enterococcus faecalis (11.5%), and Lactobacillus rhamnosus (9.4%). After TBO-mediated PAD, P. gingivalis strains, the most resistance microorganisms, were recovered in 41.7% of the samples using molecular approach (P > 0.05). As the results shown, multiplex real-time PCR as an accurate detection approach with high-throughput and TBO-mediated PAD as an efficient antimicrobial strategy due to the significant reduction of the endopathogenic count can be used for detection and treatment of microbiota involved in infected root canals, respectively. Copyright © 2018. Published by Elsevier B.V.

  18. Multiplex one-step Real-time PCR by Taqman-MGB method for rapid detection of pan and H5 subtype avian influenza viruses.

    Directory of Open Access Journals (Sweden)

    Zhujun Zhang

    Full Text Available Avian influenza virus (AIV can infect a variety of avian species and mammals, leading to severe economic losses in poultry industry and posing a substantial threat to public health. Currently, traditional virus isolation and identification is inadequate for the early diagnosis because of its labor-intensive and time-consuming features. Real-time RT-PCR (RRT-PCR is an ideal method for the detection of AIV since it is highly specific, sensitive and rapid. In addition, as the new quencher MGB is used in RRT-PCR, it only needs shorter probe and helps the binding of target gene and probe. In this study, a pan-AIV RRT-PCR for the detection of all AIVs and H5-AIV RRT-PCR for detection of H5 AIV based on NP gene of AIV and HA gene of H5 AIV were successfully established using Taqman-MGB method. We tested 14 AIV strains in total and the results showed that the pan-AIV RRT-PCR can detect AIV of various HA subtypes and the H5-AIV RRT-PCR can detect H5 AIV circulating in poultry in China in recent three years, including H5 viruses of clade 7.2, clade 2.3.4.4 and clade 2.3.2.1. Furthermore, the multiplex detection limit for pan-AIV and H5-AIV RRT-PCR was 5 copies per reaction. When this multiplex method was applied in the detection of experimental and live poultry market samples, the detection rates of pan-AIV and H5 AIV in RRT-PCR were both higher than the routine virus isolation method with embryonated chicken eggs. The multiplex RRT-PCR method established in our study showed high sensitivity, reproducibility and specificity, suggesting the promising application of our method for surveillance of both pan AIV and prevalent H5 AIV in live poultry markets and clinical samples.

  19. Multiplex Solid-Phase PCR for Rapid Detection and Identification of Salmonella spp. at Sub-species

    DEFF Research Database (Denmark)

    Cao, Cuong; Høgberg, Jonas; Wolff, Anders

    -PCR gel electrophoresis. The method will be useful for development of point-of-care devices for rapid detection and identification of Salmonella spp. A solid-phase PCR for rapid detection and identification of S. enteritidis, S. typhimurium and S. dublin is developed. The method offers advantages......This study presents a solid-phase PCR (SP-PCR) for rapid detection, identification, and sub-typing of various Salmonella species, the major food-borne cause of salmonellosis. The target DNA is firstly amplified with PCR primers (one primer is labeled with fluorophores) in the liquid phase...... by the liquid phase primer thus generating new templates for the SP-PCR. After the reaction, PCR products labeled with fluorophores remain attached to the substrate and can be visualized directly by fluorescence readout devices. Using this method, S. enteritidis, S. typhimurium and S. dublin can be detected...

  20. Rapid detection and differentiation of Campylobacter jejuni, Campylobacter coli, and Campylobacter lari in food, using multiplex real-time PCR.

    Science.gov (United States)

    Mayr, A M; Lick, S; Bauer, J; Thärigen, D; Busch, U; Huber, I

    2010-02-01

    A multiplex real-time PCR assay based on four differently labeled TaqMan probes for detection and differentiation of the thermophilic Campylobacter species C. jejuni, C. coli, and C. lari was established and validated in food products. This assay combines two previously published PCR assays for C. jejuni and C. coli with a newly developed detection assay for C. lari and an internal amplification control system. The selectivity of the method was determined by analyzing 70 Campylobacter strains and 43 strains of other bacteria. The sensitivity was 50 fg of C. jejuni and C. lari DNA and 500 fg of C. coli DNA per PCR. It was possible to detect 1 to 10 CFU/25 g of food before preenrichment of all three species. More than 400 samples of various foods (poultry, seafood, and meat) were analyzed after 48 h of preenrichment parallel to the conventional diagnostic method of culture and biochemical identification. Using the established real-time PCR assay, 55.4% of the samples were recognized as positive for thermophilic Campylobacter species, whereas with the conventional method only 40.3% of the samples were positive. The real-time PCR assay also detected contaminations with two different Campylobacter species in 32.6% of the analyzed poultry samples, a finding of epidemiological interest. Compared with the original PCR method, which was established for the differentiation of bacterial isolates of C. jejuni and C. coli, this new method also detects and distinguishes C. lari, was validated as an analytical tool for food analysis, and provides reliable and extensive results within 2 days.

  1. Rapid Multiplex Microbial Detector Project

    Data.gov (United States)

    National Aeronautics and Space Administration — ORBITEC, in collaboration with Lucigen, proposes a rapid nucleic acid-based detector for spaceflight water systems to enable simultaneous quantification of multiple...

  2. Rapid Detection of Campylobacter jejuni, Campylobacter coli, and Campylobacter lari in Fresh Chicken Meat and By-Products in Bangkok, Thailand, Using Modified Multiplex PCR.

    Science.gov (United States)

    Saiyudthong, S; Phusri, K; Buates, S

    2015-07-01

    A multiplex PCR assay for simultaneous detection and differentiation of Campylobacter jejuni, Campylobacter coli, and Campylobacter lari was developed and validated to assess the occurrence of these bacteria in fresh chicken meat and by-products in Bangkok, Thailand, by using a new combination of four previously published PCR primers for C. jejuni, C. coli, C. lari, and a universal 16S rDNA gene as an internal control. The specificity was determined by using 13 strains of other bacteria. With pure culture DNA, the detection limit was 0.017 ng/PCR for C. jejuni and C. coli and was 0.016 ng/PCR for C. lari. It can detect 10 CFU of C. jejuni, C. coli, and C. lari in 2 g of chicken meat within a 16-h enrichment time. Our multiplex PCR assay was applied for identification of Campylobacter spp. in 122 supermarket samples and 108 fresh market samples. Of the 230 samples evaluated by multiplex PCR, 54.0, 3.3, and 10.7% of supermarket samples were positive for C. jejuni, C. coli, and mixed C. jejuni and C. coli, respectively, and 56.5 and 33.3% of fresh market samples were positive for C. jejuni and mixed C. jejuni and C. coli, respectively. No sample was positive for C. lari. Fresh market samples had significantly higher C. jejuni and C. coli contamination than those from supermarkets (relative risk: 1.3; P = 0.0001). Compared with the culture method (a gold standard), the sensitivity, specificity, positive predictive value, negative predictive value, and diagnostic accuracy of multiplex PCR were 97.7, 86.8, 96.1, 92.0, and 95.2%, respectively. No significant difference was observed between results from two methods (P = 0.55). Therefore, the established multiplex PCR was not only rapid and easy to perform but had a high sensitivity and specificity to distinguish between C. jejuni, C. coli, and C. lari, even in samples containing mixed contamination. Our study indicated that fresh chicken meat and by-products from fresh markets were significantly less hygienic than those

  3. Multiplex PCR system for rapid detection of pathogens in patients with presumed sepsis - a systemic review and meta-analysis.

    Directory of Open Access Journals (Sweden)

    Shy-Shin Chang

    Full Text Available BACKGROUND: Blood culture is viewed as the golden standard for the diagnosis of sepsis but suffers from low sensitivity and long turnaround time. LightCycler SeptiFast (LC-SF is a real-time multiplex polymerase chain reaction test able to detect 25 common pathogens responsible for bloodstream infections within hours. We aim to assess the accuracy of LC-SF by systematically reviewing the published studies. METHOD: Related literature on Medline, Embase, and Cochrane databases was searched up to October 2012 for studies utilizing LC-SF to diagnose suspected sepsis and that provided sufficient data to construct two-by-two tables. RESULTS: A total of 34 studies enrolling 6012 patients of suspected sepsis were included. The overall sensitivity and specificity for LC-SF to detect bacteremia or fungemia was 0·75 (95% CI: 0·65-0·83 and 0·92 (95%CI:0·90-0·95, respectively. LC-SF had a high positive likelihood ratio (10·10 and a moderate negative likelihood ratio (0·27. Specifically, LC-SF had a sensitivity of 0·80 (95%CI: 0·70-0·88 and a specificity of 0·95(95%CI: 0·93-0·97 for the bacteremia outcome, and a sensitivity of 0·61 (95%CI: 0·48-0·72 and a specificity of 0·99 (95%CI: 0·99-0·99 for the fungemia outcome. High heterogeneity was found in the bacteremia outcome subgroup but not in the fungemia outcome subgroup. CONCLUSION: LC-SF is of high rule-in value for early detection of septic patients. In a population with low pretest probability, LC-SF test can still provide valuable information for ruling out bacteremia or fungemia.

  4. Metal Affinity-Enabled Capture and Release Antibody Reagents Generate a Multiplex Biomarker Enrichment System that Improves Detection Limits of Rapid Diagnostic Tests.

    Science.gov (United States)

    Bauer, Westley S; Gulka, Christopher P; Silva-Baucage, Lidalee; Adams, Nicholas M; Haselton, Frederick R; Wright, David W

    2017-10-03

    Multi-antigen rapid diagnostic tests (RDTs) are highly informative, simple, mobile, and inexpensive, making them valuable point-of-care (POC) diagnostic tools. However, these RDTs suffer from several technical limitations-the most significant being the failure to detect low levels of infection. To overcome this, we have developed a magnetic bead-based multiplex biomarker enrichment strategy that combines metal affinity and immunospecific capture to purify and enrich multiple target biomarkers. Modifying antibodies to contain histidine-rich peptides enables reversible loading onto immobilized metal affinity magnetic beads, generating a novel class of antibodies coined "Capture and Release" (CaR) antibody reagents. This approach extends the specificity of immunocapture to metal affinity magnetic beads while also maintaining a common trigger for releasing multiple biomarkers. Multiplex biomarker enrichment is accomplished by adding magnetic beads equipped with CaR antibody reagents to a large sample volume to capture biomarkers of interest. Once captured, these biomarkers are magnetically purified, concentrated, and released into a RDT-compatible volume. This system was tailored to enhance a popular dual-antigen lateral flow malaria RDT that targets Plasmodium falciparum histidine-rich protein-II (HRPII) and Plasmodium lactate dehydrogenase (pLDH). A suite of pLDH CaR antibody reagents were synthesized, characterized, and the optimal CaR antibody reagent was loaded onto magnetic beads to make a multiplex magnetic capture bead that simultaneously enriches pLDH and HRPII from Plasmodium falciparum parasitized blood samples. This system achieves a 17.5-fold improvement in the dual positive HRPII/pan-pLDH detection limits enabling visual detection of both antigens at levels correlating to 5 p/μL. This front-end sample processing system serves as an efficient strategy to improve the sensitivity of RDTs without the need for modifications or remanufacturing.

  5. A Rapid Multiplex Real-Time PCR High-Resolution Melt Curve Assay for the Simultaneous Detection of Bacillus cereus, Listeria monocytogenes, and Staphylococcus aureus in Food.

    Science.gov (United States)

    Forghani, Fereidoun; Wei, Shuai; Oh, Deog-Hwan

    2016-05-01

    Three important foodborne pathogens, Bacillus cereus, Listeria monocytogenes, and Staphylococcus aureus, are of great concern for food safety. They may also coexist in food matrices and, in the case of B. cereus and S. aureus, the resulting illnesses can resemble each other owing to similar symptoms. Therefore, their simultaneous detection may have advantages in terms of cost savings and rapidity. Given this context, a rapid multiplex real-time PCR high-resolution melt curve assay for the simultaneous detection of these three pathogens in food was developed. The assay successfully detected B. cereus (gyrB), L. monocytogenes (hly), and S. aureus (nuc) in a single reaction, and the average melting temperatures were 76.23, 80.19, and 74.01°C, respectively. The application of SYTO9 dye and a slow melt curve analysis ramp rate (0.1°C/s) enabled the production of sharp, high-resolution melt curve peaks that were easily distinguishable from each other. The detection limit in food (milk, rice, and lettuce) was 3.7 × 10(3) CFU/g without an enrichment step and 3.7 × 10(1) CFU/g following the 10-h enrichment. Hence, the assay developed here is specific and sensitive, providing an efficient tool for implementation in food for the simultaneous detection of B. cereus, L. monocytogenes, and S. aureus .

  6. Performance of the Real Fungus-ID kit based on multiplex RT-PCR assay for the rapid detection and identification of Trichophyton spp. and Microsporum spp. in clinical specimens with suspected dermatophyte infection.

    Science.gov (United States)

    Wang, H-Y; Kim, H; Choi, E H; Lee, H

    2016-01-01

    The aim of this study was to evaluate the performance of a commercially available multiplex RT-PCR assay for the rapid detection and identification of dermatophytes directly from clinical samples and cultures. The multiplex RT-PCR assay was used to evaluate 118 clinical isolates from various specimen types and a total of 140 known specimens were compared with both conventional methods, commercially available PCR-REBA, and ITS sequence analysis. In this study, multiplex RT-PCR assay yield significantly more positive results than culture (91·9 vs 39·5%) and conventional methods including KOH microscopy (91·9 vs 71·3%). Although the results among the multiplex RT-PCR, PCR-REBA and ITS sequence analysis were concordant (100%) in 118 clinical isolates, concordant results between multiplex RT-PCR assay and culture were at 66% (78/118). The overall positive rates for the PCR-REBA, multiplex RT-PCR assay and ITS sequence analysis were 98·8, 91·9, and 52·9% respectively. In addition, the concordance rate of multiplex RT-PCR assay and the PCR-REBA assay was 93% (95% confidence interval (CI), 89·9-96·1, P culture can take up to 2-3 weeks. The use of the multiplex RT-PCR molecular diagnostic assay was rapid and reliable for detecting pathogen infections. Even though the use of molecular diagnostic technology is more expensive than conventional methods, the clinical and economic benefit of saving time relative to expense remains to be elucidated. Therefore, the multiplex RT-PCR assay may provide the essential information to accelerate therapeutic decisions for earlier and adequate antibiotic treatment in the acute phase of fungal pathogen infections. © 2015 The Society for Applied Microbiology.

  7. Multiplex detection of respiratory pathogens

    Energy Technology Data Exchange (ETDEWEB)

    McBride, Mary [Brentwood, CA; Slezak, Thomas [Livermore, CA; Birch, James M [Albany, CA

    2012-07-31

    Described are kits and methods useful for detection of respiratory pathogens (influenza A (including subtyping capability for H1, H3, H5 and H7 subtypes) influenza B, parainfluenza (type 2), respiratory syncytial virus, and adenovirus) in a sample. Genomic sequence information from the respiratory pathogens was analyzed to identify signature sequences, e.g., polynucleotide sequences useful for confirming the presence or absence of a pathogen in a sample. Primer and probe sets were designed and optimized for use in a PCR based, multiplexed Luminex assay to successfully identify the presence or absence of pathogens in a sample.

  8. Detection and differentiation of Plum pox virus using real-time multiplex PCR with SYBR Green and melting curve analysis: a rapid method for strain typing.

    Science.gov (United States)

    Varga, Aniko; James, Delano

    2005-02-01

    A real-time multiplex PCR procedure with melting curve analysis, using the green fluorescence dye SYBR Green I, was developed for rapid and reliable identification of Plum pox virus (PPV) isolates of strains D and M. Members of the different strains were identified by their distinctive melting temperatures (T(m)s); 84.3-84.43 degrees C for D isolates, and 85.34-86.11 degrees C for M isolates. The associated amplicon sizes were 114 and 380 bp, respectively. The procedure was used for detection and identification of PPV in both herbaceous and woody hosts. The Tm for members of a particular strain was very similar, with a host effect that did not hinder strain identification. Universal primers included in the study detected all isolates of PPV tested, amplifying a 74 bp fragment. The Tm of this fragment varied from 80.12 to 81.52 degrees C and may have supplementary value for PPV identification. SYBR Green-based detection was compared to detection using a hybridization LUX fluorogenic primer. Better resolution of the melting peaks was observed with SYBR Green I, than with the LUX primers, hence strain identification with SYBR Green I was more reliable. This is a simple approach to PPV strain identification with the relatively inexpensive dye SYBR Green I, and eliminates any need for electrophoretic analysis of amplicons or RFLP patterns using ethidium bromide.

  9. Multiplex Reverse Transcription-Polymerase Chain Reaction untuk Deteksi Cepat Virus Flu Burung H5N1 (MULTIPLEX REVERSE TRANSCRIPTION-POLYMERASE CHAIN REACTION FOR RAPID DETECTION OF H5N1 AVIAN INFLUENZA VIRUS

    Directory of Open Access Journals (Sweden)

    Raden Wasito

    2015-05-01

    Full Text Available Avian influenza virus subtype H5N1 (AIV H5N1 is highly pathogenic and fatal in poultry. The virusis still endemic with low virulence rate, although it may play a critical role in causing high morbidity andmortality rates in poultry in Indonesia. In general, diagnostic approach for AIV H5N1 is based onconventional serological and viral isolation methods that have the potential to produce consumings oftime and relatively expensive cost within the laboratory without compromising test utility. Thus, amolecular approach of multiplex reverse transcription-polymerase chain reaction (mRT-PCR was developedand applied for the detection of matrix gene type A influenza viruses, AIV subtype subtype H5hemagglutinin gene with simultaneous detection of N1 nucleoprotein gene. Thirty sera specimens fromthe diseased commercial chickens that were specifically amplified positive-RT-PCR for AIV H5N1 wereselected for mRT-PCR. The mRT-PCR products were visualized by agarose gel electrophoresis and consistedof DNA fragments of AIV of 245 bp, 545 bp and 343 bp for M, H5 and N1 genes, respectively. Thus, themRT-PCR that can rapidly differentiate simultaneously between these genes is very important for thecontrol and even eradication of AIV transmission in poultry in Indonesia.

  10. Rapid and simple method by combining FTA™ card DNA extraction with two set multiplex PCR for simultaneous detection of non-O157 Shiga toxin-producing Escherichia coli strains and virulence genes in food samples.

    Science.gov (United States)

    Kim, S A; Park, S H; Lee, S I; Ricke, S C

    2017-12-01

    The aim of this research was to optimize two multiplex polymerase chain reaction (PCR) assays that could simultaneously detect six non-O157 Shiga toxin-producing Escherichia coli (STEC) as well as the three virulence genes. We also investigated the potential of combining the FTA™ card-based DNA extraction with the multiplex PCR assays. Two multiplex PCR assays were optimized using six primer pairs for each non-O157 STEC serogroup and three primer pairs for virulence genes respectively. Each STEC strain specific primer pair only amplified 155, 238, 321, 438, 587 and 750 bp product for O26, O45, O103, O111, O121 and O145 respectively. Three virulence genes were successfully multiplexed: 375 bp for eae, 655 bp for stx1 and 477 bp for stx2. When two multiplex PCR assays were validated with ground beef samples, distinctive bands were also successfully produced. Since the two multiplex PCR examined here can be conducted under the same PCR conditions, the six non-O157 STEC and their virulence genes could be concurrently detected with one run on the thermocycler. In addition, all bands clearly appeared to be amplified by FTA card DNA extraction in the multiplex PCR assay from the ground beef sample, suggesting that an FTA card could be a viable sampling approach for rapid and simple DNA extraction to reduce time and labour and therefore may have practical use for the food industry. Two multiplex polymerase chain reaction (PCR) assays were optimized for discrimination of six non-O157 Shiga toxin-producing Escherichia coli (STEC) and identification of their major virulence genes within a single reaction, simultaneously. This study also determined the successful ability of the FTA™ card as an alternative to commercial DNA extraction method for conducting multiplex STEC PCR assays. The FTA™ card combined with multiplex PCR holds promise for the food industry by offering a simple and rapid DNA sample method for reducing time, cost and labour for detection of STEC in

  11. Rapid detection of coliforms in drinking water of Arak city using multiplex PCR method in comparison with the standard method of culture (Most Probably Number

    Directory of Open Access Journals (Sweden)

    Dehghan fatemeh

    2014-05-01

    Conclusions: Multiplex PCR method with shortened operation time was used for the simultaneous detection of total coliforms and Escherichia coli in distribution system of Arak city. It's recommended to be used at least as an initial screening test, and then the positive samples could be randomly tested by MPN.

  12. Rapid detection and grouping of porcine bocaviruses by an EvaGreen(®) based multiplex real-time PCR assay using melting curve analysis.

    Science.gov (United States)

    Zheng, Xiaowen; Liu, Gaopeng; Opriessnig, Tanja; Wang, Zining; Yang, Zongqi; Jiang, Yonghou

    2016-08-01

    Several novel porcine bocaviruses (PBoVs) have been identified in pigs in recent years and association of these viruses with respiratory signs or diarrhea has been suggested. In this study, an EvaGreen(®)-based multiplex real-time PCR (EG-mPCR) with melting curve analysis was developed for simultaneous detection and grouping of novel PBoVs into the same genogroups G1, G2 and G3. Each target produced a specific amplicon with a melting peak of 81.3 ± 0.34 °C for PBoV G1, 78.2 ± 0.37 °C for PBoV G2, and 85.0 ± 0.29 °C for PBoV G3. Non-specific reactions were not observed when other pig viruses were used to assess the EG-mPCR assay. The sensitivity of the EG-mPCR assay using purified plasmid constructs containing the specific viral target fragments was 100 copies for PBoV G1, 50 for PBoV G2 and 100 for PBoV G3. The assay is able to detect and distinguish three PBoV groups with intra-assay and inter-assay variations ranging from 0.13 to 1.59%. The newly established EG-mPCR assay was validated with 227 field samples from pigs. PBoV G1, G2 and G3 was detected in 15.0%, 25.1% and 41.9% of the investigated samples and coinfections of two or three PBoV groups were also detected in 25.1% of the cases, indicating that all PBoV groups are prevalent in Chinese pigs. The agreement of the EG-mPCR assay with an EvaGreen-based singleplex real-time PCR (EG-sPCR) assay was 99.1%. This EG-mPCR will serve as a rapid, sensitive, reliable and cost effective alternative for routine surveillance testing of multiple PBoVs in pigs and will enhance our understanding of the epidemiological features and possible also pathogenetic changes associated with these viruses in pigs. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. Multiple Endonuclease Restriction Real-Time Loop-Mediated Isothermal Amplification: A Novel Analytically Rapid, Sensitive, Multiplex Loop-Mediated Isothermal Amplification Detection Technique.

    Science.gov (United States)

    Wang, Yi; Wang, Yan; Lan, Ruiting; Xu, Huaqing; Ma, Aijing; Li, Dongxun; Dai, Hang; Yuan, Xuejiao; Xu, Jianguo; Ye, Changyun

    2015-07-01

    Loop-mediated isothermal amplification (LAMP) is restricted to detecting a single target, limiting the usefulness of this method. To achieve multiplex LAMP-based detection, we developed a novel approach we called the multiple endonuclease restriction real-time-LAMP assay. In this system, the LAMP forward or backward inner primers contain 5' end short sequences that are recognized by the restriction endonuclease Nb.BsrDI, and the new forward or backward inner primers were modified at the 5' end with a fluorophore and in the middle with a dark quencher. Nb.BsrDI digests the newly synthesized double-stranded terminal sequences (5' end short sequences and their complementary sequences), which releases the quenching, resulting in a gain of signal. The assay permitted real-time detection of single or multiple target sequences in a single tube, and the positive results can be obtained in as short as 12 minutes. The novel methodology is highly efficient and specific, detecting down to 250 fg of DNA per reaction of Listeria DNA tested, and was successful in evaluating raw meat samples. The multiple endonuclease restriction real-time-LAMP technology, which is an extension of LAMP to accommodate robust, target-specific, and multiplex detection, provides a molecular diagnostic tool with less detection time and high sensitivity and specificity compared with those of LAMP and quantitative real-time PCR. Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

  14. A multiplex PCR for detection of six viruses in ducks.

    Science.gov (United States)

    Wang, Yongjuan; Zhu, Shanyuan; Hong, Weiming; Wang, Anping; Zuo, Weiyong

    2017-10-01

    In this study, six pairs of specific primers that can amplify DNA fragments of different sizes were designed and synthesized according to viral protein gene sequences published in GenBank. Then, a multiplex PCR method was established for rapid detection of duck hepatitis virus 1, duck plague virus, duck Tembusu virus, muscovy duck parvovirus, muscovy duck reovirus, and duck H9N2 avian influenza virus, and achieve simple and rapid detection of viral diseases in ducks. Single PCR was used to confirm primer specificity, and PCR conditions were optimized to construct a multiplex PCR system. Specificity and sensitivity assays were also developed. The multiplex PCR was used to detect duck embryos infected with mixed viruses and those with clinically suspected diseases to verify the feasibility of the multiplex PCR. Results show that the primers can specifically amplify target fragments, without any cross-amplification with other viruses. The multiplex PCR system can amplify six DNA fragments from the pooled viral genomes and specifically detect nucleic acids of the six duck susceptible viruses when the template amount is 10 2 copies/μl. In addition, the system can be used to detect viral nucleic acids in duck embryos infected with the six common viruses. The detection results for clinical samples are consistent with those detected by single PCR. Therefore, the established multiplex PCR method can perform specific, sensitive, and high-throughput detection of six duck-infecting viruses and can be applied to clinical identification and diagnosis of viral infection in ducks. Copyright © 2017. Published by Elsevier B.V.

  15. Automated Methods for Multiplexed Pathogen Detection

    Energy Technology Data Exchange (ETDEWEB)

    Straub, Tim M.; Dockendorff, Brian P.; Quinonez-Diaz, Maria D.; Valdez, Catherine O.; Shutthanandan, Janani I.; Tarasevich, Barbara J.; Grate, Jay W.; Bruckner-Lea, Cindy J.

    2005-09-01

    Detection of pathogenic microorganisms in environmental samples is a difficult process. Concentration of the organisms of interest also co-concentrates inhibitors of many end-point detection methods, notably, nucleic acid methods. In addition, sensitive, highly multiplexed pathogen detection continues to be problematic. The primary function of the BEADS (Biodetection Enabling Analyte Delivery System) platform is the automated concentration and purification of target analytes from interfering substances, often present in these samples, via a renewable surface column. In one version of BEADS, automated immunomagnetic separation (IMS) is used to separate cells from their samples. Captured cells are transferred to a flow-through thermal cycler where PCR, using labeled primers, is performed. PCR products are then detected by hybridization to a DNA suspension array. In another version of BEADS, cell lysis is performed, and community RNA is purified and directly labeled. Multiplexed detection is accomplished by direct hybridization of the RNA to a planar microarray. The integrated IMS/PCR version of BEADS can successfully purify and amplify 10 E. coli O157:H7 cells from river water samples. Multiplexed PCR assays for the simultaneous detection of E. coli O157:H7, Salmonella, and Shigella on bead suspension arrays was demonstrated for the detection of as few as 100 cells for each organism. Results for the RNA version of BEADS are also showing promising results. Automation yields highly purified RNA, suitable for multiplexed detection on microarrays, with microarray detection specificity equivalent to PCR. Both versions of the BEADS platform show great promise for automated pathogen detection from environmental samples. Highly multiplexed pathogen detection using PCR continues to be problematic, but may be required for trace detection in large volume samples. The RNA approach solves the issues of highly multiplexed PCR and provides ''live vs. dead

  16. Rapid Detection of Pathogens

    Energy Technology Data Exchange (ETDEWEB)

    David Perlin

    2005-08-14

    Pathogen identification is a crucial first defense against bioterrorism. A major emphasis of our national biodefense strategy is to establish fast, accurate and sensitive assays for diagnosis of infectious diseases agents. Such assays will ensure early and appropriate treatment of infected patients. Rapid diagnostics can also support infection control measures, which monitor and limit the spread of infectious diseases agents. Many select agents are highly transmissible in the early stages of disease, and it is critical to identify infected patients and limit the risk to the remainder of the population and to stem potential panic in the general population. Nucleic acid-based molecular approaches for identification overcome many of the deficiencies associated with conventional culture methods by exploiting both large- and small-scale genomic differences between organisms. PCR-based amplification of highly conserved ribosomal RNA (rRNA) genes, intergenic sequences, and specific toxin genes is currently the most reliable approach for bacterial, fungal and many viral pathogenic agents. When combined with fluorescence-based oligonucleotide detection systems, this approach provides real-time, quantitative, high fidelity analysis capable of single nucleotide allelic discrimination (4). These probe systems offer rapid turn around time (<2 h) and are suitable for high throughput, automated multiplex operations that are critical for clinical diagnostic laboratories. In this pilot program, we have used molecular beacon technology invented at the Public health Research Institute to develop a new generation of molecular probes to rapidly detect important agents of infectious diseases. We have also developed protocols to rapidly extract nucleic acids from a variety of clinical specimen including and blood and tissue to for detection in the molecular assays. This work represented a cooperative research development program between the Kramer-Tyagi/Perlin labs on probe development

  17. Development of a multiplexed microsphere PCR for rapid, culture-free detection and Gram-typing of bacteria in human blood samples.

    Science.gov (United States)

    Liang, Fang; Browne, Daniel J; Gray, Megan J; Gartlan, Kate H; Smith, David D; Barnard, Ross; Hill, Geoff R; Corrie, Simon Robert; Markey, Kate

    2018-01-19

    Blood stream infection is a significant clinical problem, particularly in vulnerable patient groups such as those undergoing chemotherapy and bone marrow transplantation. Clinical diagnostics for suspected blood stream infection remain centered around blood culture (highly variable timing, hours to days), and empiric use of broad-spectrum antibiotics is often employed for patients presenting with febrile neutropenia. Gram-typing provides the first opportunity to target therapy (e.g. combinations containing vancomycin or teicoplanin for Gram-positives; piperacillin-tazobactam or a carbapenem for Gram-negatives), however current approaches require blood culture. In this study, we describe a multiplexed microsphere-PCR assay with flow cytometry readout, which can distinguish Gram-positive from Gram-negative bacterial DNA in a 3.5-hour time period. The combination of a simple assay design (amplicon-dependent release of Gram-type specific Cy3-labelled oligonucleotides) and the Luminex-based readout (for quantifying each specific Cy3-labelled sequence) opens opportunities for further multiplexing. We demonstrate the feasibility of detecting common Gram-positive and Gram-negative organisms after spiking whole bacteria into healthy human blood prior to DNA extraction. Further development of DNA extraction methods is required to reach detection limits comparable to blood culture.

  18. Rapid and sensitive suspension array for multiplex detection of organophosphorus pesticides and carbamate pesticides based on silica–hydrogel hybrid microbeads

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Xuan [Key Laboratory of Environmental Medicine Engineering, Ministry of Education, School of Public Health, Southeast University, Nanjing, Jiangsu (China); Mu, Zhongde; Shangguan, Fengqi [State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing, Jiangsu (China); Liu, Ran; Pu, Yuepu [Key Laboratory of Environmental Medicine Engineering, Ministry of Education, School of Public Health, Southeast University, Nanjing, Jiangsu (China); Yin, Lihong, E-mail: lhyin@seu.edu.cn [Key Laboratory of Environmental Medicine Engineering, Ministry of Education, School of Public Health, Southeast University, Nanjing, Jiangsu (China)

    2014-05-01

    Highlights: • Silica–hydrogel hybrid microbeads were used to develop suspension array. • The results in detecting pesticides agree well with those from LC–MS/MS. • The method showed the good capability for multiplex analysis of pesticides residues. - Abstract: A technique for multiplex detection of organophosphorus pesticides and carbamate pesticides has been developed using a suspension array based on silica–hydrogel hybrid microbeads (SHHMs). The main advantage of SHHMs, which consist of both silica and hydrogel materials, is that they not only could be distinguished by their characteristic reflection peak originating from the stop-band of the photonic crystal but also have low non-specific adsorption of proteins. Using fluorescent immunoassay, the LODs for fenitrothion, chlorpyrifos-methyl, fenthion, carbaryl and metolcarb were measured to be 0.02 ng/mL, 0.012 ng/mL, 0.04 ng/mL, 0.05 ng/mL and 0.1 ng/mL, respectively, all of which are much lower than the maximum residue limits, as reported in the European Union pesticides database. All the determination coefficients for these five pesticides were greater than 0.99, demonstrating excellent correlations. The suspension array was specific and had no significant cross-reactivity with other chemicals. The results for the detection of pesticide residues collected from agricultural samples using this method agree well with those from liquid chromatography–tandem mass spectrometry. Our results showed that this simple method is suitable for simultaneous detection of these five pesticides residues in fruits and vegetables.

  19. Rapid multiplex detection of 10 foodborne pathogens with an up-converting phosphor technology-based 10-channel lateral flow assay.

    Science.gov (United States)

    Zhao, Yong; Wang, Haoran; Zhang, Pingping; Sun, Chongyun; Wang, Xiaochen; Wang, Xinrui; Yang, Ruifu; Wang, Chengbin; Zhou, Lei

    2016-02-17

    The rapid high-throughput detection of foodborne pathogens is essential in controlling food safety. In this study, a 10-channel up-converting phosphor technology-based lateral flow (TC-UPT-LF) assay was established for the rapid and simultaneous detection of 10 epidemic foodborne pathogens. Ten different single-target UPT-LF strips were developed and integrated into one TC-UPT-LF disc with optimization. Without enrichment the TC-UPT-LF assay had a detection sensitivity of 10(4) CFU mL(-1) or 10(5) CFU mL(-1) for each pathogen, and after sample enrichment it was 10 CFU/0.6 mg. The assay also showed good linearity, allowing quantitative detection, with a linear fitting coefficient of determination (R(2)) of 0.916-0.998. The 10 detection channels did not cross-react, so multiple targets could be specifically detected. When 279 real food samples were tested, the assay was highly consistent (100%) with culture-based methods. The results for 110 food samples artificially contaminated with single or multiple targets showed a high detection rate (≥ 80%) for most target bacteria. Overall, the TC-UPT-LF assay allows the rapid, quantitative, and simultaneous detection of 10 kinds of foodborne pathogens within 20 min, and is especially suitable for the rapid detection and surveillance of foodborne pathogens in food and water.

  20. Comparison of multiplex real-time PCR and PCR-reverse blot hybridization assay for the direct and rapid detection of bacteria and antibiotic resistance determinants in positive culture bottles.

    Science.gov (United States)

    Wang, Hye-Young; Kim, Seoyong; Kim, Jungho; Park, Soon Deok; Kim, Hyo Youl; Uh, Young; Lee, Hyeyoung

    2016-09-01

    The aim of this study was to evaluate the performance of a commercially available multiplex real-time PCR assay and a PCR-reverse blot hybridization assay (PCR-REBA) for the rapid detection of bacteria and identification of antibiotic resistance genes directly from blood culture bottles and to compare the results of these molecular assays with conventional culture methods. The molecular diagnostic methods were used to evaluate 593 blood culture bottles from patients with bloodstream infections. The detection positivity of multiplex real-time PCR assay for Gram-positive bacteria, Gram-negative bacteria and Candida spp. was equivalent to PCR-REBA as 99.6 %, 99.1 % and 100 %, respectively. Using conventional bacterial cultures as the gold standard, the sensitivity, specificity, positive predictive value and negative predictive value of these two molecular methods were 99.5 % [95 % confidence interval (CI), 0.980-1.000; PReal-methicillin-resistant Staphylococcusaureus multiplex real-time PCR assay targeting the mecA gene to detect methicillin resistance was lower than that of the PCR-REBA method, detecting an overall positivity of 98.4 % (n=182; 95 % CI, 0.964-1.000; P<0.009) and 99.5 % (n=184; 95 % CI, 0.985-1.000; P<0.0001), respectively. The entire two methods take about 3 h, while results from culture can take up to 48-72 h. Therefore, the use of these two molecular methods was rapid and reliable for the characterization of causative pathogens in bloodstream infections.

  1. Multiplex Detection of Food-Borne Pathogens.

    Science.gov (United States)

    Villamizar-Rodríguez, Germán; Lombó, Felipe

    2017-01-01

    Detection of food-borne pathogens is traditionally carried out by plating out techniques in selective or differential media using Petri agar dishes or other culture-dependent methods, usually designed for each pathogen to be detected. These classical methods are time and personnel consuming and also may last for up to 5 days in the case of final confirmation of some specific pathogens.Here we describe a method for fast multiplex detection of nine food-borne pathogens (all species usually required under most countrylegislations) by means of a single multiplex PCR reaction coupled to a capillary electrophoresis detection, in just 2-2.5 h and with a minimum cost of around 2 € per sample and nine pathogens. This method saves consumables and personnel time and allows a faster detection of any possible contaminated food batches at industrial level, therefore helping to prevent future food-borne outbreaks at clinical level.

  2. Rapid diagnosis of sepsis with TaqMan-Based multiplex real-time PCR.

    Science.gov (United States)

    Liu, Chang-Feng; Shi, Xin-Ping; Chen, Yun; Jin, Ye; Zhang, Bing

    2018-02-01

    The survival rate of septic patients mainly depends on a rapid and reliable diagnosis. A rapid, broad range, specific and sensitive quantitative diagnostic test is the urgent need. Thus, we developed a TaqMan-Based Multiplex real-time PCR assays to identify bloodstream pathogens within a few hours. Primers and TaqMan probes were designed to be complementary to conserved regions in the 16S rDNA gene of different kinds of bacteria. To evaluate accurately, sensitively, and specifically, the known bacteria samples (Standard strains, whole blood samples) are determined by TaqMan-Based Multiplex real-time PCR. In addition, 30 blood samples taken from patients with clinical symptoms of sepsis were tested by TaqMan-Based Multiplex real-time PCR and blood culture. The mean frequency of positive for Multiplex real-time PCR was 96% at a concentration of 100 CFU/mL, and it was 100% at a concentration greater than 1000 CFU/mL. All the known blood samples and Standard strains were detected positively by TaqMan-Based Multiplex PCR, no PCR products were detected when DNAs from other bacterium were used in the multiplex assay. Among the 30 patients with clinical symptoms of sepsis, 18 patients were confirmed positive by Multiplex real-time PCR and seven patients were confirmed positive by blood culture. TaqMan-Based Multiplex real-time PCR assay with highly sensitivity, specificity and broad detection range, is a rapid and accurate method in the detection of bacterial pathogens of sepsis and should have a promising usage in the diagnosis of sepsis. © 2017 Wiley Periodicals, Inc.

  3. Rapid Detection of Bloodstream Pathogens in Liver Transplantation Patients With FilmArray Multiplex Polymerase Chain Reaction Assays: Comparison With Conventional Methods.

    Science.gov (United States)

    Otlu, B; Bayindir, Y; Ozdemir, F; Ince, V; Cuglan, S; Hopoglu, M; Yakupogullari, Y; Kizilkaya, C; Kuzucu, C; Isık, B; Yilmaz, S

    2015-01-01

    Bloodstream infection (BSI) is an important concern in transplant patients. Early intervention with appropriate antimicrobial therapy is critical to better clinical outcome; however, there is significant delay when conventional identification methods are used. We aimed to determine the diagnostic performance of the FilmArray Blood Culture Identification Panel, a recently approved multiplex polymerase chain reaction assay detecting 24 BSI pathogens and 3 resistance genes, in comparison with the performances of conventional identification methods in liver transplant (LT) patients. A total of 52 defined sepsis episodes (signal-positive by blood culture systems) from 45 LT patients were prospectively studied. The FilmArray successfully identified 37 of 39 (94.8%) bacterial and 3 of 3 (100%) yeast pathogens in a total of 42 samples with microbial growth, failing to detect only 2 of 39 (5.1%) bacterial pathogens that were not covered by the test panel. The FilmArray could also detect additional pathogens in 3 samples that had been reported as having monomicrobial growth, and it could detect Acinetobacter baumannii in 2 samples suspected of skin flora contamination. The remaining 8 blood cultures showing a positive signal but yielding no growth were also negative by this assay. Results of MecA, KPC, and VanA/B gene detection were in high accordance. The FilmArray produced results with significantly shorter turnaround times (1.33 versus 36.2, 23.6, and 19.5 h; P FilmArray appeared as a reliable alternative diagnostic method with the potential to mitigate problems with protracted diagnosis of the BSI pathogens in LT patients. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. Rapid detection of aneuploidy and unbalanced chromosomal rearrangements by subtelomeric multiplex ligation-dependent probe amplification in fetuses with congenital heart disease.

    Science.gov (United States)

    Wang, Jing; Liu, Zhen; Liu, Hongqian; Li, Nana; Li, Shengli; Chen, Xinlin; Lin, Yuan; Wang, He; Zhu, Jun; Liu, Shanling

    2013-01-01

    To validate multiplex ligation-dependent probe amplification (MLPA) with subtelomeric probe mixes as a tool for diagnosis of aneuploidy and unbalanced terminal chromosomal rearrangements in fetuses with congenital heart disease. A prospective study of 117 fetuses found to have structural heart defects by ultrasound at 17-40 weeks' gestation. MLPA with P036E and P070B probe mixes was performed and compared to traditional karyotyping by cell culture and to findings of quantitative fluorescence-polymerase chain reaction (QF-PCR). MLPA was able to define the fetal karyotype in 99% of cases whereas cell culture only defined the karyotype in 64% of cases. Consequently, the overall number of chromosomal abnormalities that were detected increased. The majority of these affected chromosomes, 21, 18, 13, X or Y, were also confirmed by QF-PCR. Two (5%) cases had atypical aneuploidy that was confirmed by MLPA but not by QF-PCR. In 4 cases, structural rearrangements or mosaicism were not detected by MLPA. Subtelomeric MLPA may be a valuable adjunct to QF-PCR and/or conventional cytogenetics for the investigation of chromosomal abnormalities in fetuses with congenital heart disease. Copyright © 2013 S. Karger AG, Basel.

  5. The FilmArray® respiratory panel: an automated, broadly multiplexed molecular test for the rapid and accurate detection of respiratory pathogens.

    Science.gov (United States)

    Babady, N Esther

    2013-11-01

    The FilmArray Respiratory Panel (RP) (BioFire(™) Diagnostics, Inc., Salt Lake City, UT, USA) is the first multiplex molecular panel cleared by the US FDA for the detection of both bacterial and viral respiratory pathogens in nasopharygeal swabs. The FilmArray RP targets 20 pathogens including 17 viruses and subtypes and three bacteria, and is performed with minimal sample manipulation. The FilmArray RP has a fully automated sample-to-answer workflow with a turn-around-time of approximately 1 h. The reported sensitivity and specificity of the assay ranges from 80 to 100 and 100%, respectively, with the sensitivity for the adenovirus as low as 46%. A new version of the FilmArray RP assay (version 1.7) with improved sensitivity for the adenovirus was released in 2013. The performance characteristics and simplified workflow have allowed its implementation in a wide range of laboratories. The FilmArray RP has changed the diagnostic landscape and will have a significant impact on the care of patients with respiratory tract infection.

  6. Mach-Zehnder interferometer (MZI) point-of-care system for rapid multiplexed detection of microRNAs in human urine specimens.

    Science.gov (United States)

    Liu, Qing; Shin, Yong; Kee, Jack Sheng; Kim, Kyoung Woo; Mohamed Rafei, Siti Rafeah; Perera, Agampodi Promoda; Tu, Xiaoguang; Lo, Guo-Qiang; Ricci, Estelle; Colombel, Marc; Chiong, Edmund; Thiery, Jean Paul; Park, Mi Kyoung

    2015-09-15

    MicroRNAs have been identified as promising biomarkers for human diseases. The development of a point-of-care (POC) test for the disease-associated miRNAs would be especially beneficial, since miRNAs are unexpectedly well preserved in various human specimens, including urine. Here, we present the Mach-Zehnder interferometer-miRNA detection system capable of detecting multiple miRNAs in clinical urine samples rapidly and simultaneously in a label-free and real-time manner. Through measurement of the light phase change, the MZI sensor provides an optical platform for fast profiling of small molecules with improved accuracy. We demonstrate that this system could specifically detect target miRNAs (miR-21, and let-7a), and even identify the single nucleotide polymorphism of the let-7 family of miRNAs from synthetic and cell line samples. The clinical applicability of this system is confirmed by simultaneously detecting two types of miRNAs in urine samples of bladder cancer patients in a single reaction, with a detection time of 15 min. The POC system can be expanded to detect a number of miRNAs of different species and should be useful for a variety of clinical applications requiring at or near the site of patient care. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. Detection of diarrheagenic Escherichia coli by multiplex PCR

    Directory of Open Access Journals (Sweden)

    A Hegde

    2012-01-01

    Full Text Available Background: Diarrheagenic E.coli (DEC are an important cause of childhood diarrhea.Identification of DEC strains needs to detect factors that determine the virulence of these organisms. There is not much data regarding the importance of DEC as a cause of diarrhea in children in India.The prevalence of DEC in children belowfive years with and without diarrhea was studied using two multiplex PCR assays. Materials and Methods: Two multiplex polymerase chain reaction assays were used to detect genes of five types of DEC.The targets selected for each category were eae and bfpA (bundle-forming pilus forEnteropathogenic E.coli (EPEC, hlyA for Enterohemorrhagic E.coli (EHEC, elt and stla for Enterotoxigenic E.coli (ETEC, CVD432 for Enteroaggregative E.coli (EAEC and ial for Enteroinvasive E.coli (EIEC. Results: In 200 children with diarrhea 52 (26% DEC infections were found. Among 100 controls 8 (8% DEC infections were found. EAEC was the most common DEC by multiplex PCR both in cases (26, 13%and controls (5,5%, followed byEPEC seen in 16% cases and 3% controls. ETEC and EIEC were found in 7 (3.5% and 3 (1.5% of the diarrheal cases. EIEC and ETEC were not detected in the control cases. EHEC was not isolated from either the diarrheal or control cases. Conclusion: DEC strains are a significant cause of diarrhea in children. The two Multiplex PCR assays can be used for the detection of DEC in routine diagnostic laboratories. These assays are specific and sensitive for the rapid detection of DEC. EAEC was the most frequent pathotype in the population under study.

  8. Rapid and Simultaneous Detection of Mycobacterium tuberculosis Complex and Beijing/W Genotype in Sputum by an Optimized DNA Extraction Protocol and a Novel Multiplex Real-Time PCR ▿

    Science.gov (United States)

    Leung, Eric T. Y.; Zheng, L.; Wong, Rity Y. K.; Chan, Edward W. C.; Au, T. K.; Chan, Raphael C. Y.; Lui, Grace; Lee, Nelson; Ip, Margaret

    2011-01-01

    Rapid diagnosis and genotyping of Mycobacterium tuberculosis by molecular methods are often limited by the amount and purity of DNA extracted from body fluids. In this study, we evaluated 12 DNA extraction methods and developed a highly sensitive protocol for mycobacterial DNA extraction directly from sputa using surface-coated magnetic particles. We have also developed a novel multiplex real-time PCR for simultaneous identification of M. tuberculosis complex and the Beijing/W genotype (a hypervirulent sublineage of M. tuberculosis) by using multiple fluorogenic probes targeting both the M. tuberculosis IS6110 and the Rv0927c-pstS3 intergenic region. With reference strains and clinical isolates, our real-time PCR accurately identified 20 non-Beijing/W and 20 Beijing/W M. tuberculosis strains from 17 different species of nontuberculosis Mycobacterium (NTM). Further assessment of our DNA extraction protocol and real-time PCR with 335 nonduplicate sputum specimens correctly identified all 74 M. tuberculosis culture-positive specimens. In addition, 15 culture-negative specimens from patients with confirmed tuberculosis were also identified. No cross-reactivity was detected with NTM specimens (n = 31). The detection limit of the assay is 10 M. tuberculosis bacilli, as determined by endpoint dilution analysis. In conclusion, an optimized DNA extraction protocol coupled with a novel multiprobe multiplex real-time PCR for the direct detection of M. tuberculosis, including Beijing/W M. tuberculosis, was found to confer high sensitivity and specificity. The combined procedure has the potential to compensate for the drawbacks of conventional mycobacterial culture in routine clinical laboratory setting, such as the lengthy incubation period and the limitation to viable organisms. PMID:21593264

  9. Multiplex detection of food allergens and gluten.

    Science.gov (United States)

    Cho, Chung Y; Nowatzke, William; Oliver, Kerry; Garber, Eric A E

    2015-05-01

    To help safeguard the food supply and detect the presence of undeclared food allergens and gluten, most producers and regulatory agencies rely on commercial test kits. Most of these are ELISAs with a few being PCR-based. These methods are very sensitive and analyte specific, requiring different assays to detect each of the different food allergens. Mass spectrometry offers an alternative approach whereby multiple allergens may be detected simultaneously. However, mass spectrometry requires expensive equipment, highly trained analysts, and several years before a quantitative approach can be achieved. Using multianalyte profiling (xMAP®) technology, a commercial multiplex test kit based on the use of established antibodies was developed for the simultaneous detection of up to 14 different food allergens plus gluten. The assay simultaneously detects crustacean seafood, egg, gluten, milk, peanut, soy, and nine tree nuts (almond, Brazil nut, cashew, coconut, hazelnut, macadamia, pine nut, pistachio, and walnut). By simultaneously performing multiple tests (typically two) for each analyte, this magnetic bead-based assay offers built-in confirmatory analyses without the need for additional resources. Twenty-five of the assays were performed on buffer extracted samples, while five were conducted on samples extracted using reduced-denatured conditions. Thus, complete analysis for all 14 allergens and gluten requires only two wells of a 96-well microtiter plate. This makes it possible to include in a single analytical run up to 48 samples. All 30 bead sets in this multiplex assay detected 5 ng/mL of food allergen and gluten with responses greater than background. In addition, 26 of the bead sets displayed signal/noise ratios of five or greater. The bead-based design makes this 30-plex assay expandable to incorporate new antibodies and capture/detector methodologies by ascribing these new detectors to any of the unassigned bead sets that are commercially available.

  10. [Detection of Staphylococcus aureus, Shigella spp., Salmonella spp. in food by multiplex PCR].

    Science.gov (United States)

    Li, Bo; Chen, Fusheng; Wang, Xiaohong; Shao, Yanchun

    2008-07-01

    To establish a multiplex PCR method for simultaneous detection of Staphylococcus aureus, Shigella spp., Salmonella spp. in food. Staphylococcus aureus was enriched by 7.5% NaCl broth while Shigella spp. and Salmonella spp. were enriched by GN medium . The primers were designed according to the gene nuc of Staphylococcus aureus, the gene ipaH of Shigella spp. and the gene invA of Salmonella spp. The target genes of these pathogens in food were amplified by multiplex PCR, which reaction conditions were optimized specifically. The multiplex PCR method established in this experment was of high specificity, which detection limit was 1 cfu/ml of Staphylococcus aureus, Shigella spp. and Salmonella spp. when the milk samples contaminated with these pathogens. The multiplex PCR method, which was rapid, convenient, and with high sensitivity, could be suitable for rapid detection of Staphylococcus aureus, Shigella spp., Salmonella spp. in food, and could have a great prospect.

  11. Development of a multiplex real-time PCR for the rapid detection of the predominant beta-lactamase genes CTX-M, SHV, TEM and CIT-type AmpCs in Enterobacteriaceae.

    Directory of Open Access Journals (Sweden)

    Nicole Roschanski

    Full Text Available Beta-lactamase resistant bacteria and especially ESBL producing Enterobacteriaceae are an increasing problem worldwide. For this reason a major interest in efficient and reliable methods for rapid screening of high sample numbers is recognizable. Therefore, a multiplex real-time PCR was developed to detect the predominant class A beta-lactamase genes blaCTX-M, blaSHV, blaTEM and CIT-type AmpCs in a one-step reaction. A set of 114 Enterobacteriaceae containing previously identified resistance gene subtypes and in addition 20 undefined animal and environmental isolates were used for the validation of this assay. To confirm the accessibility in variable settings, the real-time runs were performed analogous in two different laboratories using different real-time cyclers. The obtained results showed complete accordance between the real-time data and the predetermined genotypes. Even if sequence analyses are further necessary for a comprehensive characterization, this method was proofed to be reliable for rapid screening of high sample numbers and therefore could be an important tool for e. g. epidemiological purposes or support infection control measures.

  12. Development and validation of a multiplex reverse transcription quantitative PCR (RT-qPCR) assay for the rapid detection of Citrus tristeza virus, Citrus psorosis virus, and Citrus leaf blotch virus.

    Science.gov (United States)

    Osman, Fatima; Hodzic, Emir; Kwon, Sun-Jung; Wang, Jinbo; Vidalakis, Georgios

    2015-08-01

    A single real-time multiplex reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay for the simultaneous detection of Citrus tristeza virus (CTV), Citrus psorosis virus (CPsV), and Citrus leaf blotch virus (CLBV) was developed and validated using three different fluorescently labeled minor groove binding qPCR probes. To increase the detection reliability, coat protein (CP) genes from large number of different isolates of CTV, CPsV and CLBV were sequenced and a multiple sequence alignment was generated with corresponding CP sequences from the GenBank and a robust multiplex RT-qPCR assay was designed. The capacity of the multiplex RT-qPCR assay in detecting the viruses was compared to singleplex RT-qPCR designed specifically for each virus and was assessed using multiple virus isolates from diverse geographical regions and citrus species as well as graft-inoculated citrus plants infected with various combination of the three viruses. No significant difference in detection limits was found and specificity was not affected by the inclusion of the three assays in a multiplex RT-qPCR reaction. Comparison of the viral load for each virus using singleplex and multiplex RT-qPCR assays, revealed no significant differences between the two assays in virus detection. No significant difference in Cq values was detected when using one-step and two-step multiplex RT-qPCR detection formats. Optimizing the RNA extraction technique for citrus tissues and testing the quality of the extracted RNA using RT-qPCR targeting the cytochrome oxidase citrus gene as an RNA specific internal control proved to generate better diagnostic assays. Results showed that the developed multiplex RT-qPCR can streamline viruses testing of citrus nursery stock by replacing three separate singleplex assays, thus reducing time and labor while retaining the same sensitivity and specificity. The three targeted RNA viruses are regulated pathogens for California's mandatory "Section 3701

  13. Multiplex detection of plant pathogens using a microsphere immunoassay technology.

    Directory of Open Access Journals (Sweden)

    Ratthaphol Charlermroj

    Full Text Available Plant pathogens are a serious problem for seed export, plant disease control and plant quarantine. Rapid and accurate screening tests are urgently required to protect and prevent plant diseases spreading worldwide. A novel multiplex detection method was developed based on microsphere immunoassays to simultaneously detect four important plant pathogens: a fruit blotch bacterium Acidovorax avenae subsp. citrulli (Aac, chilli vein-banding mottle virus (CVbMV, potyvirus, watermelon silver mottle virus (WSMoV, tospovirus serogroup IV and melon yellow spot virus (MYSV, tospovirus. An antibody for each plant pathogen was linked on a fluorescence-coded magnetic microsphere set which was used to capture corresponding pathogen. The presence of pathogens was detected by R-phycoerythrin (RPE-labeled antibodies specific to the pathogens. The assay conditions were optimized by identifying appropriate antibody pairs, blocking buffer, concentration of RPE-labeled antibodies and assay time. Once conditions were optimized, the assay was able to detect all four plant pathogens precisely and accurately with substantially higher sensitivity than enzyme-linked immunosorbent assay (ELISA when spiked in buffer and in healthy watermelon leaf extract. The assay time of the microsphere immunoassay (1 hour was much shorter than that of ELISA (4 hours. This system was also shown to be capable of detecting the pathogens in naturally infected plant samples and is a major advancement in plant pathogen detection.

  14. Solid-phase PCR for rapid multiplex detection of Salmonella spp. at the subspecies level, with amplification efficiency comparable to conventional PCR.

    Science.gov (United States)

    Chin, Wai Hoe; Sun, Yi; Høgberg, Jonas; Hung, Tran Quang; Wolff, Anders; Bang, Dang Duong

    2017-04-01

    Solid-phase PCR (SP-PCR) has attracted considerable interest in different research fields since it allows parallel DNA amplification on the surface of a solid substrate. However, the applications of SP-PCR have been hampered by the low efficiency of the solid-phase amplification. In order to increase the yield of the solid-phase amplification, we studied various parameters including the length, the density, as well as the annealing position of the solid support primer. A dramatic increase in the signal-to-noise (S/N) ratio was observed when increasing the length of solid support primers from 45 to 80 bp. The density of the primer on the surface was found to be important for the S/N ratio of the SP-PCR, and the optimal S/N was obtained with a density of 1.49 × 1011 molecules/mm2. In addition, the use of solid support primers with a short overhang at the 5' end would help improve the S/N ratio of the SP-PCR. With optimized conditions, SP-PCR can achieve amplification efficiency comparable to conventional PCR, with a limit of detection of 1.5 copies/μl (37.5 copies/reaction). These improvements will pave the way for wider applications of SP-PCR in various fields such as clinical diagnosis, high-throughput DNA sequencing, and single-nucleotide polymorphism analysis. Graphical abstract Schematic representation of solid-phase PCR.

  15. Multiplexed paper test strip for quantitative bacterial detection.

    Science.gov (United States)

    Hossain, S M Zakir; Ozimok, Cory; Sicard, Clémence; Aguirre, Sergio D; Ali, M Monsur; Li, Yingfu; Brennan, John D

    2012-06-01

    Rapid, sensitive, on-site detection of bacteria without a need for sophisticated equipment or skilled personnel is extremely important in clinical settings and rapid response scenarios, as well as in resource-limited settings. Here, we report a novel approach for selective and ultra-sensitive multiplexed detection of Escherichia coli (non-pathogenic or pathogenic) using a lab-on-paper test strip (bioactive paper) based on intracellular enzyme (β-galactosidase (B-GAL) or β-glucuronidase (GUS)) activity. The test strip is composed of a paper support (0.5 × 8 cm), onto which either 5-bromo-4-chloro-3-indolyl-β-D: -glucuronide sodium salt (XG), chlorophenol red β-galactopyranoside (CPRG) or both and FeCl(3) were entrapped using sol-gel-derived silica inks in different zones via an ink-jet printing technique. The sample was lysed and assayed via lateral flow through the FeCl(3) zone to the substrate area to initiate rapid enzyme hydrolysis of the substrate, causing a change from colorless-to-blue (XG hydrolyzed by GUS, indication of nonpathogenic E. coli) and/or yellow to red-magenta (CPRG hydrolyzed by B-GAL, indication of total coliforms). Using immunomagnetic nanoparticles for selective preconcentration, the limit of detection was ~5 colony-forming units (cfu) per milliliter for E. coli O157:H7 and ~20 cfu/mL for E. coli BL21, within 30 min without cell culturing. Thus, these paper test strips could be suitable for detection of viable total coliforms and pathogens in bathing water samples. Moreover, inclusion of a culturing step allows detection of less than 1 cfu in 100 mL within 8 h, making the paper tests strips relevant for detection of multiple pathogens and total coliform bacteria in beverage and food samples.

  16. Rapid identification and differentiation of Saccharomyces cerevisiae, Saccharomyces bayanus and their hybrids by multiplex PCR.

    Science.gov (United States)

    Torriani, S; Zapparoli, G; Malacrinò, P; Suzzi, G; Dellaglio, F

    2004-01-01

    To develop a multiplex PCR assay for the specific identification and differentiation of Saccharomyces cerevisiae, S. bayanus and their hybrids. Two sets of primers with sequences complementary to the region YBR033w were used. A single amplicon of 1710 bp or 329 bp was obtained with species S. cerevisiae and S. bayanus, respectively, while the presence of both bands was observed in S. pastorianus because of its hybrid nature. Both amplification products were also obtained after amplification from DNA of several laboratory S. cerevisiae x S. bayanus hybrid strains. Multiplex PCR was optimized for the rapid and reliable identification of S. cerevisiae, S. bayanus and their hybrids. The procedure may be used for routine detection of the most common Saccharomyces sensu stricto yeasts involved in industrial fermentation processes, overcoming the problems of conventional techniques.

  17. A novel capillary electrophoresis-based multiplex PCR assay for detection of respiratory pathogens.

    Science.gov (United States)

    Stevenson, Jeffery B; Hymas, Weston C; Hillyard, David R

    2011-01-01

    The field of infectious disease testing has recently experienced rapid expansion in the number of multiplexed PCR-based assays available for detecting respiratory pathogens. This study provides a preliminary evaluation of a multiplex assay from Seegene that uses capillary electrophoresis as the detection platform for viral and bacterial respiratory pathogens. We compared this technology to a real-time PCR assay for 3 viral targets. Thirty respiratory samples were collected that had previously tested positive for either Flu A, Flu B, or RSV (ten of each). The Seegene assay detected 9/10 Flu A samples, 9/10 Flu B, and 10/10 RSV, for a total detection rate of 93%. The two samples that were undetected by the Seegene assay both generated late-crossing thresholds on the real-time platform, consistent with low viral loads. The Seeplex assay provides a promising alternative for multiplex respiratory testing.

  18. Multiplex polymerase chain reaction for detection and ...

    African Journals Online (AJOL)

    Moreover, 20 strains of E. coli (25%) were isolated from milk samples collected from 80 animals suffering from mastitis and subclinical mastitis. E. coli serovars yielded from bacteriological examination of milk samples were similar to that of fecal samples. Serogroup-specific multiplex polymerase chain reaction (PCR) assay ...

  19. Rapid DNA haplotyping using a multiplex heteroduplex approach: application to Duchenne muscular dystrophy carrier testing.

    Science.gov (United States)

    Prior, T W; Wenger, G D; Papp, A C; Snyder, P J; Sedra, M S; Bartolo, C; Moore, J W; Highsmith, W E

    1995-01-01

    A new strategy has been developed for rapid haplotype analysis based on an initial multiplex amplification of several polymorphic sites, followed by heteroduplex detection. Heteroduplexes formed between two different alleles are detected because they migrate differently than the corresponding homoduplexes in Hydrolink-MDE gel. This simple, rapid method does not depend on specific sequences such as restriction enzyme sites or CA boxes and does not require the use of isotope. This approach has been tested using commonly occurring polymorphisms spanning the dystrophin gene as a model. We describe the use of the method to assign the carrier status of females in Duchenne muscular dystrophy (DMD) pedigrees. The method may be used for other genetic diseases when mutations are unknown or there are few dinucleotide markers in the gene proximity, and for the identification of haplotype backgrounds of mutant alleles.

  20. A novel multiplex assay for simultaneously analysing 13 rapidly mutating Y-STRs

    NARCIS (Netherlands)

    R. Alghafri (Rashed); W. Goodwin (Will); A. Ralf (Arwin); M.H. Kayser (Manfred); S. Hadi (Sibte)

    2015-01-01

    textabstractAbstract A multiplex polymerase chain reaction (PCR) assay (RM-Yplex) was developed which is capable of simultaneously amplifying 13 recently introduced rapidly mutating Y-STR markers (RM Y-STRs). This multiplex assay is expected to aid human identity testing in forensic and other

  1. New multiplex PCR methods for rapid screening of genetically modified organisms in foods.

    Science.gov (United States)

    Datukishvili, Nelly; Kutateladze, Tamara; Gabriadze, Inga; Bitskinashvili, Kakha; Vishnepolsky, Boris

    2015-01-01

    We present novel multiplex PCR methods for rapid and reliable screening of genetically modified organisms (GMOs). New designed PCR primers targeting four frequently used GMO specific sequences permitted identification of new DNA markers, in particular 141 bp fragment of cauliflower mosaic virus (CaMV) 35S promoter, 224 bp fragment of Agrobacterium tumefaciens nopaline synthase (NOS) terminator, 256 bp fragment of 5-enolppyruvylshikimate-phosphate synthase (epsps) gene and 258 bp fragment of Cry1Ab delta-endotoxin (cry1Ab) gene for GMO screening. The certified reference materials containing Roundup Ready soybean (RRS) and maize MON 810 were applied for the development and optimization of uniplex and multiplex PCR systems. Evaluation of amplification products by agarose gel electrophoresis using negative and positive controls confirmed high specificity and sensitivity at 0.1% GMO for both RRS and MON 810. The fourplex PCR was developed and optimized that allows simultaneous detection of three common transgenic elements, such as: CaMV 35S promoter, NOS terminator, epsps gene together with soybean-specific lectin gene. The triplex PCR developed enables simultaneous identification of transgenic elements, such as: 35S promoter and cry1Ab gene together with maize zein gene. The analysis of different processed foods demonstrated that multiplex PCR methods developed in this study are useful for accurate and fast screening of GM food products.

  2. New multiplex PCR methods for rapid screening of genetically modified organisms in foods

    Directory of Open Access Journals (Sweden)

    Nelly eDatukishvili

    2015-07-01

    Full Text Available We present novel multiplex PCR methods for rapid and reliable screening of genetically modified organisms (GMOs. New designed PCR primers targeting four frequently used GMO specific sequences permitted identification of new DNA markers, in particular 141 bp fragment of cauliflower mosaic virus (CaMV 35S promoter, 224 bp fragment of Agrobacterium tumefaciens nopaline synthase (NOS terminator, 256 bp fragment of 5-enolppyruvylshikimate-phosphate synthase (epsps gene and 258 bp fragment of Cry1Ab delta-endotoxin (cry1Ab gene for GMO screening. The certified reference materials containing Roundup Ready soybean (RRS and maize MON 810 were applied for the development and optimization of uniplex and multiplex PCR systems. Evaluation of amplification products by agarose gel electrophoresis using negative and positive controls confirmed high specificity and sensitivity at 0.1% GMO for both RRS and MON 810. The fourplex PCR was developed and optimized that allows simultaneous detection of three common transgenic elements, such as: CaMV 35S promoter, NOS terminator, epsps gene together with soybean-specific lectin gene. The triplex PCR developed enables simultaneous identification of transgenic elements, such as: 35S promoter and cry1Ab gene together with maize zein gene. The analysis of different processed foods demonstrated that multiplex PCR methods developed in this study are useful for accurate and fast screening of GM food products.

  3. Rapid, Time-Division Multiplexed, Direct Absorption- and Wavelength Modulation-Spectroscopy

    Directory of Open Access Journals (Sweden)

    Alexander Klein

    2014-11-01

    Full Text Available We present a tunable diode laser spectrometer with a novel, rapid time multiplexed direct absorption- and wavelength modulation-spectroscopy operation mode. The new technique allows enhancing the precision and dynamic range of a tunable diode laser absorption spectrometer without sacrificing accuracy. The spectroscopic technique combines the benefits of absolute concentration measurements using calibration-free direct tunable diode laser absorption spectroscopy (dTDLAS with the enhanced noise rejection of wavelength modulation spectroscopy (WMS. In this work we demonstrate for the first time a 125 Hz time division multiplexed (TDM-dTDLAS-WMS spectroscopic scheme by alternating the modulation of a DFB-laser between a triangle-ramp (dTDLAS and an additional 20 kHz sinusoidal modulation (WMS. The absolute concentration measurement via the dTDLAS-technique allows one to simultaneously calibrate the normalized 2f/1f-signal of the WMS-technique. A dTDLAS/WMS-spectrometer at 1.37 µm for H2O detection was built for experimental validation of the multiplexing scheme over a concentration range from 50 to 3000 ppmV (0.1 MPa, 293 K. A precision of 190 ppbV was achieved with an absorption length of 12.7 cm and an averaging time of two seconds. Our results show a five-fold improvement in precision over the entire concentration range and a significantly decreased averaging time of the spectrometer.

  4. Rapid multiplex PCR assay to identify respiratory viral pathogens: moving forward diagnosing the common cold.

    Science.gov (United States)

    Layman, Clifton P; Gordon, Sarah M; Elegino-Steffens, Diane U; Agee, Willie; Barnhill, Jason; Hsue, Gunther

    2013-09-01

    Upper respiratory tract infections (URIs) can be a serious burden to the healthcare system. The majority of URIs are viral in etiology, but definitive diagnosis can prove difficult due to frequently overlapping clinical presentations of viral and bacterial infections, and the variable sensitivity, and lengthy turn-around time of viral culture. We tested new automated nested multiplex PCR technology, the FilmArray(®) system, in the TAMC department of clinical investigations, to determine the feasibility of replacing the standard viral culture with a rapid turn-around system. We conducted a feasibility study using a single-blinded comparison study, comparing PCR results with archived viral culture results from a convenience sample of cryopreserved archived nasopharyngeal swabs from acutely ill ED patients who presented with complaints of URI symptoms. A total of 61 archived samples were processed. Viral culture had previously identified 31 positive specimens from these samples. The automated nested multiplex PCR detected 38 positive samples. In total, PCR was 94.5% concordant with the previously positive viral culture results. However, PCR was only 63.4% concordant with the negative viral culture results, owing to PCR detection of 11 additional viral pathogens not recovered on viral culture. The average time to process a sample was 75 minutes. We determined that an automated nested multiplex PCR is a feasible alternative to viral culture in an acute clinical setting. We were able to detect at least 94.5% as many viral pathogens as viral culture is able to identify, with a faster turn-around time.

  5. A new trilocus sequence-based multiplex-PCR to detect major Acinetobacter baumannii clones.

    Science.gov (United States)

    Martins, Natacha; Picão, Renata Cristina; Cerqueira-Alves, Morgana; Uehara, Aline; Barbosa, Lívia Carvalho; Riley, Lee W; Moreira, Beatriz Meurer

    2016-08-01

    A collection of 163 Acinetobacter baumannii isolates detected in a large Brazilian hospital, was potentially related with the dissemination of four clonal complexes (CC): 113/79, 103/15, 109/1 and 110/25, defined by University of Oxford/Institut Pasteur multilocus sequence typing (MLST) schemes. The urge of a simple multiplex-PCR scheme to specify these clones has motivated the present study. The established trilocus sequence-based typing (3LST, for ompA, csuE and blaOXA-51-like genes) multiplex-PCR rapidly identifies international clones I (CC109/1), II (CC118/2) and III (CC187/3). Thus, the system detects only one (CC109/1) out of four main CC in Brazil. We aimed to develop an alternative multiplex-PCR scheme to detect these clones, known to be present additionally in Africa, Asia, Europe, USA and South America. MLST, performed in the present study to complement typing our whole collection of isolates, confirmed that all isolates belonged to the same four CC detected previously. When typed by 3LST-based multiplex-PCR, only 12% of the 163 isolates were classified into groups. By comparative sequence analysis of ompA, csuE and blaOXA-51-like genes, a set of eight primers was designed for an alternative multiplex-PCR to distinguish the five CC 113/79, 103/15, 109/1, 110/25 and 118/2. Study isolates and one CC118/2 isolate were blind-tested with the new alternative PCR scheme; all were correctly clustered in groups of the corresponding CC. The new multiplex-PCR, with the advantage of fitting in a single reaction, detects five leading A. baumannii clones and could help preventing the spread in healthcare settings. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. Development of a multiplex PCR assay detecting 52 autosomal SNPs

    DEFF Research Database (Denmark)

    Sanchez Sanchez, Juan Jose; Phillips, C.; Børsting, Claus

    2006-01-01

    for amplifying 52 genomic DNA fragments, each containing one SNP, in a single tube, and accurately genotyping the PCR product mixture using two single base extension reactions. This multiplex approach reduces the cost of SNP genotyping and requires as little as 0.5 ng of genomic DNA to detect 52 SNPs. We used...

  7. Development of multiplex real-time PCR assay for the detection of Brucella spp., Leptospira spp. and Campylobacter foetus

    Directory of Open Access Journals (Sweden)

    Abdelfattah M. Selim

    2014-12-01

    Full Text Available Abortion among dairy cattle is one of the major causes of economic losses in the livestock industry. This study describes a 1-step multiplex real-time polymerase chain reaction (PCR to detect Brucella spp., Leptospira spp. and Campylobacter foetus, these are significant bacteria commonly implicated in bovine abortion. ß-actin was added to the same PCR reaction as an internal control to detect any extraction failure or PCR inhibition. The detection limit of multiplex real-time PCR using purified DNA from cultured organisms was set to 5 fg for Leptospira spp. and C. foetus and to 50 fg for Brucella spp. The multiplex real-time PCR did not produce any non-specific amplification when tested with different strains of the 3 pathogens. This multiplex real-time PCR provides a valuable tool for diagnosis, simultaneous and rapid detection for the 3 pathogens causing abortion in bovine.

  8. Direct object detection from field-of-view multiplexed measurements

    Science.gov (United States)

    Shilling, Richard Z.; Muise, Robert R.

    2017-04-01

    A compressive imaging model is proposed that multiplexes segments of the field of view (FOV) onto an infrared focal plane array (FPA). Similar to compound imaging, our model is based on combining pixels from a surface comprising of the different parts of the FOV. We formalize this superposition of pixels in a global multiplexing process reducing the number of detectors required for the FPA. We present an analysis of the signal-to-noise ratio for the full rank and compressive collection paradigms for a target detection and tracking scenario. We then apply automated target detection algorithms directly on the measurement sequence for this multiplexing model. We extend the target training and detection processes for the application directly on the encoded measurements. Optimal measurement codes for this application may imply abandoning the ability to reconstruct the actual scene in favor of reconstructing the locations of interesting objects. We present a simulated case study as well as real data results from a visible FOV multiplexing camera.

  9. A high-throughput multiplex method adapted for GMO detection.

    Science.gov (United States)

    Chaouachi, Maher; Chupeau, Gaëlle; Berard, Aurélie; McKhann, Heather; Romaniuk, Marcel; Giancola, Sandra; Laval, Valérie; Bertheau, Yves; Brunel, Dominique

    2008-12-24

    A high-throughput multiplex assay for the detection of genetically modified organisms (GMO) was developed on the basis of the existing SNPlex method designed for SNP genotyping. This SNPlex assay allows the simultaneous detection of up to 48 short DNA sequences (approximately 70 bp; "signature sequences") from taxa endogenous reference genes, from GMO constructions, screening targets, construct-specific, and event-specific targets, and finally from donor organisms. This assay avoids certain shortcomings of multiplex PCR-based methods already in widespread use for GMO detection. The assay demonstrated high specificity and sensitivity. The results suggest that this assay is reliable, flexible, and cost- and time-effective for high-throughput GMO detection.

  10. Interferometric Reflectance Imaging Sensor (IRIS—A Platform Technology for Multiplexed Diagnostics and Digital Detection

    Directory of Open Access Journals (Sweden)

    Oguzhan Avci

    2015-07-01

    Full Text Available Over the last decade, the growing need in disease diagnostics has stimulated rapid development of new technologies with unprecedented capabilities. Recent emerging infectious diseases and epidemics have revealed the shortcomings of existing diagnostics tools, and the necessity for further improvements. Optical biosensors can lay the foundations for future generation diagnostics by providing means to detect biomarkers in a highly sensitive, specific, quantitative and multiplexed fashion. Here, we review an optical sensing technology, Interferometric Reflectance Imaging Sensor (IRIS, and the relevant features of this multifunctional platform for quantitative, label-free and dynamic detection. We discuss two distinct modalities for IRIS: (i low-magnification (ensemble biomolecular mass measurements and (ii high-magnification (digital detection of individual nanoparticles along with their applications, including label-free detection of multiplexed protein chips, measurement of single nucleotide polymorphism, quantification of transcription factor DNA binding, and high sensitivity digital sensing and characterization of nanoparticles and viruses.

  11. Multiplex PCR for Detection of Botulinum Neurotoxin-Producing Clostridia in Clinical, Food, and Environmental Samples▿

    Science.gov (United States)

    De Medici, Dario; Anniballi, Fabrizio; Wyatt, Gary M.; Lindström, Miia; Messelhäußer, Ute; Aldus, Clare F.; Delibato, Elisabetta; Korkeala, Hannu; Peck, Michael W.; Fenicia, Lucia

    2009-01-01

    Botulinum neurotoxin (BoNT), the most toxic substance known, is produced by the spore-forming bacterium Clostridium botulinum and, in rare cases, also by some strains of Clostridium butyricum and Clostridium baratii. The standard procedure for definitive detection of BoNT-producing clostridia is a culture method combined with neurotoxin detection using a standard mouse bioassay (SMB). The SMB is highly sensitive and specific, but it is expensive and time-consuming and there are ethical concerns due to use of laboratory animals. PCR provides a rapid alternative for initial screening for BoNT-producing clostridia. In this study, a previously described multiplex PCR assay was modified to detect all type A, B, E, and F neurotoxin genes in isolated strains and in clinical, food, environmental samples. This assay includes an internal amplification control. The effectiveness of the multiplex PCR method for detecting clostridia possessing type A, B, E, and F neurotoxin genes was evaluated by direct comparison with the SMB. This method showed 100% inclusivity and 100% exclusivity when 182 BoNT-producing clostridia and 21 other bacterial strains were used. The relative accuracy of the multiplex PCR and SMB was evaluated using 532 clinical, food, and environmental samples and was estimated to be 99.2%. The multiplex PCR was also used to investigate 110 freshly collected food and environmental samples, and 4 of the 110 samples (3.6%) were positive for BoNT-encoding genes. PMID:19684163

  12. Multiplex PCR for detection of botulinum neurotoxin-producing clostridia in clinical, food, and environmental samples.

    Science.gov (United States)

    De Medici, Dario; Anniballi, Fabrizio; Wyatt, Gary M; Lindström, Miia; Messelhäusser, Ute; Aldus, Clare F; Delibato, Elisabetta; Korkeala, Hannu; Peck, Michael W; Fenicia, Lucia

    2009-10-01

    Botulinum neurotoxin (BoNT), the most toxic substance known, is produced by the spore-forming bacterium Clostridium botulinum and, in rare cases, also by some strains of Clostridium butyricum and Clostridium baratii. The standard procedure for definitive detection of BoNT-producing clostridia is a culture method combined with neurotoxin detection using a standard mouse bioassay (SMB). The SMB is highly sensitive and specific, but it is expensive and time-consuming and there are ethical concerns due to use of laboratory animals. PCR provides a rapid alternative for initial screening for BoNT-producing clostridia. In this study, a previously described multiplex PCR assay was modified to detect all type A, B, E, and F neurotoxin genes in isolated strains and in clinical, food, environmental samples. This assay includes an internal amplification control. The effectiveness of the multiplex PCR method for detecting clostridia possessing type A, B, E, and F neurotoxin genes was evaluated by direct comparison with the SMB. This method showed 100% inclusivity and 100% exclusivity when 182 BoNT-producing clostridia and 21 other bacterial strains were used. The relative accuracy of the multiplex PCR and SMB was evaluated using 532 clinical, food, and environmental samples and was estimated to be 99.2%. The multiplex PCR was also used to investigate 110 freshly collected food and environmental samples, and 4 of the 110 samples (3.6%) were positive for BoNT-encoding genes.

  13. Nested-multiplex PCR detection of Orthopoxvirus and Parapoxvirus directly from exanthematic clinical samples

    Directory of Open Access Journals (Sweden)

    Trindade Giliane S

    2009-09-01

    Full Text Available Abstract Background Orthopoxvirus (OPV and Parapoxvirus (PPV have been associated with worldwide exanthematic outbreaks. Some species of these genera are able to infect humans and domestic animals, causing serious economic losses and public health impact. Rapid, useful and highly specific methods are required to detect and epidemiologically monitor such poxviruses. In the present paper, we describe the development of a nested-multiplex PCR method for the simultaneous detection of OPV and PPV species directly from exanthematic lesions, with no previous viral isolation or DNA extraction. Methods and Results The OPV/PPV nested-multiplex PCR was developed based on the evaluation and combination of published primer sets, and was applied to the detection of the target pathogens. The method showed high sensitivity, and the specificity was confirmed by amplicon sequencing. Exanthematic lesion samples collected during bovine vaccinia or contagious ecthyma outbreaks were submitted to OPV/PPV nested-multiplex PCR and confirmed its applicability. Conclusion These results suggest that the presented multiplex PCR provides a highly robust and sensitive method to detect OPV and PPV directly from clinical samples. The method can be used for viral identification and monitoring, especially in areas where OPV and PPV co-circulate.

  14. Diagnostic accuracy study of multiplex PCR for detecting tuberculosis drug resistance.

    Science.gov (United States)

    Molina-Moya, B; Lacoma, A; Prat, C; Pimkina, E; Diaz, J; García-Sierra, N; Haba, L; Maldonado, J; Samper, S; Ruiz-Manzano, J; Ausina, V; Dominguez, J

    2015-08-01

    To study the diagnostic accuracy of a multiplex real-time PCR (Anyplex II MTB/MDR/XDR, Seegene, Corea) that detects Mycobacterium tuberculosis resistant to isoniazid (INH), rifampicin (RIF), fluoroquinolones (FLQ) and injectable drugs (kanamycin [KAN], amikacin [AMK] and capreomycin [CAP]) in isolates and specimens. One hundred fourteen cultured isolates and 73 sputum specimens were retrospectively selected. Results obtained with multiplex PCR were compared with those obtained with BACTEC. Discordant results between multiplex PCR and BACTEC were tested by alternative molecular methods. Sensitivity and specificity of multiplex PCR for detecting drug resistance in isolates were 76.5% and 100%, respectively, for INH; 97.2% and 96.0%, respectively, for RIF; 70.4% and 87.9%, respectively, for FLQ; 81.5% and 84.8%, respectively, for KAN; 100% and 60%, respectively, for AMK, and 100% and 72.3%, respectively, for CAP. Sensitivity and specificity of Anyplex for detecting drug resistance in specimens were 93.3% and 100%, respectively, for INH; 100% and 100%, respectively, for RIF; 50.0% and 100%, respectively, for FLQ; and 100% and 94.4%, respectively, for both KAN and CAP. Among the discordant results, 87.7% (71/81) of results obtained with the multiplex PCR were concordant with at least one of the alternative molecular methods. This multiplex PCR may be a useful tool for the rapid identification of drug resistant tuberculosis in isolates and specimens, thus allowing an initial therapeutic approach. Nevertheless, for a correct management of patients, results should be confirmed by a phenotypic method. Copyright © 2015 The British Infection Association. Published by Elsevier Ltd. All rights reserved.

  15. New Multiplexing Tools for Reliable GMO Detection

    NARCIS (Netherlands)

    Pla, M.; Nadal, A.; Baeten, V.; Bahrdt, C.; Berben, G.; Bertheau, Y.; Coll, A.; Dijk, van J.P.; Dobnik, D.; Fernandez-Pierna, J.A.; Gruden, K.; Hamels, S.; Holck, A.; Holst-Jensen, A.; Janssen, E.; Kok, E.J.; Paz, La J.L.; Laval, V.; Leimanis, S.; Malcevschi, A.; Marmiroli, N.; Morisset, D.; Prins, T.W.; Remacle, J.; Ujhelyi, G.; Wulff, D.

    2012-01-01

    Among the available methods for GMO detection, enforcement and routine laboratories use in practice PCR, based on the detection of transgenic DNA. The cost required for GMO analysis is constantly increasing due to the progress of GMO commercialization, with inclusion of higher diversity of species,

  16. Quantitative multiplex detection of pathogen biomarkers

    Science.gov (United States)

    Mukundan, Harshini; Xie, Hongzhi; Swanson, Basil I; Martinez, Jennifer; Grace, Wynne K

    2014-10-14

    The present invention addresses the simultaneous detection and quantitative measurement of multiple biomolecules, e.g., pathogen biomarkers through either a sandwich assay approach or a lipid insertion approach. The invention can further employ a multichannel, structure with multi-sensor elements per channel.

  17. Quantitative multiplex detection of pathogen biomarkers

    Energy Technology Data Exchange (ETDEWEB)

    Mukundan, Harshini; Xie, Hongzhi; Swanson, Basil I.; Martinez, Jennifer; Grace, Wynne K.

    2016-02-09

    The present invention addresses the simultaneous detection and quantitative measurement of multiple biomolecules, e.g., pathogen biomarkers through either a sandwich assay approach or a lipid insertion approach. The invention can further employ a multichannel, structure with multi-sensor elements per channel.

  18. Hybrid hydrogel photonic barcodes for multiplex detection of tumor markers.

    Science.gov (United States)

    Xu, Yueshuang; Zhang, Xiaoping; Luan, Chengxin; Wang, Huan; Chen, Baoan; Zhao, Yuanjin

    2017-01-15

    Barcodes-based suspension array have for demonstrated values in multiplex assay of tumor markers. Photonic barcodes which are encoded by their characteristic reflection peaks are the important supports for suspension array due to their stable code, low fluorescent background and high surface-volume ratio. Attempts to develop this technology tend to improve the function of the photonic barcodes. Here, we present a new type of hybrid hydrogel photonic barcodes for efficient multiplex assays. This photonic barcodes are hybrid inverse opal hydrogel composed of poly(ethylene glycol) diacrylate (PEG-DA) and agarose. The polymerized PEG-DA hydrogel could guarantee the stabilities of the inverse opal structure and its resultant code, while the agarose could offer active chemical groups for the probe immobilization and homogeneous water surrounding for the bioassay. In addition, the interconnected pores inverse opal structure could provide channels for biomolecules diffusing and reaction into the voids of barcodes. These features imparted the hybrid hydrogel photonic barcodes with limits of detection (LOD) of 0.78ng/mL for carcinoembryonic antigen (CEA) and 0.21ng/mL for α-fetoprotein (AFP), respectively. It was also demonstrated that the proposed barcodes showed acceptable accuracy and detection reproducibility, and the results were in acceptable agreement with those from common clinic method for the detections of practical clinical samples. Thus, our technique provides a new platform for simultaneous multiplex immunoassay. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Detection of respiratory bacterial pathogens causing atypical pneumonia by multiplex Lightmix®RT-PCR.

    Science.gov (United States)

    Wagner, Karoline; Springer, Burkard; Imkamp, Frank; Opota, Onya; Greub, Gilbert; Keller, Peter M

    2018-01-31

    Pneumonia is a severe infectious disease. In addition to common viruses and bacterial pathogens (e.g. Streptococcus pneumoniae), fastidious respiratory pathogens like Chlamydia pneumoniae, Mycoplasma pneumoniae and Legionella spp. can cause severe atypical pneumonia. They do not respond to penicillin derivatives, which may cause failure of antibiotic empirical therapy. The same applies for infections with B. pertussis and B. parapertussis, the cause of pertussis disease, that may present atypically and need to be treated with macrolides. Moreover, these fastidious bacteria are difficult to identify by culture or serology, and therefore often remain undetected. Thus, rapid and accurate identification of bacterial pathogens causing atypical pneumonia is crucial. We performed a retrospective method evaluation study to evaluate the diagnostic performance of the new, commercially available Lightmix ® multiplex RT-PCR assay that detects these fastidious bacterial pathogens causing atypical pneumonia. In this retrospective study, 368 clinical respiratory specimens, obtained from patients suffering from atypical pneumonia that have been tested negative for the presence of common agents of pneumonia by culture and viral PCR, were investigated. These clinical specimens have been previously characterized by singleplex RT-PCR assays in our diagnostic laboratory and were used to evaluate the diagnostic performance of the respiratory multiplex Lightmix ® RT-PCR. The multiplex RT-PCR displayed a limit of detection between 5 and 10 DNA copies for different in-panel organisms and showed identical performance characteristics with respect to specificity and sensitivity as in-house singleplex RT-PCRs for pathogen detection. The Lightmix ® multiplex RT-PCR assay represents a low-cost, time-saving and accurate diagnostic tool with high throughput potential. The time-to-result using an automated DNA extraction device for respiratory specimens followed by multiplex RT-PCR detection was

  20. Novel methods for improving rapid paper-based protein assays with gold nanoparticle detection

    OpenAIRE

    Lama, Lara

    2017-01-01

    This thesis describes methods for improving sensitivity in rapid singleplex and multiplex microarray assays. The assays utilize the optical characteristics of colloidal gold nanoparticles for the colorimetric detection of proteins. Multiplexed detection in sandwich immunoassays is limited by cross-reactivity between different detection antibodies. The cross-reactivity between antibodies can contribute to increased background noise - decreasing the Limit-of-Detection of the assay - or generate...

  1. Multiplex PCR to detect four different tomato-infecting pathogens.

    Science.gov (United States)

    Quintero-Vásquez, Gabriela Alejandra; Bazán-Tejeda, María Luisa; Martínez-Peñafiel, Eva; Kameyama-Kawabe, Luis; Bermúdez-Cruz, Rosa María

    2013-07-01

    This work was aimed to develop a multiplex PCR assay to detect infectious agents such as Clavibacter michiganensis subsp. michiganensis, Fusarium sp, Leveillula taurica, and begomoviruses in tomato (Solanum lycopersicum) plants. Specific primer sets of each pathogen were designed based on intergenic ribosomal RNA sequences for the first three, whereas for begomoviruses, primers were designed based on conserved regions. The design also considered that the length (200-800 bp) of the PCR products was resolvable by electrophoresis; thus 296, 380, 457, and 731 bp fragments for Clavibacter, Fusarium, Leveillula, and begomoviruses, respectively, were considered. PCR conditions were optimized to amplify all the products in a single tube from genomic DNA and circumvent PCR inhibitors from infected plants. Finally, when the multiplex PCR assay was tested with tomato plants infected with any of the four pathogens, specific PCR products confirmed the presence of the pathogens. Optimized PCR multiplex allowed for the accurate and simultaneous detection of Clavibacter, Fusarium, Leveillula, and begomoviruses in infected plants or seeds from tomato.

  2. Rapid screening of pyogenic Staphylococcus aureus for confirmation of genus and species, methicillin resistance and virulence factors by using two novel multiplex PCR.

    Science.gov (United States)

    Haque, Abdul; Haque, Asma; Saeed, Muhammad; Azhar, Aysha; Rasool, Samreen; Shan, Sidra; Ehsan, Beenish; Nisar, Zohaib

    2017-01-01

    Emergence of methicillin resistant Staphylococcus aureus (MRSA) is a major medical problem of current era. These bacteria are resistant to most drugs and rapid diagnosis can provide a clear guideline to clinicians. They possess specific virulence factors and relevant information can be very useful. We designed this study to develop multiplex PCRs to provide rapid information. We studied 60 Staphylococcus aureus isolates and detected methicillin resistance by cefoxitin sensitivity and targeting of mecA gene. After initial studies with uniplex PCRs we optimized two multiplex PCRs with highly reproducible results. The first multiplex PCR was developed to confirm genus, species and methicillin resistance simultaneously, and the second multiplex PCR was for screening of virulence factors. We found 38.33% isolates as methicillin resistant. α -toxin, the major cytotoxic factor, was detected in 40% whereas β-hemolysin was found in 25% cases. Panton Valentine leucocidin was detected in 8.33% and toxic shock syndrome toxin in5% cases. The results of uniplex and multiplex PCRs were highly compatible. These two multiplex PCRs when run simultaneously can provide vital information about methicillin resistance and virulence status of the isolate within a few hours as compared to several days needed by routine procedures.

  3. A multiplex PCR assay for the detection and quantification of Sclerotinia sclerotiorum and Botrytis cinerea.

    Science.gov (United States)

    Reich, J D; Alexander, T W; Chatterton, S

    2016-05-01

    Traditional culture methods for identifying the plant fungal pathogens Sclerotinia sclerotiorum (Lib.) de Bary and Botrytis cinerea Pers.:Fr. are slow and laborious. The goal of this study was to develop a multiplex real-time PCR (qPCR) assay to detect and quantify DNA from S. sclerotiorum and B. cinerea. A primer set (SsIGS_5) for S. sclerotiorum was designed that targeted the intergenic spacer (IGS) regions of the ribosomal DNA. Addition of a probe to the assay increased its specificity: when the primer/probe set was tested against 21 fungal species (35 strains), amplification was detected from all S. sclerotiorum strains and no other species. For qPCR, the SsIGS_5 primer and probe set exhibited a linear range from 7·0 ng to 0·07 pg target DNA (R(2)  = 0·99). SsIGS_5 was then multiplexed with a previously published primer/probe set for B. cinerea to develop a high-throughput method for the detection and quantification of DNA from both pathogens. When multiplexed, the sensitivity and specificity of both assays were not different from individual qPCR reactions. The multiplex assay is currently being used to detect and quantify S. sclerotiorum and B. cinerea DNA from aerosol samples collected in commercial seed alfalfa fields. A primer and probe set for the quantification of Sclerotinia sclerotiorum DNA in a PCR assay was developed. The probe-based nature of this assay signifies an improvement over previous assays for this species by allowing multiplex reactions while maintaining high sensitivity. The primer/probe set was used in a multiplex real-time PCR assay for the quantification of S. sclerotiorum and Botrytis cinerea DNA, enabling rapid analysis of environmental samples. In crops susceptible to both pathogens, this multiplex assay can be used to quickly quantify the presence of each pathogen. © 2016 Her Majesty the Queen in Right of Canada © 2016 The Society for Applied Microbiology. Reproduced with the permission of the Office of the

  4. Development of a Multiplex PCR Assay for Rapid Molecular Serotyping of Haemophilus parasuis

    Science.gov (United States)

    Peters, Sarah E.; Wang, Jinhong; Hernandez-Garcia, Juan; Weinert, Lucy A.; Luan, Shi-Lu; Chaudhuri, Roy R.; Angen, Øystein; Aragon, Virginia; Williamson, Susanna M.; Langford, Paul R.; Rycroft, Andrew N.; Wren, Brendan W.; Maskell, Duncan J.; Tucker, Alexander W.

    2015-01-01

    Haemophilus parasuis causes Glässer's disease and pneumonia in pigs. Indirect hemagglutination (IHA) is typically used to serotype this bacterium, distinguishing 15 serovars with some nontypeable isolates. The capsule loci of the 15 reference strains have been annotated, and significant genetic variation was identified between serovars, with the exception of serovars 5 and 12. A capsule locus and in silico serovar were identified for all but two nontypeable isolates in our collection of >200 isolates. Here, we describe the development of a multiplex PCR, based on variation within the capsule loci of the 15 serovars of H. parasuis, for rapid molecular serotyping. The multiplex PCR (mPCR) distinguished between all previously described serovars except 5 and 12, which were detected by the same pair of primers. The detection limit of the mPCR was 4.29 × 105 ng/μl bacterial genomic DNA, and high specificity was indicated by the absence of reactivity against closely related commensal Pasteurellaceae and other bacterial pathogens of pigs. A subset of 150 isolates from a previously sequenced H. parasuis collection was used to validate the mPCR with 100% accuracy compared to the in silico results. In addition, the two in silico-nontypeable isolates were typeable using the mPCR. A further 84 isolates were analyzed by mPCR and compared to the IHA serotyping results with 90% concordance (excluding those that were nontypeable by IHA). The mPCR was faster, more sensitive, and more specific than IHA, enabling the differentiation of 14 of the 15 serovars of H. parasuis. PMID:26424843

  5. [Evaluation of Seeplex Pneumobacter multiplex PCR kit for the detection of respiratory bacterial pathogens in pediatric patients].

    Science.gov (United States)

    Park, Joowon; Kim, Jae Kyoung; Rheem, Insoo; Kim, Jongwan

    2009-08-01

    Rapid identification of the causative agent among potential bacterial and viral pathogens is important for the management of acute respiratory disease. In this study, we evaluated the analytical performance and clinical usefulness of a recently-introduced multiplex PCR assay, Seeplex Pneumobacter detection kit (Seegene Inc., Korea) for the identification of respiratory bacterial pathogens. One hundred and eighty one nasopharyngeal aspirates were collected from pediatric patients with respiratory symptoms and analysed by multiplex PCR for the detection of Streptococcus pneumoniae (S.P), Haemophilus influenzae (H.I), Mycoplasma pneumoniae (M.P), Chlamydophila pneumoniae (C.P), Bordetella pertussis (B.P) and Legionella pneumophila (L.P). A comparison of multiplex PCR with conventional culture for the isolation of S.P and H.I was performed on 112 specimens. The cross reactivity of multiplex PCR was also evaluated. Of 181 cases, 81 cases were positive by multiplex PCR (44.8%): 52 cases for S.P (28.7%), 47 cases for H.I (26.0%), 9 cases for M.P (5.0%), 3 cases for B.P (1.7%) and 1 case for C.P (0.6%) including multiple infection cases. The agreement rates between multiplex PCR and culture for S.P and H.I were 92.9% (kappa index=0.84, P<0.001) and 91.1% (kappa index=0.75, P<0.001), respectively. There was no cross reactivity with common bacterial and viral pathogens. Seeplex Pneumobacter detection kit could be a useful screening tool for the rapid detection of respiratory bacterial pathogens. Further studies with lower respiratory tract specimens would be needed for the clinical evaluation of S. pneumoniae and H. influenzae detected by multiplex PCR.

  6. Multiplex RT-PCR method for the simultaneous detection of nine grapevine viruses.

    Science.gov (United States)

    Gambino, Giorgio

    2015-01-01

    Viral diseases are a serious pathological problem for grapevines, and in recent years the need for increasingly specific and rapid diagnostic methods for the selection of propagation materials has grown. Arabis mosaic virus, Grapevine fanleaf virus, Grapevine virus A, Grapevine virus B, Grapevine rupestris stem pitting-associated virus, Grapevine fleck virus, and Grapevine leafroll-associated viruses 1, 2, and 3 are nine of the most widespread viruses that naturally infect grapevines. A multiplex RT-PCR was developed for simultaneous detection of these nine grapevine viruses, in combination with a plant RNA internal control used as an indicator of the effectiveness of the reaction. One to ten fragments specific for the viruses and an internal control were simultaneously amplified from infected samples and identified by their specific molecular sizes in agarose gel. The protocol reported is an update of previously published protocols for RNA extraction and multiplex diagnosis of viruses. After several years of use and hundreds of samples tested, and following validation in several laboratories, this multiplex RT-PCR provides a reliable and rapid method for detecting grapevine viruses from a large number of samples.

  7. Detection of Bacillus sphaericus mosquitocidal toxin genes by multiplex colony PCR.

    Science.gov (United States)

    Jagtap, Santosh C; Jagtap, Chandrakant B; Kumar, Pradeep; Srivastava, R B

    2009-02-01

    A multiplex colony PCR assay was developed for the detection of 5 genes encoding Bacillus sphaericus mosquito larvicidal toxins, namely binA, binB, mtx1, mtx2, and mtx3. Primers designed for these 5 genes yielded specific PCR amplicons of the expected size from type cultures of B. sphaericus. This method of detecting multiple toxin genes by colony PCR in a single tube reaction is a simple, rapid, and economical technique for identification of highly toxic environmental B. sphaericus isolates.

  8. Label-Free Multiplex DNA Detection Utilizing Projected Capacitive Touchscreen.

    Science.gov (United States)

    Lee, Joon Young; Won, Byoung Yeon; Park, Hyun Gyu

    2017-10-13

    A novel strategy to achieve label-free multiplex DNA detection system based on the projected capacitive touchscreen is developed. Touchscreen panel is first fabricated by patterning the ITO (indium-tin-oxide) electrode array on the glass wafer, and the electrodes are modified with the respective capture probe DNA sequences complementary to hemagglutinin1 (H1), neuraminidase1 (N1), and matrix1 (M1) DNA to demonstrate the molecular diagnosis of H1N1 influenza virus as a model pathogen. DNA sample is applied to the electrodes to allow hybridization of target DNA with the corresponding complementary capture probe. As a result, the hybridization event significantly enhanced the capacitance on the electrode, which can be very conveniently detected by the projected capacitive touchscreen. Based on this design principle, the authors have successfully detected target regions of H1N1 influenza virus in a label-free multiplexed manner. This touchscreen-based detecting system would greatly benefit the point-of-care testing (POCT) in various diagnostic applications. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Simultaneous Detection of 13 Key Bacterial Respiratory Pathogens by Combination of Multiplex PCR and Capillary Electrophoresis.

    Science.gov (United States)

    Jiang, Lu Xi; Ren, Hong Yu; Zhou, Hai Jian; Zhao, Si Hong; Hou, Bo Yan; Yan, Jian Ping; Qin, Tian; Chen, Yu

    2017-08-01

    Lower respiratory tract infections continue to pose a significant threat to human health. It is important to accurately and rapidly detect respiratory bacteria. To compensate for the limits of current respiratory bacteria detection methods, we developed a combination of multiplex polymerase chain reaction (PCR) and capillary electrophoresis (MPCE) assay to detect thirteen bacterial pathogens responsible for lower respiratory tract infections, including Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, Staphylococcus aureus, Mycoplasma pneumoniae, Legionella spp., Bordetella pertussis, Mycobacterium tuberculosis complex, Corynebacterium diphtheriae, and Streptococcus pyogenes. Three multiplex PCR reactions were built, and the products were analyzed by capillary electrophoresis using the high-throughput DNA analyzer. The specificity of the MPCE assay was examined and the detection limit was evaluated using DNA samples from each bacterial strain and the simulative samples of each strain. This assay was further evaluated using 152 clinical specimens and compared with real-time PCR reactions. For this assay, three nested-multiplex-PCRs were used to detect these clinical specimens. The detection limits of the MPCE assay for the 13 pathogens were very low and ranged from 10-7 to 10-2 ng/μL. Furthermore, analysis of the 152 clinical specimens yielded a specificity ranging from 96.5%-100.0%, and a sensitivity of 100.0% for the 13 pathogens. This study revealed that the MPCE assay is a rapid, reliable, and high-throughput method with high specificity and sensitivity. This assay has great potential in the molecular epidemiological survey of respiratory pathogens. Copyright © 2017 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.

  10. A multiplex PCR for rapid identification of Brassica species in the triangle of U.

    Science.gov (United States)

    Koh, Joshua C O; Barbulescu, Denise M; Norton, Sally; Redden, Bob; Salisbury, Phil A; Kaur, Sukhjiwan; Cogan, Noel; Slater, Anthony T

    2017-01-01

    Within the Brassicaceae, six species from the genus Brassica are widely cultivated throughout the world as oilseed, condiment, fodder or vegetable crops. The genetic relationships among the six Brassica species are described by U's triangle model. Extensive shared traits and diverse morphotypes among Brassica species make identification and classification based on phenotypic data alone challenging and unreliable, especially when dealing with large germplasm collections. Consequently, a major issue for genebank collections is ensuring the correct identification of species. Molecular genotyping based on simple sequence repeat (SSR) marker sequencing or the Illumina Infinium Brassica napus 60K single nucleotide polymorphism (SNP) array has been used to identify species and assess genetic diversity of Brassica collections. However, these methods are technically challenging, expensive and time-consuming, making them unsuitable for routine or rapid screening of Brassica accessions for germplasm management. A cheaper, faster and simpler method for Brassica species identification is described here. A multiplex polymerase chain reaction (MPCR) consisting of new and existing primers specific to the Brassica A, B and C genomes was able to reliably distinguish all six Brassica species in the triangle of U with 16 control samples of known species identity. Further validation against 120 Brassica accessions previously genotyped showed that the MPCR is highly accurate and comparable to more advanced techniques such as SSR marker sequencing or the Illumina Infinium B. napus 60K SNP array. In addition, the MPCR was sensitive enough to detect seed contaminations in pooled seed samples of Brassica accessions. A cheap and fast multiplex PCR assay for identification of Brassica species in the triangle of U was developed and validated in this study. The MPCR assay can be readily implemented in any basic molecular laboratory and should prove useful for the management of Brassica

  11. Short communication: Detection of stx2 and elt genes in bovine milk by using a multiplex PCR system.

    Science.gov (United States)

    Nandi, R D S; Campos, A C; Puño-Sarmiento, J J; Maluta, R P; Rocha, S P D; Kobayashi, R K T; Nakazato, G

    2017-10-01

    The aim of this study was to detect 2 important toxin genes from diarrheagenic Escherichia coli (DEC) in bovine milk using a new multiplex PCR. To standardize the multiplex PCR, the stx2 and elt genes were investigated for the detection of Shiga toxin-producing Escherichia coli (STEC) and enterotoxigenic E. coli (ETEC), respectively. The DNA template was prepared with a thermal procedure (boiling) and a commercial kit. Samples consisted of UHT and pasteurized milk, both skimmed, and STEC and ETEC were tested in concentrations between 10 1 and 10 9 cfu/mL. With the thermal procedure, the multiplex PCR system detected both pathotypes of E. coli at 10 9 cfu/mL in UHT and pasteurized milk. When the commercial kit was used for template preparation, STEC and ETEC could be detected at concentrations as low as 10 4 cfu/mL in UHT and pasteurized milk. Negative controls (Listeria monocytogenes, Salmonella Typhimurium, Salmonella Enteritidis, and Escherichia coli strain APEC 13) were not amplified with the multiplex PCR. These results indicate that the multiplex PCR was a rapid (less than 6 h) and efficient method to detect STEC and ETEC in milk using different methods for DNA preparation; however, the commercial kit was more sensitive than the thermal procedure. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  12. Detecting and genotyping Escherichia coli O157:H7 using multiplexed PCR and nucleic acid microarrays

    Energy Technology Data Exchange (ETDEWEB)

    Call, Douglas R.; Brockman, Fred J.; Chandler, Darrell P.

    2001-07-05

    Rapid detection and characterization of food borne pathogens such as Escherichia coli O157:H7 is crucial for epidemiological investigations and food safety surveillance. As an alternative to conventional technologies, we examined the sensitivity and specificity of nucleic acid microarrays for detecting and genotyping E. coli O157:H7. The array was composed of oligonucleotide probes (25-30 mer) complementary to four virulence loci (intimin, Shiga-like toxins I and II, and hemolysin A). Target DNA was amplified from whole cells or from purified DNA via single or multiplexed polymerase chain reaction (PCR), and PCR products were hybridized to the array without further modification or purification. The array was 32-fold more sensitive than gel electrophoresis and capable of detecting amplification products from < 1 cell equivalent of genomic DNA (1 fg). Immunomagnetic capture, PCR and a microarray were subsequently used to detect 55 CFUs ml-1 (E. coli O157:H7) from chicken rinsate without the aid of pre-enrichment. Four isolates of E. coli O157:H7 and one isolate of O91:H2, for which genotypic data were available, were unambiguously genotyped with this array. Glass based microarrays are relatively simple to construct and provide a rapid and sensitive means to detect multiplexed PCR products and the system is amenable to automation.

  13. Detecting and Genotyping Escherichia coli O157:H7 using multiplexed PCR and nucleic acid microarrays

    Energy Technology Data Exchange (ETDEWEB)

    Call, Douglas R.(Michigan, Univ Of - Ann Arbor); Brockman, Fred J.(BATTELLE (PACIFIC NW LAB)); Chandler, Darrell P.(Pacific Northwest National Laboratory)

    2000-12-01

    Rapid detection and characterization of food borne pathogens such as Escherichia coli O157:H7 is crucial for epidemiological investigations and food safety surveillance. As an alternative to conventional technologies, we examined the sensitivity and specificity of nucleic acid microarrays for detecting and genotyping E. coli O157:H7. The array was composed of oligonucleotide probes (25-30 mer) complementary to four virulence loci (intimin, Shiga-like toxins I and II, and hemolysin A). Target DNA was amplified from whole cells or from purified DNA via single or multiplexed polymerase chain reaction (PCR), and PCR products were hybridized to the array without further modification or purification. The array was 32-fold more sensitive than gel electrophoresis and capable of detecting amplification products from < 1 cell equivalent of genomic DNA (1 fg). Immunomagnetic capture, PCR and a microarray were subsequently used to detect 55 CFU ml-1 (E. coli O157:H7) from chicken rinsate without the aid of pre-enrichment. Four isolates of E. coli O157:H7 and one isolate of O91:H2, for which genotypic data were available, were unambiguously genotyped with this array. Glass based microarrays are relatively simple to construct and provide a rapid and sensitive means to detect multiplexed PCR products and the system is amenable to automation.

  14. Colorimetric aptasensor using unmodified gold nanoparticles for homogeneous multiplex detection.

    Directory of Open Access Journals (Sweden)

    Shucao Niu

    Full Text Available Colorimetric aptasensors using unmodified gold nanoparticles (AuNPs have attracted much attention because of their low cost, simplicity, and practicality, and they have been developed for various targets in the past several years. However, previous research has focused on developing single-target assays. Here, we report the development of a homogeneous multiplex aptasensor by using more than one class of aptamers to stabilize AuNPs. Using sulfadimethoxine (SDM, kanamycin (KAN and adenosine (ADE as example targets, a KAN aptamer (750 nM, an SDM aptamer (250 nM and an ADE aptamer (500 nM were mixed at a 1∶1∶1 volume ratio and adsorbed directly onto the surface of unmodified AuNPs by electrostatic interaction. Upon the addition of any of the three targets, the conformation of the corresponding aptamer changed from a random coil structure to a rigid folded structure, which could not adsorb and stabilize AuNPs. The AuNPs aggregated in a specific reaction buffer (20 mM Tris-HCl containing 20 mM NaCl and 5 mM KCl, which led to a color change from red to purple/blue. These results demonstrate that the multiplex colorimetric aptasensor detected three targets simultaneously while maintaining the same sensitivity as a single-target aptasensor for each individual target. The multiplex aptasensor could be extended to other aptamers for various molecular detection events. Due to its simple design, easy operation, fast response, cost effectiveness and lack of need for sophisticated instrumentation, the proposed strategy provides a powerful tool to examine large numbers of samples to screen for a small number of potentially positive samples containing more than one analyte, which can be further validated using sophisticated instruments.

  15. Graphite nanocomposites sensor for multiplex detection of antioxidants in food.

    Science.gov (United States)

    Ng, Khan Loon; Tan, Guan Huat; Khor, Sook Mei

    2017-12-15

    Butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and tert-butylhydroquinone (TBHQ) are synthetic antioxidants used in the food industry. Herein, we describe the development of a novel graphite nanocomposite-based electrochemical sensor for the multiplex detection and measurement of BHA, BHT, and TBHQ levels in complex food samples using a linear sweep voltammetry technique. Moreover, our newly established analytical method exhibited good sensitivity, limit of detection, limit of quantitation, and selectivity. The accuracy and reliability of analytical results were challenged by method validation and comparison with the results of the liquid chromatography method, where a linear correlation of more than 0.99 was achieved. The addition of sodium dodecyl sulfate as supporting additive further enhanced the LSV response (anodic peak current, Ipa) of BHA and BHT by 2- and 20-times, respectively. Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. SCAR makers and multiplex PCR-based rapid molecular typing of Lentinula edodes strains.

    Science.gov (United States)

    Wu, Xueqian; Li, Haibo; Zhao, Weiwei; Fu, Lizhong; Peng, Huazheng; He, Liang; Cheng, Junwen; Wei, Hailong; Wu, Qingqi

    2010-11-01

    Lentinula edodes is the second most important cultivated mushroom worldwide, the most commercial strains have been identified only through traditional phenotypic analysis. In this study, a simple rapid PCR-based molecular method was developed for distinguishing commercial strains of L. edodes by developing specific sequence characterized amplified region (SCAR) markers and establishing multiplex PCR assays with the SCAR primers. Derived from the randomly amplified polymorphic DNA (RAPD) and sequence-related amplified polymorphism (SRAP) techniques, 10 informative SCAR markers were generated from 10 polymorphic RAPD and SRAP bands. The differences in SCAR phenotypes among different strains made these SCAR markers potentially useful to characterize 6 strains and identify them from other studied strains. Moreover, different SCAR phenotypes also made the other 17 studied strains to be divided into four distinguishable groups. The multiplex PCR assays were further established for the joint use of some SCAR markers efficiently. Compared with some identification methods reported previously, the special feature of this new molecular method is technically rapid and convenient in the practical use and suitable for analyzing large numbers of samples. Thus, the simple rapid PCR-based molecular method can be used as a helpful assistant tool for the lentinula industry. To our knowledge, this study is the first to describe a development of a new SCAR maker-based multiplex PCR assay for rapid molecular typing of edible mushroom.

  17. Electroanalytical Sensors and Devices for Multiplexed Detection of Foodborne Pathogen Microorganisms

    Directory of Open Access Journals (Sweden)

    Susana Campuzano

    2009-07-01

    Full Text Available The detection and identification of pathogen microorganisms still rely on conventional culturing techniques, which are not suitable for on-site monitoring. Therefore, a great research challenge in this field is focused on the need to develop rapid, reliable, specific, and sensitive methods to detect these bacteria at low cost. Moreover, the growing interest in biochip development for large scale screening analysis implies improved miniaturization, reduction of analysis time and cost, and multi-analyte detection, which has nowadays become a crucial challenge. This paper reviews multiplexed foodborne pathogen microorganisms detection methods based on electrochemical sensors incorporating microarrays and other platforms. These devices usually involve antibody-antigen and DNA hybridization specific interactions, although other approaches such as the monitoring of oxygen consumption are also considered.

  18. 78 FR 16513 - Application of Advances in Nucleic Acid and Protein Based Detection Methods to Multiplex...

    Science.gov (United States)

    2013-03-15

    ... Detection Methods to Multiplex Detection of Transfusion-Transmissible Agents and Blood Cell Antigens in... the following topics: (1) Blood safety and infectious agents, (2) advances in blood-borne pathogen... include presentations and discussion on the following topics: (1) Highly multiplexed technologies in blood...

  19. New multiplex PCR methods for rapid screening of genetically modified organisms in foods

    OpenAIRE

    Nelly eDatukishvili; Tamara eKutateladze; Inga eGabriadze; Kakha eBitskinashvili; Boris eVishnepolsky

    2015-01-01

    We present novel multiplex PCR methods for rapid and reliable screening of genetically modified organisms (GMOs). New designed PCR primers targeting four frequently used GMO specific sequences permitted identification of new DNA markers, in particular 141 bp fragment of cauliflower mosaic virus (CaMV) 35S promoter, 224 bp fragment of Agrobacterium tumefaciens nopaline synthase (NOS) terminator, 256 bp fragment of 5-enolppyruvylshikimate-phosphate synthase (epsps) gene and 258 bp fragment of C...

  20. Rapid Sequential in Situ Multiplexing with DNA Exchange Imaging in Neuronal Cells and Tissues.

    Science.gov (United States)

    Wang, Yu; Woehrstein, Johannes B; Donoghue, Noah; Dai, Mingjie; Avendaño, Maier S; Schackmann, Ron C J; Zoeller, Jason J; Wang, Shan Shan H; Tillberg, Paul W; Park, Demian; Lapan, Sylvain W; Boyden, Edward S; Brugge, Joan S; Kaeser, Pascal S; Church, George M; Agasti, Sarit S; Jungmann, Ralf; Yin, Peng

    2017-10-11

    To decipher the molecular mechanisms of biological function, it is critical to map the molecular composition of individual cells or even more importantly tissue samples in the context of their biological environment in situ. Immunofluorescence (IF) provides specific labeling for molecular profiling. However, conventional IF methods have finite multiplexing capabilities due to spectral overlap of the fluorophores. Various sequential imaging methods have been developed to circumvent this spectral limit but are not widely adopted due to the common limitation of requiring multirounds of slow (typically over 2 h at room temperature to overnight at 4 °C in practice) immunostaining. We present here a practical and robust method, which we call DNA Exchange Imaging (DEI), for rapid in situ spectrally unlimited multiplexing. This technique overcomes speed restrictions by allowing for single-round immunostaining with DNA-barcoded antibodies, followed by rapid (less than 10 min) buffer exchange of fluorophore-bearing DNA imager strands. The programmability of DEI allows us to apply it to diverse microscopy platforms (with Exchange Confocal, Exchange-SIM, Exchange-STED, and Exchange-PAINT demonstrated here) at multiple desired resolution scales (from ∼300 nm down to sub-20 nm). We optimized and validated the use of DEI in complex biological samples, including primary neuron cultures and tissue sections. These results collectively suggest DNA exchange as a versatile, practical platform for rapid, highly multiplexed in situ imaging, potentially enabling new applications ranging from basic science, to drug discovery, and to clinical pathology.

  1. Large scale multiplex PCR improves pathogen detection by DNA microarrays

    Directory of Open Access Journals (Sweden)

    Krönke Martin

    2009-01-01

    Full Text Available Abstract Background Medium density DNA microchips that carry a collection of probes for a broad spectrum of pathogens, have the potential to be powerful tools for simultaneous species identification, detection of virulence factors and antimicrobial resistance determinants. However, their widespread use in microbiological diagnostics is limited by the problem of low pathogen numbers in clinical specimens revealing relatively low amounts of pathogen DNA. Results To increase the detection power of a fluorescence-based prototype-microarray designed to identify pathogenic microorganisms involved in sepsis, we propose a large scale multiplex PCR (LSplex PCR for amplification of several dozens of gene-segments of 9 pathogenic species. This protocol employs a large set of primer pairs, potentially able to amplify 800 different gene segments that correspond to the capture probes spotted on the microarray. The LSplex protocol is shown to selectively amplify only the gene segments corresponding to the specific pathogen present in the analyte. Application of LSplex increases the microarray detection of target templates by a factor of 100 to 1000. Conclusion Our data provide a proof of principle for the improvement of detection of pathogen DNA by microarray hybridization by using LSplex PCR.

  2. Development of multiplex PCR assay for simultaneous detection of Salmonella genus, Salmonella subspecies I, Salm. Enteritidis, Salm. Heidelberg and Salm. Typhimurium.

    Science.gov (United States)

    Park, S H; Ricke, S C

    2015-01-01

    The aim of this research was to develop multiplex PCR assay that could simultaneously detect Salmonella genus, Salmonella subsp. I, Salm. Enteritidis, Heidelberg and Typhimurium because these Salmonella serovars are the most common isolates associated with poultry products. Five primers were utilized to establish multiplex PCR and applied to Salmonella isolates from chickens and farm environments. These isolates were identified as Salmonella subsp. I and 16 of 66 isolates were classified as Salm. Enteritidis, while Heidelberg or Typhimurium was not detected. We also spiked three Salmonella strains on chicken breast meat to evaluate the specificity and sensitivity of multiplex PCR as well as qPCR to optimize quantification of Salmonella in these samples. The optimized multiplex PCR and qPCR could detect approx. 2·2 CFU of Salmonella per gram after 18 h enrichment. The multiplex PCR and qPCR would provide rapid and consistent results. Also, these techniques would be useful for the detection and quantification of Salmonella in contaminated poultry, foods and environmental samples. The strategy for the rapid detection of Salmonella serovars in poultry is needed to further reduce the incidence of salmonellosis in humans. The optimized multiplex PCR will be useful to detect prevalent Salmonella serovars in poultry products. © 2014 The Society for Applied Microbiology.

  3. Multiplex polymerase chain reaction for detection of Campylobacter from stool specimen.

    Science.gov (United States)

    Sarkar, S R; Ray, N C; Hossain, M A; Paul, S K; Sarkar, S; Kobayashi, N

    2014-07-01

    This was a study to prospectively evaluate the sensitivity and specificity of multiplex polymerase chain reaction (PCR) to identify Campylobacter. Multiplex polymerase chain reaction (PCR) based on cadF, hipO & asp gene for Campylobacter genus, C. jejuni & C. coli were tested for detection of Campylobacter jejuni & C. coli in naturally infected faecal samples of human. All the samples were subjected to the cultural isolation of organism and biochemical characterization. The samples resulted in the amplification of a DNA fragment of size 400 bp, 500 bp &735 bp in PCR assay. Two hundred faecal samples comprising diarrheal stools, 23(11.5%) could be detected by isolation whereas 24(12.0%) were found positive by PCR. All culture positive cases were positive by PCR and among 01 culture negative case, were positive by PCR. PCR was found to be more sensitive for Campylobacter detection in faecal samples 12.0% as relative to culture isolation which could detect the organism in 11.5% samples. Sensitivity and specificity of PCR were 100% and 99.4% respectively taking Culture as gold standard. The results depicted the superior efficacy of PCR for rapid screening of samples owing to its high sensitivity, specificity and automation potential.

  4. Quantitative multiplex detection of biomarkers on a waveguide-based biosensor using quantum dots

    Energy Technology Data Exchange (ETDEWEB)

    Xie, Hongzhi [Los Alamos National Laboratory; Mukundan, Harshini [Los Alamos National Laboratory; Martinez, Jennifer S [Los Alamos National Laboratory; Swanson, Basil I [Los Alamos National Laboratory; Anderson, Aaron S [Los Alamos National Laboratory; Grace, Kevin [Los Alamos National Laboratory

    2009-01-01

    color imaging of live cells using QD-bioconjugates [Jaiswal 2003]. Gao [Gao 2004] and So [So 2006] have used QDs as probes for in-vivo cancer targeting and imaging. Medintz et al. reported self-assembled QD-based biosensors for detection of analytes based on energy transfer [Medintz 2003]. Others have developed an approach for multiplex optical encoding of biomolecules using QDs [Han 2001]. Immunoassays have also benefited from the advantages of QDs. Recently, dihydrolipoic acid (DHLA) capped-QDs have been attached to antibodies and used as fluorescence reporters in plate-based multiplex immunoassays [Goodman 2004]. However, DHLA-QDs are associated with low quantum efficiency and are unstable at neutral pH. These problems limit the application of this technology to the sensitive detection of biomolecules, especially in complex biological samples. Thus, the development of a rapid, sensitive, quantitative, and specific multiplex platform for the detection of biomarkers in difficult samples remains an elusive target. The goal stated above has applications in many fields including medical diagnostics, biological research, and threat reduction. The current decade alone has seen the development of a need to rapidly and accurately detect potential biological warfare agents. For example, current methods for the detection of anthrax are grossly inadequate for a variety of reasons including long incubation time (5 days from time of exposure to onset of symptoms) and non-specific ('flu-like') symptoms. When five employees of the United State Senate were exposed to B. anthracis in the mail (2001), only one patient had a confirmed diagnosis before death. Since then, sandwich immunoassays using both colorimetric and fluorescence detectors have been developed for key components of the anthrax lethal toxin, namely protective antigen (PA), lethal factor (LF), and the edema factor [Mourez 2001]. While these platforms were successful in assays against anthrax toxins, the

  5. A Multiplexed Diagnostic Platform for Point-of-Care Pathogen Detection

    Energy Technology Data Exchange (ETDEWEB)

    Regan, J F; Letant, S E; Adams, K L; Mahnke, R C; Nguyen, N T; Dzenitis, J M; Hindson, B J; Hadley, D R; Makarewicz, T J; Henderer, B D; Breneman, J W; Tammero, L F; Ortiz, J I; Derlet, R W; Cohen, S; Colston, W W; McBride, M T; Birch, J M

    2008-02-04

    We developed an automated point-of-care diagnostic instrument that is capable of analyzing nasal swab samples for the presence of respiratory diseases. This robust instrument, called FluIDx, performs autonomous multiplexed RT-PCR reactions that are analyzed by microsphere xMAP technology. We evaluated the performance of FluIDx, in comparison rapid tests specific for influenza and respiratory syncytial virus, in a clinical study performed at the UC Davis Medical Center. The clinical study included samples positive for RSV (n = 71), influenza A (n = 16), influenza B (n = 4), adenovirus (n = 5), parainfluenza virus (n = 2), and 44 negative samples, according to a composite reference method. FluIDx and the rapid tests detected 85.9% and 62.0% of the RSV positive samples, respectively. Similar sensitivities were recorded for the influenza B samples; whereas the influenza A samples were poorly detected, likely due to the utilization of an influenza A signature that did not accurately match currently circulating influenza A strains. Data for all pathogens were compiled and indicate that FluIDx is more sensitive than the rapid tests, detecting 74.2% (95% C.I. of 64.7-81.9%) of the positive samples in comparison to 53.6% (95% C.I. of 43.7-63.2%) for the rapid tests. The higher sensitivity of FluIDx was partially offset by a lower specificity, 77.3% versus 100.0%. Overall, these data suggest automated flow-through PCR-based instruments that perform multiplexed assays can successfully screen clinical samples for infectious diseases.

  6. Rapid genetically modified organism (GMO screening of various food products and animal feeds using multiplex polymerase chain reaction (PCR

    Directory of Open Access Journals (Sweden)

    Lisha, V.

    2017-01-01

    Full Text Available modified crops which brought up a controversy on the safety usage of genetically modified organisms (GMOs. It has been implemented globally that all GMO products and its derived ingredients should have regulations on the usage and labelling. Thus, it is necessary to develop methods that allow rapid screening of GMO products to comply with the regulations. This study employed a reliable and flexible multiplex polymerase chain reaction (PCR method for the rapid detection of transgenic elements in genetically modified soy and maize along with the soybean LECTIN gene and maize ZEIN gene respectively. The selected four common transgenic elements were 35S promoter (35S; Agrobacterium tumefaciens nopaline synthase terminator (NOS; 5-enolypyruvylshikimate-3-phosphate synthase (epsps gene; and Cry1Ab delta-endotoxin (cry1Ab gene. Optimization of the multiplex PCR methods were carried out by using 1% Roundup ReadyTM Soybean (RRS as the certified reference material for soybean that produced fourplex PCR method detecting 35S promoter, NOS terminator, epsps gene and soybean LECTIN gene and by using 1% MON810 as the certified reference material for maize that produced triplex PCR method detecting 35S promoter, cry1Ab gene and maize ZEIN gene prior to screening of the GMO traits in various food products and animal feeds. 1/9 (11.1% of the animal feed contained maize and 1/15 (6.7% of the soybean food products showed positive results for the detection of GMO transgenic gene. None of the maize food products showed positive results for GMO transgenic gene. In total, approximately 4% of the food products and animal feed were positive as GMO. This indicated GMOs have not widely entered the food chain. However, it is necessary to have an appropriate screening method due to GMOs’ unknown potential risk to humans and to animals. This rapid screening method will provide leverage in terms of being economically wise, time saving and reliable.

  7. Design of a multiplex PCR assay for the simultaneous detection and confirmation of Neisseria gonorrhoeae.

    LENUS (Irish Health Repository)

    O'Callaghan, Isabelle

    2010-05-01

    To improve the detection of Neisseria gonorrhoeae by designing a multiplex PCR assay using two N gonorrhoeae-specific genes as targets, thereby providing detection and confirmation of a positive result simultaneously.

  8. A multiplex chemiluminescent biosensor for type B-fumonisins and aflatoxin B1 quantitative detection in maize flour.

    Science.gov (United States)

    Zangheri, Martina; Di Nardo, Fabio; Anfossi, Laura; Giovannoli, Cristina; Baggiani, Claudio; Roda, Aldo; Mirasoli, Mara

    2015-01-07

    A multiplex chemiluminescent biosensor for simple, rapid and ultrasensitive on-site quantification of aflatoxin B1 and type B-fumonisins in maize samples has been developed. The biosensor integrates a multiplex indirect competitive lateral flow immunoassay (LFIA) based on enzyme-catalyzed chemiluminescence detection and a highly sensitive portable charge-coupled device (CCD) camera, employed in a lensless "contact" imaging configuration. The developed assay requires a simple extraction of the analytes from maize flour samples followed by their detection with a 30 min assay time. The use of chemiluminescence detection allowed accurate and objective analytes quantification, enabling simultaneous detection of type B-fumonisins and aflatoxin B1 down to 6 μg kg(-1) and 1.5 μg kg(-1), respectively, thus fulfilling the standards imposed by the legislation of European Union. Maize flour samples spiked with both analytes were subjected to multiplex analysis obtaining recoveries ranging from 80 to 115% and the coefficient of variation below 20%. Finally, analysis of naturally contaminated maize samples resulted in a good agreement between CL-LFIA and a validated confirmatory HPLC-UV and commercial ELISA kit, obtaining recoveries in the range 88-120%. The proposed CL-LFIA protocol is rapid, inexpensive, easy-to-use, and fit for the purpose of rapid screening of mycotoxins in maize flour.

  9. A sensitive and a rapid multiplex polymerase chain reaction for the identification of Candida species in concentrated oral rinse specimens in patients with diabetes.

    Science.gov (United States)

    Sampath, Asanga; Weerasekera, Manjula; Gunasekara, Chinthika; Dilhari, Ayomi; Bulugahapitiya, Uditha; Fernando, Neluka

    2017-03-01

    Oral candidiasis is being frequently recognized in patients with diabetes, and is associated with multiple pathogens including Candida albicans, Candida parapsilosis, Candida glabrata and Candida tropicalis. The aim of this study was to evaluate a usefulness of a Multiplex Polymerase Chain Reaction as a rapid diagnostic tool for identification of four oral Candida pathogens in patients with diabetes. A multiplex PCR was optimized to identify four Candida species in concentrated oral rinse samples. Common reverse primer, ITS4 and four species-specific forward primers targeting ITS1 and ITS2 regions of yeast genome were used. Species-specific single amplicon were detected by agarose gel electrophoresis. Performance efficacy of multiplex PCR was compared with phenotypic identification. Out of 100 oral rinse samples, 72 were culture positive and of these 43 were at risk of oral Candida infection (>600cfu/ml). Multiple Candida species including C. albicans, C. parapsilosis and C. tropicalis were identified in 22 samples which had risk of oral Candida infection. In total, 85 patients were positive for Candida by multiplex PCR and of them 49 had multiple Candida species. All 43 colonized specimens were also positive by multiplex PCR. C. albicans was the most predominant organism (75/85) followed by C. parapsilosis (47/85), C. tropicalis (17/85) and C. glabrata (6/85). In specimens with multiple species, the two most common organisms were C. albicans and C. parapsilosis. Multiplex PCR yielded a sensitivity of 10 Candida cells/ml of oral rinse sample. Multiplex PCR is found to be rapid, sensitive and specific than phenotypic identification methods in discriminating multiple Candida species in oral rinse specimens.

  10. Development of a GeXP-multiplex PCR assay for the simultaneous detection and differentiation of six cattle viruses.

    Directory of Open Access Journals (Sweden)

    Qing Fan

    Full Text Available Foot-and-mouth disease virus (FMDV, Bluetongue virus (BTV, Vesicular stomatitis Virus (VSV, Bovine viral diarrheal (BVDV, Bovine rotavirus (BRV, and Bovine herpesvirus 1 (IBRV are common cattle infectious viruses that cause a great economic loss every year in many parts of the world. A rapid and high-throughput GenomeLab Gene Expression Profiler (GeXP analyzer-based multiplex PCR assay was developed for the simultaneous detection and differentiation of these six cattle viruses. Six pairs of chimeric primers consisting of both the gene-specific primer and a universal primer were designed and used for amplification. Then capillary electrophoresis was used to separate the fluorescent labeled PCR products according to the amplicons size. The specificity of GeXP-multiplex PCR assay was examined with samples of the single template and mixed template of six viruses. The sensitivity was evaluated using the GeXP-multiplex PCR assay on serial 10-fold dilutions of ssRNAs obtained via in vitro transcription. To further evaluate the reliability, 305 clinical samples were tested by the GeXP-multiplex PCR assay. The results showed that the corresponding virus specific fragments of genes were amplified. The detection limit of the GeXP-multiplex PCR assay was 100 copies/μL in a mixed sample of ssRNAs containing target genes of six different cattle viruses, whereas the detection limit for the Gexp-mono PCR assay for a single target gene was 10 copies/μL. In detection of viruses in 305 clinical samples, the results of GeXP were consistent with simplex real-time PCR. Analysis of positive samples by sequencing demonstrated that the GeXP-multiplex PCR assay had no false positive samples of nonspecific amplification. In conclusion, this GeXP-multiplex PCR assay is a high throughput, specific, sensitive, rapid and simple method for the detection and differentiation of six cattle viruses. It is an effective tool that can be applied for the rapid differential diagnosis

  11. Multiplex hydrolysis probe real-time PCR for simultaneous detection of hepatitis A virus and hepatitis E virus.

    Science.gov (United States)

    Qiu, Feng; Cao, Jingyuan; Su, Qiudong; Yi, Yao; Bi, Shengli

    2014-05-30

    Detection of hepatitis viral infections has traditionally relied on the circulating antibody test using the enzyme-linked immunosorbent assay. However, multiplex real-time PCR has been increasingly used for a variety of viral nucleic acid detections and has proven to be superior to traditional methods. Hepatitis A virus (HAV) and hepatitis E virus (HEV) are the major causes of acute hepatitis worldwide; both HAV and HEV infection are a main public health problem. In the present study, a one-step multiplex reverse transcriptase quantitative polymerase chain reaction assay using hydrolysis probes was developed for simultaneously detecting HAV and HEV. This novel detection system proved specific to the target viruses, to be highly sensitive and to be applicable to clinical sera samples, making it useful for rapid, accurate and feasible identification of HAV and HEV.

  12. Multiplex PCR for the detection and differentiation of Vibrio parahaemolyticus strains using the groEL, tdh and trh genes.

    Science.gov (United States)

    Hossain, Muhammad Tofazzal; Kim, Young-Ok; Kong, In-Soo

    2013-01-01

    Vibrio parahaemolyticus is a significant cause of human gastrointestinal disorders worldwide, transmitted primarily by ingestion of raw or undercooked contaminated seafood. In this study, a multiplex PCR assay for the detection and differentiation of V. parahaemolyticus strains was developed using primer sets for a species-specific marker, groEL, and two virulence markers, tdh and trh. Multiplex PCR conditions were standardised, and extracted genomic DNA of 70 V. parahaemolyticus strains was used for identification. The sensitivity and efficacy of this method were validated using artificially inoculated shellfish and seawater. The expected sizes of amplicons were 510 bp, 382 bp, and 171 bp for groEL, tdh and trh, respectively. PCR products were sufficiently different in size, and the detection limits of the multiplex PCR for groEL, tdh and trh were each 200 pg DNA. Specific detection and differentiation of virulent from non-virulent strains in shellfish homogenates and seawater was also possible after artificial inoculation with various V. parahaemolyticus strains. This newly developed multiplex PCR is a rapid assay for detection and differentiation of pathogenic V. parahaemolyticus strains, and could be used to prevent disease outbreaks and protect public health by helping the seafood industry maintain a safe shellfish supply. Copyright © 2013 Elsevier Ltd. All rights reserved.

  13. Evaluation of dual priming oligonucleotide-based multiplex PCR for detection of HBV YMDD mutants.

    Science.gov (United States)

    Woo, H Y; Park, H; Kim, B I; Jeon, W K; Kim, Y J

    2008-01-01

    We evaluated the usefulness of dual priming oligonucleotide (DPO)-based multiplex PCR, Seeplex HBV Lami-DR assay (Seegene Institute of Life Sciences, Seoul, Korea), to detect lamivudine-resistant HBV mutants in a comparison with the use of TRUGENE HBV genotyping and restriction fragment mass polymorphism (RFMP). Sera from 44 chronic hepatitis B patients were analyzed for the presence of mutations at codons 180 and 204 by performing DPO-based multiplex PCR, RFMP, and TRUGENE. The overall concordance rate among the three assays was 40.9% (18/44). Concordance rates between multiplex PCR and RFMP or multiplex PCR and TRUGENE were 61.4% (27/44) and 50.0% (22/44), respectively. In ten patients, multiplex PCR identified additional mutants not found using the other two methods. DPO-based multiplex PCR is a highly sensitive method to identify minor mutant populations and could be a practical tool in the monitoring of lamivudine resistance.

  14. Simultaneous detection of papaya ringspot virus, papaya leaf distortion mosaic virus, and papaya mosaic virus by multiplex real-time reverse transcription PCR.

    Science.gov (United States)

    Huo, P; Shen, W T; Yan, P; Tuo, D C; Li, X Y; Zhou, P

    2015-12-01

    Both the single infection of papaya ringspot virus (PRSV), papaya leaf distortion mosaic virus (PLDMV) or papaya mosaic virus (PapMV) and double infection of PRSV and PLDMV or PapMV which cause indistinguishable symptoms, threaten the papaya industry in Hainan Island, China. In this study, a multiplex real-time reverse transcription PCR (RT-PCR) was developed to detect simultaneously the three viruses based on their distinctive melting temperatures (Tms): 81.0±0.8°C for PRSV, 84.7±0.6°C for PLDMV, and 88.7±0.4°C for PapMV. The multiplex real-time RT-PCR method was specific and sensitive in detecting the three viruses, with a detection limit of 1.0×10(1), 1.0×10(2), and 1.0×10(2) copies for PRSV, PLDMV, and PapMV, respectively. Indeed, the reaction was 100 times more sensitive than the multiplex RT-PCR for PRSV, and 10 times more sensitive than multiplex RT-PCR for PLDMV. Field application of the multiplex real-time RT-PCR demonstrated that some non-symptomatic samples were positive for PLDMV by multiplex real-time RT-PCR but negative by multiplex RT-PCR, whereas some samples were positive for both PRSV and PLDMV by multiplex real-time RT-PCR assay but only positive for PLDMV by multiplex RT-PCR. Therefore, this multiplex real-time RT-PCR assay provides a more rapid, sensitive and reliable method for simultaneous detection of PRSV, PLDMV, PapMV and their mixed infections in papaya.

  15. Multiplexed Enrichment and Detection of Malarial Biomarkers Using a Stimuli-Responsive Iron Oxide and Gold Nanoparticle Reagent System

    OpenAIRE

    Nash, Michael A.; Waitumbi, John N.; Hoffman, Allan S.; Yager, Paul; Stayton, Patrick S.

    2012-01-01

    There is a need for simple yet robust biomarker and antigen purification and enrichment strategies that are compatible with current rapid diagnostic modalities. Here, a stimuli-responsive nanoparticle system is presented for multiplexed magneto-enrichment and non-instrumented lateral flow strip detection of model antigens from spiked pooled plasma. The integrated reagent system allows purification and enrichment of the gold-labeled biomarker half-sandwich that can be applied directly to later...

  16. Multiplex autoantibody detection for autoimmune liver diseases and autoimmune gastritis.

    Science.gov (United States)

    Vanderlocht, Joris; van der Cruys, Mart; Stals, Frans; Bakker-Jonges, Liesbeth; Damoiseaux, Jan

    2017-09-01

    Autoantibody detection for autoimmune hepatitis (AIH), primary biliary cirrhosis (PBC) and autoimmune gastritis (AIG) is traditionally performed by IIF on a combination of tissues. Multiplex line/dot blots (LIA/DIA) offer multiple advantages, i.e. automation, objective reading, no interfering reactivities, no coincidental findings. In the current study we evaluated automated DIA (D-Tek) for detecting autoantibodies related to autoimmune diseases of the gastrointestinal tract. We tested samples of the Dutch EQC program and compared the results with the consensus of the participating labs. For the autoimmune liver diseases and AIG, respectively, 64 and 36 samples were tested. For anti-mitochondrial and anti-smooth muscle antibodies a concordance rate of 97% and 88% was observed, respectively. The concordance rate for anti-parietal cell antibodies was 92% when samples without EQC consensus (n=15) were excluded. For antibodies against intrinsic factor a concordance of 96% was observed. For all these antibodies discrepancies were identified that relate to the different test characteristics and the preponderance of IIF utilizing labs in the EQC program. In conclusion, we observed good agreement of the tested DIA blots with the consensus results of the Dutch EQC program. Taken together with the logistic advantages these blots are a good alternative for autoantibody detection in the respective diseases. A large prospective multicenter study is warranted to position these novel tests further in the whole spectrum of assays for the detection of these antibodies in a routine autoimmune laboratory. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. A multiplex ligation detection assay for the characterization of Salmonella enterica strains

    DEFF Research Database (Denmark)

    Aarts, Henk J.M.; Vos, Pieter; Larsson, Jonas T.

    2011-01-01

    of four serovars each serovar was characterized by a unique virulence associated gene repertoire. The LDR microarray platform proved to be a convenient, rapid and easy to use tool with potential in tracing a Salmonella contamination in the food chain, for outbreak studies, and to provide data for risk....... The feasibility of the developed assay was verified in a method comparison study with conventional PCR using 16 Salmonella ‘test’ strains comprising eight serovars. Subsequently, the feasibility of the LDR microarray assay was also tested by analyzing 41 strains belonging to 23 serovars. With the exception......A proof of principle of a multi-target assay for genotyping Salmonella has been developed targeting 62 genomic marker sequences of Salmonella related to pathogenicity. The assay is based on multiplex ligation detection reaction (LDR) followed by customized ArrayTube® microarray detection...

  18. A Multiplex PCR-coupled Liquid Bead Array for the Simultaneous Detection of Four Biothreat Agents

    Energy Technology Data Exchange (ETDEWEB)

    Wilson, W J; Erler, A M; Nasarabadi, S L; Skowronski, E W; McCready, P M

    2004-02-04

    We have developed a 10-plexed PCR assay coupled to a 12-plexed liquid bead array to rapidly screen environmental samples for B. anthracis, Y. pestis, F. tularensis, and B. melitensis. Highly validated species -specific primer sets were used to simultaneously amplify multiple diagnostic regions unique to each individual pathogen. Resolution of the mix of amplified products was achieved by PCR product hybridization to corresponding probe sequences, attached to unique sets of fluorescent beads. The hybridized beads were processed through a flow cytometer, which detected presence and quantity of each PCR product. The assay was optimized to allow for maximum sensitivity in a multiplexed format. A high- throughput demonstration was performed where 384 simulated environmental samples were spiked with different amounts of B. thuringensis spores and pathogen DNA. The samples were robotically processed to extract DNA and arrayed for multiplexed PCR-liquid bead detection. The assay correctly identified the presence or absence of each pathogen and collected over 3,000 individual data points within a single 8-hour shift for approximately $1.20 per sample in a 10-plexed assay.

  19. Interferometric biosensing platform for multiplexed digital detection of viral pathogens and biomarkers

    Science.gov (United States)

    Daaboul, George

    of SP-IRIS we developed was single molecule counting of biomarkers utilizing a sandwich assay with detection probes labeled with gold nanoparticles. We demonstrated the use of single molecule counting in a nucleic acid assay for melanoma biomarker detection. We showed that a single molecule counting assay can lead to detection limits in the attomolar range. The improved sensitivity of IRIS utilizing single nanoparticle detection holds promise for a simple and low-cost technology for rapid virus detection and multiplexed molecular screening for clinical applications.

  20. Detection and differentiation of genotype I and III Japanese encephalitis virus in mosquitoes by multiplex reverse transcriptase-polymerase chain reaction.

    Science.gov (United States)

    Chen, Y Y; Lin, J W; Fan, Y C; Chiou, S S

    2014-02-01

    Japanese encephalitis (JE) is a disease that threatens both human and animal populations in Asian countries, and the causative agent of JE, Japanese encephalitis virus (JEV), has recently changed from genotype III (GIII) to genotype I (GI). However, a test for the rapid differentiation of GI and GIII JEV is still unavailable, especially one that can be used for mosquito-based surveillance. We have designed GI- and GIII-specific primer sets for the rapid detection and differentiation of GI and GIII JEV by multiplex reverse transcriptase-polymerase chain reaction (multiplex RT-PCR). The GI-specific and GIII-specific primer sets were able to specifically amplify the target gene from GI and GIII JEV, respectively. The limitations of detection were 0.00225 and 0.225 pfu for the GI-specific and GIII-specific primers, respectively. Using a mixture of GI-specific and GIII-specific primers, the multiplex RT-PCR was able to specifically detect and differentiate GI and GIII JEV. The multiplex RT-PCR was able to successfully differentiate GI and GIII virus in JEV-infected mosquitoes. Thus, a sensitive and specific multiplex RT-PCR system for the rapid detection and differentiation of GI and GIII JEV has been developed, and this test is likely to be valuable when carrying out mosquito-based JEV surveillance. © 2012 Blackwell Verlag GmbH.

  1. Quantitative multiplex assay for simultaneous detection and identification of Indiana and New Jersey serotypes of vesicular stomatitis virus

    DEFF Research Database (Denmark)

    Rasmussen, Thomas Bruun; Uttenthal, Åse; Fernandez, Jovita

    2005-01-01

    In order to establish a rapid and reliable system for the detection of vesicular stomatitis virus (VSV), we developed a quantitative reverse transcription-PCR assay for the detection, quantification, and differentiation of the major serotypes, VSV Indiana and VSV New Jersey, using a closed......-tube multiplex format. The detection system is based on the recently invented primer-probe energy transfer (PriProET) system. A region of the gene encoding the RNA-dependent RNA polymerase was amplified by using VSV-specific primers in the presence of two serotype-specific fluorescent probes. By incorporating...... identification of VSV....

  2. Quantum-dot-tagged photonic crystal beads for multiplex detection of tumor markers.

    Science.gov (United States)

    Li, Juan; Wang, Huan; Dong, Shujun; Zhu, Peizhi; Diao, Guowang; Yang, Zhanjun

    2014-12-04

    Novel quantum-dot-tagged photonic crystal beads were fabricated for multiplex detection of tumor markers via self-assembly of quantum dot-embedded polystyrene nanospheres into photonic crystal beads through a microfluidic device.

  3. Development and Validation of a Multiplex Reverse Transcription PCR Assay for Simultaneous Detection of Three Papaya Viruses

    Directory of Open Access Journals (Sweden)

    Decai Tuo

    2014-10-01

    Full Text Available Papaya ringspot virus (PRSV, Papaya leaf distortion mosaic virus (PLDMV, and Papaya mosaic virus (PapMV produce similar symptoms in papaya. Each threatens commercial production of papaya on Hainan Island, China. In this study, a multiplex reverse transcription PCR assay was developed to detect simultaneously these three viruses by screening combinations of mixed primer pairs and optimizing the multiplex RT-PCR reaction conditions. A mixture of three specific primer pairs was used to amplify three distinct fragments of 613 bp from the P3 gene of PRSV, 355 bp from the CP gene of PLDMV, and 205 bp from the CP gene of PapMV, demonstrating the assay’s specificity. The sensitivity of the multiplex RT-PCR was evaluated by showing plasmids containing each of the viral target genes with 1.44 × 103, 1.79 × 103, and 1.91 × 102 copies for the three viruses could be detected successfully. The multiplex RT-PCR was applied successfully for detection of three viruses from 341 field samples collected from 18 counties of Hainan Island, China. Rates of single infections were 186/341 (54.5%, 93/341 (27.3%, and 3/341 (0.9%, for PRSV, PLDMV, and PapMV, respectively; 59/341 (17.3% of the samples were co-infected with PRSV and PLDMV, which is the first time being reported in Hainan Island. This multiplex RT-PCR assay is a simple, rapid, sensitive, and cost-effective method for detecting multiple viruses in papaya and can be used for routine molecular diagnosis and epidemiological studies in papaya.

  4. Application of Multiplex RT-PCR for Detection of Cucurbit-infecting Tobamovirus

    OpenAIRE

    Daryono, Budi Setiadi; Natsuaki, Keiko T.

    2016-01-01

    Cucumber green mottle mosaic virus (CGMMV) and Kyuri green mottle mosaic virus (KGMMV) are seed borne viruses and they are also transmitted mechanically during agricultural practice and through water. Hence, these viruses have potential diseases widely distributed throughout the world. To detect different strains of CGMMV and KGMMV, several specific primers for each virus were designed for single and multiplex RT-PCR. The results of single and multiplex RT-PCR showed that CGMMV was detected i...

  5. Multiplex real-time PCR for detection of Campylobacter, Salmonella, and Shigella.

    Science.gov (United States)

    Barletta, F; Mercado, E H; Lluque, A; Ruiz, J; Cleary, T G; Ochoa, T J

    2013-09-01

    Infectious diarrhea can be classified based on its clinical presentation as noninflammatory or inflammatory disease. In developing countries, among inflammatory diarrhea cases, Shigella is the most common cause, followed by Campylobacter and Salmonella. Because the time frame in which treatment choices must be made is short and conventional stool cultures lack good sensitivity, there is a need for a rapid, sensitive, and inexpensive detection technique. The purpose of our study was to develop a multiplex real-time PCR procedure to simultaneously identify Campylobacter spp., Salmonella spp., and Shigella spp. Primers were designed to amplify the invA, ipaH, and 16S rRNA genes simultaneously in a single reaction to detect Salmonella, Shigella, and Campylobacter, respectively. Using this approach, we correctly identified 102 of 103 strains of the targeted enteropathogens and 34 of 34 other pathogens. The melting temperatures were 82.96 ± 0.05 °C for invA, 85.56 ± 0.28 °C for ipaH, and 89.21 ± 0.24 °C for 16S rRNA. The limit of accurate quantification for the assay in stool samples was 10(4) CFU g(-1); however, the limit of detection was 10(3) CFU g(-1). This assay is a simple, rapid, inexpensive, and reliable system for the practical detection of these three enteropathogens in clinical specimens.

  6. Multiplexed lateral flow microarray assay for detection of citrus pathogens Xylella fastidiosa and Xanthomonas axonopodis pv citri

    Science.gov (United States)

    Cary,; Bruce, R [Santa Fe, NM; Stubben, Christopher J [Los Alamos, NM

    2011-03-22

    The invention provides highly sensitive and specific assays for the major citrus pathogens Xylella fastidiosa and Xanthomonas axonopodis, including a field deployable multiplexed assay capable of rapidly assaying for both pathogens simultaneously. The assays are directed at particular gene targets derived from pathogenic strains that specifically cause the major citrus diseases of citrus variegated chlorosis (Xylella fastidiosa 9a5c) and citrus canker (Xanthomonas axonopodis pv citri). The citrus pathogen assays of the invention offer femtomole sensitivity, excellent linear dynamic range, and rapid and specific detection.

  7. Simultaneous detection of bovine arboviruses using single-tube multiplex reverse transcription-polymerase chain reaction.

    Science.gov (United States)

    Ohashi, Seiichi; Yoshida, Kazuo; Yanase, Tohru; Kato, Tomoko; Tsuda, Tomoyuki

    2004-09-01

    Single-tube multiplex reverse transcription-polymerase chain reaction (mRT-PCR) assay was developed to detect and identify arboviruses in infected cell-culture fluids and field specimens. The technique was equally sensitive for detecting five different viruses in cell cultures, namely the Chuzan, Ibaraki, and Bluetongue viruses belonging to Orbivirus, and the Akabane virus and Peaton virus belonging to Orthobunyavirus, and was less sensitive than former viruses for detecting Aino virus belonging to Orthobunyavirus. The mRT-PCR reliably detected 0.6-10(3.1) median tissue culture infective doses. The mRT-PCR readily identified viruses by discriminating the size of their amplified gene products. The technique was as sensitive as virus isolation in detecting single infected plasma in five plasmas from sentinel cattle and in detecting two infectious homogenates in eight homogenates of Culicoides biting midges. The mRT-PCR may be a sensitive and rapid assay for surveillance of bovine arboviruses in field specimens.

  8. [Multiplex quantitative PCR detection for female carrier in an X-linked ichthyosis family].

    Science.gov (United States)

    Zhu, Hai-yan; Li, Hai-bo; Wu, Ling-qian; Zhu, Xiang-yu; Li, Jie; Yang, Ying; Zhu, Rui-fang; Wu, Xing; Duan, Hong-lei; Zhang, Ying; Hu, Ya-li

    2008-12-16

    To analyze the pathogenic mutation of an X-linked ichthyosis (XLI) family, and identify the genetic diagnosis of three probable female carriers in this family. To evaluate the availability of different detect methods for steroid sulfatase (STS) gene mutation. Peripheral blood samples were collected from the family, including the proband, proband's mother, younger sister, and younger female cousin, and 10 males and 10 females as controls. Ordinary PCR was used to detect whether there was STS gene deletion in the male proband. Then, multiplex quantitative fluorescent PCR (QF-PCR) was used to detect the STS gene in the proband and his 3 female family members. Fluorescence in situ hybridization (FISH) was used to authenticate the results of multiplex QF-PCR method. No amplified product of the exons 1-10 of STS gene deletion was detected by ordinary PCR in the proband. The proband's mother was diagnosed as a carrier, but his sister and cousin were diagnosed as normal females by multiplex QF-PCR. FISH confirmed the results of multiplex QF-PCR. Both multiplex QF-PCR and FISH are effective to detect the complete deletion mutation of STS gene and identify the female carrier, and multiplex QF-PCR is more convenient and automatic compared with FISH.

  9. Rapid spectro-polarimetry to probe molecular symmetry in multiplex coherent anti-Stokes Raman scattering.

    Science.gov (United States)

    Würthwein, Thomas; Brinkmann, Maximilian; Hellwig, Tim; Fallnich, Carsten

    2017-11-21

    We present the simultaneous detection of the spectrum and the complete polarization state of a multiplex coherent anti-Stokes Raman scattering signal with a fast division-of-amplitude spectro-polarimeter. The spectro-polarimeter is based on a commercial imaging spectrograph, a birefringent wedge prism, and a segmented polarizer. Compared to the standard rotating-retarder fixed-analyzer spectro-polarimeter, only a single measurement is required and an up to 21-fold reduced acquisition time is shown. The measured Stokes parameters allow us to differentiate between vibrational symmetries and to determine the depolarization ratio ρ by data post-processing.

  10. Evaluation of the multiplex PCR Allplex-GI assay in the detection of bacterial pathogens in diarrheic stool samples.

    Science.gov (United States)

    Martín, Ariadna; Pérez-Ayala, Ana; Chaves, Fernando; Lora, David; Orellana, M Ángeles

    2017-10-31

    Rapid and accurate detection of the pathogens that cause gastrointestinal infection is important for appropriate therapy and proper infection control. This study assesses the performance of a new molecular assay for simultaneous detection of 13 different gastrointestinal bacteria in stool specimens. Using the Allplex GI-Bacteria (AGI-BI/AGI-BII) assay, a total of 394 stool samples were tested and the results were compared with culturing on selective differential followed by identification by mass spectroscopy. Discordant results were analyzed by a different multiplex PCR method, the Fast-Track Diagnostics Bacterial gastroenteritis (FTD-BG). The routine method (RM) detected 109 (27.7%) positive samples and the Allplex-GI assay, 261 (66.2%). Analysis of discordant results revealed that the molecular assay detected 44 pathogens that were not detected by the RM, including 23 Campylobacter spp., 11 Salmonella spp, 3 Y. enterocolitica, 2 EIEC/Shigella spp, 2 E. coli 0157, 2 C. difficile and 1 Aeromonas spp. Five cases not detected by the molecular method were detected by the RM (3 Aeromonas spp, 1 Salmonella spp and 1 Y. enterocolitica). For all targets, the percentages of sensitivity and specificity were >95%, except for Aeromonas spp., which were 81% and 99% respectively. This study suggests that Allplex-GI multiplex PCR is a sensitive and specific assay that enables a rapid and accurate diagnosis of bacterial gastrointestinal infections. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Comparison of two multiplex PCR assays for the detection of respiratory viral infections.

    Science.gov (United States)

    Kim, Hanah; Hur, Mina; Moon, Hee-Won; Yun, Yeo-Min; Cho, Hyun Chan

    2014-10-01

    Respiratory viruses are the main causes of upper and lower respiratory tract diseases. Rapid and accurate detection of respiratory viruses is crucial for appropriate patient treatment and prevention of endemic spread. We compared two multiplex polymerase chain reaction (PCR) assays for the detection of respiratory viral pathogens. A total of 245 respiratory specimens (229 sputum samples, 14 bronchoalveolar lavage samples, 6 nasal swabs, 3 throat swabs, 7 unknown) were analyzed using two multiplex assays: One-step RV real-time PCR (BioSewoom, Seoul, Korea) and Seeplex RV 12 Detection kit (Seegene, Seoul, Korea). The results were further confirmed using sequencing as a reference. Among 245 samples (265 identifications including co-infections), the identification of respiratory viruses was 44.9% (119/265), 44.2% (117/265) and 45.3% (120/265) by One-step RV assay, Seeplex RV assay and sequencing, respectively. The concordance rate between One-step RV assay and sequencing was 95.5% (253/265), and that between Seeplex RV assay and sequencing was 89.8% (238/265) (P = 0.0189). The sensitivities of One-step RV and Seeplex RV assays were 94.1% [95% confidential interval (CI), 88.3%-97.6%] and 83.3% (95% CI, 75.4%-89.5%), respectively (P = 0.0002). The specificities of One-step RV and Seeplex RV assays were 96.6% (95% CI, 92.2%-98.9%) and 95.2% (95% CI, 90.3%-98.0%), respectively. Although the performances of One-step RV and Seeplex RV assays were overall comparable, One-step RV assay showed better sensitivity and concordance with sequencing. One-step RV assay can be a useful option for respiratory virus testing in clinical laboratories. © 2013 John Wiley & Sons Ltd.

  12. Modified multiplex PCR methods for comprehensive detection of Pectinatus and beer-spoilage cocci.

    Science.gov (United States)

    Iijima, Kazumaru; Asano, Shizuka; Suzuki, Koji; Ogata, Tomoo; Kitagawa, Yasushi

    2008-10-01

    Specific PCR primers were designed based on the 16S rRNA genes of recently proposed beer-spoilage species, Pectinatus haikarae, Megasphaera sueciensis, and M. paucivorans, and two sets of our previously reported multiplex PCR methods for Pectinatus spp. and beer-spoilage cocci were reconstructed. Each modified multiplex PCR method was found specifically to detect beer-spoilage species of Pectinatus and cocci, including new species.

  13. Multiplexed Molecular Assays for Rapid Rule-Out of Foot-and-Mouth Disease

    Energy Technology Data Exchange (ETDEWEB)

    Lenhoff, R; Naraghi-Arani, P; Thissen, J; Olivas, J; Carillo, C; Chinn, C; Rasmussen, M; Messenger, S; Suer, L; Smith, S M; Tammero, L; Vitalis, E; Slezak, T R; Hullinger, P J; Hindson, B J; Hietala, S; Crossley, B; Mcbride, M

    2007-06-26

    A nucleic acid-based multiplexed assay was developed that combines detection of foot-and-mouth disease virus (FMDV) with rule-out assays for two other foreign animal diseases and four domestic animal diseases that cause vesicular or ulcerative lesions indistinguishable from FMDV infection in cattle, sheep and swine. The FMDV 'look-alike' diagnostic assay panel contains five PCR and twelve reverse transcriptase PCR (RT-PCR) signatures for a total of seventeen simultaneous PCR amplifications for seven diseases plus incorporating four internal assay controls. It was developed and optimized to amplify both DNA and RNA viruses simultaneously in a single tube and employs Luminex{trademark} liquid array technology. Assay development including selection of appropriate controls, a comparison of signature performance in single and multiplex testing against target nucleic acids, as well of limits of detection for each of the individual signatures is presented. While this assay is a prototype and by no means a comprehensive test for FMDV 'look-alike' viruses, an assay of this type is envisioned to have benefit to a laboratory network in routine surveillance and possibly for post-outbreak proof of freedom from foot-and-mouth disease.

  14. [Evaluation of the usefulness of selective chromogenic agar medium (chromID VRE) and multiplex PCR method for the detection of vancomycin-resistant enterococci].

    Science.gov (United States)

    Kim, Do-Hoon; Lee, Jae-Hee; Ha, Jung-Sook; Ryoo, Nam-Hee; Jeon, Dong-Seok; Kim, Jae-Ryong

    2010-12-01

    Accurate and early detection of vancomycin-resistant enterococci (VRE) is critical for controlling nosocomial infection. In this study, we evaluated the usefulness of a selective chromogenic agar medium and of multiplex PCR for detection of VRE, and both these techniques were compared with the conventional culture method for VRE detection. We performed the following 3 methods for detecting VRE infection in stool specimens: the routine culture method, culturing in selective chromogenic agar medium (chromID VRE, bioMérieux, France), and multiplex PCR using the Seeplex® VRE ACE Detection kit (Seegene Inc., Korea) with additional PCR for vanC genes. We isolated 109 VRE strains from 100 stool specimens by the routine culture method. In chromID VRE, all the isolates showed purple colonies, including Enterococcus gallinarum and E. raffinosus, which were later identified using the Vitek card. All VRE isolates were identified by the multiplex PCR method; 100 were vanA-positive E. faecium, 8 were vanA- and vanC-1-positive E. gallinarum, and 1 was vanA-positive E. raffinosus. For VRE surveillance, culturing the isolates in chromID VRE after broth enrichment appears to be an accurate, rapid, and easy method for routine screening test. Multiplex PCR is relatively expensive and needs skilled techniques for detecting VRE, but it can be an auxiliary tool for rapid detection of genotype during a VRE outbreak.

  15. Quantitative and ultrasensitive detection of multiplex cardiac biomarkers in lateral flow assay with core-shell SERS nanotags.

    Science.gov (United States)

    Zhang, Di; Huang, Li; Liu, Bing; Ni, Haibin; Sun, Liangdong; Su, Enben; Chen, Hongyuan; Gu, Zhongze; Zhao, Xiangwei

    2018-05-30

    Rapid and sensitive quantification of multiplex proteins in a wide concentration range is challenging in high throughput analysis. Herein, we proposed a lateral flow assay (LFA) based on core-shell surface enhanced Raman scattering (SERS) nanotags for multiplex and quantitative detection of cardiac biomarkers for the early diagnosis of acute myocardial infarction (AMI). In practice, Raman dyes (RDs) were embedded into the interior-gap of silver core and gold shell nanoparticles (NPs) to form SERS nanotags as labels instead of gold colloids and three test lines were employed in the strip for the detection of three cardiac biomarkers, Myo, cTnI, and CK-MB, respectively. Due to the amplified signal of the SERS nanotags and the high surface area to volume ratio (SVR) of porous nitrocellulose (NC) membrane, ultrasensitive quantification of protein markers with wide linear dynamic range (LDR) was realized, which is crucial for the quick detection of multiplex biomarkers in the same sample without pretreatments at bedsides. This method makes it possible for LFA in point of care testing (POCT) to be comparable with chemiluminescence immunoassay (CLIA) used in labs. Copyright © 2018 Elsevier B.V. All rights reserved.

  16. A multiplex RT-PCR assay for the detection of fish picornaviruses.

    Science.gov (United States)

    Mor, Sunil K; Phelps, Nicholas B D; Barbknecht, Marisa; Hoffman, Michael A; Goyal, Sagar M

    2015-09-01

    With the emergence of high profile fish diseases in the Great Lakes region, surveillance and regulatory inspections of fish populations have increased. This has resulted in a better understanding of known pathogens and isolation of many new pathogens of fish. In this study, a multiplex RT-PCR assay was developed for the detection of three newly discovered fish picornaviruses: bluegill picornavirus-1 (BGPV-1), fathead minnow picornavirus (FHMPV), and eel picornavirus-1 (EPV-1). This assay was found to be very sensitive with a detection limit of 81.9pg/μl of extracted RNA from a pool of FHMPV and BGPV-1 and was able to detect 501 and 224 gene copies/μl of BGPV-1 and FHMPV, respectively. The assay was highly reproducible and did not cross react with other closely related pathogens. We believe that this new assay provides a rapid and cost effective tool for confirming cell culture isolates and conducting prevalence studies of these newly detected fish picornaviruses. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. Multiplex PCR Tests for Detection of Pathogens Associated with Gastroenteritis

    OpenAIRE

    Zhang, Hongwei; Morrison, Scott; Tang, Yi-Wei

    2015-01-01

    A wide range of enteric pathogens can cause infectious gastroenteritis. Conventional diagnostic algorithms including culture, biochemical identification, immunoassay and microscopic examination are time consuming and often lack sensitivity and specificity. Advances in molecular technology have as allowed its use as clinical diagnostic tools. Multiplex PCR based testing has made its way to gastroenterology diagnostic arena in recent years. In this article we present a review of recent laborato...

  18. A Multiplex PCR for Detection of Mycoplasma pneumoniae, Chlamydophila pneumoniae, Legionella pneumophila, and Bordetella pertussis in Clinical Specimens

    National Research Council Canada - National Science Library

    McDonough, E. A; Barrozo, C. P; Russell, K. L; Metzgar, D

    2005-01-01

    A multiplex PCR was developed that is capable of detecting four of the most important bacterial agents of atypical pneumophia, Mycaplasma pneumoniae, Chlamydophia pneumoniae, Legionella pneumophila...

  19. A Multiplex SYBR Green Real-Time PCR Assay for the Detection of Three Colistin Resistance Genes from Cultured Bacteria, Feces, and Environment Samples

    Directory of Open Access Journals (Sweden)

    Jiyun Li

    2017-10-01

    Full Text Available The aim of the study was to develop a multiplex assay for rapid detection of mcr-1, mcr-2, and mcr-3, a group of genes of conferring resistance to colistin mediated by plasmid in Enterobacteriaceae. A SYBR Green based real-time PCR assay has been designed to detect the mcr genes, and applied to cultured bacteria, feces and soil samples. All three mcr genes could be detected with a lower limit of 102 cultured bacteria. This test was highly specific and sensitive, and generated no false-positive results. The assay was also conclusive when applied to feces and soil samples containing mcr-1-positive Escherichia coli, which could facilitate the screening of mcr genes not only in the bacteria, but also directly from the environment. This simple, rapid, sensitive, and specific multiplex assay will be useful for rapid screening of the colistin resistance in both clinical medicine and animal husbandry.

  20. Development of a multiplex PCR assay for rapid identification of Burkholderia pseudomallei, Burkholderia thailandensis, Burkholderia mallei and Burkholderia cepacia complex.

    Science.gov (United States)

    Koh, Seng Fook; Tay, Sun Tee; Sermswan, Rasana; Wongratanacheewin, Surasakdi; Chua, Kek Heng; Puthucheary, Savithri D

    2012-09-01

    We have developed a multiplex PCR assay for rapid identification and differentiation of cultures for Burkholderia pseudomallei, Burkholderia thailandensis, Burkholderia mallei and Burkholderia cepacia complex. The assay is valuable for use in clinical and veterinary laboratories, and in a deployable laboratory during outbreaks. Copyright © 2012 Elsevier B.V. All rights reserved.

  1. In vivo magnetic enrichment and multiplex photoacoustic detection of circulating tumour cells

    Science.gov (United States)

    Galanzha, Ekaterina I.; Shashkov, Evgeny V.; Kelly, Thomas; Kim, Jin-Woo; Yang, Lily; Zharov, Vladimir P.

    2009-12-01

    The spread of cancer cells between organs, a process known as metastasis, is the cause of most cancer deaths. Detecting circulating tumour cells-a common marker for the development of metastasis-is difficult because ex vivo methods are not sensitive enough owing to limited blood sample volume and in vivo diagnosis is time-consuming as large volumes of blood must be analysed. Here, we show a way to magnetically capture circulating tumour cells in the bloodstream of mice followed by rapid photoacoustic detection. Magnetic nanoparticles, which were functionalized to target a receptor commonly found in breast cancer cells, bound and captured circulating tumour cells under a magnet. To improve detection sensitivity and specificity, gold-plated carbon nanotubes conjugated with folic acid were used as a second contrast agent for photoacoustic imaging. By integrating in vivo multiplex targeting, magnetic enrichment, signal amplification and multicolour recognition, our approach allows circulating tumour cells to be concentrated from a large volume of blood in the vessels of tumour-bearing mice, and this could have potential for the early diagnosis of cancer and the prevention of metastasis in humans.

  2. Comparison of multiplex-PCR and antigen detection for differential diagnosis of Entamoeba histolytica

    Directory of Open Access Journals (Sweden)

    Helena Lúcia Carneiro Santos

    Full Text Available Amebiasis is an infection caused by Entamoeba histolytica. However, differentiation between E. histolytica and Entamoeba dispar, which are morphologically identical species, is essential for treatment decision, precaution of the invasive disease and public health. The purpose of the present study was to evaluate a Multiplex -PCR for detection and differentiation of E. histolytica from E. dispar from fresh stool samples in comparison with the coproantigen commercial ELISA. Microscopic examination of stools using the Coprotest method, detection of stool antigen by enzyme-linked immunosorbent assay kit and a home made Multiplex-PCR, were used for the diagnosis of amoebiasis infection. Analysis of the 127 stools samples by microscopy examination demonstrated that only 27 (21% samples were positive for E. histolytica/E. dispar complex. Among these stool samples, 11 were positive by Multiplex-PCR, with nine presenting the diagnostic fragment characteristic of E. dispar (96 bp and two presenting diagnostic fragment of E. histolytica (132 bp. Among negative samples detected by microscopic examination, three positive samples for E. dispar and one positive for E. histolytica by Multiplex-PCR was observed. This denotes a low sensibility of microscopic examination when a single stool sample is analyzed. Assay for detection of E. histolytica antigen was concordant with multiplex-PCR in relation to E. histolytica. Statistical analysis comparing the sensibility tests was not done because of the low number of E. histolytica cases. The results demonstrate the importance of the specific techniques use for the differentiation between E. histolytica and E. dispar.

  3. A multiplex PCR for detection of Listeria monocytogenes and its lineages.

    Science.gov (United States)

    Rawool, Deepak B; Doijad, Swapnil P; Poharkar, Krupali V; Negi, Mamta; Kale, Satyajit B; Malik, S V S; Kurkure, Nitin V; Chakraborty, Trinad; Barbuddhe, Sukhadeo B

    2016-11-01

    A novel multiplex PCR assay was developed to identify genus Listeria, and discriminate Listeria monocytogenes and its major lineages (LI, LII, LIII). This assay is a rapid and inexpensive subtyping method for screening and characterization of L. monocytogenes. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Detection of three porcine vesicular viruses using multiplex real-time primer-probe energy transfer

    DEFF Research Database (Denmark)

    Rasmussen, Thomas Bruun; Uttenthal, Åse; Aguero, M.

    2006-01-01

    Rapid identification of the etiologic agent in infected animals is important for the control of an outbreak of vesicular disease in livestock. We have in the present study developed a multiplex real-time reverse transcription-PCR, based on primer-probe energy transfer (PriProET), for simultaneous...

  5. Multiplex polymerase chain reaction of genetic markers for detection of potentially pathogenic environmental Legionella pneumophila isolates

    Directory of Open Access Journals (Sweden)

    Arvind Valavane

    2017-01-01

    Interpretation & conclusions: The findings of this study showed the presence of gene markers of pathogenic potential of the bacterium L. pneumophila. However, the genomic constitution of the environmental isolates should be correlated with clinical isolates to prove their pathogenic potential. Rapid diagnostic methods such as multiplex PCR reported here, for elucidating gene markers, could help in future epidemiological studies of bacterium L. pneumophila.

  6. High throughput plasma N-glycome profiling using multiplexed labelling and UPLC with fluorescence detection

    DEFF Research Database (Denmark)

    Kneževic, Ana; Bones, Jonathan; Kracun, Stjepan Kresimir

    2011-01-01

    A rapid glycomic profiling method is described wherein N-glycans from plasma samples individually labelled with aniline, 2-aminobenzamide and 2-aminoacridone are mixed, co-injected and separated in the same HILIC-fluorescence run. Transfer of the multiplexed method to UPLC-fluorescence permits...

  7. Narcissus yellow stripe virus and Narcissus mosaic virus detection in Narcissus via multiplex TaqMan-based reverse transcription-PCR assay.

    Science.gov (United States)

    Jin, J; Shen, J G; Cai, W; Xie, G H; Liao, F R; Gao, F L; Ma, J F; Chen, X H; Wu, Z J

    2017-05-01

    Development of a multiplex TaqMan RT-qPCR assay to simultaneously detect Narcissus yellow stripe virus (NYSV) and Narcissus mosaic virus (NMV), frequently causing mixed narcissus infection. Feasibility verification was confirmed in natural samples. Primers and probes were designed based on the conserved CP gene regions of NYSV or NMV and their suitability for singleplex and multiplex TaqMan RT-qPCR assays as well as for conventional RT-PCR. Conventional RT-PCR, singleplex and multiplex TaqMan RT-qPCR assays proved to be NYSV and NMV specific. P-values and coefficients of variation of TaqMan RT-qPCR assays indicated high reproducibility. Significantly increased sensitivity was achieved compared to conventional RT-PCR. The detection limit of both viruses was 103 copies with superior correlation coefficients and linear standard curve responses between plasmid concentrations and Ct values. NYSV and NMV infection of narcissus leaves, petals and bulbs could successfully be detected via our multiplex RT-qPCR method at 1·25 mg. Our multiplex TaqMan RT-qPCR assay provides rapid, specific, sensitive and reliable testing to simultaneously detect NYSV and NMV, supplying useful routine monitoring for different narcissus samples. Efficient identification and discrimination of the narcissus viruses provides reliable information for scientists and conventional growers. Furthermore, it enriches the information of NYSV, NMV and other narcissus viruses. © 2017 The Society for Applied Microbiology.

  8. Rapid methods for detection of bacteria

    DEFF Research Database (Denmark)

    Corfitzen, Charlotte B.; Andersen, B.Ø.; Miller, M.

    2006-01-01

    Traditional methods for detection of bacteria in drinking water e.g. Heterotrophic Plate Counts (HPC) or Most Probable Number (MNP) take 48-72 hours to give the result. New rapid methods for detection of bacteria are needed to protect the consumers against contaminations. Two rapid methods...

  9. Detection of Chlamydia trachomatis and Mycoplasma genitalium in semen samples of infertile men using multiplex PCR

    Directory of Open Access Journals (Sweden)

    Sadrpour P

    2013-01-01

    Full Text Available Background: Chlamydia trachomatis is the most common bacterial sexually transmitted infection in the world, but the effect of this infection on male fertility is still controversial. Despite reports of interaction between Mycoplasma genitalium and sperm, this pathogen in semen samples of infertile men is less studied. We studied, the prevalence of Chlamydia trachomatis and Mycoplasma genitalium infection in infertile men.Methods: Among attending Avicenna Infertility Center, 120 men who had abnormal semen analysis tests were selected and the samples were taken. After detailed analysis of semen quality, DNA was extracted from each sample by chelex. Samples were evaluated for these two pathogens by multiplex PCR. Results were statistically analyzed.Results: Chlamydia trachomatis and Mycoplasma genitalium was detected in 23/3% and 12/5% of the samples, respectively. Although, Mycoplasma genitalium infection rises by increasing (P=0.640 and decreasing in age of first sexually activity (P=0.203, and also positive cases of Chlamydia trachomatis infection showed increase regarding age increase (P=0.619 and age decrease in first sexually activity (P=0.511, but these differences were not statistically significant. Conclusion: All in all, regarding to the increased prevalence of Chlamydia trachomatis infection compared with the only similar study in Iran and high prevalence of Mycoplasma genitalium infection in infertile men, this assessment was done. A multiplex PCR protocol rapidly and simultaneously identify these organisms in comparison with uniplex from clinical samples. Based on our results screening for Chlamydia trachomatis and Mycoplasma genitalium infection among infertile men seems to be valuable.

  10. Evaluation of an Internally Controlled Multiplex Tth Endonuclease Cleavage Loop-Mediated Isothermal Amplification (TEC-LAMP Assay for the Detection of Bacterial Meningitis Pathogens

    Directory of Open Access Journals (Sweden)

    Owen Higgins

    2018-02-01

    Full Text Available Bacterial meningitis infection is a leading global health concern for which rapid and accurate diagnosis is essential to reduce associated morbidity and mortality. Loop-mediated isothermal amplification (LAMP offers an effective low-cost diagnostic approach; however, multiplex LAMP is difficult to achieve, limiting its application. We have developed novel real-time multiplex LAMP technology, TEC-LAMP, using Tth endonuclease IV and a unique LAMP primer/probe. This study evaluates the analytical specificity, limit of detection (LOD and clinical application of an internally controlled multiplex TEC-LAMP assay for detection of leading bacterial meningitis pathogens: Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae. Analytical specificities were established by testing 168 bacterial strains, and LODs were determined using Probit analysis. The TEC-LAMP assay was 100% specific, with LODs for S. pneumoniae, N. meningitidis and H. influenzae of 39.5, 17.3 and 25.9 genome copies per reaction, respectively. Clinical performance was evaluated by testing 65 archived PCR-positive samples. Compared to singleplex real-time PCR, the multiplex TEC-LAMP assay demonstrated diagnostic sensitivity and specificity of 92.3% and 100%, respectively. This is the first report of a single-tube internally controlled multiplex LAMP assay for bacterial meningitis pathogen detection, and the first report of Tth endonuclease IV incorporation into nucleic acid amplification diagnostic technology.

  11. Detection and quantification of Campylobacter jejuni and Campylobacter coli using real-time multiplex PCR.

    Science.gov (United States)

    Toplak, N; Kovač, M; Piskernik, S; Možina, S Smole; Jeršek, B

    2012-04-01

    We describe a real-time quantitative multiplex polymerase chain reaction (qmPCR) assay to identify and discriminate between isolates of Campylobacter jejuni and Campylobacter coli. Two novel sets of primers and hydrolysis probes were designed to amplify the unique DNA sequences within the hipO, ccoN and cadF genes that are specific to Camp. jejuni and Camp. coli. Using the designed optimized qmPCR assay conditions, the amplification efficiency is in range from 108 to 116%. These qmPCR assays are highly specific for Camp. jejuni and Camp. coli, as seen through testing of 40 Campylobacter strains and 17 non-Campylobacter strains. In chicken juice and tap water models spiked with known quantities of Camp. jejuni, qmPCR detected 10(2) -10(3) CFU ml(-1) within 4 h. The qmPCR assays developed in this study provide reliable and simultaneous detection and quantification of Camp. jejuni and Camp. coli, with good amplification reaction parameters. Following further validation, the qmPCR assay reported here has the potential to be applied to various sample types as an alternative and rapid methodology. © 2012 The Authors. Journal of Applied Microbiology © 2012 The Society for Applied Microbiology.

  12. A multiplex PCR for detection of genes encoding exfoliative toxins from Staphylococcus hyicus

    DEFF Research Database (Denmark)

    Andresen, Lars Ole; Ahrens, Peter

    2004-01-01

    Aims: To develop a multiplex PCR for detection of genes encoding the exfoliative toxins ExhA, ExhB, ExhC and ExhD from Staphylococcus hyicus and to estimate the prevalence of exfoliative toxins among Staph. hyicus isolates from Danish pig herds with exudative epidermitis (EE). Methods and Results......: A multiplex PCR employing specific primers for each of the genes encoding four different exfoliative toxins was developed and evaluated using a collection of Staph. hyicus with known toxin type and a number of other staphylococcal species. A total of 314 Staph. hyicus isolates from pigs with EE were screened...... by multiplex PCR and the combined results of the present and previous investigations showed that ExhA, ExhB, ExhC and ExhD was found in 20, 33, 18 and 22%, respectively, of 60 cases of EE investigated. Conclusions: This study has provided a new tool for detection of toxigenic Staph. hyicus and a more...

  13. Sensitive multiplex detection of serological liver cancer biomarkers using SERS-active photonic crystal fiber probe.

    Science.gov (United States)

    Dinish, U S; Balasundaram, Ghayathri; Chang, Young Tae; Olivo, Malini

    2014-11-01

    Surface-enhanced Raman scattering (SERS) spectroscopy possesses the most promising advantage of multiplex detection for biosensing applications, which is achieved due to the narrow 'fingerprint' Raman spectra from the analyte molecules. We developed an ultrasensitive platform for the multiplex detection of cancer biomarkers by combining the SERS technique with a hollow-core photonic crystal fiber (HCPCF). Axially aligned air channels inside the HCPCF provide an excellent platform for optical sensing using SERS. In addition to the flexibility of optical fibers, HCPCF provides better light confinement and a larger interaction length for the guided light and the analyte, resulting in an improvement in sensitivity to detect low concentrations of bioanalytes in extremely low sample volumes. Herein, for the first time, we demonstrate the sensitive multiplex detection of biomarkers immobilized inside the HCPCF using antibody-conjugated SERS-active nanoparticles (SERS nanotags). As a proof-of-concept for targeted multiplex detection, initially we carried out the sensing of epidermal growth factor receptor (EGFR) biomarker in oral squamous carcinoma cell lysate using three different SERS nanotags. Subsequently, we also achieved simultaneous detection of hepatocellular carcinoma (HCC) biomarkers-alpha fetoprotein (AFP) and alpha-1-antitrypsin (A1AT) secreted in the supernatant from Hep3b cancer cell line. Using a SERS-HCPCF sensing platform, we could successfully demonstrate the multiplex detection in an extremely low sample volume of ∼20 nL. In future, this study may lead to sensitive biosensing platform for the low concentration detection of biomarkers in an extremely low sample volume of body fluids to achieve early diagnosis of multiple diseases. (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim). Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Multiplexed Lateral Flow Test for Detection and Differentiation ofCronobacter sakazakiiSerotypes O1 and O2.

    Science.gov (United States)

    Scharinger, Eva J; Dietrich, Richard; Wittwer, Tobias; Märtlbauer, Erwin; Schauer, Kristina

    2017-01-01

    The ubiquitous and opportunistic pathogen Cronobacter sakazakii is responsible for severe meningitis, sepsis, and necrotizing enterocolitis in neonates and infants associated with ingestion of contaminated powdered infant formula (PIF). The current ISO method for isolation and detection of Cronobacter spp. is laborious, time-consuming and expensive. In this study, a multiplexed lateral flow test strip was developed to rapidly detect and simultaneously serotype O1 and O2 C. sakazakii serotypes. The assay is based on two monoclonal antibodies (MAb) that specifically bind to the lipopolysaccharides (LPS) of these pathogens. The test strip provides results very quickly; C. sakazakii could be detected in pure culture within 15 min with a sensitivity of 10 7 CFU/ml. After non-selective enrichment for 18 h as low as one Cronobacter cell per g PIF could be detected. Moreover, the established lateral flow assay (LFA) offers excellent specificity showing no cross-reactivity with other C. sakazakii serotypes, Cronobacter species or Enterobacteriaceae tested. These characteristics, together with several advantages such as speed, simplicity in performance, low analysis cost, and no requirement of specialized skills or sophisticated equipment make the developed multiplexed LFA suitable for reliable detection and serotyping of C. sakazakii serotypes O1 and O2.

  15. Multiplexed Lateral Flow Test for Detection and Differentiation of Cronobacter sakazakii Serotypes O1 and O2

    Directory of Open Access Journals (Sweden)

    Eva J. Scharinger

    2017-09-01

    Full Text Available The ubiquitous and opportunistic pathogen Cronobacter sakazakii is responsible for severe meningitis, sepsis, and necrotizing enterocolitis in neonates and infants associated with ingestion of contaminated powdered infant formula (PIF. The current ISO method for isolation and detection of Cronobacter spp. is laborious, time-consuming and expensive. In this study, a multiplexed lateral flow test strip was developed to rapidly detect and simultaneously serotype O1 and O2 C. sakazakii serotypes. The assay is based on two monoclonal antibodies (MAb that specifically bind to the lipopolysaccharides (LPS of these pathogens. The test strip provides results very quickly; C. sakazakii could be detected in pure culture within 15 min with a sensitivity of 107 CFU/ml. After non-selective enrichment for 18 h as low as one Cronobacter cell per g PIF could be detected. Moreover, the established lateral flow assay (LFA offers excellent specificity showing no cross-reactivity with other C. sakazakii serotypes, Cronobacter species or Enterobacteriaceae tested. These characteristics, together with several advantages such as speed, simplicity in performance, low analysis cost, and no requirement of specialized skills or sophisticated equipment make the developed multiplexed LFA suitable for reliable detection and serotyping of C. sakazakii serotypes O1 and O2.

  16. Detection of sexually transmitted infection and human papillomavirus in negative cytology by multiplex-PCR

    Directory of Open Access Journals (Sweden)

    Chung Hyun-Jae

    2010-09-01

    Full Text Available Abstract Background The aim of this study was to determine the prevalence of human papillomavirus (HPV and 15 species that cause sexually transmitted infections (STIs in negative cytology. In addition, we compared the diagnostic performance of multiplex polymerase chain reaction (PCR with widely available techniques used to detect HPV. Methods We recruited 235 women of reproductive age who had negative cytology findings in a liquid-based cervical smear. STIs were identified by multiplex PCR, and HPV genotypes by multiplex PCR, hybrid capture 2, and DNA microaray; discordant results were analyzed by direct sequencing. Results Approximately 96.6% of patients with negative cytology results were positive for pathogens that cause STIs. The pathogens most frequently detected were Gardnerella vaginalis, Ureaplasma urealyticum. The incidence of HPV in negative cytology was 23.3%. Low-risk HPV infection was significantly correlated with Chalmaydia trachomatis, and high-risk HPV infection was significantly correlated with Group β streptococcus. The analytical sensitivities of the multiplex PCR and DNA microarray were higher than 80%, and the analytical specificity was nearly 100% for all tests. Conclusions Multiplex PCR yielded results that most of patients with negative cytology were positive for pathogens that cause STIs, and were more similar to that of DNA microarray, than that of hybrid capture 2 in terms of analytical sensitivity and prediction value of HPV infection.

  17. Rapid detection of threshold VEPs.

    Science.gov (United States)

    Mackay, Alison M; Bradnam, Michael S; Hamilton, Ruth

    2003-06-01

    To determine whether a one-dimensional (1D) Laplacian analysis detects steady-state visual evoked potentials (ssVEPs) faster than the standard O(z)-F(z) montage and to establish the optimum position of Laplacian reference electrodes. Twenty-two normal adults were shown reversing checks ranging from 1.5' to 60'. Three electrode montages were investigated: O(z)-F(z), LO-F(z) and a 1D Laplacian analysis of 3 occipital electrodes (2O(z)-(RO+LO)). RO and LO were placed symmetrically and horizontally about O(z). Five different locations for RO and LO were investigated. Recordings were analysed in the frequency domain and the presence (and detection time, DT) or absence of a ssVEP defined statistically. Effects of individual, reference electrode site and check size on DT and phase differences between recording montages were investigated. Laplacian analysis detected ssVEPs to small (3') checks faster than O(z)-F(z), by 12.3 and 4.1s on average with Laplacian reference electrodes at 15 and 20% of half-head circumference, respectively. The optimum position of reference electrodes was governed by the instantaneous spatial spread of the response and the noise coherence between midline and lateral electrodes. A 1D Laplacian analysis can reduce the time to statistical detection of ssVEPs compared to the traditional O(z)-F(z) recording for stimuli near the normal acuity threshold of adults. This in turn could be used to minimise the length of a VEP acuity assessment.

  18. Development of a multiplex real-time PCR assay for the rapid diagnosis of neonatal late onset sepsis.

    Science.gov (United States)

    van den Brand, Marre; Peters, Remco P H; Catsburg, Arnold; Rubenjan, Anna; Broeke, Ferdi J; van den Dungen, Frank A M; van Weissenbruch, Mirjam M; van Furth, A Marceline; Kõressaar, Triinu; Remm, Maido; Savelkoul, Paul H M; Bos, Martine P

    2014-11-01

    The diagnosis of late onset sepsis (LOS), a severe condition with high prevalence in preterm infants, is hampered by the suboptimal sensitivity and long turnaround time of blood culture. Detection of the infecting pathogen directly in blood by PCR would provide a much more timely result. Unfortunately, PCR-based assays reported so far are labor intensive and often lack direct species identification. Therefore we developed a real-time multiplex PCR assay tailored to LOS diagnosis which is easy-to-use, is applicable on small blood volumes and provides species-specific results within 4h. Species-specific PCR assays were selected from literature or developed using bioinformatic tools for the detection of the most prevalent etiologic pathogens: Enterococcus faecalis, Staphylococcus aureus, Staphylococcus spp., Streptococcus agalactiae, Escherichia coli, Pseudomonas aeruginosa, Klebsiella spp. and Serratia marcescens. The PCR assays showed 100% specificity, full coverage of the target pathogens and a limit of detection (LOD) of ≤10CFUeq./reaction. These LOD values were maintained in the multiplex format or when bacterial DNA was isolated from blood. Clinical evaluation showed high concordance between the multiplex PCR and blood culture. In conclusion, we developed a multiplex PCR that allows the direct detection of the most important bacterial pathogens causing LOS in preterm infants. Copyright © 2014 Elsevier B.V. All rights reserved.

  19. Validation of the performance of a GMO multiplex screening assay based on microarray detection

    NARCIS (Netherlands)

    Leimanis, S.; Hamels, S.; Naze, F.; Mbongolo, G.; Sneyers, M.; Hochegger, R.; Broll, H.; Roth, L.; Dallmann, K.; Micsinai, A.; Dijk, van J.P.; Kok, E.J.

    2008-01-01

    A new screening method for the detection and identification of GMO, based on the use of multiplex PCR followed by microarray, has been developed and is presented. The technology is based on the identification of quite ubiquitous GMO genetic target elements first amplified by PCR, followed by direct

  20. A novel electromagnetic apparatus for rapid multiplex single molecule force spectroscopy.

    Science.gov (United States)

    Shen, Yi; Czajkowsky, Daniel M; Li, Xiaowei; Sun, Jielin; Hu, Jun; Shao, Zhifeng

    2013-02-01

    Single-molecule force spectroscopy has revolutionized our ability to probe the details of molecular structures and interactions, but the numbers of individual measurements required for achieving a statistically reliable result can sometimes prove daunting. To overcome this problem, a number of instruments have recently been developed that are capable of monitoring the behavior of tens of individual biomolecules simultaneously. In this work, we have constructed a novel electromagnetic apparatus for multiplex single molecule force measurements utilizing magnetic microspheres. In this system, the magnetic field is generated with an electron-lens of an electron microscope mated with a high voltage flash light circuit to rapidly attain a stable magnetic field. We show that this instrument can generate a uniform magnetic force of up to -20 pN within 5 ms, over a region spanning 1 mm. The successful application of this apparatus to the force-dependent extension of dsDNA fully validates this approach. Furthermore, the lens-like design of the pole piece is fully compatible with optical imaging, thus allowing for the integration of single molecule fluorescence capabilities that should make this system a particularly powerful apparatus for multi-dimensional characterization of fast processes within interacting single molecules.

  1. Detection and typing of highly pathogenic porcine reproductive and respiratory syndrome virus by multiplex real-time rt-PCR.

    Directory of Open Access Journals (Sweden)

    Kerstin Wernike

    Full Text Available Porcine reproductive and respiratory syndrome (PRRS causes economic losses in the pig industry worldwide, and PRRS viruses (PRRSV are classified into the two distinct genotypes "North American (NA, type 2" and "European (EU, type 1". In 2006, a highly pathogenic NA strain of PRRSV (HP-PRRSV, characterized by high fever as well as high morbidity and mortality, emerged in swine farms in China. Therefore, a real-time reverse transcription polymerase chain reaction (RT-qPCR assay specific for HP-PRRSV was developed and combined with type 1- and type 2-specific RT-qPCR systems. Furthermore, an internal control, based on a heterologous RNA, was successfully introduced. This final multiplex PRRSV RT-qPCR, detecting and typing PRRSV, had an analytical sensitivity of less than 200 copies per µl for the type 1-assay and 20 copies per µl for the type 2- and HP assays and a high diagnostic sensitivity. A panel of reference strains and field isolates was reliably detected and samples from an animal trial with a Chinese HP-PRRS strain were used for test validation. The new multiplex PRRSV RT-qPCR system allows for the first time the highly sensitive detection and rapid differentiation of PRRSV of both genotypes as well as the direct detection of HP-PRRSV.

  2. Detection of diarrheagenic Escherichia coli by use of melting-curve analysis and real-time multiplex PCR.

    Science.gov (United States)

    Guion, Chase E; Ochoa, Theresa J; Walker, Christopher M; Barletta, Francesca; Cleary, Thomas G

    2008-05-01

    Diarrheagenic Escherichia coli strains are important causes of diarrhea in children from the developing world and are now being recognized as emerging enteropathogens in the developed world. Current methods of detection are too expensive and labor-intensive for routine detection of these organisms to be practical. We developed a real-time fluorescence-based multiplex PCR for the detection of all six of the currently recognized classes of diarrheagenic E. coli. The primers were designed to specifically amplify eight different virulence genes in the same reaction: aggR for enteroaggregative E. coli, stIa/stIb and lt for enterotoxigenic E. coli, eaeA for enteropathogenic E. coli and Shiga toxin-producing E. coli (STEC), stx(1) and stx(2) for STEC, ipaH for enteroinvasive E. coli, and daaD for diffusely adherent E. coli (DAEC). Eighty-nine of ninety diarrheagenic E. coli and 36/36 nonpathogenic E. coli strains were correctly identified using this approach (specificity, 1.00; sensitivity, 0.99). The single false negative was a DAEC strain. The total time between preparation of DNA from E. coli colonies on agar plates and completion of PCR and melting-curve analysis was less than 90 min. The cost of materials was low. Melting-point analysis of real-time multiplex PCR is a rapid, sensitive, specific, and inexpensive method for detection of diarrheagenic E. coli.

  3. Detection of Diarrheagenic Escherichia coli by Use of Melting-Curve Analysis and Real-Time Multiplex PCR ▿

    Science.gov (United States)

    Guion, Chase E.; Ochoa, Theresa J.; Walker, Christopher M.; Barletta, Francesca; Cleary, Thomas G.

    2008-01-01

    Diarrheagenic Escherichia coli strains are important causes of diarrhea in children from the developing world and are now being recognized as emerging enteropathogens in the developed world. Current methods of detection are too expensive and labor-intensive for routine detection of these organisms to be practical. We developed a real-time fluorescence-based multiplex PCR for the detection of all six of the currently recognized classes of diarrheagenic E. coli. The primers were designed to specifically amplify eight different virulence genes in the same reaction: aggR for enteroaggregative E. coli, stIa/stIb and lt for enterotoxigenic E. coli, eaeA for enteropathogenic E. coli and Shiga toxin-producing E. coli (STEC), stx1 and stx2 for STEC, ipaH for enteroinvasive E. coli, and daaD for diffusely adherent E. coli (DAEC). Eighty-nine of ninety diarrheagenic E. coli and 36/36 nonpathogenic E. coli strains were correctly identified using this approach (specificity, 1.00; sensitivity, 0.99). The single false negative was a DAEC strain. The total time between preparation of DNA from E. coli colonies on agar plates and completion of PCR and melting-curve analysis was less than 90 min. The cost of materials was low. Melting-point analysis of real-time multiplex PCR is a rapid, sensitive, specific, and inexpensive method for detection of diarrheagenic E. coli. PMID:18322059

  4. Scalable multiplexed detector system for high-rate telecom-band single-photon detection.

    Science.gov (United States)

    Brida, G; Degiovanni, I P; Piacentini, F; Schettini, V; Polyakov, S V; Migdall, A

    2009-11-01

    We present an actively multiplexed photon-counting detection system at telecom wavelengths that overcomes the difficulties of photon-counting at high rates. We find that for gated detectors, the heretofore unconsidered deadtime associated with the detector gate is a critical parameter, that limits the overall scalability of the scheme to just a few detectors. We propose and implement a new scheme that overcomes this problem and restores full scalability that allows an order of magnitude improvement with systems with as few as 4 detectors. When using just two multiplexed detectors, our experimental results show a 5x improvement over a single detector and a greater than 2x improvement over multiplexed schemes that do not consider gate deadtime.

  5. Polarization-interleave-multiplexed discrete multi-tone modulation with direct detection utilizing MIMO equalization.

    Science.gov (United States)

    Zhou, Xian; Zhong, Kangping; Gao, Yuliang; Sui, Qi; Dong, Zhenghua; Yuan, Jinhui; Wang, Liang; Long, Keping; Lau, Alan Pak Tao; Lu, Chao

    2015-04-06

    Discrete multi-tone (DMT) modulation is an attractive modulation format for short-reach applications to achieve the best use of available channel bandwidth and signal noise ratio (SNR). In order to realize polarization-multiplexed DMT modulation with direct detection, we derive an analytical transmission model for dual polarizations with intensity modulation and direct diction (IM-DD) in this paper. Based on the model, we propose a novel polarization-interleave-multiplexed DMT modulation with direct diction (PIM-DMT-DD) transmission system, where the polarization de-multiplexing can be achieved by using a simple multiple-input-multiple-output (MIMO) equalizer and the transmission performance is optimized over two distinct received polarization states to eliminate the singularity issue of MIMO demultiplexing algorithms. The feasibility and effectiveness of the proposed PIM-DMT-DD system are investigated via theoretical analyses and simulation studies.

  6. A nested, multiplex, PCR assay for the simultaneous detection and differentiation of Entamoeba histolytica and Entamoeba dispar in faeces.

    Science.gov (United States)

    Evangelopoulos, A; Spanakos, G; Patsoula, E; Vakalis, N; Legakis, N

    2000-04-01

    The detection of and differentiation between Entamoeba histolytica and Entamoeba dispar are of great importance, both for diagnosis and for epidemiological studies. Most PCR-based methods for the discrimination of these two species employ complex procedures for DNA extraction and require different protocols for E. histolytica and E. dispar, leading to relatively high expenditure, labour costs and turnaround times. A simple, rapid, cost-effective and yet sensitive and specific multiplex PCR technique has now been developed for the simultaneous detection and differentiation of E. histolytica and E. dispar in faecal samples. The detection limit is 200 trophozoites of E. dispar or 1000 trophozoites of E. histolytica/g stool sample. The sensitivity of the assay remains practically unchanged, even in the presence of 20,000 trophozoites of the other species/g stool sample. Thus, this technique may also easily reveal mixed infections, without the danger of misdiagnosis caused by one strain displacing the other in culture.

  7. High throughput multiplex PCR and probe-based detection with Luminex beads for seven intestinal parasites.

    Science.gov (United States)

    Taniuchi, Mami; Verweij, Jaco J; Noor, Zannatun; Sobuz, Shihab U; Lieshout, Lisette van; Petri, William A; Haque, Rashidul; Houpt, Eric R

    2011-02-01

    Polymerase chain reaction (PCR) assays for intestinal parasites are increasingly being used on fecal DNA samples for enhanced specificity and sensitivity of detection. Comparison of these tests against microscopy and copro-antigen detection has been favorable, and substitution of PCR-based assays for the ova and parasite stool examination is a foreseeable goal for the near future. One challenge is the diverse list of protozoan and helminth parasites. Several existing real-time PCR assays for the major intestinal parasites-Cryptosporidium spp., Giardia intestinalis, Entamoeba histolytica, Ancylostoma duodenale, Ascaris lumbricoides, Necator americanus, and Strongyloides stercoralis-were adapted into a high throughput protocol. The assay involves two multiplex PCR reactions, one with specific primers for the protozoa and one with specific primers for the helminths, after which PCR products are hybridized to beads linked to internal oligonucleotide probes and detected on a Luminex platform. When compared with the parent multiplex real-time PCR assays, this multiplex PCR-bead assay afforded between 83% and 100% sensitivity and specificity on a total of 319 clinical specimens. In conclusion, this multiplex PCR-bead protocol provides a sensitive diagnostic screen for a large panel of intestinal parasites.

  8. A cytolethal distending toxin gene-based multiplex PCR assay for detection of Campylobacter spp. in stool specimens and comparison with culture method.

    Science.gov (United States)

    Shiramaru, Sachi; Asakura, Masahiro; Inoue, Haruna; Nagita, Akira; Matsuhisa, Akio; Yamasaki, Shinji

    2012-07-01

    In this study, we evaluated the applicability of cytolethal distending toxin (cdt) gene-based species-specific multiplex PCR for the direct detection and identification of Campylobacter jejuni, C. coli and C. fetus from stool specimens of patients with gastroenteritis in comparison to culture methods. A total of 711 stool specimens were examined for the isolation or detection of campylobacters by using Skirrow's selective agar culture plates, a filtration method and the multiplex PCR assay. Forty-one and 36 C. jejuni strains were isolated by culture and filtration methods, respectively. In addition, 2 and 3 C. coli strains were isolated by Skirrow and the filtration methods, respectively. However, when the multiplex PCR was employed, the cdtB genes of C. jejuni and C. coli were detected in 45 and 4 stool samples, respectively, and 9 C. jejuni PCR-positive samples by multiplex PCR were negative by culture method. Sequence analysis of the PCR products obtained from 8 stool specimens from which campylobacters were not isolated by culture method but the sequences exactly matched with that of the cdtB gene of C. jejuni strain 81-176. None of the remaining stool samples which were culture negative for campylobacters produced any amplicon. Stool samples were defined as Campylobacter-positive if detected by any method. The sensitivity of the multiplex PCR was 83%, which was higher than Skirrow (74%) and filtration method (66%). These data indicate that cdtB gene-based multiplex PCR is a rapid and more sensitive method to identify the most important species of Campylobacter for human diseases. (248).

  9. Rapid and inexpensive body fluid identification by RNA profiling-based multiplex High Resolution Melt (HRM) analysis.

    Science.gov (United States)

    Hanson, Erin K; Ballantyne, Jack

    2013-01-01

    Positive identification of the nature of biological material present on evidentiary items can be crucial for understanding the circumstances surrounding a crime. However, traditional protein-based methods do not permit the identification of all body fluids and tissues, and thus molecular based strategies for the conclusive identification of all forensically relevant biological fluids and tissues need to be developed. Messenger RNA (mRNA) profiling is an example of such a molecular-based approach. Current mRNA body fluid identification assays involve capillary electrophoresis (CE) or quantitative RT-PCR (qRT-PCR) platforms, each with its own limitations. Both platforms require the use of expensive fluorescently labeled primers or probes. CE-based assays require separate amplification and detection steps thus increasing the analysis time. For qRT-PCR assays, only 3-4 markers can be included in a single reaction since each requires a different fluorescent dye. To simplify mRNA profiling assays, and reduce the time and cost of analysis, we have developed single- and multiplex body fluid High Resolution Melt (HRM) assays for the identification of common forensically relevant biological fluids and tissues. The incorporated biomarkers include IL19 (vaginal secretions), IL1F7 (skin), ALAS2 (blood), MMP10 (menstrual blood), HTN3 (saliva) and TGM4 (semen).  The HRM assays require only unlabeled PCR primers and a single saturating intercalating fluorescent dye (Eva Green). Each body-fluid-specific marker can easily be identified by the presence of a distinct melt peak. Usually, HRM assays are used to detect variants or isoforms for a single gene target. However, we have uniquely developed duplex and triplex HRM assays to permit the simultaneous detection of multiple targets per reaction. Here we describe the development and initial performance evaluation of the developed HRM assays. The results demonstrate the potential use of HRM assays for rapid, and relatively inexpensive

  10. Evaluation of a modular multiplex-PCR methicillin-resistant Staphylococcus aureus detection assay adapted for mecC detection

    NARCIS (Netherlands)

    Becker, Karsten; Larsen, Anders R; Skov, Robert L; Paterson, Gavin K; Holmes, Mark A; Sabat, Artur J; Friedrich, Alexander W; Köck, Robin; Peters, Georg; Kriegeskorte, André

    A mecC (mecA(LGA251))-adapted multiplex PCR-based methicillin-resistant Staphylococcus aureus (MRSA) detection assay was evaluated using an international, spa-typed Staphylococcus aureus collection comprising 51 mecC-positive MRSA, 240 mecA-positive MRSA, and 50 mecA-and mecC-negative

  11. High throughput multiplex-PCR for direct detection and diagnosis of dermatophyte species, Candida albicans and Candida parapsilosis in clinical specimen.

    Science.gov (United States)

    Vahidnia, Ali; Bekers, Wouter; Bliekendaal, Harry; Spaargaren, Joke

    2015-06-01

    We have developed and validated a multiplex-PCR method for detection of dermatophyte spp., Candida albicans and parapsilosis for routine diagnostics. Our m-PCR showed excellent concordance with culture results in 475 clinical samples. Through the rapid diagnosis by our m-PCR, clinicians are able to initiate adequate antimycotic therapy much earlier. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. High-throughput biosensors for multiplexed foodborne pathogen detection

    Science.gov (United States)

    Incidental contamination of foods by harmful bacteria (such as E. coli and Salmonella) and the toxins that they produce is a serious threat to public health and the economy in the United States. The presence of such bacteri and toxins in foods must be rapidly determined at various stages of food pr...

  13. Impact of Early Detection of Respiratory Viruses by Multiplex PCR Assay on Clinical Outcomes in Adult Patients.

    Science.gov (United States)

    Rappo, Urania; Schuetz, Audrey N; Jenkins, Stephen G; Calfee, David P; Walsh, Thomas J; Wells, Martin T; Hollenberg, James P; Glesby, Marshall J

    2016-08-01

    Rapid and definitive diagnosis of viral respiratory infections is imperative in patient triage and management. We compared the outcomes for adult patients with positive tests for respiratory viruses at a tertiary care center across two consecutive influenza seasons (winters of 2010-2011 and 2012). Infections were diagnosed by conventional methods in the first season and by multiplex PCR (FilmArray) in the second season. FilmArray decreased the time to diagnosis of influenza compared to conventional methods (median turnaround times of 1.7 h versus 7.7 h, respectively; P = 0.015); FilmArray also decreased the time to diagnosis of non-influenza viruses (1.5 h versus 13.5 h, respectively; P FilmArray was associated with significantly lower odds ratios (ORs) for admission (P = 0.046), length of stay (P = 0.040), duration of antimicrobial use (P = 0.032), and number of chest radiographs (P = 0.005), when controlling for potential confounders. We conclude that the rapid turnaround time, multiplex nature of the test (allowing simultaneous detection of an array of viruses), and superior sensitivity of FilmArray may improve the evaluation and management of patients suspected of having respiratory virus infections. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  14. Rapid analysis of Saccharomyces cerevisiae genome rearrangements by multiplex ligation-dependent probe amplification.

    Directory of Open Access Journals (Sweden)

    Jason E Chan

    Full Text Available Aneuploidy and gross chromosomal rearrangements (GCRs can lead to genetic diseases and the development of cancer. We previously demonstrated that introduction of the repetitive retrotransposon Ty912 onto a nonessential chromosome arm of Saccharomyces cerevisiae led to increased genome instability predominantly due to increased rates of formation of monocentric nonreciprocal translocations. In this study, we adapted Multiplex Ligation-dependent Probe Amplification (MLPA to analyze a large numbers of these GCRs. Using MLPA, we found that the distribution of translocations induced by the presence of Ty912 in a wild-type strain was nonrandom and that the majority of these translocations were mediated by only six translocation targets on four different chromosomes, even though there were 254 potential Ty-related translocation targets in the S. cerevisiae genome. While the majority of Ty912-mediated translocations resulted from RAD52-dependent recombination, we observed a number of nonreciprocal translocations mediated by RAD52-independent recombination between Ty1 elements. The formation of these RAD52-independent translocations did not require the Rad51 or Rad59 homologous pairing proteins or the Rad1-Rad10 endonuclease complex that processes branched DNAs during recombination. Finally, we found that defects in ASF1-RTT109-dependent acetylation of histone H3 lysine residue 56 (H3K56 resulted in increased accumulation of both GCRs and whole-chromosome duplications, and resulted in aneuploidy that tended to occur simultaneously with GCRs. Overall, we found that MLPA is a versatile technique for the rapid analysis of GCRs and can facilitate the genetic analysis of the pathways that prevent and promote GCRs and aneuploidy.

  15. The increasing application of multiplex nucleic acid detection tests to the diagnosis of syndromic infections.

    Science.gov (United States)

    Gray, J; Coupland, L J

    2014-01-01

    On 14 January 2013, the US Food and Drug Administration (FDA) announced permission for a multiplex nucleic acid test, the xTAG® Gastrointestinal Pathogen Panel (GPP) (Luminex Corporation, USA), which simultaneously detects 11 common viral, bacterial and parasitic causes of infectious gastroenteritis, to be marketed in the USA. This announcement reflects the current move towards the development and commercialization of detection technologies based on nucleic acid amplification techniques for diagnosis of syndromic infections. We discuss the limitations and advantages of nucleic acid amplification techniques and the recent advances in Conformité Européene - in-vitro diagnostic (CE-IVD)-approved multiplex real-time PCR kits for the simultaneous detection of multiple targets within the clinical diagnostics market.

  16. Simultaneous Detection of Genetically Modified Organisms in a Mixture by Multiplex PCR-Chip Capillary Electrophoresis.

    Science.gov (United States)

    Patwardhan, Supriya; Dasari, Srikanth; Bhagavatula, Krishna; Mueller, Steffen; Deepak, Saligrama Adavigowda; Ghosh, Sudip; Basak, Sanjay

    2015-01-01

    An efficient PCR-based method to trace genetically modified food and feed products is in demand due to regulatory requirements and contaminant issues in India. However, post-PCR detection with conventional methods has limited sensitivity in amplicon separation that is crucial in multiplexing. The study aimed to develop a sensitive post-PCR detection method by using PCR-chip capillary electrophoresis (PCR-CCE) to detect and identify specific genetically modified organisms in their genomic DNA mixture by targeting event-specific nucleotide sequences. Using the PCR-CCE approach, novel multiplex methods were developed to detect MON531 cotton, EH 92-527-1 potato, Bt176 maize, GT73 canola, or GA21 maize simultaneously when their genomic DNAs in mixtures were amplified using their primer mixture. The repeatability RSD (RSDr) of the peak migration time was 0.06 and 3.88% for the MON531 and Bt176, respectively. The RSD (RSDR) of the Cry1Ac peak ranged from 0.12 to 0.40% in multiplex methods. The method was sensitive in resolving amplicon of size difference up to 4 bp. The PCR-CCE method is suitable to detect multiple genetically modified events in a composite DNA sample by tagging their event specific sequences.

  17. Simultaneous Detection of Mycoplasma pneumoniae, Mycoplasma hominis and Mycoplasma arthritidis in Synovial Fluid of Patients with Rheumatoid Arthritis by Multiplex PCR.

    Science.gov (United States)

    Ataee, Ramezan Ali; Golmohammadi, Reza; Alishiri, Gholam Hossein; Mirnejad, Reza; Najafi, Ali; Esmaeili, Davood; Jonaidi-Jafari, Nematollah

    2015-06-01

    It has been recognized that infectious agents, such as different bacteria and viruses, may play a role in the developing of rheumatoid arthritis (RA). Recently, the mycoplasma species has been implicated in the pathogenesis of RA. The aim of this study was to design a multiplex PCR for rapid and simultaneous detection of Mycoplasma pneumoniae, Mycoplasma hominis, and Mycoplasma arthritidis in the synovial fluid of patients with rheumatoid arthritis (RA). A total of 131 synovial fluid (SF) samples from patients with RA were assayed. Mycoplasma pneumoniae (ATCC: 29342), M. hominis (native strain), and the synthetic complete genome of M. arthritidis mitogen (MAM) superantigen were used as controls. All SF samples were subjected to DNA extraction separately and multiplex PCR was performed. The PCR products were confirmed by sequencing. The designed multiplex PCR was able to detect M. pneumoniae, M. hominis, and M. arthritidis in the SF of patients with RA with a frequency of 30 (22.9%), 23 (17.5%) and 13 (9.9%), respectively. In this study, the overall detection of the Mycoplasma species in RA patients was 53.4%; thus, we recommend the application of multiplex PCR assays when searching for a specific anti mycoplasma treatment for RA patients.

  18. Evaluation of a multiplex selective enrichment broth SEL for simultaneous detection of injured Salmonella, Escherichia coli O157:H7 and Listeria monocytogenes.

    Science.gov (United States)

    Suo, Biao; Wang, Yuexia

    2013-01-01

    Although many rapid and high throughput molecular methods have been developed in the recent years for the multiplex detection of foodborne pathogens, the simultaneous recovery and enrichment of sublethally injured cells is still a problem that needs to be considered. Combined with previous established multiplex real-time PCR assay, the capability of simultaneous recovery and enrichment of sublethally injured Salmonella, E. coli O157:H7 and L. monocytogenes cells was evaluated in a multiplex selective enrichment broth SEL. The injured cells were obtained by heat shock. After evaluation of different procedures, 1 h of recovery period prior to 20 h of enrichment was proved to be necessary for the detection of less than 10 CFU/5 mL broth of injured L. monocytogenes. When the detection method was applied to artificially contaminated ground beef, all the three injured pathogens could be simultaneously detected without discrimination by real-time PCR combined with SEL broth, the detection limit was SEL broth herein appears to be a promising tool for high-throughput screening of a large number of processed food samples, which require either single or multiple pathogen detection. More important, the sublethally injured foodborne pathogen cells were also detectable.

  19. Multiplex real-time PCR assays for detection of four seedborne spinach pathogens.

    Science.gov (United States)

    Feng, C; Mansouri, S; Bluhm, B H; du Toit, L J; Correll, J C

    2014-08-01

    To develop multiplex TaqMan real-time PCR assays for detection of spinach seedborne pathogens that cause economically important diseases on spinach. Primers and probes were designed from conserved sequences of the internal transcribed spacer (for Peronospora farinosa f. sp. spinaciae and Stemphylium botryosum), the intergenic spacer (for Verticillium dahliae) and the elongation factor 1 alpha (for Cladosporium variabile) regions of DNA. The TaqMan assays were tested on DNA extracted from numerous isolates of the four target pathogens, as well as a wide range of nontarget, related fungi or oomycetes and numerous saprophytes commonly found on spinach seed. Multiplex real-time PCR assays were evaluated by detecting two or three target pathogens simultaneously. Singular and multiplex real-time PCR assays were also applied to DNA extracted from bulked seed and single spinach seed. The real-time PCR assays were species-specific and sensitive. Singular or multiplex real-time PCR assays could detect target pathogens from both bulked seed samples as well as single spinach seed. The freeze-blotter assay that is currently routinely used in the spinach seed industry to detect and quantify three fungal seedborne pathogens of spinach (C. variabile, S. botryosum and V. dahliae) is quite laborious and takes several weeks to process. The real-time PCR assays developed in this study are more sensitive and can be completed in a single day. As the assays can be applied easily for routine seed inspections, these tools could be very useful to the spinach seed industry. © 2014 The Society for Applied Microbiology.

  20. Simultaneous differential detection of Chlamydophila abortus, Chlamydophila pecorum and Coxiella burnetii from aborted ruminant's clinical samples using multiplex PCR

    Directory of Open Access Journals (Sweden)

    Rodolakis Annie

    2009-07-01

    Full Text Available Abstract Background Chlamydiosis and Q fever, two zoonosis, are important causes of ruminants' abortion around the world. They are caused respectively by strictly intracellular and Gram negative bacterium Chlamydophila abortus (Cp. abortus and Coxiella burnetii (C. burnetii. Chlamydophila pecorum (Cp. pecorum is commonly isolated from the digestive tract of clinically inconspicuous ruminants but the abortive and zoonotic impact of this bacterium is still unknown because Cp. pecorum is rarely suspected in abortion cases of small ruminants. We have developed a multiplex PCR (m-PCR for rapid simultaneous differential detection of Cp. abortus, Cp. pecorum and C. burnetii in clinical samples taken from infected animals. Results Specific PCR primers were designed and a sensitive and specific m-PCR was developed to detect simultaneously, in one tube reaction, three specific fragments of 821, 526 and 687-bp long for Cp. abortus, Cp. pecorum and C. burnetii respectively. This m-PCR assay was performed on 253 clinical samples taken from infected ruminant's flocks that have showed problems of abortion diseases. Thus, 67 samples were infected by either one of the three pathogens: 16 (13 vaginal swabs and 3 placentas were positive for Cp. abortus, 2 were positive for Cp. pecorum (1 vaginal swab and 1 placenta and 49 samples (33 vaginal swabs, 11 raw milks, 4 faeces and 1 placenta were positive for C. burnetii. Two vaginal swabs were m-PCR positive of both Cp. abortus and C. burnetii and none of the tested samples was shown to be infected simultaneously with the three pathogens. Conclusion We have successfully developed a rapid multiplex PCR that can detect and differentiate Cp. abortus, Cp. pecorum and C. burnetii; with a good sensitivity and specificity. The diagnosis of chlamydiosis and Q fever may be greatly simplified and performed at low cost. In addition, the improvement in diagnostic techniques will enhance our knowledge regarding the prevalence and

  1. Label-free, real-time multiplexed DNA detection using fluorescent conjugated polymers.

    Science.gov (United States)

    Zheng, Weiming; He, Lin

    2009-03-18

    A label-free, multiplexed DNA assay using fluorescent conjugated polymers as a detection probe to illustrate hybridization on metallic striped nanorods is demonstrated. Different DNA capture probes were encoded by the different reflectivities of Au and Ag stripe patterns. Successful DNA hybridization induced an optically detectable conformational change in conjugated polythiophene derivatives from forming single-stranded DNA-polymer complexes to forming double-stranded DNA-polymer ones. The results show attomole detection sensitivity and single-mutation specificity comparable to those of single-element assays but with much improved throughput.

  2. Non-labeling multiplex surface enhanced Raman scattering (SERS) detection of volatile organic compounds (VOCs)

    DEFF Research Database (Denmark)

    Wong, Chi Lok; Dinish, U. S.; Schmidt, Michael Stenbæk

    2014-01-01

    -pillars. In this experiment, detections of acetone and ethanol vapor at different concentrations were demonstrated. The detection limits were found to be 0.0017 ng and 0.0037 ng for ethanol and acetone vapor molecules respectively. Our approach is a non-labeling method such that it does not require the incorporation of any...... of acetone and ethanol vapor was also successfully demonstrated. The vibrational fingerprints of molecular structures provide specific Raman peaks for different VOCs contents. To the best of our knowledge, this is the first multiplex VOCs detection using SERS. We believe that this work may lead to a portable...

  3. A novel multiplex PCR for the simultaneous detection of Salmonella enterica and Shigella species

    Directory of Open Access Journals (Sweden)

    M. Radhika

    2014-06-01

    Full Text Available Salmonella enterica and Shigella species are commonly associated with food and water borne infections leading to gastrointestinal diseases. The present work was undertaken to develop a sensitive and reliable PCR based detection system for simultaneous detection of Salmonella enterica and Shigella at species level. For this the conserved regions of specific genes namely ipaH1, ipaH, wbgZ, wzy and invA were targeted for detection of Shigella genus, S. flexneri, S. sonnei, S. boydii and Salmonella enterica respectively along with an internal amplification control (IAC. The results showed that twenty Salmonella and eleven Shigella spp., were accurately identified by the assay without showing non-specificity against closely related other Enterobacteriaceae organisms and also against other pathogens. Further evaluation of multiplex PCR was undertaken on 50 natural samples of chicken, eggs and poultry litter and results compared with conventional culture isolation and identification procedure. The multiplex PCR identified the presence of Salmonella and Shigella strains with a short pre-enrichment step of 5 h in peptone water and the same samples were processed by conventional procedures for comparison. Therefore, this reported multiplex PCR can serve as an alternative to the tedious time-consuming procedure of culture and identification in food safety laboratories.

  4. Spectrally efficient polarization multiplexed direct-detection OFDM system without frequency gap.

    Science.gov (United States)

    Wei, Chia-Chien; Zeng, Wei-Siang; Lin, Chun-Ting

    2016-01-25

    We experimentally demonstrate a spectrally efficient direct-detection orthogonal frequency-division multiplexing (DD-OFDM) system. In addition to polarization-division multiplexing, removing the frequency gap further improves the spectral efficiency of the OFDM system. The frequency gap between a reference carrier and OFDM subcarriers avoids subcarrier-to-subcarrier beating interference (SSBI) in traditional DD-OFDM systems. Without dynamic polarization control, the resulting interference after square-law direct detection in the proposed gap-less system is polarization-dependent and composed of linear inter-carrier interference (ICI) and nonlinear SSBI. Thus, this work proposes an iterative multiple-input multiple-output detection scheme to remove the mixed polarization-dependent interference. Compared to the previous scheme, which only removes ICI, the proposed scheme can further eliminate SSBI to achieve the improvement of ∼ 7 dB in signal-to-noise ratio. Without the need for polarization control, we successfully utilize 7-GHz bandwidth to transmit a 39.5-Gbps polarization multiplexed OFDM signal over 100 km.

  5. Detection of stress resistance genes in transgenic maize by multiplex and touchdown polymerase chain reaction

    Directory of Open Access Journals (Sweden)

    Bannikova M. A.

    2015-10-01

    Full Text Available Aim. To develop a methodology for detection of the genes of resistance to the stress factors in transgenic maize by multiplex (mPCR and touchdown polymerase chain reactions. Methods. isolation of total DNA by CTAB method, purification of DNA from RNA and proteins, electrophoresis of total DNA and amplification products in agarose gel, polymerase chain reaction. Results. The protocol of multiplex and touchdown polymerase chain reactions has been developed for simultaneous verification of the quality of total DNA extracted from the studied maize plant samples and detection of the following genes that determine resistance to the stress factors in the transgenic maize and maize transformation events: BT176, MON810, MON88017, DAS1507, DAS59122, MIR604, GA21, NK603 (mPCR, Bt11, MON863, MON89034, T25 (touchdown PCR. The multiplex PCR and touchdown PCR were developed using the reference samples. Conclusions. The proposed protocol of mPCR and touchdown PCR reactions can be used for mass analysis of maize samples to detect the genes of tolerance/resistance to herbicides and genes of resistance to insects reliably, authentically, quickly and cheaply.

  6. A novel multiplex PCR for the simultaneous detection of Salmonella enterica and Shigella species.

    Science.gov (United States)

    Radhika, M; Saugata, Majumder; Murali, H S; Batra, H V

    2014-01-01

    Salmonella enterica and Shigella species are commonly associated with food and water borne infections leading to gastrointestinal diseases. The present work was undertaken to develop a sensitive and reliable PCR based detection system for simultaneous detection of Salmonella enterica and Shigella at species level. For this the conserved regions of specific genes namely ipaH1, ipaH, wbgZ, wzy and invA were targeted for detection of Shigella genus, S. flexneri, S. sonnei, S. boydii and Salmonella enterica respectively along with an internal amplification control (IAC). The results showed that twenty Salmonella and eleven Shigella spp., were accurately identified by the assay without showing non-specificity against closely related other Enterobacteriaceae organisms and also against other pathogens. Further evaluation of multiplex PCR was undertaken on 50 natural samples of chicken, eggs and poultry litter and results compared with conventional culture isolation and identification procedure. The multiplex PCR identified the presence of Salmonella and Shigella strains with a short pre-enrichment step of 5 h in peptone water and the same samples were processed by conventional procedures for comparison. Therefore, this reported multiplex PCR can serve as an alternative to the tedious time-consuming procedure of culture and identification in food safety laboratories.

  7. Semiconductor quantum dots for multiplexed bio-detection on solid-state microarrays.

    Science.gov (United States)

    Rousserie, Gilles; Sukhanova, Alyona; Even-Desrumeaux, Klervi; Fleury, Fabrice; Chames, Patrick; Baty, Daniel; Oleinikov, Vladimir; Pluot, Michel; Cohen, Jacques H M; Nabiev, Igor

    2010-04-01

    Understanding cellular systems requires identification and analysis of their multiple components and determination of how they act together and are regulated. Microarray technology is one of the few tools that is able to solve such problems. It is based on high-throughput recognition of a target to the probe and has the potential to simultaneously measure the presence of numerous molecules in multiplexed tests, all contained in a small drop of test fluid. Microarrays allow the parallel analysis of genomic or proteomic content in healthy versus disease-affected or altered tissues or cells. The signal read-out from the microarrays is done with organic dyes which often suffer of photobleaching, low brightness and background fluorescence. Recent data show that the use of fluorescent nanocrystals named "quantum dots" (QDs) allows to push these limits away. QDs are sufficiently bright to be detected as individual particles, extremely resistant against photobleaching and provide unique possibilities for multiplexing, thus supplying the microarray technology with a novel read-out option enabling the sensitivity of detection to reach the single-molecule level. This paper reviews QDs applications to microarray-based detection and demonstrates how the combination of microarray and QDs technologies may increase sensitivity and highly parallel capacities of multiplexed microarrays. Such a combination should provide the breakthrough results in drug discovery, cancer diagnosis and establish new therapeutic approaches through the identification of binding target molecules and better understanding of cell signalling pathways.

  8. Development of a One-Step Multiplex PCR Assay for Differential Detection of Major Mycobacterium Species.

    Science.gov (United States)

    Chae, Hansong; Han, Seung Jung; Kim, Su-Young; Ki, Chang-Seok; Huh, Hee Jae; Yong, Dongeun; Koh, Won-Jung; Shin, Sung Jae

    2017-09-01

    The prevalence of tuberculosis continues to be high, and nontuberculous mycobacterial (NTM) infection has also emerged worldwide. Moreover, differential and accurate identification of mycobacteria to the species or subspecies level is an unmet clinical need. Here, we developed a one-step multiplex PCR assay using whole-genome analysis and bioinformatics to identify novel molecular targets. The aims of this assay were to (i) discriminate between the Mycobacterium tuberculosis complex (MTBC) and NTM using rv0577 or RD750, (ii) differentiate M. tuberculosis ( M. tuberculosis ) from MTBC using RD9, (iii) selectively identify the widespread M. tuberculosis Beijing genotype by targeting mtbk_20680 , and (iv) simultaneously detect five clinically important NTM ( M. avium , M. intracellulare , M. abscessus , M. massiliense , and M. kansasii ) by targeting IS 1311 , DT1, mass_3210 , and mkan_rs12360 An initial evaluation of the multiplex PCR assay using reference strains demonstrated 100% specificity for the targeted Mycobacterium species. Analytical sensitivity ranged from 1 to 10 pg for extracted DNA and was 10 3 and 10 4 CFU for pure cultures and nonhomogenized artificial sputum cultures, respectively, of the targeted species. The accuracy of the multiplex PCR assay was further evaluated using 55 reference strains and 94 mycobacterial clinical isolates. Spoligotyping, multilocus sequence analysis, and a commercial real-time PCR assay were employed as standard assays to evaluate the multiplex PCR assay with clinical M. tuberculosis and NTM isolates. The PCR assay displayed 100% identification agreement with the standard assays. Our multiplex PCR assay is a simple, convenient, and reliable technique for differential identification of MTBC, M. tuberculosis , M. tuberculosis Beijing genotype, and major NTM species. Copyright © 2017 American Society for Microbiology.

  9. A Multiplex real-time PCR for detection of Mycoplasma gallisepticum and Mycoplasma synoviae in clinical samples from Brazilian commercial poultry flocks

    Directory of Open Access Journals (Sweden)

    Aline Padilha Fraga

    2013-01-01

    Full Text Available Mycoplasma gallisepticum (MS and Mycoplasma synoviae (MS are important avian pathogens and cause economic losses to the poultry industry. Molecular biology techniques are currently used for a rapid detection of these pathogens and the adoption of control measures of the diseases. The aim of this study was to develop and validate a technique for simultaneous detection of MG and MS by multiplex real time polymerase chain reaction (PCR. The complete assay (Multiplex MGMS was designed with primers and probes specific for each pathogen and developed to be carried out in a single tube reaction. Vaccines, MG and MS isolates and DNA from other Mycoplasma species were used for the development and validation of the method. Further, 78 pooled clinical samples from different poultry flocks in Brazil were obtained and used to determine the sensitivity and specificity of the technique in comparison to 2 real time PCR assays specific for MG (MG PCR and MS (MS PCR. The results demonstrated an agreement of 100% (23 positive and 44 negative samples between Multiplex MGMS and MG PCR in the analysis of 67 samples from MG positive and negative poultry flocks, and an agreement of 96.9% between Multiplex MGMS and MS PCR in the analysis of 64 samples from MS positive and negative poultry flocks. Considering the single amplification tests as the gold standard, the Multiplex MGMS showed 100% of specificity and sensitivity in the MG analysis and 94.7% sensitivity and 100% specificity in the MS analysis. This new assay could be used for rapid analysis of MG and MS in the poultry industry laboratories.

  10. Ultrarapid mutation detection by multiplex, solid-phase chemical cleavage

    Energy Technology Data Exchange (ETDEWEB)

    Rowley, G.; Saad, S.; Giannelli, F.; Green, P.M. [Guy`s & St. Thomas`s Hospitals, London (United Kingdom)

    1995-12-10

    The chemical cleavage of mismatches in heteroduplexes formed by probe and test DNA detects and locates any sequence change in long DNA segments ({approximately}1.8 kb), and its efficiency has been well tested in the analysis of both average (e.g., coagulation factor IX) and large, complex genes (e.g., coagulation factor VIII and dystrophin). In the latter application RT/PCR products allow the examination of all essential sequences of the gene in a minimum number of reactions. We use two specific chemical reactants (hydroxylamine and osmium tetroxide) and piperidine cleavage of the above procedure to develop a very fast mutation screening method. This is based on: (1) 5{prime} or internal fluorescent labeling to allow concurrent screening of three to four DNA fragments and (2) solid-phase chemistry to use a microliter format and reduce the time required for the procedure, from amplification of sequence to gel loading inclusive, to one person-working-day. We test the two variations of the method, one entailing 5{prime} labeling of probe DNA and the other uniform labeling of both probe and target DNA, by detecting 114 known hemophilia B (coagulation factor IX) mutations and by analyzing 129 new patients. Uniform labeling of both probe and target DNA prior to formation of the heteroduplexes leads to almost twofold redundancy in the ability to detect mutations. Alternatively, the latter procedure may offer very efficient though less than 100% screening for sequence changes with only hydroxylamine. The full method with two chemical reactions (hydroxylamine and osmium tetroxide) should allow one person to screen with virtually 100% accuracy more than 300 kb of sequence in three ABI 373 gels in 1 day. 26 refs., 7 figs., 1 tab.

  11. Evaluation of a PCR multiplex for detection and differentiation of Mycoplasma synoviae, M. gallisepticum, and M. gallisepticum strain F-vaccine

    Directory of Open Access Journals (Sweden)

    Elena Mettifogo

    2015-01-01

    Full Text Available Mycoplasma gallisepticum (MG and Mycoplasma synoviae (MS are the mycoplasma infections of most concern for commercial poultry industry. MG infection is commonly designated as chronic respiratory disease (CRD of chickens and infections sinusitis of turkeys. MS causes sub clinical upper respiratory infection and tenosynovitis or bursitis in chickens and turkeys. The multiplex PCR was standardized to detect simultaneously the MS, MG field strains and MG F-vaccine strain specific. The generic PCR for detection of any species of Mollicutes Class was performed and compared to the multiplex PCR and to PCR using species-specific primers. A total of 129 avian tracheal swabs were collected from broiler-breeders, layer hens and broilers in seven different farms and were examined by multiplex PCR methods. The system (multiplex PCR demonstrated to be very rapid, sensitive, and specific. Therefore, the results showed a high prevalence of MS in the flocks examined (27.9%, and indicate that the MS is a recurrent pathogen in Brazilian commercial poultry flocks.

  12. A Rapid, Multiplexed, High-Throughput Flow-Through Membrane Immunoassay: A Convenient Alternative to ELISA

    Directory of Open Access Journals (Sweden)

    Gonzalo J. Domingo

    2013-04-01

    Full Text Available This paper describes a rapid, high-throughput flow-through membrane immunoassay (FMIA platform. A nitrocellulose membrane was spotted in an array format with multiple capture and control reagents for each sample detection area, and assay steps were carried out by sequential aspiration of sample and reagents through each detection area using a 96-well vacuum manifold. The FMIA provides an alternate assay format with several advantages over ELISA. The high surface area of the membrane permits high label concentration using gold labels, and the small pores and vacuum control provide rapid diffusion to reduce total assay time to ~30 min. All reagents used in the FMIA are compatible with dry storage without refrigeration. The results appear as colored spots on the membrane that can be quantified using a flatbed scanner. We demonstrate the platform for detection of IgM specific to lipopolysaccharides (LPS derived from Salmonella Typhi. The FMIA format provides analytical results comparable to ELISA in less time, provides integrated assay controls, and allows compensation for specimen-to-specimen variability in background, which is a particular challenge for IgM assays.

  13. Development of Fault Detection System using Wavelength Division Multiplexing Transmission of Optical Fiber Current Sensor

    Science.gov (United States)

    Kayaki, Masahiro; Hirata, Toshinari; Kurosawa, Kiyoshi; Kondo, Reishi; Yamada, Toshiharu; Itakura, Eiji

    A fault detection system is applied to power lines consisting of both overhead power line and underground power cable in order to detect a fault on the underground power cable section and prevent the automatic reclosing. The fault detection system using optical fiber current sensor has two subjects. The fist subject is that we have to use wound-type current transformer together, when applying the fault detection system to single-core underground power cable. The second subject is that we are not able to detect three-phase short-circuit fault in using the fault detection system. This paper describes that we developed a new fault detection system using optical fiber current sensor by applying the multiplex transmission technology of optical fiber current sensor signal in order to solve these subjects.

  14. One-Step Multiplex RT-qPCR Assay for the Detection of Peste des petits ruminants virus, Capripoxvirus, Pasteurella multocida and Mycoplasma capricolum subspecies (ssp.) capripneumoniae.

    Science.gov (United States)

    Settypalli, Tirumala Bharani Kumar; Lamien, Charles Euloge; Spergser, Joachim; Lelenta, Mamadou; Wade, Abel; Gelaye, Esayas; Loitsch, Angelika; Minoungou, Germaine; Thiaucourt, Francois; Diallo, Adama

    2016-01-01

    , PPRV in 45, and PM in six samples. In addition, three samples showed a co-infection of PPRV and PM. Overall, the one-step multiplex RT-qPCR assay developed will be a valuable tool for rapid detection of individual and co-infections of the targeted pathogens with high specificity and sensitivity.

  15. One-Step Multiplex RT-qPCR Assay for the Detection of Peste des petits ruminants virus, Capripoxvirus, Pasteurella multocida and Mycoplasma capricolum subspecies (ssp. capripneumoniae.

    Directory of Open Access Journals (Sweden)

    Tirumala Bharani Kumar Settypalli

    17 samples, PPRV in 45, and PM in six samples. In addition, three samples showed a co-infection of PPRV and PM. Overall, the one-step multiplex RT-qPCR assay developed will be a valuable tool for rapid detection of individual and co-infections of the targeted pathogens with high specificity and sensitivity.

  16. Detection of virulence genes in Uropathogenic E. coli (UPEC strains by Multiplex-PCR method

    Directory of Open Access Journals (Sweden)

    Javad Mohammadi

    2017-06-01

    Full Text Available Background & Objectives: Urinary tract infection caused by E. coli is one of the most common illnesses in all age groups worldwide. Presence of virulence genes is a key factor in bacterial pathogens in uroepithelial cells. The present study was performed to detect iha, iroN, ompT genes in the Uropathogenic E.coli isolates from clinical samples using multiplex-PCR method in Kerman. Materials & Methods: In this descriptive cross-sectional study, 200 samples of patients with urinary tract infections in Kerman hospitals were collected. After biochemical and microbiological tests, all strains were tested with regard to the presence of iha, iroN, and ompT genes using multiplex-PCR method. Results: The results of Multiplex-PCR showed that all specimens had one, two, or three virulence genes simultaneously. The highest and lowest frequency distribution of genes was related to iha (56.7% and iroN (20% respectively. Conclusion: According to the prevalence of urinary tract infection in the community and distribution of resistance and virulence factors, the fast and accurate detection of the strains and virulence genes is necessary

  17. Multiplex fluorescence melting curve analysis for mutation detection with dual-labeled, self-quenched probes.

    Directory of Open Access Journals (Sweden)

    Qiuying Huang

    2011-04-01

    Full Text Available Probe-based fluorescence melting curve analysis (FMCA is a powerful tool for mutation detection based on melting temperature generated by thermal denaturation of the probe-target hybrid. Nevertheless, the color multiplexing, probe design, and cross-platform compatibility remain to be limited by using existing probe chemistries. We hereby explored two dual-labeled, self-quenched probes, TaqMan and shared-stem molecular beacons, in their ability to conduct FMCA. Both probes could be directly used for FMCA and readily integrated with closed-tube amplicon hybridization under asymmetric PCR conditions. Improved flexibility of FMCA by using these probes was illustrated in three representative applications of FMCA: mutation scanning, mutation identification and mutation genotyping, all of which achieved improved color-multiplexing with easy probe design and versatile probe combination and all were validated with a large number of real clinical samples. The universal cross-platform compatibility of these probes-based FMCA was also demonstrated by a 4-color mutation genotyping assay performed on five different real-time PCR instruments. The dual-labeled, self-quenched probes offered unprecedented combined advantage of enhanced multiplexing, improved flexibility in probe design, and expanded cross-platform compatibility, which would substantially improve FMCA in mutation detection of various applications.

  18. Fluorescent multiplex linkage analysis and carrier detection for Duchenne/Becker muscular dystrophy

    Energy Technology Data Exchange (ETDEWEB)

    Schwartz, L.S.; Hoffman, E.P. (Univ. of Pittsburgh Schoool of Medicine, Pittsburgh, PA (United States)); Tarleton, J. (Self Memorial Hospital, Greenwood, SC (United States)); Popovich, B. (Children' s Hosptial and Health Center, San Diego, CA (United States)); Seltzer, W.K. (Univ. of Colorado Health Sciences Center, Denver, CO (United States))

    1992-10-01

    The authors have developed a fast and accurate PCR-based linkage and carrier detection protocol for families of Duchenne muscular dystrophy (DMD)/Becker muscular dystrophy (BMD) patients with or without detectable deletions of the dystrophin gene, using fluorescent PCR products analyzed on an automated sequencer. When a deletion is found in the affected male DMD/BMD patient by standard multiplex PCR, fluorescently labeled primers specific for the deleted and nondeleted exon(s) are used to amplify the DNA of at-risk female relatives by using multiplex PCR at low cycle number (20 cycles). The products are then quantitatively analyzed on an automatic sequencer to determine whether they are heterozygous for the deletion and thus are carriers. As a confirmation of the deletion data, and in cases in which a deletion is not found in the proband, fluorescent multiplex PCR linkage is done by using four previously described polymorphic dinucleotide sequences. The four (CA)[sub n] repeats are located throughout the dystrophin gene, making the analysis highly informative and accurate. The authors present the successful application of this protocol in families who proved refractory to more traditional analyses. 22 refs., 3 figs.

  19. Comparison of the BAX System with a multiplex PCR method for simultaneous detection and identification of Campylobacter jejuni and Campylobacter coli in environmental samples.

    Science.gov (United States)

    Manfreda, Gerardo; De Cesare, Alessandra; Bondioli, Valentina; Franchini, Achille

    2003-11-01

    The Campylobacter detection is performed by conventional culture methods and the identification of Campylobacter jejuni and Campylobacter coli is principally based on the hippurate hydrolysis test. The two major drawbacks of this biochemical test for species identification include the inconsistency of the results and the presence of atypical strains, which can lead to the misidentification of an isolate. As an alternative, multiplex polymerase chain reaction (mPCR) protocols for the simultaneous detection and identification of different Campylobacter species have been developed. This study examined the performances of an experimental BAX System assay for the C. jejuni and C. coli identification in comparison to a multiplex PCR protocol recently published. The samples tested were represented by 106 environmental swabs collected on Teflon strips and tables, stainless steel saws, hooks and trays, ceramic floors and walls, as well as equipment surfaces, located in a swine (N=50) and a poultry (N=56) slaughterhouse. The highest Campylobacter detection rate was obtained after 48 h of enrichment by using both the PCR procedures. After 24 h, the BAX System provides a more rapid and accurate Campylobacter detection and identification assay than the multiplex PCR. Except for two samples, all the broths where Campylobacter cells were detected after 24 or 48 h of enrichment, with at least one of the PCR protocols, gave Campylobacter colonies using the culture method.

  20. Detection of Lymnaea columella infection by Fasciola hepatica through Multiplex-PCR

    Directory of Open Access Journals (Sweden)

    Kelly Grace Magalhães

    2004-06-01

    Full Text Available From complete mitochondrial DNA sequence of Fasciola hepatica available in Genbank, specific primers were designed for a conserved and repetitive region of this trematode. A pair of primers was used for diagnosis of infected Lymnaea columella by F. hepatica during the pre-patent period simultaneously with another pair of primers which amplified the internal transcribed spacer (ITS region of rDNA from L. columella in a single Multiplex-PCR. The amplification generated a ladder band profile specific for F. hepatica. This profile was observed in positive molluscs at different times of infection, including adult worms from the trematode. The Multiplex-PCR technique showed to be a fast and safe tool for fascioliasis diagnosis, enabling the detection of F. hepatica miracidia in L. columella during the pre-patent period and identification of transmission areas.

  1. Toward photostable multiplex analyte detection on a single mode planar optical waveguide

    Energy Technology Data Exchange (ETDEWEB)

    Mukundan, Harshini [Los Alamos National Laboratory; Xei, Hongshi [Los Alamos National Laboratory; Anderson, Aaron S [Los Alamos National Laboratory; Grace, Wynne K [Los Alamos National Laboratory; Martinez, Jennifer S [NON LANL; Swanson, Basil [Los Alamos National Laboratory

    2009-01-01

    We have developed a waveguide-based optical biosensor for the sensitive and specific detection of biomarkers associated with disease. Our technology combines the superior optical properties of single-mode planar waveguides, the robust nature of functionalized self-assembled monolayer sensing films and the specificity of fluorescence sandwich immunoassays to detect biomarkers in complex biological samples such as serum, urine and sputum. We have previously reported the adaptation of our technology to the detection of biomarkers associated with breast cancer and anthrax. However, these approaches primarily used phospholipid bilayers as the functional film and organic dyes (ex: AlexaFluors) as the fluorescence reporter. Organic dyes are easily photodegraded and are not amenable to multiplexing because of their narrow Stokes' shift. Here we have developed strategies for conjugation of the detector antibodies with quantum dots for use in a multiplex detection platform. We have previously evaluated dihydroxylipoic acid quantum dots for the detection of a breast cancer biomarker. In this manuscript, we investigate the detection of the Bacillus anthracis protective antigen using antibodies conjugated with polymer-coated quantum dots. Kinetics of binding on the waveguide-based biosensor is reported. We compare the sensitivity of quantum dot labeled antibodies to those labeled with AlexaFluor and demonstrate the photostability of the former in our assay platform. In addition, we compare sulfydryl labeling of the antibody in the hinge region to that of nonspecific amine labeling. This is but the first step in developing a multiplex assay for such biomarkers on our waveguide platform.

  2. Multiplex nested PCR for detection of Xanthomonas axonopodis pv. allii from onion seeds.

    Science.gov (United States)

    Robène-Soustrade, Isabelle; Legrand, Delphine; Gagnevin, Lionel; Chiroleu, Frédéric; Laurent, Annie; Pruvost, Olivier

    2010-05-01

    Bacterial blight of onion (BBO) is an emerging disease that is present in many onion-producing areas. The causal agent, Xanthomonas axonopodis pv. allii, is seed transmitted. A reliable and sensitive diagnostic tool for testing seed health is needed. Detection of X. axonopodis pv. allii was achieved using a multiplex nested PCR assay developed using two randomly amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) sequences corresponding to pilus assembly genes (pilW and pilX) and the avrRxv gene, respectively. The multiplex nested PCR was used with a large collection of X. axonopodis pv. allii strains pathogenic to onion and/or other Allium species isolated in different regions of the world. The internal primers used in the multiplex PCR assay directed amplification for all 86 X. axonopodis pv. allii strains tested, resulting in a 401-bp amplicon, a 444- to 447-bp amplicon, or both amplicons, depending on the strain. No amplification was obtained for 41 unrelated phytopathogenic bacteria and for 14 saprophytic bacteria commonly isolated from onion leaves and seeds. Most Xanthomonas strains also did not produce amplicons, except for nine strains classified in X. axonopodis genetic subgroup 9.1 or 9.2 and not pathogenic to onion. Nevertheless, sequence signatures distinguished most of these strains from X. axonopodis pv. allii. The assay detected X. axonopodis pv. allii in seed lots with contamination levels of 5 x 10(2) CFU g(-1) or higher. The sensitivity threshold of the multiplex nested PCR assay was found to be 1 infected seed in 27,340 seeds. This PCR-based assay should be useful for certifying that commercial seed lots are free of this important seed-borne pathogen.

  3. Multiplexed Detection and Quantitation of Extracellular Vesicle RNA Expression Using NanoString.

    Science.gov (United States)

    Shukla, Neha; Yan, Irene K; Patel, Tushar

    2018-01-01

    Several different types of RNA molecules such as microRNAs (miRNAs) have been detected within extracellular vesicles in the circulation. The detection and potential utility of these as disease biomarkers requires the ability to detect their presence with adequate sensitivity and to quantitate their expression. The potential for circulating miRNA to serve as biomarkers can be evaluated through their detection in association with specific disease states. Multiplexed detection of several miRNA simultaneously can be useful for discovery studies. We describe the analysis of miRNA from biological fluids like plasma and serum using the Nanostring nCounter platform. Assays can be used to quantitate the expression of miRNA using direct detection based on hybridization to target specific color-coded probes followed by counting each color-coded barcode digitally.

  4. Multiplex tumor marker detection with new chemiluminescent immunoassay based on silica colloidal crystal beads.

    Science.gov (United States)

    Pei, Xiaoping; Chen, Baoan; Li, Li; Gao, Feng; Jiang, Zhi

    2010-01-01

    A new multiplex chemiluminescent immunoassay (CLIA) based on silica colloidal crystal beads (SCCBs) was developed for tumor marker detection. As the code is the characteristic reflection peak originating from the stop-band of colloid crystal, they avoid photobleaching, the potential interference of encoding fluorescence with analyte-detection fluorescence and chemical instability. Meanwhile our SCCBs suspension array improved the luminescence analysis efficiency by using chemiluminescent detection of enzyme labels. By forming a sandwich immunocomplex on SCCBs, the proposed suspension array was used for simultaneous multiplex detection of tumor markers in one test tube. The results showed that the linear range was 0.5-100ng ml(-1) and 1.0-120ng ml(-1) for carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP) with a detection limit of 0.12ng ml(-1) and 0.16ng ml(-1) at 3sigma. The proposed array showed the storage stability and the accuracy for sample detection were acceptable, and the results were in acceptable agreement with the reference electrochemiluminescence method. This technique provided an automated, simple, sensitive and low-cost approach for multianalyte immunoassay.

  5. Improved detection of episomal Banana streak viruses by multiplex immunocapture PCR.

    Science.gov (United States)

    Le Provost, Grégoire; Iskra-Caruana, Marie-Line; Acina, Isabelle; Teycheney, Pierre-Yves

    2006-10-01

    Banana streak viruses (BSV) are currently the main viral constraint to Musa germplasm movement, genetic improvement and mass propagation. Therefore, it is necessary to develop and implement BSV detection strategies that are both reliable and sensitive, such as PCR-based techniques. Unfortunately, BSV endogenous pararetrovirus sequences (BSV EPRVs) are present in the genome of Musa balbisiana. They interfere with PCR-based detection of episomal BSV in infected banana and plantain, such as immunocapture PCR. Therefore, a multiplex, immunocapture PCR (M-IC-PCR) was developed for the detection of BSV. Musa sequence tagged microsatellite site (STMS) primers were selected and used in combination with BSV species-specific primers in order to monitor possible contamination by Musa genomic DNA, using multiplex PCR. Furthermore, immunocapture conditions were optimized in order to prevent Musa DNA from interfering with episomal BSV DNA during the PCR step. This improved detection method successfully allowed the accurate, specific and sensitive detection of episomal DNA only from distinct BSV species. Its implementation should benefit PCR-based detection of viruses for which homologous sequences are present in the genome of their hosts, including transgenic plants expressing viral sequences.

  6. Community Detection for Multiplex Social Networks Based on Relational Bayesian Networks

    DEFF Research Database (Denmark)

    Jiang, Jiuchuan; Jaeger, Manfred

    2014-01-01

    . In this paper we propose to use relational Bayesian networks for the specification of probabilistic network models, and develop inference techniques that solve the community detection problem based on these models. The use of relational Bayesian networks as a flexible high-level modeling framework enables us......Many techniques have been proposed for community detection in social networks. Most of these techniques are only designed for networks defined by a single relation. However, many real networks are multiplex networks that contain multiple types of relations and different attributes on the nodes...

  7. Development of a multiplex autoantibody test for detection of lung cancer.

    Science.gov (United States)

    Jia, Jing; Wang, Wenzhe; Meng, Wen; Ding, Mingjian; Ma, Shenglin; Wang, Xiaoju

    2014-01-01

    Lung cancer is the leading cause of cancer-related deaths for both men and women. Early diagnosis of lung cancer has a 5-year survival rate of 48.8%, however, nearly 35% of stage I patients relapses after surgical resection, thus portending a poor prognosis. Therefore, detecting lung cancer in early stage and further identifying the high-risk patients would allow the opportunity to provide adjuvant therapy and possibly increase survival. There is considerable evidence that the immune system produces an autoantibody response to neoplastic cells. The detection of such autoantibodies has been shown to have diagnostic and prognostic value. Here we took advantage of the high-throughput Luminex technique to multiplex a total of 14 tumor-associated autoantigens to detect the autoantibody from the patients sera. The 14 antigens were expressed by in vitro transcription/translation system with HaloTag at N-terminus. The fusion proteins were then covalently immobilized onto the Luminex microspheres conjugated by the halo-link ligand, thus eliminating the protein purification procedure. Sera samples from cancer patients and healthy controls were interacted with the microsphere-antigen complex to measure the autoantibodies. We have developed a quick multiplex detection system for measuring autoantibody signature from patient sera with minimal cross-reaction. A panel of seven autoantibody biomarkers has generated an AUC>80% in distinguishing the lung cancers from healthy controls. This study is the first report by combining Luminex platform and HaloTag technology to detect humoral immune response in cancer patients. Due to the flexibility of the Luminex technology, this approach can be applied to others conditions such as infectious, neurological, and metabolic diseases. One can envision that this multiplex Luminex system as well as the panel of seven biomarkers could be used to screen the high-risk population with subsequent CT test based on the blood test result.

  8. Development of a multiplex autoantibody test for detection of lung cancer.

    Directory of Open Access Journals (Sweden)

    Jing Jia

    Full Text Available Lung cancer is the leading cause of cancer-related deaths for both men and women. Early diagnosis of lung cancer has a 5-year survival rate of 48.8%, however, nearly 35% of stage I patients relapses after surgical resection, thus portending a poor prognosis. Therefore, detecting lung cancer in early stage and further identifying the high-risk patients would allow the opportunity to provide adjuvant therapy and possibly increase survival. There is considerable evidence that the immune system produces an autoantibody response to neoplastic cells. The detection of such autoantibodies has been shown to have diagnostic and prognostic value. Here we took advantage of the high-throughput Luminex technique to multiplex a total of 14 tumor-associated autoantigens to detect the autoantibody from the patients sera. The 14 antigens were expressed by in vitro transcription/translation system with HaloTag at N-terminus. The fusion proteins were then covalently immobilized onto the Luminex microspheres conjugated by the halo-link ligand, thus eliminating the protein purification procedure. Sera samples from cancer patients and healthy controls were interacted with the microsphere-antigen complex to measure the autoantibodies. We have developed a quick multiplex detection system for measuring autoantibody signature from patient sera with minimal cross-reaction. A panel of seven autoantibody biomarkers has generated an AUC>80% in distinguishing the lung cancers from healthy controls. This study is the first report by combining Luminex platform and HaloTag technology to detect humoral immune response in cancer patients. Due to the flexibility of the Luminex technology, this approach can be applied to others conditions such as infectious, neurological, and metabolic diseases. One can envision that this multiplex Luminex system as well as the panel of seven biomarkers could be used to screen the high-risk population with subsequent CT test based on the blood test

  9. A bead-based multiplex assay for the detection of DNA viruses infecting laboratory rodents.

    Directory of Open Access Journals (Sweden)

    Daniela Höfler

    Full Text Available The Federation of European Laboratory Animal Science Association (FELASA recommends screening of laboratory rodents and biological materials for a broad variety of bacterial agents, viruses, and parasites. Methods commonly used to date for pathogen detection are neither cost-effective nor time- and animal-efficient or uniform. However, an infection even if silent alters experimental results through changing the animals' physiology and increases inter-individual variability. As a consequence higher numbers of animals and experiments are needed for valid and significant results. We developed a novel high-throughput multiplex assay, called rodent DNA virus finder (rDVF for the simultaneous identification of 24 DNA viruses infecting mice and rats. We detected all 24 DNA viruses with high specificity and reproducibility. Detection limits for the different DNA viruses varied between 10 and 1000 copies per PCR. The validation of rDVF was done with DNA isolated from homogenised organs amplified by pathogen specific primers in one multiplex PCR. The biotinylated amplicons were detected via hybridisation to specific oligonucleotide probes coupled to spectrally distinct sets of fluorescent Luminex beads. In conclusion, rDVF may have the potential to replace conventional testing and may simplify and improve routine detection of DNA viruses infecting rodents.

  10. A bead-based multiplex assay for the detection of DNA viruses infecting laboratory rodents.

    Science.gov (United States)

    Höfler, Daniela; Nicklas, Werner; Mauter, Petra; Pawlita, Michael; Schmitt, Markus

    2014-01-01

    The Federation of European Laboratory Animal Science Association (FELASA) recommends screening of laboratory rodents and biological materials for a broad variety of bacterial agents, viruses, and parasites. Methods commonly used to date for pathogen detection are neither cost-effective nor time- and animal-efficient or uniform. However, an infection even if silent alters experimental results through changing the animals' physiology and increases inter-individual variability. As a consequence higher numbers of animals and experiments are needed for valid and significant results. We developed a novel high-throughput multiplex assay, called rodent DNA virus finder (rDVF) for the simultaneous identification of 24 DNA viruses infecting mice and rats. We detected all 24 DNA viruses with high specificity and reproducibility. Detection limits for the different DNA viruses varied between 10 and 1000 copies per PCR. The validation of rDVF was done with DNA isolated from homogenised organs amplified by pathogen specific primers in one multiplex PCR. The biotinylated amplicons were detected via hybridisation to specific oligonucleotide probes coupled to spectrally distinct sets of fluorescent Luminex beads. In conclusion, rDVF may have the potential to replace conventional testing and may simplify and improve routine detection of DNA viruses infecting rodents.

  11. Gold-silver-alloy nanoprobes for one-pot multiplex DNA detection

    Energy Technology Data Exchange (ETDEWEB)

    Doria, G; Larguinho, M; Dias, J T; Baptista, P V [Centro de Investigacao em Genetica Molecular Humana (CIGMH), Departamento de Ciencias da Vida, Faculdade de Ciencias e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica (Portugal); Pereira, E [Rede de Quimica e Tecnologia (REQUIMTE), Departamento de Quimica, Faculdade de Ciencias, Universidade do Porto, 4169-007 Porto (Portugal); Franco, R, E-mail: pmvb@fct.unl.pt [Rede de Quimica e Tecnologia (REQUIMTE), Departamento de Quimica, Faculdade de Ciencias e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica (Portugal)

    2010-06-25

    A specific colorimetric DNA detection method based on oligonucleotide functionalized gold-silver-alloy nanoparticles (AuAg-alloy-nanoprobes) is presented. The AuAg-alloy-nanoprobes were then used for the specific detection of a DNA sequence from TP53-a gene involved in cancer development. The AuAg-alloy-nanoprobes were then used in combination with Au-nanoprobes for a one-pot dual-colour detection strategy that allowed for the simultaneous differential detection of two distinct target sequences. This system poses an unprecedented opportunity to explore the combined use of metal nanoparticles with different composition towards the development of a multiplex one-pot colorimetric assay for DNA detection.

  12. Multiplex strand displacement amplification (SDA) and detection of DNA sequences from Mycobacterium tuberculosis and other mycobacteria.

    Science.gov (United States)

    Walker, G T; Nadeau, J G; Spears, P A; Schram, J L; Nycz, C M; Shank, D D

    1994-07-11

    Strand Displacement Amplification (SDA) is an isothermal, in vitro method of amplifying a DNA target sequence prior to detection [Walker et al (1992) Nucleic Acids Res., 20, 1691-1693]. Here we describe a multiplex form of SDA that allows two target sequences and an internal amplification control to be co-amplified by a single pair of primers after common priming sequences are spontaneously appended to the ends of target fragments. Multiplex SDA operates at a single temperature, under the same simple protocol previously developed for single-target SDA. We applied multiplex SDA to co-amplification of a target sequence (IS6110) that is specific to members of the Mycobacterium tuberculosis-complex and a target (16S ribosomal gene) that is common to most clinically relevant species of mycobacteria. Both targets are amplified 10(8)-fold during a 2 hour, single temperature incubation. The relative sensitivity of the system was evaluated across a number of clinically relevant mycobacteria and checked for crossreactivity against organisms that are closely related to mycobacteria.

  13. A multiplex method for detection of glucose-6-phosphate dehydrogenase (G6PD) gene mutations.

    Science.gov (United States)

    Zhang, L; Yang, Y; Liu, R; Li, Q; Yang, F; Ma, L; Liu, H; Chen, X; Yang, Z; Cui, L; He, Y

    2015-12-01

    Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common human enzyme defect caused by G6PD gene mutations. This study aimed to develop a cost-effective, multiplex, genotyping method for detecting common mutations in the G6PD gene. We used a SNaPshot approach to genotype multiple G6PD mutations that are common to human populations in South-East Asia. This assay is based on multiplex PCR coupled with primer extension reactions. Different G6PD gene mutations were determined by peak retention time and colors of the primer extension products. We designed PCR primers for multiplex amplification of the G6PD gene fragments and for primer extension reactions to genotype 11 G6PD mutations. DNA samples from a total of 120 unrelated G6PD-deficient individuals from the China-Myanmar border area were used to establish and validate this method. Direct sequencing of the PCR products demonstrated 100% concordance between the SNaPshot and the sequencing results. The SNaPshot method offers a specific and sensitive alternative for simultaneously interrogating multiple G6PD mutations. © 2015 John Wiley & Sons Ltd.

  14. Indigenous people's detection of rapid ecological change.

    Science.gov (United States)

    Aswani, Shankar; Lauer, Matthew

    2014-06-01

    When sudden catastrophic events occur, it becomes critical for coastal communities to detect and respond to environmental transformations because failure to do so may undermine overall ecosystem resilience and threaten people's livelihoods. We therefore asked how capable of detecting rapid ecological change following massive environmental disruptions local, indigenous people are. We assessed the direction and periodicity of experimental learning of people in the Western Solomon Islands after a tsunami in 2007. We compared the results of marine science surveys with local ecological knowledge of the benthos across 3 affected villages and 3 periods before and after the tsunami. We sought to determine how people recognize biophysical changes in the environment before and after catastrophic events such as earthquakes and tsunamis and whether people have the ability to detect ecological changes over short time scales or need longer time scales to recognize changes. Indigenous people were able to detect changes in the benthos over time. Detection levels differed between marine science surveys and local ecological knowledge sources over time, but overall patterns of statistically significant detection of change were evident for various habitats. Our findings have implications for marine conservation, coastal management policies, and disaster-relief efforts because when people are able to detect ecological changes, this, in turn, affects how they exploit and manage their marine resources. © 2014 Society for Conservation Biology.

  15. Targeted deposition of antibodies on a multiplex CMOS microarray and optimization of a sensitive immunoassay using electrochemical detection.

    Directory of Open Access Journals (Sweden)

    John Cooper

    Full Text Available BACKGROUND: The CombiMatrix ElectraSense microarray is a highly multiplex, complementary metal oxide semiconductor with 12,544 electrodes that are individually addressable. This platform is commercially available as a custom DNA microarray; and, in this configuration, it has also been used to tether antibodies (Abs specifically on electrodes using complementary DNA sequences conjugated to the Abs. METHODOLOGY/PRINCIPAL FINDINGS: An empirical method is described for developing and optimizing immunoassays on the CombiMatrix ElectraSense microarray based upon targeted deposition of polypyrrole (Ppy and capture Ab. This process was automated using instrumentation that can selectively apply a potential or current to individual electrodes and also measure current generated at the electrodes by an enzyme-enhanced electrochemical (ECD reaction. By designating groups of electrodes on the array for different Ppy deposition conditions, we determined that the sensitivity and specificity of a sandwich immunoassay for staphylococcal enterotoxin B (SEB is influenced by the application of different voltages or currents and the application time. The sandwich immunoassay used a capture Ab adsorbed to the Ppy and a reporter Ab labeled for fluorescence detection or ECD, and results from these methods of detection were different. CONCLUSIONS/SIGNIFICANCE: Using Ppy deposition conditions for optimum results, the lower limit of detection for SEB using the ECD assay was between 0.003 and 0.01 pg/ml, which represents an order of magnitude improvement over a conventional enzyme-linked immunosorbant assay. In the absence of understanding the variables and complexities that affect assay performance, this highly multiplexed electrode array provided a rapid, high throughput, and empirical approach for developing a sensitive immunoassay.

  16. Detection of Neisseria meningitidis in cerebrospinal fluid using a multiplex PCR and the Luminex detection technology

    DEFF Research Database (Denmark)

    Møller, Jens Kjølseth

    2012-01-01

    laser. Reporter signals from each reaction are simultaneously quantified by fluorescence generated by a green reporter laser. This nonculture, multiplex assay may prove to be an important tool for optimal laboratory diagnosis, not only of meningococcal meningitis, but also of meningitis caused by other...... bacterial or viral pathogens....

  17. Economic analysis of rapid multiplex polymerase chain reaction testing for meningitis/encephalitis in pediatric patients.

    Science.gov (United States)

    Duff, Steve; Hasbun, Rodrigo; Ginocchio, Christine C; Balada-Llasat, Joan-Miquel; Zimmer, Louise; Bozzette, Samuel A

    2018-01-10

    We assessed the possible economic impact of a rapid test in pediatric patients with suspected community-acquired meningitis/encephalitis. Modeling simulated diagnosis, clinical decisions, resource use/costs of standard of care (SOC) and two cerebrospinal fluid testing strategies using FilmArray ® (FA), a US FDA-cleared system that provides results in approximately 1 h. Pathogens detected by FA caused approximately 75% of cases, 97% of which would be accurately diagnosed with FA.  Mean cost/case ranged from $17,599 to $22,025.  Syndromic testing is less expensive than SOC. Testing all suspected cases yielded greater savings ($3481/case) than testing only those with abnormal cerebrospinal fluid ($2157/case). Greater economic benefits are achievable with syndromic testing of all cases, rather than SOC or targeted syndromic testing.

  18. Rapid diagnostic multiplex PCR (RD-PCR) to discriminate Schistosoma haematobium and S. bovis.

    Science.gov (United States)

    Webster, B L; Rollinson, D; Stothard, J R; Huyse, T

    2010-03-01

    Schistosoma haematobium and S. bovis are widespread schistosome species causing human and cattle schistosomiasis, respectively, in Africa. The sympatric occurrence of these two species and their ability to infect the same Bulinus intermediate snail hosts necessitates precise methods of identification of the larval stages. A rapid diagnostic 'mulitplex' one-step polymerase chain reaction protocol (RD-PCR) was developed using cytochrome oxidase subunit 1 (COX1) mitochondrial DNA (mtDNA) to discriminate between S. haematobium and S. bovis. A single forward primer and two species-specific reverse primers were used to produce a polymerase chain reaction (PCR) fragment of 306 bp and 543 bp for S. bovis and S. haematobium, respectively. Serial dilutions were carried out on various lifecycle stages and species combinations to test the sensitivity and specificity of the primers. This RD-PCR proved highly sensitive, detecting a single larval stage and as little as 0.78 ng of genomic DNA (gDNA) from an adult schistosome, providing a cost-effective, rapid and robust molecular tool for high-throughput screening of S. haematobium and S. bovis populations. In areas where human and cattle schistosomiasis overlap and are transmitted in close proximity, this mitochondrial assay will be a valuable identification tool for epidemiological studies, especially when used in conjunction with other nuclear diagnostic markers.

  19. Simultaneous detection of peanut and hazelnut allergens in food matrices using multiplex PCR method

    Directory of Open Access Journals (Sweden)

    Eva Renčová

    2014-01-01

    Full Text Available Multiplex PCR analysis for the detection of two targeting segments of genes coding major food protein allergens as peanut (Arachis hypogaea Ara h 1 gene and hazelnut (Corylus avellana Cor a 1 gene was developed. Two sets of primers were designed and tested to their specificity on a broad range of ingredients. The identity of amplicons (Ara h 1- 180 bp, Cor a 1 – 258 bp by sequencing and alignment of sequences with sequences deposited in Genbank was confirmed. When testing the specificity of designed primer pairs on a spectrum of food ingredients, no cross reactions were detected. A potential inhibition of PCR reaction was eliminated using the universal plant primers of chloroplast gene 124 bp for the plant matrices confirmation. The intrinsic detection limit was 10 pg·ml-1 and the practical detection limit was 0.001% w/w (10 mg·kg-1 for both peanuts and hazelnuts. The method was applied to the investigation of 60 commercial food samples. The developed multiplex PCR method is cheap, specific and sensitive enough and can be used as a simple, one day procedure for the checking of undeclared peanut and hazelnut major allergens in food.

  20. Multiplexing of ChIP-Seq Samples in an Optimized Experimental Condition Has Minimal Impact on Peak Detection.

    Directory of Open Access Journals (Sweden)

    Thadeous J Kacmarczyk

    Full Text Available Multiplexing samples in sequencing experiments is a common approach to maximize information yield while minimizing cost. In most cases the number of samples that are multiplexed is determined by financial consideration or experimental convenience, with limited understanding on the effects on the experimental results. Here we set to examine the impact of multiplexing ChIP-seq experiments on the ability to identify a specific epigenetic modification. We performed peak detection analyses to determine the effects of multiplexing. These include false discovery rates, size, position and statistical significance of peak detection, and changes in gene annotation. We found that, for histone marker H3K4me3, one can multiplex up to 8 samples (7 IP + 1 input at ~21 million single-end reads each and still detect over 90% of all peaks found when using a full lane for sample (~181 million reads. Furthermore, there are no variations introduced by indexing or lane batch effects and importantly there is no significant reduction in the number of genes with neighboring H3K4me3 peaks. We conclude that, for a well characterized antibody and, therefore, model IP condition, multiplexing 8 samples per lane is sufficient to capture most of the biological signal.

  1. Reprogrammable multiplexed detection of circulating oncomiRs using hybridization chain reaction.

    Science.gov (United States)

    Rana, Muhit; Balcioglu, Mustafa; Kovach, Maya; Hizir, Mustafa Salih; Robertson, Neil M; Khan, Irfan; Yigit, Mehmet V

    2016-02-28

    In this study, we have coupled the DNA polymerization capability of hybridization chain reaction (HCR) with the plasmonic properties of gold nanoparticles to develop a reprogrammable and multiplexed detection of three circulating oncomiRs (miR-10b, miR-21 and miR-141) dysregulated in various disease states of breast cancer. We have demonstrated that by simply changing the initiator (label-free short single stranded DNA) content of the HCR, while keeping everything else unchanged, the same nanoparticle assembly can be reprogrammed for the detection of the target oncomiRs individually or simultaneously in all possible combinations. We have shown that as little as 20 femtomoles of each oncomiR can be detected visually without using any analytical instrument. Furthermore, we demonstrated that the target oncomiR can be detected in an RNA pool isolated from a liquid biopsy mimic of breast cancer.

  2. Rapid methods for the detection of foodborne bacterial pathogens: principles, applications, advantages and limitations

    Science.gov (United States)

    Law, Jodi Woan-Fei; Ab Mutalib, Nurul-Syakima; Chan, Kok-Gan; Lee, Learn-Han

    2015-01-01

    The incidence of foodborne diseases has increased over the years and resulted in major public health problem globally. Foodborne pathogens can be found in various foods and it is important to detect foodborne pathogens to provide safe food supply and to prevent foodborne diseases. The conventional methods used to detect foodborne pathogen are time consuming and laborious. Hence, a variety of methods have been developed for rapid detection of foodborne pathogens as it is required in many food analyses. Rapid detection methods can be categorized into nucleic acid-based, biosensor-based and immunological-based methods. This review emphasizes on the principles and application of recent rapid methods for the detection of foodborne bacterial pathogens. Detection methods included are simple polymerase chain reaction (PCR), multiplex PCR, real-time PCR, nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP) and oligonucleotide DNA microarray which classified as nucleic acid-based methods; optical, electrochemical and mass-based biosensors which classified as biosensor-based methods; enzyme-linked immunosorbent assay (ELISA) and lateral flow immunoassay which classified as immunological-based methods. In general, rapid detection methods are generally time-efficient, sensitive, specific and labor-saving. The developments of rapid detection methods are vital in prevention and treatment of foodborne diseases. PMID:25628612

  3. Rapid Methods for the Detection of Foodborne Bacterial Pathogens: Principles, Applications, Advantages and Limitations

    Directory of Open Access Journals (Sweden)

    Law eJodi Woan-Fei

    2015-01-01

    Full Text Available The incidence of foodborne diseases has increased over the years and resulted in major public health problem globally. Foodborne pathogens can be found in various foods and it is important to detect foodborne pathogens to provide safe food supply and to prevent foodborne diseases. The conventional methods used to detect foodborne pathogen are time consuming and laborious. Hence, a variety of methods have been developed for rapid detection of foodborne pathogens as it is required in many food analyses. Rapid detection methods can be categorized into nucleic acid-based, biosensor-based and immunological-based methods. This review emphasizes on the principles and application of recent rapid methods for the detection of foodborne bacterial pathogens. Detection methods included are simple polymerase chain reaction (PCR, multiplex PCR, real-time PCR, nucleic acid sequence-based amplification (NASBA, loop-mediated isothermal amplification (LAMP and oligonucleotide DNA microarray which classified as nucleic acid-based methods; optical, electrochemical and mass-based biosensors which classified as biosensor-based methods; enzyme-linked immunosorbent assay (ELISA and lateral flow immunoassay which classified as immunological-based methods. In general, rapid detection methods are generally time-efficient, sensitive, specific and labor-saving. The developments of rapid detection methods are vital in prevention and treatment of foodborne diseases.

  4. Multiplex-PCR for simultaneous detection of 3 bacterial fish pathogens, Flavobacterium columnare, Edwardsiella ictaluri, and Aeromonas hydrophila.

    Science.gov (United States)

    Panangala, Victor S; Shoemaker, Craig A; Van Santen, Vicky L; Dybvig, Kevin; Klesius, Phillip H

    2007-03-13

    A multiplex PCR (m-PCR) method was developed for simultaneous detection of 3 important fish pathogens in warm water aquaculture. The m-PCR to amplify target DNA fragments from Flavobacterium columnare (504 bp), Edwardsiella ictaluri (407 bp) and Aeromonas hydrophila (209 bp) was optimized by adjustment of reaction buffers and a touchdown protocol. The lower detection limit for each of the 3 bacteria was 20 pg of nucleic acid template from each bacteria per m-PCR reaction mixture. The sensitivity threshold for detection of the 3 bacteria in tissues ranged between 3.4 x 10(2) and 2.5 x 10(5) cells g(-1) of tissue (channel catfish Ictalurus punctatus Rafinesque). The diagnostic sensitivity and specificity of the m-PCR was evaluated with 10 representative isolates of each of the 3 bacteria and 11 other Gram-negative and 2 Gram-positive bacteria that are taxonomically related or ubiquitous in the aquatic environment. Except for a single species (A. salmonicida subsp. salmonicida), each set of primers specifically amplified the target DNA of the cognate species of bacteria. m-PCR was compared with bacteriological culture for identification of bacteria in experimentally infected fish. The m-PCR appears promising for the rapid, sensitive and simultaneous detection of Flavobacterium columnare, E. ictaluri and A. hydrophila in infected fish compared to the time-consuming traditional bacteriological culture techniques.

  5. Analysis of hybrid subcarrier multiplexing of OCDMA based on single photodiode detection

    Science.gov (United States)

    Ahmad, N. A. A.; Junita, M. N.; Aljunid, S. A.; Rashidi, C. B. M.; Endut, R.

    2017-11-01

    This paper analyzes the performance of subcarrier multiplexing (SCM) of spectral amplitude coding optical code multiple access (SAC-OCDMA) by applying Recursive Combinatorial (RC) code based on single photodiode detection (SPD). SPD is used in the receiver part to reduce the effect of multiple access interference (MAI) which contributes as a dominant noise in incoherent SAC-OCDMA systems. Results indicate that the SCM OCDMA network performance could be improved by using lower data rates and higher number of weight. Total number of users can also be enhanced by adding lower data rates and higher number of subcarriers.

  6. Rapid Detection of the Varicella Zoster Virus

    Science.gov (United States)

    Lewis, Michelle P.; Harding, Robert

    2011-01-01

    1.Technology Description-Researchers discovered that when the Varicella Zoster Virus (VZV) reactivates from latency in the body, the virus is consistently present in saliva before the appearance of skin lesions. A small saliva sample is mixed with a specialized reagent in a test kit. If the virus is present in the saliva sample, the mixture turns a red color. The sensitivity and specificity emanates from an antibody-antigen reaction. This technology is a rapid, non-invasive, point of-of-care testing kit for detecting the virus from a saliva sample. The device is easy to use and can be used in clinics and in remote locations to quickly detect VZV and begin treatment with antiviral drugs. 2.Market Opportunity- RST Bioscience will be the first and only company to market a rapid, same day test kit for the detection of VZV in saliva. The RST detection test kit will have several advantages over existing, competitive technology. The test kit is self contained and laboratory equipment is not required for analysis of the sample. Only a single saliva sample is required to be taken instead of blood or cerebral spinal fluid. The test kit is portable, sterile and disposable after use. RST detection test kits require no electrical power or expensive storage equipment and can be used in remote locations. 3.Market Analysis- According to the CDC, it is estimated that 1 million cases of shingles occur each year in the U.S. with more than half over the age of sixty. There is a high demand for rapid diagnostics by the public. The point-of-care testing (POCT) market is growing faster than other segments of in vitro diagnostics. According to a July 2007 InteLab Corporation industry report the overall market for POCT was forecast to increase from $10.3 billion in 2005 to $18.7 billion by 2011. The market value of this test kit has not been determined. 4.Competition- The VZV vaccine prevents 50% of cases and reduces neuralgia by 66%. The most popular test detects VZV-specific IgM antibody

  7. Rapid detection and characterization of Salmonella enterica ...

    African Journals Online (AJOL)

    Multiplex polymerase chain reaction (PCR) was used for molecular typing of Salmonella enterica serovars in Egypt. During the summer of 2010, a total of 1075 samples were collected from cattle, sheep and poultry farms to be subjected for isolation of Salmonella (290 rectal swabs from cattle, 335 rectal swabs from sheep ...

  8. Multiplex real-time PCR SYBR Green for detection and typing of group III Clostridium botulinum.

    Science.gov (United States)

    Anniballi, Fabrizio; Auricchio, Bruna; Delibato, Elisabetta; Antonacci, Monia; De Medici, Dario; Fenicia, Lucia

    2012-01-27

    Clostridium botulinum type C and type D belonging to the group III organisms, are mainly responsible for animal botulism outbreaks. Clinical signs alone are often insufficient to make a diagnosis of botulism and a laboratory confirmation is required. Laboratory confirmation can be performed by demonstrating the presence of botulinum neurotoxins in serum, gastrointestinal contents, liver, wound of sick or dead animals, or by demonstrating the presence of C. botulinum in gastrointestinal contents, liver, and wound. Demonstration of spores in gastrointestinal contents or tissue of animals with clinical signs indicative of botulism reinforces the clinical diagnosis. With the aim of detecting and typing C. botulinum group III organisms, a multiplex real-time PCR SYBR Green was developed and in-house validated. Selectivity, limit of detection, relative accuracy, relative specificity, relative sensitivity, and repeatability of the method were investigated. The multiplex real-time PCR SYBR green used showed a 100% selectivity, 100% relative accuracy, 100% relative specificity, 100% relative sensitivity and a limit of detection of 277 and 580 DNA copies for C. botulinum type C and C. botulinum type D, respectively. The method reported here represents a suitable tool for laboratory diagnosis of type C and D botulism and for testing a large number of samples collected during the animal botulism surveillance and prevention activities. Copyright © 2011 Elsevier B.V. All rights reserved.

  9. Detection of virulence-associated genes of Pasteurella multocida isolated from cases of fowl cholera by multiplex-PCR

    Directory of Open Access Journals (Sweden)

    Thales Q. Furian

    2013-02-01

    Full Text Available The current systems of breeding poultry, based on high population density, increase the risk of spreading pathogens, especially those causing respiratory diseases and those that have more than one host. Fowl Cholera (FC is one such pathogen, and even though it represents one of several avian diseases that should be considered in the differential diagnosis of notifiable diseases that present with sudden death, the pathogenesis and virulence factors involved in FC are still poorly understood. The objective of this study was to investigate twelve genes related to virulence in 25 samples of Pasteurella multocida isolated from FC cases in the southern region of Brazil through the development of multiplex PCR protocols. The protocols developed were capable of detecting all of the proposed genes. The ompH, oma87, sodC, hgbA, hgbB, exBD-tonB and nanB genes were present in 100% of the samples (25/25, the sodA and nanH genes were present in 96% (24/25, ptfA was present in 92% (23/25, and pfhA was present in 60% (15/25. Gene toxA was not identified in any of the samples studied (0/25. Five different genetic profiles were obtained, of which P1 (negative to toxA was the most common. We concluded that the multiplex-PCR protocols could be useful tools for rapid and simultaneous detection of virulence genes. Despite the high frequency of the analyzed genes and the fact that all samples belonged to the same subspecies of P. multocida, five genetic profiles were observed, which should be confirmed in a study with a larger number of samples.

  10. A simple approach for ultrasensitive detection of bisphenols by multiplexed surface-enhanced Raman scattering

    Energy Technology Data Exchange (ETDEWEB)

    De Bleye, C., E-mail: cdebleye@ulg.ac.be; Dumont, E.; Hubert, C.; Sacré, P.-Y.; Netchacovitch, L.; Chavez, P.-F.; Hubert, Ph.; Ziemons, E.

    2015-08-12

    Bisphenol A (BPA) is well known for its use in plastic manufacture and thermal paper production despite its risk of health toxicity as an endocrine disruptor in humans. Since the publication of new legislation regarding the use of BPA, manufacturers have begun to replace BPA with other phenolic molecules such as bisphenol F (BPF) and bisphenol B (BPB), but there are no guarantees regarding the health safety of these compounds at this time. In this context, a very simple, cheap and fast surface-enhanced Raman scattering (SERS) method was developed for the sensitive detection of these molecules in spiked tap water solutions. Silver nanoparticles were used as SERS substrates. An original strategy was employed to circumvent the issue of the affinity of bisphenols for metallic surfaces and the silver nanoparticles surface was functionalized using pyridine in order to improve again the sensitivity of the detection. Semi-quantitative detections were performed in tap water solutions at a concentrations range from 0.25 to 20 μg L{sup −1} for BPA and BPB and from 5 to 100 μg L{sup −1} for BPF. Moreover, a feasibility study for performing a multiplex-SERS detection of these molecules was also performed before successfully implementing the developed SERS method on real samples. - Highlights: • Development of a simple, fast and ultrasensitive SERS method to detect bisphenols. • Multiplexed-SERS detection of bisphenol A, bisphenol B and bisphenol F. • Implementation of the SERS developed method on real samples to detect bisphenols.

  11. Simultaneous detection of four foodborne viruses in food samples using a one-step multiplex reverse transcription PCR.

    Science.gov (United States)

    Lee, Shin-Young; Kim, Mi-Ju; Kim, Hyun-Joong; Jeong, KwangCheol Casey; Kim, Hae-Yeong

    2017-11-15

    A one-step multiplex reverse transcription PCR (RT-PCR) method comprising six primer sets (for the detection of norovirus GI and GII, hepatitis A virus, rotavirus, and astrovirus) was developed to simultaneously detect four kinds of pathogenic viruses. The size of the PCR products for norovirus GI and GII, hepatitis A virus (VP3/VP1 and P2A regions), rotavirus, and astrovirus were 330, 164, 244, 198, 629, and 449 bp, respectively. The RT-PCR with the six primer sets showed specificity for the pathogenic viruses. The detection limit of developed multiplex RT-PCR, as evaluated using serially diluted viral RNAs, was comparable to that of one-step single RT-PCR. Also, this multiplex RT-PCR was evaluated using food samples such as water, oysters, lettuce, and vegetable product. These food samples were artificially spiked with four kinds of viruses in diverse combinations, and the spiked viruses in all food samples were detected successfully.

  12. High throughput, multiplexed pathogen detection authenticates plague waves in medieval Venice, Italy.

    Directory of Open Access Journals (Sweden)

    Thi-Nguyen-Ny Tran

    Full Text Available BACKGROUND: Historical records suggest that multiple burial sites from the 14th-16th centuries in Venice, Italy, were used during the Black Death and subsequent plague epidemics. METHODOLOGY/PRINCIPAL FINDINGS: High throughput, multiplexed real-time PCR detected DNA of seven highly transmissible pathogens in 173 dental pulp specimens collected from 46 graves. Bartonella quintana DNA was identified in five (2.9% samples, including three from the 16th century and two from the 15th century, and Yersinia pestis DNA was detected in three (1.7% samples, including two from the 14th century and one from the 16th century. Partial glpD gene sequencing indicated that the detected Y. pestis was the Orientalis biotype. CONCLUSIONS: These data document for the first time successive plague epidemics in the medieval European city where quarantine was first instituted in the 14th century.

  13. High throughput, multiplexed pathogen detection authenticates plague waves in medieval Venice, Italy.

    Science.gov (United States)

    Tran, Thi-Nguyen-Ny; Signoli, Michel; Fozzati, Luigi; Aboudharam, Gérard; Raoult, Didier; Drancourt, Michel

    2011-03-10

    Historical records suggest that multiple burial sites from the 14th-16th centuries in Venice, Italy, were used during the Black Death and subsequent plague epidemics. High throughput, multiplexed real-time PCR detected DNA of seven highly transmissible pathogens in 173 dental pulp specimens collected from 46 graves. Bartonella quintana DNA was identified in five (2.9%) samples, including three from the 16th century and two from the 15th century, and Yersinia pestis DNA was detected in three (1.7%) samples, including two from the 14th century and one from the 16th century. Partial glpD gene sequencing indicated that the detected Y. pestis was the Orientalis biotype. These data document for the first time successive plague epidemics in the medieval European city where quarantine was first instituted in the 14th century.

  14. Snapping shrimp noise mitigation based on statistical detection in underwater acoustic orthogonal frequency division multiplexing systems

    Science.gov (United States)

    Kim, Hyeonsu; Seo, Jongpil; Ahn, Jongmin; Chung, Jaehak

    2017-07-01

    We propose a mitigation scheme for snapping shrimp noise when it corrupts an orthogonal frequency division multiplexing (OFDM) signal in underwater acoustic communication systems. The OFDM signal distorted by the snapping shrimp noise is filtered by a band-stop filter. The snapping shrimp noises in the filtered signal are detected by a detector with a constant false alarm rate whose threshold is derived theoretically from the statistics of the background noise. The detected signals are reconstructed by a simple reconstruction method. The proposed scheme has a higher detection capability and a lower mean square error of the channel estimation for simulated data and a lower bit error rate for practical ocean OFDM data collected in northern East China Sea than the conventional noise-mitigating methods.

  15. Multiplexed polarization OTDR system with high DOP and ability of multi-event detection.

    Science.gov (United States)

    Wang, Xuefeng; Wang, Chaodong; Tang, Ming; Fu, Songnian; Shum, Perry

    2017-05-01

    A novel polarization optical time domain reflectometry (POTDR) with high degree of polarization is proposed for multi-event detection. By employing multiple 2×2 optical fiber couplers and fiber mirrors, an arbitrary number and customized length of sensing fiber can be multiplexed into the system without modification of the other components, e.g., the light source, photodetector, signal processing device, etc. More importantly, the signal-to-noise ratio of this system is significantly improved, and the temporal depolarization effect can be almost completely suppressed. Additionally, the system response time is considerably reduced by dispensing with data averaging, so that intrusion events such as touching and moving fiber can be detected instantaneously and precisely located. Experiments have been conducted that proved the capability of multi-event simultaneous detection and vibration frequency measurement. This system promises application potential in multi-zone perimeter security and physical field measurement.

  16. Multiplex detection of pancreatic cancer biomarkers using a SERS-based immunoassay

    Science.gov (United States)

    Banaei, Nariman; Foley, Anne; Houghton, Jean Marie; Sun, Yubing; Kim, Byung

    2017-11-01

    Early diagnosis of pancreatic cancer (PC) is critical to reduce the mortality rate of this disease. Current biological analysis approaches cannot robustly detect several low abundance PC biomarkers in sera, limiting the clinical application of these biomarkers. Enzyme linked immunosorbent assay and radioimmunoassay are two common platforms for detection of biomarkers; however, they suffer from some limitation. This study demonstrates a novel system for multiplex detection of pancreatic biomarkers CA19-9, MMP7 and MUC4 in sera samples with high sensitivity using surface enhanced Raman spectroscopy. Measuring the levels of these biomarkers in PC patients, pancreatitis patients, and healthy individuals reveals the unique expression pattern of these markers in PC patients, suggesting the great potential of using this approach for early diagnostics of PCs.

  17. One-step multiplex real time RT-PCR for the detection of bovine respiratory syncytial virus, bovine herpesvirus 1 and bovine parainfluenza virus 3.

    Science.gov (United States)

    Thonur, Leenadevi; Maley, Madeleine; Gilray, Janice; Crook, Tara; Laming, Ellie; Turnbull, Dylan; Nath, Mintu; Willoughby, Kim

    2012-03-28

    Detection of respiratory viruses in veterinary species has traditionally relied on virus detection by isolation or immunofluorescence and/or detection of circulating antibody using ELISA or serum neutralising antibody tests. Multiplex real time PCR is increasingly used to diagnose respiratory viruses in humans and has proved to be superior to traditional methods. Bovine respiratory disease (BRD) is one of the most common causes of morbidity and mortality in housed cattle and virus infections can play a major role. We describe here a one step multiplex reverse transcriptase quantitative polymerase chain reaction (mRT-qPCR) to detect the viruses commonly implicated in BRD. A mRT-qPCR assay was developed and optimised for the simultaneous detection of bovine respiratory syncytial virus (BRSV), bovine herpes virus type 1 (BoHV-1) and bovine parainfluenza virus type 3 (BPI3 i & ii) nucleic acids in clinical samples from cattle. The assay targets the highly conserved glycoprotein B gene of BoHV-1, nucleocapsid gene of BRSV and nucleoprotein gene of BPI3. This mRT-qPCR assay was assessed for sensitivity, specificity and repeatability using in vitro transcribed RNA and recent field isolates. For clinical validation, 541 samples from clinically affected animals were tested and mRT-qPCR result compared to those obtained by conventional testing using virus isolation (VI) and/or indirect fluorescent antibody test (IFAT). The mRT-qPCR assay was rapid, highly repeatable, specific and had a sensitivity of 97% in detecting 102 copies of BRSV, BoHV-1 and BPI3 i & ii. This is the first mRT-qPCR developed to detect the three primary viral agents of BRD and the first multiplex designed using locked nucleic acid (LNA), minor groove binding (MGB) and TaqMan probes in one reaction mix. This test was more sensitive than both VI and IFAT and can replace the aforesaid methods for virus detection during outbreaks of BRD.

  18. Simultaneous detection and differentiation of four closely related sweet potato potyviruses by a multiplex one-step RT-PCR.

    Science.gov (United States)

    Li, Fan; Zuo, Ruijuan; Abad, Jorge; Xu, Donglin; Bao, Gaili; Li, Ruhui

    2012-12-01

    Four closely related potyviruses, Sweet potato feathery mottle virus (SPFMV), Sweet potato virus C (SPVC), Sweet potato virus G (SPVG) and/or Sweet potato virus 2 (SPV2), are involved in sweet potato virus disease complexes worldwide. Identification and detection of these viruses are complicated by high similarity among their genomic sequences, frequent occurrence as mixed infections and low titer in many sweet potato cultivars. A one-tube multiplex reverse transcription-PCR (mRT-PCR) assay was developed for simultaneous detection and differentiation of SPFMV, SPVC, SPVG and SPV2. Four specific forward primers unique to each virus and one reverse primer based on the region conserved in all four viruses were selected and used in the assay. The mRT-PCR assay was optimized for primer concentration and cycling conditions. It was tested using sweet potato plants infected naturally with one to four target viruses and then evaluated using field samples collected from southwestern China. The mRT-PCR assay is reliable and sensitive as a simple, rapid and cost-effective method to detect these pathogens in sweet potato. This assay will be useful to quarantine and certification programs and virus surveys when large numbers of samples are tested. Published by Elsevier B.V.

  19. Multiplex loop-mediated isothermal amplification detection by sequence-based barcodes coupled with nicking endonuclease-mediated pyrosequencing.

    Science.gov (United States)

    Liang, Chao; Chu, Yanan; Cheng, Sijia; Wu, Haiping; Kajiyama, Tomoharu; Kambara, Hideki; Zhou, Guohua

    2012-04-17

    The loop-mediated isothermal amplification (LAMP) is a well-developed method for replicating a targeted DNA sequence with a high specificity, but multiplex LAMP detection is difficult because LAMP amplicons are very complicated in structure. To allow simultaneous detection of multiple LAMP products, a series of target-specific barcodes were designed and tagged in LAMP amplicons by FIP primers. The targeted barcodes were decoded by pyrosequencing on nicked LAMP amplicons. To enable the nicking reaction to occur just near the barcode regions, the recognition sequence of the nicking endonuclease (NEase) was also introduced into the FIP primer. After the nicking reaction, pyrosequencing started at the nicked 3' end when the added deoxyribonucleoside triphosphate (dNTP) was complementary to the non-nicked strand. To efficiently encode multiple targets, the barcodes were designed with a reporter base and two stuffer bases, so that the decoding of a target-specific barcode only required a single peak in a pyrogram. We have successfully detected the four kinds of pathogens including hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), and Treponema pallidum (TP), which are easily infected in blood, by a 4-plex LAMP in a single tube, indicating that barcoded LAMP coupled with NEase-mediated pyrosequencing is a simple, rapid, and reliable way in multiple target identification.

  20. A Two-Tube Multiplex Reverse Transcription PCR Assay for Simultaneous Detection of Viral and Bacterial Pathogens of Infectious Diarrhea

    Directory of Open Access Journals (Sweden)

    Ji Wang

    2014-01-01

    Full Text Available Diarrhea caused by viral and bacterial infections is a major health problem in developing countries. The purpose of this study is to develop a two-tube multiplex PCR assay using automatic electrophoresis for simultaneous detection of 13 diarrhea-causative viruses or bacteria, with an intended application in provincial Centers for Diseases Control and Prevention, China. The assay was designed to detect rotavirus A, norovirus genogroups GI and GII, human astrovirus, enteric adenoviruses, and human bocavirus (tube 1, and Salmonella, Vibrio parahaemolyticus, diarrheagenic Escherichia coli, Campylobacter jejuni, Shigella, Yersinia, and Vibrio cholera (tube 2. The analytical specificity was examined with positive controls for each pathogen. The analytical sensitivity was evaluated by performing the assay on serial tenfold dilutions of in vitro transcribed RNA, recombinant plasmids, or bacterial culture. A total of 122 stool samples were tested by this two-tube assay and the results were compared with those obtained from reference methods. The two-tube assay achieved a sensitivity of 20–200 copies for a single virus and 102-103 CFU/mL for bacteria. The clinical performance demonstrated that the two-tube assay had comparable sensitivity and specificity to those of reference methods. In conclusion, the two-tube assay is a rapid, cost-effective, sensitive, specific, and high throughput method for the simultaneous detection of enteric bacteria and virus.

  1. Detection of Gastrointestinal Pathogens from Stool Samples on Hemoccult Cards by Multiplex PCR

    Directory of Open Access Journals (Sweden)

    Martin Alberer

    2017-01-01

    Full Text Available Purpose. Up to 30% of international travelers are affected by travelers’ diarrhea (TD. Reliable data on the etiology of TD is lacking. Sufficient laboratory capacity at travel destinations is often unavailable and transporting conventional stool samples to the home country is inconvenient. We evaluated the use of Hemoccult cards for stool sampling combined with a multiplex PCR for the detection of model viral, bacterial, and protozoal TD pathogens. Methods. Following the creation of serial dilutions for each model pathogen, last positive dilution steps (LPDs and thereof calculated last positive sample concentrations (LPCs were compared between conventional stool samples and card samples. Furthermore, card samples were tested after a prolonged time interval simulating storage during a travel duration of up to 6 weeks. Results. The LPDs/LPCs were comparable to testing of conventional stool samples. After storage on Hemoccult cards, the recovery rate was 97.6% for C. jejuni, 100% for E. histolytica, 97.6% for norovirus GI, and 100% for GII. Detection of expected pathogens was possible at weekly intervals up to 42 days. Conclusion. Stool samples on Hemoccult cards stored at room temperature can be used in combination with a multiplex PCR as a reliable tool for testing of TD pathogens.

  2. Quantum dot-assembled nanoparticles with polydiacetylene supramolecule toward label-free, multiplexed optical detection.

    Science.gov (United States)

    Kyeong, San; Kang, Homan; Yim, Joonhyuk; Jeon, Su-Ji; Jeong, Cheol Hwan; Lee, Yoon-Sik; Jun, Bong-Hyun; Kim, Jong-Ho

    2013-03-15

    Quantum dot (QD)-assembled silica nanoparticles bearing a polydiacetylene (PDA) supramolecule on their surface (SiO(2)@QDs@PDA NPs) were developed for label-free and multiplexed detection of biological molecules. Two types of QD-assembled silica NPs (SiO(2)@QDs NPs) were prepared and coated with the PDA supramolecule via photo-induced polymerization of 10,12-pentacosadiynoic acid. One of the SiO(2)@QDs NPs was embedded with blue-QDs, and the other was embedded with green-QDs for encoding. The resulting SiO(2)@QDs@PDA NPs showed discrete QD photoluminescence for encoding as well as PDA fluorescence for sensing a target without interference or overlap. Under heating stress of the SiO(2)@QDs@PDA NPs, the color of the PDA changed from blue to red, which allowed us to observe the fluorescence emitted from red PDA. The mixture of two different SiO(2)@QDs@PDA NPs, SiO(2)@QDs@blue-PDA NPs not emitting the fluorescence of PDA and SiO(2)@QDs@red-PDA NPs where stress was brought onto turn on the PDA fluorescence, was effectively imaged and readily distinguished via fluorescence microscopy, indicating their potential for label-free and multiplexed detection of target molecules. Copyright © 2012 Elsevier Inc. All rights reserved.

  3. Gold nanoparticle-enhanced multiplexed imaging surface plasmon resonance (iSPR) detection of Fusarium mycotoxins in wheat

    Science.gov (United States)

    A rapid, sensitive and multiplexed imaging surface plasmon resonance (iSPR) biosensor assay was developed and validated for three Fusarium toxins, deoxynivalenol (DON), zearalenone (ZEA) and T-2 toxin. The iSPR assay was based on a competitive inhibition format with secondary antibodies (Ab2) conjug...

  4. Rapid genetic detection of ingested Amanita phalloides.

    Science.gov (United States)

    Gausterer, Christian; Penker, Martina; Krisai-Greilhuber, Irmgard; Stein, Christina; Stimpfl, Thomas

    2014-03-01

    Mushrooms are often poorly digested by humans. Thus, their remains (tissues, spores) may persist in the gastrointestinal tract and can be detected in feces several days after mushroom consumption. In this report, we present protocols for the rapid PCR-based detection of fungal traces in a variety of complex samples. Novel primers were designed to amplify portions of ribosomal DNA from deadly poisonous European members of the genus Amanita, namely the death cap (A. phalloides), the destroying angel (A. virosa) and the fool's mushroom (A. verna), respectively. Assay sensitivity was sufficient to discover diluted DNA traces in amounts below the genomic content of a single target mushroom cell. Specificity testing was performed with DNA extracts from a variety of mushroom species. Template amplification was exclusively observed with intended targets and it was not compromised by a vast excess of non-target DNA (i.e. DNA from human and human fecal origin, respectively). A series of experiments was conducted with prepared specimens in order to follow the course of mushroom food processing and digestion. Amplification by direct PCR was successful with raw, fried and digested mixed mushrooms. To improve assay performance with fecal samples, a rapid protocol for sample pre-processing (including water-ether sedimentation and bead beating) and a modified PCR reaction mix were applied. Thereby, it was possible to detect the presence of A. phalloides DNA in spiked feces as well as in clinical samples (vomit, stool) from two independent cases of suspected mushroom poisoning. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  5. Comparison of Seeplex PneumoBacter aCE detection assay and in-house multiplex PCR for the identification of Streptococcus pneumoniae.

    Science.gov (United States)

    Luznik, Dane; Kosnik, Mitja; Tomic, Viktorija

    2015-01-01

    Rapid and accurate identification of Streptococcus pneumoniae is important for appropriate and prudent antimicrobial use in the treatment of lower respiratory tract infection. It is difficult to separate S. pneumoniae from commensal viridans group streptococci either by classical techniques or molecular methods. Aim of this study was to compare a commercially available multiplex PCR assay Seeplex PneumoBacter ACE Detection assay (Seegene, Seoul, South Korea), and in-house multiplex PCR using primer sets for lytA and cpsA for ability to differentiate S. pneumoniae in a known set of bacteria (S. pneumoniae and viridans group streptococci) and clinical samples. Of 20 viridans streptococcal isolates, 8 were misidentified as S. pneumoniae by commercial PCR test. Of 209 throat swabs tested with Seeplex PneumoBacter ACE Detection assay, 122 (58,4%) were positive for S. pneumoniae while only 11 (5.3%) samples were positive with lytA and cpsA primers. Therefore, the commercial multiplex PCR test appears to have low specificity in diagnosing S. pneumoniae.

  6. Universal primers for rapid detection of hytrosaviruses.

    Science.gov (United States)

    Abd-Alla, Adly M M; Salem, Tamer Z; Parker, Andrew G; Wang, Yongjie; Jehle, Johannes A; Vreysen, Marc J B; Boucias, Drion

    2011-01-01

    Hytrosaviridae is a proposed virus family encompassing viruses that cause salivary gland hypertrophy (SGH) syndrome in infected insects and reduce the fertility in their dipteran insect hosts. They contain a large, double stranded DNA genome of 120-190 kbp. To date, these viruses have been detected only in adult Diptera. These include hytrosaviruses detected in various tsetse fly species (Glossina spp.), the narcissus bulb fly Merodon equestris and the house fly Musca domestica. The limited number of hytrosaviruses reported to date may be a reflection of the frequent absence of external symptoms in infected adult flies and the fact that the virus does not cause rapid mortality. Based on the complete genome sequence of Glossinia pallidipes (GpSGHV) and Musca domestica (MdSGHV) salivary gland hypertrophy viruses, a PCR based methodology was developed to detect the viruses in these species. To be able to detect hytrosaviruses in other Diptera, five degenerate primer pairs were designed and tested on GpSGHV and MdSGHV DNA using gradient PCR with annealing temperatures from 37 to 61°C. Two pairs of primers were selected from p74, two pairs from PIF-1 and one pair from ODV-e66 homologous proteins. Four primer pairs generated a virus specific PCR product on both MdSGHV and GpSGHV at all tested annealing temperatures, while the ODV-e66 based primers did not generate a virus specific product with annealing temperatures higher that 47°C. No non-specific PCR product was found when using genomic DNA of infected flies as template DNA. These results offer new sets of primers that could be used to detect hytrosaviruses in other insects. Copyright © 2010 Elsevier B.V. All rights reserved.

  7. Use of multiplex allele-specific polymerase chain reaction (MAS-PCR to detect multidrug-resistant tuberculosis in Panama.

    Directory of Open Access Journals (Sweden)

    Bing-Shao Chia

    Full Text Available The frequency of individual genetic mutations conferring drug resistance (DR to Mycobacterium tuberculosis has not been studied previously in Central America, the place of origin of many immigrants to the United States. The current gold standard for detecting multidrug-resistant tuberculosis (MDR-TB is phenotypic drug susceptibility testing (DST, which is resource-intensive and slow, leading to increased MDR-TB transmission in the community. We evaluated multiplex allele-specific polymerase chain reaction (MAS-PCR as a rapid molecular tool to detect MDR-TB in Panama. Based on DST, 67 MDR-TB and 31 drug-sensitive clinical isolates were identified and cultured from an archived collection. Primers were designed to target five mutation hotspots that confer resistance to the first-line drugs isoniazid and rifampin, and MAS-PCR was performed. Whole-genome sequencing confirmed DR mutations identified by MAS-PCR, and provided frequencies of genetic mutations. DNA sequencing revealed 70.1% of MDR strains to have point mutations at codon 315 of the katG gene, 19.4% within mabA-inhA promoter, and 98.5% at three hotspots within rpoB. MAS-PCR detected each of these mutations, yielding 82.8% sensitivity and 100% specificity for isoniazid resistance, and 98.4% sensitivity and 100% specificity for rifampin resistance relative to DST. The frequency of individual DR mutations among MDR strains in Panama parallels that of other TB-endemic countries. The performance of MAS-PCR suggests that it may be a relatively inexpensive and technically feasible method for rapid detection of MDR-TB in developing countries.

  8. Nanostructured bioluminescent sensor for rapidly detecting thrombin.

    Science.gov (United States)

    Chen, Longyan; Bao, Yige; Denstedt, John; Zhang, Jin

    2016-03-15

    Thrombin plays a key role in thrombosis and hemostasis. The abnormal level of thrombin in body fluids may lead to different diseases, such as rheumatoid arthritis, glomerulonephritis, etc. Detection of thrombin level in blood and/or urine is one of important methods for medical diagnosis. Here, a bioluminescent sensor is developed for non-invasively and rapidly detecting thrombin in urine. The sensor is assembled through conjugating gold nanoparticles (Au NPs) and a recombinant protein containing Renilla luciferase (pRluc) by a peptide, which is thrombin specific substrate. The luciferase-catalyzed bioluminescence can be quenched by peptide-conjugating Au NPs. In the presence of thrombin, the short peptide conjugating luciferase and Au NPs is digested and cut off, which results in the recovery of bioluminescence due to the release of luciferase from Au NPs. The bioluminescence intensity at 470 nm is observed, and increases with increasing concentration of thrombin. The bioluminescence intensity of this designed sensor is significantly recovered when the thrombin digestion time lasts for 10 min. In addition, a similar linear relationship between luminescence intensity and the concentration of thrombin is found in the range of 8 nM to 8 μM in both buffer and human urine spiked samples. The limit of detection is as low as 80 pM. It is anticipated that our nanosensor could be a promising tool for clinical diagnosis of thrombin in human urine. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. A handheld flow genetic analysis system (FGAS): towards rapid, sensitive, quantitative and multiplex molecular diagnosis at the point-of-care level.

    Science.gov (United States)

    Shu, Bowen; Zhang, Chunsun; Xing, Da

    2015-06-21

    A handheld flow genetic analysis system (FGAS) is proposed for rapid, sensitive, multiplex and real-time quantification of nucleic acids at the point-of-care (POC) level. The FGAS includes a helical thermal-gradient microreactor and a microflow actuator, as well as control circuitry for temperature, fluid and power management, and smartphone fluorescence imaging. All of these features are integrated into a field-portable and easy-to-use molecular diagnostic platform powered by lithium batteries. Due to the unique design of the microreactor, not only steady temperatures for denaturation and annealing/extension but also a linear thermal gradient for spatial high-resolution melting can be achieved through simply maintaining a single heater at constant temperature. The smartphone fluorescence imaging system has a wide field of view that captures all PCR channels of the microreactor in a single snapshot without the need for any mechanical scanning. By these designs, the FGAS enables real-time monitoring of the temporal and spatial fluorescence signatures of amplicons during continuous-flow amplification. On the current FGAS, visual detection of as little as 10 copies per μL of genomic DNA of Salmonella enterica was achieved in 15 min, with real-time quantitative detection of the DNA over 6 orders of magnitude concentration from 10(6) to 10(1) copies per μL also completed in 7.5-15 min. In addition, multiple pathogenic DNA targets could be simultaneously discriminated with direct bar-chart readout or multiplex spatial melting in serial flow. We anticipate that the FGAS has great potential to become a next-generation gene analyzer for POC molecular diagnostics.

  10. Detection of Salmonella spp, Salmonella Enteritidis and Typhimurium in naturally infected broiler chickens by a multiplex PCR-based assay

    Directory of Open Access Journals (Sweden)

    F.G. Paião

    2013-01-01

    Full Text Available The presence of Salmonella in the intestinal tract, on the chickens skin and among their feathers, may cause carcasses contamination during slaughtering and processing and possibly it is responsible by the introduction of this microorganism in the slaughterhouses. A rapid method to identify and monitor Salmonella and their sorovars in farm is becoming necessary. A pre-enriched multiplex polymerase chain reaction (m-PCR assay employing specific primers was developed and used to detect Salmonella at the genus level and to identify the Salmonella enterica serovar Enteritidis (S. Enteritidis and Salmonella enterica serovar Typhimurium (S. Typhimurium in broiler chicken swab samples. The method was validated by testing DNA extract from 90 fresh culture cloacal swab samples from poultry chicken cultured in phosphate buffer peptone water at 37 ºC for 18 h. The final results showed the presence of Salmonella spp. in 25% of samples, S. Enteritidis was present in 12% of the Salmonella-positive samples and S. Typhimurium in 3% of the samples. The m-PCR assay developed in this study is a specific and rapid alternative method for the identification of Salmonella spp. and allowed the observation of specific serovar contamination in the field conditions within the locations where these chickens are typically raised.

  11. Detection of Salmonella spp, Salmonella Enteritidis and Typhimurium in naturally infected broiler chickens by a multiplex PCR-based assay.

    Science.gov (United States)

    Paião, F G; Arisitides, L G A; Murate, L S; Vilas-Bôas, G T; Vilas-Boas, L A; Shimokomaki, M

    2013-01-01

    The presence of Salmonella in the intestinal tract, on the chickens skin and among their feathers, may cause carcasses contamination during slaughtering and processing and possibly it is responsible by the introduction of this microorganism in the slaughterhouses. A rapid method to identify and monitor Salmonella and their sorovars in farm is becoming necessary. A pre-enriched multiplex polymerase chain reaction (m-PCR) assay employing specific primers was developed and used to detect Salmonella at the genus level and to identify the Salmonella enterica serovar Enteritidis (S. Enteritidis) and Salmonella enterica serovar Typhimurium (S. Typhimurium) in broiler chicken swab samples. The method was validated by testing DNA extract from 90 fresh culture cloacal swab samples from poultry chicken cultured in phosphate buffer peptone water at 37 °C for 18 h. The final results showed the presence of Salmonella spp. in 25% of samples, S. Enteritidis was present in 12% of the Salmonella-positive samples and S. Typhimurium in 3% of the samples. The m-PCR assay developed in this study is a specific and rapid alternative method for the identification of Salmonella spp. and allowed the observation of specific serovar contamination in the field conditions within the locations where these chickens are typically raised.

  12. Multiplexed Enrichment and Detection of Malarial Biomarkers Using a Stimuli-Responsive Iron Oxide and Gold Nanoparticle Reagent System

    Science.gov (United States)

    Nash, Michael A.; Waitumbi, John N.; Hoffman, Allan S.; Yager, Paul; Stayton, Patrick S.

    2014-01-01

    There is a need for simple yet robust biomarker and antigen purification and enrichment strategies that are compatible with current rapid diagnostic modalities. Here, a stimuli-responsive nanoparticle system is presented for multiplexed magneto-enrichment and non-instrumented lateral flow strip detection of model antigens from spiked pooled plasma. The integrated reagent system allows purification and enrichment of the gold-labeled biomarker half-sandwich that can be applied directly to lateral flow test strips. A linear diblock copolymer with a thermally-responsive poly(N-isopropylacrylamide) (pNIPAm) segment and a gold-binding block composed of NIPAm-co-N,N-dimethylaminoethylacrylamide (DMAEAm) was prepared by reversible addition fragmentation chain transfer (RAFT) polymerization. The diblock copolymer was used to functionalize gold nanoparticles (AuNPs), with subsequent bioconjugation to yield thermally-responsive pNIPAm-AuNPs that were co-decorated with streptavidin. These AuNPs efficiently complexed biotinylated capture antibody reagents that were bound to picomolar quantities of pan-aldolase and Plasmodium Falciparum histidine rich protein 2 (PfHRP2) in spiked pooled plasma samples. The gold-labeled biomarker half-sandwich was then purified and enriched using 10 nm thermally-responsive magnetic nanoparticles that were similarly decorated with pNIPAm. When a thermal stimulus was applied in conjunction with a magnetic field, co-aggregation of the AuNP-half-sandwiches with the pNIPAm-coated iron oxide nanoparticles created large aggregates that were efficiently magnetophoresed and separated from bulk serum. The purified biomarkers from a spiked pooled plasma sample could be concentrated 50-fold into a small volume and applied directly to a commercial multiplexed lateral flow strip to dramatically improve the signal-to-noise ratio and test sensitivity. PMID:22804625

  13. Multiplexed enrichment and detection of malarial biomarkers using a stimuli-responsive iron oxide and gold nanoparticle reagent system.

    Science.gov (United States)

    Nash, Michael A; Waitumbi, John N; Hoffman, Allan S; Yager, Paul; Stayton, Patrick S

    2012-08-28

    There is a need for simple yet robust biomarker and antigen purification and enrichment strategies that are compatible with current rapid diagnostic modalities. Here, a stimuli-responsive nanoparticle system is presented for multiplexed magneto-enrichment and non-instrumented lateral flow strip detection of model antigens from spiked pooled plasma. The integrated reagent system allows purification and enrichment of the gold-labeled biomarker half-sandwich that can be applied directly to lateral flow test strips. A linear diblock copolymer with a thermally responsive poly(N-isopropylacrylamide) (pNIPAm) segment and a gold-binding block composed of NIPAm-co-N,N-dimethylaminoethylacrylamide was prepared by reversible addition-fragmentation chain transfer polymerization. The diblock copolymer was used to functionalize gold nanoparticles (AuNPs), with subsequent bioconjugation to yield thermally responsive pNIPAm-AuNPs that were co-decorated with streptavidin. These AuNPs efficiently complexed biotinylated capture antibody reagents that were bound to picomolar quantities of pan-aldolase and Plasmodium falciparum histidine-rich protein 2 (PfHRP2) in spiked pooled plasma samples. The gold-labeled biomarker half-sandwich was then purified and enriched using 10 nm thermally responsive magnetic nanoparticles that were similarly decorated with pNIPAm. When a thermal stimulus was applied in conjunction with a magnetic field, coaggregation of the AuNP half-sandwiches with the pNIPAm-coated iron oxide nanoparticles created large aggregates that were efficiently magnetophoresed and separated from bulk serum. The purified biomarkers from a spiked pooled plasma sample could be concentrated 50-fold into a small volume and applied directly to a commercial multiplexed lateral flow strip to dramatically improve the signal-to-noise ratio and test sensitivity.

  14. A microfluidic device for multiplexed protein detection in nano-liter volumes.

    Science.gov (United States)

    Diercks, Alan H; Ozinsky, Adrian; Hansen, Carl L; Spotts, James M; Rodriguez, David J; Aderem, Alan

    2009-03-01

    We describe a microfluidic immunoassay device that permits sensitive and quantitative multiplexed protein measurements on nano-liter-scale samples. The device exploits the combined power of integrated microfluidics and optically encoded microspheres to create an array of approximately 100-microm(2) sensors functionalized with capture antibodies directed against distinct targets. This strategy overcomes the need for performing biochemical coupling of affinity reagents to the device substrate, permits multiple proteins to be detected in a nano-liter-scale sample, is scalable to large numbers of samples, and has the required sensitivity to measure the abundance of proteins derived from single mammalian cells. The sensitivity of the device is sufficient to detect 1000 copies of tumor necrosis factor (TNF) in a volume of 4.7nl.

  15. Autonomous Detection of Aerosolized Biological Agents by Multiplexed Immunoassay with PCR Confirmation

    Energy Technology Data Exchange (ETDEWEB)

    Hindson, B J; McBride, M T; Makarewicz, A J; Henderer, B D; Setlur, U S; Smith, S M; Gutierrez, D M; Metz, T R; Nasarabadi, S L; Venkateswaran, K S; Farrow, S W; Colston, Jr., B W; Dzenitis, J M

    2004-05-27

    The autonomous pathogen detection system (APDS) is an automated, podium-sized instrument that continuously monitors the air for biological threat agents (bacteria, viruses, and toxins). The system has been developed to warn of a biological attack in critical or high-traffic facilities and at special events. The APDS performs continuous aerosol collection, sample preparation, and detection using multiplexed immunoassay followed by confirmatory PCR using real-time TaqMan assays. We have integrated completely reusable flow-through devices that perform DNA extraction and PCR amplification. The fully integrated system was challenged with aerosolized Bacillus anthracis, Yersinia pestis, Bacillus globigii and botulinum toxoid. By coupling highly selective antibody and DNA based assays, the probability of an APDS reporting a false positive is extremely low.

  16. Development of one-tube multiplex polymerase chain reaction (PCR) for detecting Mycobacterium bovis.

    Science.gov (United States)

    Quan, Zhang; Haiming, Tan; Xiaoyao, Cai; Weifeng, Yuan; Hong, Jia; Hongfei, Zhu

    2017-01-10

    A multiplex PCR (m-PCR) with primers targeting the 16S rRNA, Rv3873 and a 12.7-kb fragment in the genomes of a Mycobacterium tuberculosis complex was designed for the differential diagnosis of M. tuberculosis, M. bovis, M. bovis BCG and non-tuberculosis Mycobacterium (NTM). The specificity of this assay was 100%, and the detection limit was 15 pg of genomic DNA. Of the 206 blinded clinical samples, the detection rate of M. bovis infection by m-PCR was lower than that of the interferon gamma (IFN-γ) release assay; however, the false-positive rate by the tuberculin skin test and false-negative samples in the IFN-γ release assay were reduced. Our findings indicated that our m-PCR method is a useful tool for complementation to differentiate M. bovis from M. tuberculosis and NTM species.

  17. Detection of Shiga toxins genes by Multiplex PCR in clinical samples

    Directory of Open Access Journals (Sweden)

    2013-09-01

    Full Text Available Background: Different methods have been used for detection of shiga toxins; such as,  cell culture, ELISA, and RFPLA. However, all of these methods suffer from high cost, time-consumption and relatively low sensitivity. In this study we used Multiplex PCR method for detection of genes encoding shiga toxins. Material and Methods: In this study, 63 clinical samples were obtained from positive cultures of Shigella and E. coli O157, from Bahman 1391 until Ordibehesht 1392 in Mazandaran province. Initial confirmation of shiga toxins producing bacteria was performed by biochemical and serological methods. After DNA extraction, detection of stx1 and stx2 genes was accomplished by multiplex PCR.  For confirmation of the PCR amplicon, DNA sequencing was used. Antibiotic sensitivity tests were performed by disk diffusion method. Results:  Among the positive strains, 13 strains contained stx2 genes, 4 strains contained Stx/Stx1 genes and 4 strains harbored both Stx/Stx1 and Stx2. The DNA extracted from other Gram-negative bacteria was not protected by the relevant parts of these toxins. Sequencing of the amplified fragments indicated the correct toxin sequences.  The sensitivity for identification of Stx/Stx1 gene was 1.56 pg/ µl and for Stx2 was 1.08 pg/µl. The toxin positive strains were all sensitive to Cefixime, Gentamicin, Amikacin, Ceftriaxone, and Nitrofurantoin. Conclusion: This method is fast and accurate for detection of bacteria producing shiga toxin and can be used to identify different types of shiga toxin.

  18. Multiplex Assay for Simultaneous Detection of Mycoplasma genitalium and Macrolide Resistance Using PlexZyme and PlexPrime Technology.

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    Sepehr N Tabrizi

    Full Text Available Mycoplasma genitalium is a cause of non-gonoccocal urethritis (NGU in men and cervicitis and pelvic inflammatory disease in women. Recent international data also indicated that the first line treatment, 1 gram stat azithromycin therapy, for M. genitalium is becoming less effective, with the corresponding emergence of macrolide resistant strains. Increasing failure rates of azithromycin for M. genitalium has significant implications for the presumptive treatment of NGU and international clinical treatment guidelines. Assays able to predict macrolide resistance along with detection of M. genitalium will be useful to enable appropriate selection of antimicrobials to which the organism is susceptible and facilitate high levels of rapid cure. One such assay recently developed is the MG 23S assay, which employs novel PlexZyme™ and PlexPrime™ technology. It is a multiplex assay for detection of M. genitalium and 5 mutations associated with macrolide resistance. The assay was evaluated in 400 samples from 254 (186 males and 68 females consecutively infected participants, undergoing tests of cure. Using the MG 23S assay, 83% (331/440 of samples were positive, with 56% of positives carrying a macrolide resistance mutation. Comparison of the MG 23S assay to a reference qPCR method for M. genitalium detection and high resolution melt analysis (HRMA and sequencing for detection of macrolide resistance mutations, resulted in a sensitivity and specificity for M. genitalium detection and for macrolide resistance of 99.1/98.5% and 97.4/100%, respectively. The MG 23S assay provides a considerable advantage in clinical settings through combined diagnosis and detection of macrolide resistance.

  19. A new multiplex polymerase chain reaction assay for the identification a panel of bacteria involved in bacteremia

    Directory of Open Access Journals (Sweden)

    Hossein Fazzeli

    2013-01-01

    Conclusions: The presented multiplex PCR offers a rapid and accurate molecular diagnostic tool for simultaneous detection of some pathogenic microorganisms. The IC exists in the multiplex PCR accompanied by other primers in the system, can serve as a simple, cost- effective internal control for the multiplex PCR assay.

  20. Detection of human beta defensin-1 and -2 by RT-competitive multiplex PCR.

    Science.gov (United States)

    Kippenberger, Stefan; Loitsch, Stefan; Thaci, Diamant; Kaufmann, Roland; Bernd, August

    2005-05-01

    Although, the human epithelium is constantly challenged by a broad spectrum of microorganisms, invasive infections are rather rare. Recent findings suggest the expression of antimicrobial peptides by skin cells in order to provide an innate defensive barrier. In particular, peptides of the beta-defensin family offer antimicrobial activity against different pathogens including bacteria and fungi. Within this peptide family, hBD-1 is rather constitutively expressed while hBD-2 and hBD-3 expression depends on environmental conditions. The present paper introduces RT-competitive multiplex PCR as a precise tool to detect hBD-1 and hBD-2 expression on the transcriptional level. The method makes use of co-amplification of synthetic competitors along with referring wildtype targets. Competitor- and wildtype-derived products differ in size allowing signal discrimination using agarose gel electrophoresis. Regulation of gene transcripts is evaluated by comparison of competitor and corresponding wildtype signals. It was found that primary human keratinocytes stimulated with Escherichia coli cells for 8 h offered an upregulation of hBD-2 to about 2,000 fold, while hBD-1 was only marginally regulated. RT-competitive multiplex PCR is a simple and accurate method that enables new insights into defensin regulation under physiological and pathophysiological conditions.

  1. Response surface methodology to design a selective co-enrichment broth of Escherichia coli, Salmonella spp. and Staphylococcus aureus for simultaneous detection by multiplex PCR.

    Science.gov (United States)

    Zhang, Qiao Yan; Zhou, Wen Wu; Zhou, Yu; Wang, Xiao Fu; Xu, Jun Feng

    2012-07-25

    Escherichia coli, Salmonella spp. and Staphylococcus aureus are frequent co-visitors of contaminated foods to cause food-borne diseases. To achieve rapid detection of three organisms by multiplex PCR, a selective co-enrichment broth was considered to design using response surface methodology (RSM) in this work. NaCl, LiCl and KSCN as selective bacterial inhibitors were selected to optimize their concentrations for a matched composition of bacterial biomass with uniform amplification of three targets. Central composite design was employed to collect the data and fit the responses. Three quadratic polynomial models were derived by computer simulation. A statistical analysis was carried out to explore the effects of the variables on the composition of bacterial biomass and PCR amplification yields. In the end, a novel broth (ESS-3 broth) of NaCl 1.60%, LiCl 0.70%, KSCN 0.10% was formulated to allow co-enrichment of the target pathogens and suppress growth of some non-target pathogens. The simultaneous detection of E. coli, Salmonella spp. and S. aureus was developed on a rapid, convenient and sensitive method consisting of selective co-enrichment in ESS-3 broth, DNA extraction with the boiling method and robust test by multiplex PCR. Our work provided broader application of RSM for the simultaneous detection of other combinations of multiple pathogens. Copyright © 2012 Elsevier GmbH. All rights reserved.

  2. Multiplex reverse transcription loop-mediated isothermal amplification for the simultaneous detection of CVB and CSVd in chrysanthemum.

    Science.gov (United States)

    Liu, Xing-Liang; Zhao, Xi-Ting; Muhammad, Imtiaz; Ge, Bei-Bei; Hong, Bo

    2014-12-15

    A multiplex reverse transcription loop-mediated isothermal amplification (mRT-LAMP) assay was developed for the simultaneous detection of Chrysanthemum Virus B (CVB) and Chrysanthemum stunt viroid (CSVd), which are the major viral pathogens of chrysanthemum worldwide. Two sets of mRT-LAMP primers were designed for the coat protein gene of CVB and the complete nucleotide sequence of CSVd, and a restriction enzyme cleavage site was inserted into two pairs of species-specific primers. The mRT-LAMP assay was designed by combining these two sets for a total of eight primers. The mRT-LAMP method distinguished between CVB and CSVd due to the subsequent restriction enzyme analysis. The sensitivity of the mRT-LAMP method was 10(3) times higher than classical PCR regarding the detection limits for CVB and CSVd. No positive results were observed when RNA from other chrysanthemum pathogens were used as mRT-LAMP templates. The method was verified by testing chrysanthemum samples collected from Beijing and Henan Province and showed high reliability and sensitivity. The developed mRT-LAMP assay also offers an efficient, convenient, and rapid tool for screening chrysanthemum virus and viroid, especially CVB and CSVd, and can be diagnosed in a single reaction. These results suggest that the new mRT-LAMP method may be used routinely for virus and viroid surveys. Copyright © 2014 Elsevier B.V. All rights reserved.

  3. Multiplexed Detection of Foodborne Pathogens from Contaminated Lettuces Using a Handheld Multistep Lateral Flow Assay Device.

    Science.gov (United States)

    Shin, Joong Ho; Hong, Jisoo; Go, Hyeyun; Park, Juhwan; Kong, Minsuk; Ryu, Sangryeol; Kim, Kwang-Pyo; Roh, Eunjung; Park, Je-Kyun

    2018-01-10

    This paper presents a handheld device that is capable of simplifying multistep assays to perform sensitive detection of foodborne pathogens. The device is capable of multiplexed detection of Escherichia coli (E. coli) O157:H7, Salmonella Typhimurium (S. Typhimurium), Staphylococcus aureus, and Bacillus cereus. The limit of detection for each bacterium was characterized, and then, the detection of bacteria from contaminated fresh lettuces was demonstrated for two representative foodborne pathogens. We employed a sample pretreatment protocol to recover and concentrate target bacteria from contaminated lettuces, which can detect 1.87 × 10 4 CFU of E. coli O157:H7 and 1.47 × 10 4 CFU of S. Typhimurium/1 g of lettuce without an enrichment process. Lastly, we demonstrated that the limit of detection can be reduced to 1 CFU of E. coli O157:H7 and 1 CFU of S. Typhimurium/1 g of lettuce by including a 6 h enrichment of contaminated lettuces in growth media before pretreatment.

  4. Multiplex PCR system for the rapid diagnosis of respiratory virus infection: systematic review and meta-analysis.

    Science.gov (United States)

    Huang, H-S; Tsai, C-L; Chang, J; Hsu, T-C; Lin, S; Lee, C-C

    2017-12-05

    To provide a summary of evidence for the diagnostic accuracies of three multiplex PCR systems (mPCRs)-BioFire FilmArray RP (FilmArray), Nanosphere Verigene RV+ test (Verigene RV+) and Hologic Gen-Probe Prodesse assays-on the detection of viral respiratory infections. A comprehensive search up to 1 July 2017 was conducted on Medline and Embase for studies that utilized FilmArray, Verigene RV+ and Prodesse for diagnosis of viral respiratory infections. A summary of diagnostic accuracies for the following five viruses were calculated: influenza A virus (FluA), influenza B virus, respiratory syncytial virus, human metapneumovirus and adenovirus. Hierarchical summary receiver operating curves were used for estimating the viral detection performance per assay. Twenty studies of 5510 patient samples were eligible for analysis. Multiplex PCRs demonstrated high diagnostic accuracy, with area under the receiver operating characteristic curve (AUROC) equal to or more than 0.98 for all the above viruses except for adenovirus (AUROC 0.89). FilmArray, Verigene RV+ and ProFlu+ (the only Prodesse assay with enough data) demonstrated a summary sensitivity for FluA of 0.911 (95% confidence interval, 0.848-0.949), 0.949 (95% confidence interval, 0.882-0.979) and 0.954 (95% confidence interval, 0.871-0.985), respectively. The three mPCRs were comparable in terms of detection of FluA. Point estimates calculated from eligible studies showed that the three mPCRs (FilmArray, Verigene RV+ and ProFlu+) are highly accurate and may provide important diagnostic information for early identification of respiratory virus infections. In patients with low pretest probability for FluA, these three mPCRs can predict a low possibility of infection and may justify withholding empirical antiviral treatments. Copyright © 2017 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

  5. Rapid aneuploidy diagnosis by multiplex ligation-dependent probe amplification and array comparative genomic hybridization in pregnancy with major congenital malformations

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    Chih-Ping Chen

    2011-03-01

    Conclusions: Prenatal diagnosis of major congenital malformations should alert one to the possibility of chromosomal abnormalities. Multiplex ligation-dependent probe amplification and aCGH have the advantage of rapid aneuploidy diagnosis of common aneuploidies in cases with major congenital malformations.

  6. Comparison of two multiplex PCR assays for the detection of Listeria spp. and Listeria monocytogenes in biological samples

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    Budniak Sylwia

    2016-12-01

    Full Text Available Introduction: The aim of the study was to optimise and compare two multiplex PCR assays for the detection of Listeria spp. and Listeria monocytogenes in biological samples including the liver, brain, and blood. Material and Methods: Three strains of L. monocytogenes and single strains of each of the species: L. ivanovii, L. innocua, L. grayi, L. welshimeri, and L. seeligeri were used. Additionally, five other species of bacterium were used to evaluate the specificity of the tests. Results: Specific amplification products were obtained for both multiplex PCR assays, which confirmed the tested strains as Listeria spp. and L. monocytogenes, respectively. Isolates of other species did not yield PCR products. Conclusion: Both multiplex PCR assays proved to be significantly sensitive and highly-specific methods for the detection of Listeria strains.

  7. Comparative study of colloidal gold and quantum dots as labels for multiplex screening tests for multi-mycotoxin detection

    Energy Technology Data Exchange (ETDEWEB)

    Foubert, Astrid, E-mail: astrid.foubert@hotmail.com; Beloglazova, Natalia V.; De Saeger, Sarah

    2017-02-22

    Quantum dots (QDs) and colloidal gold nanoparticles (CG) were evaluated as labels for multiplex lateral flow immunoassay (LFIA) for determination of mycotoxins deoxynivalenol (DON), zearalenone (ZEN) and T2/HT2-toxin (T2/HT2) in cereal matrices. Both developed assays were based on the same immunoreagents (except for the labels), therefore their analytical characteristics could be objectively compared. For both LFIAs antigens (DON-ovalbumin (OVA), ZEN-OVA and T2-OVA) and rabbit anti-mouse immunoglobulin were immobilized on a nitrocellulose membrane as three test lines and one control line, respectively. Depending on the LFIA, monoclonal antibodies (mAb) against DON, ZEN and T2 were conjugated with CdSeS/ZnS QDs or CG. T2 and HT2 were detected by one test line (T2-OVA) with an anti-T2 mAb which showed 110% cross-reactivity with HT2. Both tests were developed in accordance with the legal limits and were developed in such a way that they had the same cut-off limits of 1000 μg kg{sup −1}, 80 μg kg{sup −1} and 80 μg kg{sup −1} for DON, ZEN and T2/HT2, respectively in order to allow a correct comparison. Applicability of these assays was demonstrated by analysis of naturally contaminated wheat samples. The results demonstrate that both the LFIAs can be used as rapid, cost-effective and convenient qualitative tool for on-site screening for simultaneous detection of DON, ZEN and HT2/T2 in wheat without special instrumentation. However, the QD-based LFIA consumed less immunoreagents and was more sensitive and economically beneficial. In addition, the results were easier to interpret, resulting in a lower false negative rate (<5%) which was in good agreement with Commission Decision 2002/657/EC regarding the performance of analytical methods intended for screening purposes. - Highlights: • Development of colloidal gold- and quantum dot-based multiplex lateral flow immunoassay. • Lateral flow immunoassays allow simultaneous detection of four mycotoxins.

  8. Multiplex detection of homo- and heterodimerization of g protein-coupled receptors by proximity biotinylation.

    Science.gov (United States)

    Steel, Elisabeth; Murray, Victoria L; Liu, Allen P

    2014-01-01

    Dimerization of G protein-coupled receptors (GPCRs) represents a potential mechanism by which GPCR functions are regulated. Several resonance energy transfer (RET)-based methods have revealed GPCR homo- and heterodimerization. However, interpretation of an increase in FRET efficiency could be attributed to either dimerization/oligomerization events or conformational changes within an already dimerized/oligomerized receptor complex. Furthermore, RET-based methods can only measure pairwise dimerization, and cannot easily achieve multiplex detection. In this study, we applied proximity-based biotinylation for detecting receptor dimerization by utilizing a specific enzyme-substrate pair that are fused to GPCRs. The biotin ligase BirA is fused to CXCR4 and site-specifically biotinylates an acceptor peptide (AP) in the presence of biotin. As a test case for our newly developed assay, we have characterized the homo-dimerization of chemokine receptor CXCR4 and heterodimerization of CXCR4 with CCR2 or CCR5. The degree of biotinylation varies with the amount of GPCR-AP as well as biotinylation time. Using enzyme/substrate receptor pairs and measuring receptor biotinylation, we demonstrate that CXCR4 can homo-dimerize and hetero-dimerize with CCR2 and CCR5. The effect of CXCL12, agonist for CXCR4, was found to decrease surface biotinylation of CXCR4-AP. This effect is due to a combination of CXCR4 endocytosis and stabilization of CXCR4 homodimers. Finally, when CXCR4-AP, CCR2-AP, and CCR5-AP were expressed together, we observed CXCR4-CXCR4 homodimers and CXCR4-CCR2 and CXCR4-CCR5 heterodimers. The newly developed assay opens new opportunity for multiplex detection for GPCR homo- and heterodimerization within the same cellular context.

  9. Multiplex detection of homo- and heterodimerization of g protein-coupled receptors by proximity biotinylation.

    Directory of Open Access Journals (Sweden)

    Elisabeth Steel

    Full Text Available Dimerization of G protein-coupled receptors (GPCRs represents a potential mechanism by which GPCR functions are regulated. Several resonance energy transfer (RET-based methods have revealed GPCR homo- and heterodimerization. However, interpretation of an increase in FRET efficiency could be attributed to either dimerization/oligomerization events or conformational changes within an already dimerized/oligomerized receptor complex. Furthermore, RET-based methods can only measure pairwise dimerization, and cannot easily achieve multiplex detection. In this study, we applied proximity-based biotinylation for detecting receptor dimerization by utilizing a specific enzyme-substrate pair that are fused to GPCRs. The biotin ligase BirA is fused to CXCR4 and site-specifically biotinylates an acceptor peptide (AP in the presence of biotin. As a test case for our newly developed assay, we have characterized the homo-dimerization of chemokine receptor CXCR4 and heterodimerization of CXCR4 with CCR2 or CCR5. The degree of biotinylation varies with the amount of GPCR-AP as well as biotinylation time. Using enzyme/substrate receptor pairs and measuring receptor biotinylation, we demonstrate that CXCR4 can homo-dimerize and hetero-dimerize with CCR2 and CCR5. The effect of CXCL12, agonist for CXCR4, was found to decrease surface biotinylation of CXCR4-AP. This effect is due to a combination of CXCR4 endocytosis and stabilization of CXCR4 homodimers. Finally, when CXCR4-AP, CCR2-AP, and CCR5-AP were expressed together, we observed CXCR4-CXCR4 homodimers and CXCR4-CCR2 and CXCR4-CCR5 heterodimers. The newly developed assay opens new opportunity for multiplex detection for GPCR homo- and heterodimerization within the same cellular context.

  10. Multiplex surface plasmon resonance imaging platform for label-free detection of foodborne pathogens

    Science.gov (United States)

    Salmonellae are among the leading causes of foodborne outbreaks in the United States, and more rapid and efficient detection methods are needed. Surface plasmon resonance imaging (SPRi) is an emerging optical technique, which allows for rapid and label-free screening of multiple targets simultaneous...

  11. Detection of genetically modified crops using multiplex asymmetric polymerase chain reaction and asymmetric hyperbranched rolling circle amplification coupled with reverse dot blot.

    Science.gov (United States)

    Wang, Xiumin; Teng, Da; Guan, Qingfeng; Tian, Fang; Wang, Jianhua

    2015-04-15

    To meet the ever-increasing demand for detection of genetically modified crops (GMCs), low-cost, high-throughput and high-accuracy detection assays are needed. The new multiplex asymmetric polymerase chain reaction and asymmetric hyper-branched rolling circle amplification coupled with reverse dot blot (RDB) systems were developed to detect GMCs. Thirteen oligonucleotide probes were designed to identify endogenous targets (Lec1, Hmg and Sad1), event-specific targets (RRS-5C, RRS-3C, Bt176-3C and MON810-3C), screening targets (35S promoter and NOS terminator), and control targets (18S and PLX). Optimised conditions were as follows: tailed hybridization probes (1-2 pmol/l) were immobilized on a membrane by baking for 2h, and a 10:1 ratio of forward to reverse primers was used. The detection limits were 0.1 μg/l of 2% RRS and 0.5 ng/l of DNA from genetically modified (GM) soybean. These results indicate that the RDB assay could be used to detect multiplex target genes of GMCs rapidly and inexpensively. Copyright © 2014 Elsevier Ltd. All rights reserved.

  12. Multiplex real-time PCR assay for detection of Escherichia coli O157:H7 and screening for non-O157 Shiga toxin-producing E. coli.

    Science.gov (United States)

    Li, Baoguang; Liu, Huanli; Wang, Weimin

    2017-11-09

    enterica, Shigella strains, or any other pathogenic strains tested. A multiplex real-time PCR assay that can rapidly and simultaneously detect E. coli O157:H7 and screen for non-O157 STEC strains has been developed and assessed for efficacy. The inclusivity and exclusivity tests demonstrated high sensitivity and specificity of the multiplex real-time PCR assay. In addition, this multiplex assay was shown to be effective for the detection of E. coli O157:H7 from two common food matrices, beef and spinach, and may be applied for detection of E. coli O157:H7 and screening for non-O157 STEC strains from other food matrices as well.

  13. Multiplex polymerase chain reaction assays for the detection of the zearalenone chemotype of Fusarium species in white and brown rice.

    Science.gov (United States)

    Sim, Jae Ho; Tian, Fei; Jung, Soo Yeon; Auh, Joong-Hyuck; Chun, Hyang Sook

    2018-02-03

    Early detection of the zearalenone (ZEA) chemotype of Fusarium species could be a precautionary measure for preventing ZEA contamination in rice. In this study, a multiplex polymerase chain reaction (mPCR) assay for detecting ZEA-producing fungi in rice was established using a set of four primers targeting the ZEA biosynthesis genes PKS3, PKS13, ZEB1, and ZEB2. Two mPCR approaches were used: one that amplified the DNA obtained from Fusarium isolates (conventional method) and another that directly amplified the target DNA from rice samples without time-consuming DNA isolation (direct method). The two mPCR methods showed high sensitivity in detecting ZEA-producing species, with a detection limit of 1.25 pg/μL of genomic DNA and 10 2 and 10 3 spores/g of white and brown rice, respectively. Both methods were specific for ZEA-producing species and gave four band patterns. The application of the two mPCR methods to 51 Fusarium isolates and 41 rice samples revealed that 31% (16 of 51) and 24% (10 of 41) of the samples were contaminated with ZEA-producing species, respectively. The mPCR results were further evaluated using high-performance liquid chromatography; in general, the two methods yielded similar results. These findings indicate that both mPCR methods are suitable for the detection of ZEA-producing Fusarium species in white and brown rice; however, the direct method yielded more rapid results. Copyright © 2018. Published by Elsevier B.V.

  14. Detection by multiplex PCR of Staphylococcus aureus , S. intermedius and S. hyicus in artificially contaminated milk

    Directory of Open Access Journals (Sweden)

    Eliezer Avila Gandra

    2016-01-01

    Full Text Available ABSTRACT: This research aimed to detect coagulase-positive Staphylococcus (CPS directly in samples of artificially contaminated milk, using multiplex PCR (mPCR. Standard and isolated bacterial strains of S. aureus, S. epidermidis, S. hyicus, S. intermedius, Listeria monocytogenes and Escherichia coli species were used, evaluating the specificity and detection limit of mPCR, for artificially contaminated UHT milk. Primers specific for the nuc gene (NUC1-NUC2 were used for S. aureus, NUC3-NUC4 for S. hyicus and NUC5-NUC6 for S. intermedius. It was possible to detect the three target species by mPCR, directly from bovine whole milk, with adequate specificity and acceptable detention limit for identification of coagulase-positive Staphylococcus (CPS in foods. The specificity was determined by the amplification of species-specific fragments, and the detection limit was assessed by the detection thresholds obtained for the three species (103 CFU mL-1. From these results, the mPCR described, with the proposed set of primers, has the potential for use in precise identification and differentiation between CPSs in milk samples.

  15. Simultaneous detection of randomly arranged multiple barcodes using time division multiplexing technique

    Science.gov (United States)

    Haider, Saad Md. Jaglul; Islam, Md. Kafiul

    2010-02-01

    A method of detecting multiple barcodes simultaneously using time division multiplexing technique has been proposed in this paper to minimize the effective time needed for handling multiple tags of barcodes and to lessen the overall workload. Available barcode detection systems can handle multiple types of barcode but a single barcode at a time. This is not so efficient and can create large queue and chaos in places like mega shopping malls or large warehouses where we need to scan huge number of barcodes daily. Our proposed system is expected to improve the real time identification of goods or products on production lines and in automated warehouses or in mega shopping malls in a much more convenient and efficient way. For identifying of multiple barcodes simultaneously, a particular arrangement and orientation of LASER scanner and reflector have been used with a special curve shaped basement where the barcodes are placed. An effective and novel algorithm is developed to extract information from multiple barcodes which introduces starting pattern and ending pattern in barcodes with bit stuffing technique for the convenience of multiple detections. CRC technique is also used for trustworthiness of detection. The overall system enhances the existing single barcode detection system by a great amount although it is easy to implement and use.

  16. Multiplex detection of extensively drug resistant tuberculosis using binary deoxyribozyme sensors.

    Science.gov (United States)

    Bengtson, Hillary N; Homolka, Susanne; Niemann, Stefan; Reis, Ana Júlia; da Silva, Pedro Eduardo; Gerasimova, Yulia V; Kolpashchikov, Dmitry M; Rohde, Kyle H

    2017-08-15

    Current diagnostic tools for Mycobacterium tuberculosis (Mtb) have many disadvantages including low sensitivity, slow turnaround times, or high cost. Accurate, easy to use, and inexpensive point of care molecular diagnostic tests are urgently needed for the analysis of multidrug resistant (MDR) and extensively drug resistant (XDR) Mtb strains that emerge globally as a public health threat. In this study, we established proof-of-concept for a novel diagnostic platform (TB-DzT) for Mtb detection and the identification of drug resistant mutants using binary deoxyribozyme sensors (BiDz). TB-DzT combines a multiplex PCR with single nucleotide polymorphism (SNP) detection using highly selective BiDz sensors targeting loci associated with species typing and resistance to rifampin, isoniazid and fluoroquinolone antibiotics. Using the TB-DzT assay, we demonstrated accurate detection of Mtb and 5 mutations associated with resistance to three anti-TB drugs in clinical isolates. The assay also enables detection of a minority population of drug resistant Mtb, a clinically relevant scenario referred to as heteroresistance. Additionally, we show that TB-DzT can detect the presence of unknown mutations at target loci using combinatorial BiDz sensors. This diagnostic platform provides the foundation for the development of cost-effective, accurate and sensitive alternatives for molecular diagnostics of MDR- and XDR-TB. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Improved detection of Escherichia coli and coliform bacteria by multiplex PCR.

    Science.gov (United States)

    Molina, Felipe; López-Acedo, Elena; Tabla, Rafael; Roa, Isidro; Gómez, Antonia; Rebollo, José E

    2015-06-04

    The presence of coliform bacteria is routinely assessed to establish the microbiological safety of water supplies and raw or processed foods. Coliforms are a group of lactose-fermenting Enterobacteriaceae, which most likely acquired the lacZ gene by horizontal transfer and therefore constitute a polyphyletic group. Among this group of bacteria is Escherichia coli, the pathogen that is most frequently associated with foodborne disease outbreaks and is often identified by β-glucuronidase enzymatic activity or by the redundant detection of uidA by PCR. Because a significant fraction of essential E. coli genes are preserved throughout the bacterial kingdom, alternative oligonucleotide primers for specific E. coli detection are not easily identified. In this manuscript, two strategies were used to design oligonucleotide primers with differing levels of specificity for the simultaneous detection of total coliforms and E. coli by multiplex PCR. A consensus sequence of lacZ and the orphan gene yaiO were chosen as targets for amplification, yielding 234 bp and 115 bp PCR products, respectively. The assay designed in this work demonstrated superior detection ability when tested with lab collection and dairy isolated lactose-fermenting strains. While lacZ amplicons were found in a wide range of coliforms, yaiO amplification was highly specific for E. coli. Additionally, yaiO detection is non-redundant with enzymatic methods.

  18. Detection of Salmonella in Shellfish Using SYBR Green™ I-Based Real-Time Multiplexed PCR Assay Targeting invA and spvB

    KAUST Repository

    Gangwar, Maulshree

    2012-09-23

    A SYBR Green™ I-based real-time multiplexed PCR assay was developed targeting invA and spvB for the detection of Salmonella strains in shellfish after both hns and invA genes were identified in all Salmonella strains. Simultaneously, the 16S rRNA gene was used as a PCR internal amplification control (IAC). All 89 Salmonella strains tested in this study exhibited amplification of invA, whereas only 21 (23. 6 %) were PCR positive for spvB. The sensitivity of detection of all three targeted genes was 1 ng, which is equivalent to approximately 105 colony-forming unit (CFU) of Salmonella enterica. The analysis showed specific PCR products that were identified by reproducible melt temperature profiles (invA, 84. 27 ± 1. 7 °C; spvB, 88. 76 ± 1. 0 °C; and 16S rRNA gene, 87. 16 ± 0. 8 °C). The sensitivity of detection was 10 pg purified DNA (invA) or 105 CFU in 1 mL pure culture of S. enterica ATCC 14028. The above molecular detection method for Salmonella strains was successfully applied to the oyster homogenates (food matrix). An initial inoculum of 106 and 102 CFU Salmonella in 1 ml seeded oyster tissue homogenate was detected by multiplexed PCR for all three genes after 5 and 24 h of enrichment, respectively. Natural oysters isolated from Gulf of Mexico during the winter months exhibited negative PCR amplification results suggesting the absence of Salmonella. In contrast to conventional PCR, real-time multiplex PCR assay developed in this study is rapid and sensitive and will help Interstate Shellfish Sanitation Conference undertake appropriate measures to monitor Salmonella in oysters, thereby preventing disease outbreaks and consequently protecting consumer health. © 2012 Springer Science+Business Media, LLC.

  19. Evaluation of Optical Detection Platforms for Multiplexed Detection of Proteins and the Need for Point-of-Care Biosensors for Clinical Use

    Directory of Open Access Journals (Sweden)

    Samantha Spindel

    2014-11-01

    Full Text Available This review investigates optical sensor platforms for protein multiplexing, the ability to analyze multiple analytes simultaneously. Multiplexing is becoming increasingly important for clinical needs because disease and therapeutic response often involve the interplay between a variety of complex biological networks encompassing multiple, rather than single, proteins. Multiplexing is generally achieved through one of two routes, either through spatial separation on a surface (different wells or spots or with the use of unique identifiers/labels (such as spectral separation—different colored dyes, or unique beads—size or color. The strengths and weaknesses of conventional platforms such as immunoassays and new platforms involving protein arrays and lab-on-a-chip technology, including commercially-available devices, are discussed. Three major public health concerns are identified whereby detecting medically-relevant markers using Point-of-Care (POC multiplex assays could potentially allow for a more efficient diagnosis and treatment of diseases.

  20. Rapid sex identification of papaya (Carica papaya) using multiplex loop-mediated isothermal amplification (mLAMP).

    Science.gov (United States)

    Hsu, Te-Hua; Gwo, Jin-Chywan; Lin, Kuan-Hung

    2012-10-01

    Papaya (Carica papaya L.) is established as a cash crop throughout the tropical and subtropical regions due to its easy adaptation to diverse agricultural conditions, high yields, and prompt returns. The sex types of papaya plants are hermaphrodite, male, and female. Among them, hermaphroditic plants are the major type in papaya production, because the fruit has commercial advantages over that of the other sexes. Sex inheritance in papaya is determined by the M and M(h) dominant alleles in males and hermaphrodites, respectively, and a recessive m allele in females. Currently, all hermaphrodite seeds are not available due to the lethality of dominant homozygosity. Therefore, in this study, six male-hermaphrodite-specific markers were developed for a rapid sex identification using multiplex loop-mediated isothermal amplification (mLAMP) to efficiently and precisely select hermaphroditic individuals in the seedling or early growth stage. The LM1-LAMP assay consisted of two sex-LAMP reactions for amplifying two male-specific markers (T12 and Cpsm90) in one reaction, and showed several advantages in terms of a rapid reaction time (papaya production.

  1. Multiplexed microRNA detection using lanthanide-labeled DNA probes and laser ablation inductively coupled plasma mass spectrometry

    DEFF Research Database (Denmark)

    de Bang, Thomas Christian; Shah, Pratik; Cho, Seok Keun

    2014-01-01

    . The narrow size range of miRNAs (20-24 nucleotides) combined with the chemical properties of conventional reporter tags has hampered the development of multiplexed miRNA assays. In this study, we have used lanthanide-labeled DNA probes for the detection of miRNAs on membranes using laser ablation inductively...

  2. Detection of diarrhoea-causing protozoa in general practice patients in The Netherlands by multiplex real-time PCR

    NARCIS (Netherlands)

    ten Hove, R.; Schuurman, T.; Kooistra, M.; Moeller, L.; van Lieshout, L.; Verweij, J. J.

    2007-01-01

    The diagnostic value of a multiplex real-time PCR for the detection of Entamoeba histolytica, Giardia lamblia and Cryptosporidium parvum/Cryptosporidium hominis was evaluated by comparing the PCR results obtained with those of routinely performed microscopy of faecal samples from patients consulting

  3. Simultaneous detection and differentiation of Campylobacter jejuni, C. coli, and C. lari in chickens by multiplex real-time PCR

    Science.gov (United States)

    A multiplex real-time PCR (qPCR) assay was developed to detect and differentiate the three most commonly found and harmful species of Campylobacter in a single PCR reaction. The qPCR primers and TaqMan probes were designed to amplify the unique DNA sequences of hipO, cdtA, and pepT genes which are s...

  4. Multiplex detection and identification of bacterial pathogens causing potato blackleg and soft rot in Europe, using padlock probes

    NARCIS (Netherlands)

    Slawiak, M.; Doorn, van R.; Szemes, M.; Speksnijder, A.G.C.L.; Waleron, M.; Wolf, van der J.M.; Lojkowska, E.; Schoen, C.D.

    2013-01-01

    The objective of this study was to develop a multiplex detection and identification protocol for bacterial soft rot coliforms, namely Pectobacterium wasabiae (Pw), Pectobacterium atrosepticum (Pba) and Dickeya spp., responsible for potato blackleg and tuber soft rot. The procedures were derived from

  5. Simultaneous detection and differentiation of three Potyviridae viruses in sweet potato by a multiplex TaqMan real time RT-PCR assay.

    Science.gov (United States)

    Lan, Pingxiu; Li, Fan; Abad, Jorge; Pu, Lingling; Li, Ruhui

    2018-02-01

    A multiplex TaqMan real time RT-PCR was developed for detection and differentiation of Sweet potato virus G, Sweet potato latent virus and Sweet potato mild mottle virus in one tube. Amplification and detection of a fluorogenic cytochrome oxidase gene was included as an internal control. The assay was compared with a multiplex RT-PCR developed in the initial study for the detection and differentiation of the three viruses and host 18S rRNA. Primers and/or probes of the two assays were designed from conserved regions of each virus. The two assays were optimized for primers/probes and primer concentrations and thermal cycling conditions. Sensitivity and specificity of the assays were compared each other and with other assay. Both assays were evaluated by 74 field samples original from five different provinces of China. showed that the TaqMan real time RT-PCR offered rapid, sensitive, effective and reliable for the simultaneous detection and differentiation of the three viruses in sweet potato plants. The assay will be useful to quarantine and certification programs and virus surveys when large numbers of samples are tested. Copyright © 2017. Published by Elsevier B.V.

  6. Development of multiplex loop mediated isothermal amplification (m-LAMP) label-based gold nanoparticles lateral flow dipstick biosensor for detection of pathogenic Leptospira

    Energy Technology Data Exchange (ETDEWEB)

    Nurul Najian, A.B.; Engku Nur Syafirah, E.A.R.; Ismail, Nabilah [Department of Medical Microbiology & Parasitology, School of Medical Sciences, Health Campus, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan (Malaysia); Mohamed, Maizan [Faculty of Veterinary Medicine, Universiti Malaysia Kelantan, City Campus, Pengkalan Chepa, Locked Bag 36, 16100 Kota Bharu, Kelantan (Malaysia); Yean, Chan Yean, E-mail: yeancyn@yahoo.com [Department of Medical Microbiology & Parasitology, School of Medical Sciences, Health Campus, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan (Malaysia); Institute for Research in Molecular Medicine (INFORMM), Health Campus, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan (Malaysia)

    2016-01-15

    In recent years extensive numbers of molecular diagnostic methods have been developed to meet the need of point-of-care devices. Efforts have been made towards producing rapid, simple and inexpensive DNA tests, especially in the diagnostics field. We report on the development of a label-based lateral flow dipstick for the rapid and simple detection of multiplex loop-mediated isothermal amplification (m-LAMP) amplicons. A label-based m-LAMP lateral flow dipstick assay was developed for the simultaneous detection of target DNA template and a LAMP internal control. This biosensor operates through a label based system, in which probe-hybridization and the additional incubation step are eliminated. We demonstrated this m-LAMP assay by detecting pathogenic Leptospira, which causes the re-emerging disease Leptospirosis. The lateral flow dipstick was developed to detect of three targets, the LAMP target amplicon, the LAMP internal control amplicon and a chromatography control. Three lines appeared on the dipstick, indicating positive results for all representative pathogenic Leptospira species, whereas two lines appeared, indicating negative results, for other bacterial species. The specificity of this biosensor assay was 100% when it was tested with 13 representative pathogenic Leptospira species, 2 intermediate Leptospira species, 1 non-pathogenic Leptospira species and 28 other bacteria species. This study found that this DNA biosensor was able to detect DNA at concentrations as low as 3.95 × 10{sup −1} genomic equivalent ml{sup −1}. An integrated m-LAMP and label-based lateral flow dipstick was successfully developed, promising simple and rapid visual detection in clinical diagnostics and serving as a point-of-care device. - Highlights: • We develop multiplex LAMP label-based lateral flow dipstick biosensor for detection of pathogenic Leptospira. • We design primers for multiplex LAMP targeting the conserved LipL32 gene of pathogenic Leptospira and LAMP

  7. Reagent deposition for rapid multiplex pathogen identification in human blood culture samples

    DEFF Research Database (Denmark)

    Mogensen, Klaus Bo; Machado, Ana Manuel; Dufva, Martin

    2014-01-01

    viewed in a dual-color microscope configuration. The test takes 20-30 min to perform. In order to lower the cost of the test, rapid automated reagent deposition is needed. Here, ultrasonic spray coating of polyvinyl alcohol/PNA-probes on microscope glass slides is presented. Different wetting regimes...

  8. Rapid identification of mycobacteria and rapid detection of drug resistance in Mycobacterium tuberculosis in cultured isolates and in respiratory specimens.

    Science.gov (United States)

    Yam, Wing-Cheong; Siu, Kit-Hang Gilman

    2013-01-01

    Recent advances in molecular biology and better understanding of the genetic basis of drug resistance have allowed rapid identification of mycobacteria and rapid detection of drug resistance of Mycobacterium tuberculosis present in cultured isolates or in respiratory specimens. In this chapter, several simple nucleic acid amplification-based techniques are introduced as molecular approach for clinical diagnosis of tuberculosis. A one-tube nested IS6110-based polymerase chain reaction (PCR) is used for M. tuberculosis complex identification; the use of a multiplex allele-specific PCR is demonstrated to detect the isoniazid resistance; PCR-sequencing assays are applied for rifampicin and ofloxacin resistance detection and 16S rDNA sequencing is utilized for identification of mycobacterial species from cultures of acid fast bacilli (AFB). Despite the high specificity and sensitivity of the molecular techniques, mycobacterial culture remains the "Gold Standard" for tuberculosis diagnosis. Negative results of molecular tests never preclude the infection or the presence of drug resistance. These technological advancements are, therefore, not intended to replace the conventional tests, but rather have major complementary roles in tuberculosis diagnosis.

  9. Detection of viable enterotoxin-producing Bacillus cereus and analysis of toxigenicity from ready-to-eat foods and infant formula milk powder by multiplex PCR.

    Science.gov (United States)

    Zhang, Zhihong; Feng, Lixia; Xu, Hengyi; Liu, Chengwei; Shah, Nagendra P; Wei, Hua

    2016-02-01

    Bacillus cereus is responsible for several outbreaks of foodborne diseases due to its emetic toxin and enterotoxin. Enterotoxins, cytotoxin K (CytK), nonhemolytic enterotoxin (Nhe), and hemolysin BL (Hbl), have been recorded in several diarrheal cases due to food poisoning from B. cereus. The objective of this study was to develop a rapid and accurate method that combines multiplex PCR with propidium monoazide to selectively detect viable cells of enterotoxin-producing B. cereus in milk powder, noodles, and rice, and investigate the distribution of enterotoxins in 62 strains of B. cereus in Jiangxi province, China. The specificity of primers of 3 enterotoxins (i.e., cytK, nheA, and hblD) of B. cereus was verified by inclusivity and exclusivity tests using single PCR. Upon optimization of multiplex PCR conditions, it was found that the detection limit of viable cells was 10(2) cfu/mL of B. cereus in pure culture. By enrichment for 3 or 4 h and propidium monoazide pretreatment, a protocol for detection of viable cells as low as 2.2×10(1) cfu/g in spiked food (e.g., milk powder, noodles, and rice) was established and proved valid even under the interference of non-Bacillus cereus at as high as 10(5) cfu/g. Moreover, the protocol based on multiplex PCR for detection was applied for the analysis of distribution of toxin gene of B. cereus, and the results showed a regional feature for toxin gene distribution, indicating that potential toxigenicity of B. cereus should be evaluated further. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  10. Development of multiplex real-time polymerase chain reaction for detection of Entamoeba histolytica, Entamoeba dispar, and Entamoeba moshkovskii in clinical specimens.

    Science.gov (United States)

    Hamzah, Zulhainan; Petmitr, Songsak; Mungthin, Mathirut; Leelayoova, Saovanee; Chavalitshewinkoon-Petmitr, Porntip

    2010-10-01

    Multiplex real-time polymerase chain reaction (PCR) was developed for differential detection of Entamoeba histolytica, Entamoeba dispar, and Entamoeba moshkovskii. Specific primers were designed for all three species, and then differentiation of E. histolytica and E. dispar was achieved simultaneously using a hybridization probe and melting curve analysis, whereas E. moshkovskii was detected with a separate probe under the same condition. This assay detected as little as 0.2 pg of E. histolytica DNA and 2 pg each for E. dispar and E. moshkovskii DNA. Thirty-five clinical samples suspected to be E. histolytica infection by microscopy were tested. The results showed 32 positive samples; four samples were E. histolytica and 28 samples were E. dispar. Interestingly, one E. dispar positive sample showed a mixed infection with E. moshkovskii. This is the first report of E. moshkovskii infection from Thailand and this assay is currently the most rapid and sensitive method to differentiate these human amoebas.

  11. One-Step Multiplex RT-qPCR Assay for the Detection of Peste des petits ruminants virus, Capripoxvirus, Pasteurella multocida and Mycoplasma capricolum subspecies (ssp.) capripneumoniae

    National Research Council Canada - National Science Library

    Settypalli, Tirumala Bharani Kumar; Lamien, Charles Euloge; Spergser, Joachim; Lelenta, Mamadou; Wade, Abel; Gelaye, Esayas; Loitsch, Angelika; Minoungou, Germaine; Thiaucourt, Francois; Diallo, Adama

    2016-01-01

    ..., where detection of different pathogens is carried out in a single reaction. In the present study, a one-step multiplex RT-qPCR assay was developed for simultaneous detection of Capripoxvirus (CaPV...

  12. Synovial fluid multiplex PCR is superior to culture for detection of low-virulent pathogens causing periprosthetic joint infection.

    Science.gov (United States)

    Morgenstern, Christian; Cabric, Sabrina; Perka, Carsten; Trampuz, Andrej; Renz, Nora

    2018-02-01

    Analysis of joint aspirate is the standard preoperative investigation for diagnosis of periprosthetic joint infection (PJI). We compared the diagnostic performance of culture and multiplex polymerase chain reaction (PCR) of synovial fluid for diagnosis of PJI. Patients in whom aspiration of the prosthetic hip or knee joint was performed before revision arthroplasty were prospectively included. The performance of synovial fluid culture and multiplex PCR was compared by McNemar's chi-squared test. A total of 142 patients were included, 82 with knee and 60 with hip prosthesis. PJI was diagnosed in 77 patients (54%) and aseptic failure in 65 patients (46%). The sensitivity of synovial fluid culture and PCR was 52% and 60%, respectively, showing concordant results in 116 patients (82%). In patients with PJI, PCR missed 6 high-virulent pathogens (S. aureus, streptococci, E. faecalis, E. coli) which grew in synovial fluid culture, whereas synovial fluid culture missed 12 pathogens detected by multiplex PCR, predominantly low-virulent pathogens (Cutibacterium acnes and coagulase-negative staphylococci). In patients with aseptic failure, PCR detected 6 low-virulent organisms (predominantly C. acnes). While the overall performance of synovial fluid PCR was comparable to culture, PCR was superior for detection of low-virulent bacteria such as Cutibacterium spp. and coagulase-negative staphylococci. In addition, synovial fluid culture required several days for growth, whereas multiplex PCR provided results within 5hours in an automated manner. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. Early detection of Mycobacterium avium subsp. paratuberculosis infection in cattle with multiplex-bead based immunoassays.

    Directory of Open Access Journals (Sweden)

    Lingling Li

    Full Text Available Johne's Disease (JD, caused by Mycobacterium avium subspecies paratuberculosis (MAP, results in significant economic loss to livestock production. The early detection of MAP infection in animals with extant serological assays has remained challenging due to the low sensitivity of commercially available ELISA tests, a fact that has hampered the development of effective JD control programs. Our recent protein microarray-based studies identified several promising candidate antigens that are immunogenic during different stages of MAP infection. To evaluate these antigens for use in diagnostic assays and reliably identify animals with MAP infection, a multiplex (Luminex® assay was developed using color-coded flourescent beads coupled to 6 MAP recombinant proteins and applied to screen 180 serum and 90 milk samples from cows at different stages of MAP infection including negative (NL, fecal test positive/ELISA negative (F+E-, and fecal positive/ELISA positive (F+E+. The results show that while serum antibody reactivities to each of the 6 antigens were highest in F+E+ group, antibody reactivity to three of the six antigens were identified in the F+E- group, suggesting that these three antigens are expressed and provoke antibody responses during the early infection stages with MAP. Further, antibodies against all six antigens were elevated in milk samples from both the F+E- and F+E+ groups in comparison to the NL group (p<0.01. Taken together, the results of our investigation suggest that multiplex bead-based assays are able to reliably identify MAP infection, even during early stages when antibody responses in animals are undetectable with widely used commercial ELISA tests.

  14. Early detection of Mycobacterium avium subsp. paratuberculosis infection in cattle with multiplex-bead based immunoassays.

    Science.gov (United States)

    Li, Lingling; Wagner, Bettina; Freer, Heather; Schilling, Megan; Bannantine, John P; Campo, Joseph J; Katani, Robab; Grohn, Yrjo T; Radzio-Basu, Jessica; Kapur, Vivek

    2017-01-01

    Johne's Disease (JD), caused by Mycobacterium avium subspecies paratuberculosis (MAP), results in significant economic loss to livestock production. The early detection of MAP infection in animals with extant serological assays has remained challenging due to the low sensitivity of commercially available ELISA tests, a fact that has hampered the development of effective JD control programs. Our recent protein microarray-based studies identified several promising candidate antigens that are immunogenic during different stages of MAP infection. To evaluate these antigens for use in diagnostic assays and reliably identify animals with MAP infection, a multiplex (Luminex®) assay was developed using color-coded flourescent beads coupled to 6 MAP recombinant proteins and applied to screen 180 serum and 90 milk samples from cows at different stages of MAP infection including negative (NL), fecal test positive/ELISA negative (F+E-), and fecal positive/ELISA positive (F+E+). The results show that while serum antibody reactivities to each of the 6 antigens were highest in F+E+ group, antibody reactivity to three of the six antigens were identified in the F+E- group, suggesting that these three antigens are expressed and provoke antibody responses during the early infection stages with MAP. Further, antibodies against all six antigens were elevated in milk samples from both the F+E- and F+E+ groups in comparison to the NL group (p<0.01). Taken together, the results of our investigation suggest that multiplex bead-based assays are able to reliably identify MAP infection, even during early stages when antibody responses in animals are undetectable with widely used commercial ELISA tests.

  15. [Detection of complex chromosomal aberrations in patients with multiple myeloma using multiplex fluorescence in situ hybridization].

    Science.gov (United States)

    Jiang, Yuan-qiang; Chen, Li-juan; Zhu, Yu; Qiu, Hai-rong; Wang, Rong; Xu, Jia-ren; Lu, Hua; Li, Jian-yong

    2010-08-01

    To explore the value of multiplex fluorescence in situ hybridization (M-FISH) in the detection of the complex chromosomal aberrations (CCAs) in multiple myeloma (MM). M-FISH was used in 10 MM patients with CCAs detected by conventional cytogenetics (CC) using R-banding to refine the rearrangement of CCAs and identify the characteristics of marker chromosome. M-FISH confirmed the 29 structural aberrations shown by CC analysis, and also confirmed the specific source of 21 types of chromosomal aberration, which were not detected by CC analysis. Among them, t(2;15)(q33;q22), t(6;7)(q23;q34), t(8;11) (q24;q23), t(1;14)(q10;q32) and t(X;1)(q26;q25) were new chromosomal aberrations. The median survival time of 9 MM patients with CCAs was 23 months and evidently shorter than that of MM patients without CCAs, with the mean survival time being 34 months. M-FISH could refine CCAs in MM patients, find or correct the missed or misidentified abnormalities analyzed by CC. It has provided one of the essential methods for the research of chromosomal aberrations in MM.

  16. Multiplex, Quantitative, Reverse Transcription PCR Detection of Influenza Viruses Using Droplet Microfluidic Technology

    Directory of Open Access Journals (Sweden)

    Ravi Prakash

    2014-12-01

    Full Text Available Quantitative, reverse transcription, polymerase chain reaction (qRT-PCR is facilitated by leveraging droplet microfluidic (DMF system, which due to its precision dispensing and sample handling capabilities at microliter and lower volumes has emerged as a popular method for miniaturization of the PCR platform. This work substantially improves and extends the functional capabilities of our previously demonstrated single qRT-PCR micro-chip, which utilized a combination of electrostatic and electrowetting droplet actuation. In the reported work we illustrate a spatially multiplexed micro-device that is capable of conducting up to eight parallel, real-time PCR reactions per usage, with adjustable control on the PCR thermal cycling parameters (both process time and temperature set-points. This micro-device has been utilized to detect and quantify the presence of two clinically relevant respiratory viruses, Influenza A and Influenza B, in human samples (nasopharyngeal swabs, throat swabs. The device performed accurate detection and quantification of the two respiratory viruses, over several orders of RNA copy counts, in unknown (blind panels of extracted patient samples with acceptably high PCR efficiency (>94%. The multi-stage qRT-PCR assays on eight panel patient samples were accomplished within 35–40 min, with a detection limit for the target Influenza virus RNAs estimated to be less than 10 RNA copies per reaction.

  17. Detection of Coccidioides immitis in Kern County, California, by multiplex PCR.

    Science.gov (United States)

    Lauer, Antje; Baal, Joe Darryl Hugo; Baal, Jed Cyril Hugo; Verma, Mona; Chen, Jeffrey M

    2012-01-01

    Coccidioides immitis is a fungal human pathogen endemic to semiarid soils in southern California and Baja California (Mexico). Results of culture-dependent detection of C. immitis in the past indicated a spotty distribution and unreliable prediction of C. immitis growth sites and accumulation sites. In this project we investigated bulk soil samples for the presence of the pathogen in nonagricultural loamy soils at nine different locations around Bakersfield, Kern County, California, for almost 2 y (2008-2009). To detect the pathogen we used a multiplex PCR method with optimized soil handling and storage, DNA extraction procedure and PCR protocol. With this method we were able to detect C. immitis in 8.42% of our samples in 2008 (n = 285), mostly from early spring to early summer. In 2009 however the percentage of samples positive for C. immitis from the same sites declined to 2.68% (n = 261). We also were able to distinguish C. immitis growth sites from accumulation sites. One site close to a recreation area (Lake Webb, Buena Vista Lake Basin), not previously known to support the growth of C. immitis, was identified as a strong growth site of the fungus. The cultivation-independent method in this study together with soil parameters can be used to predict and confirm C. immitis growth sites and might be a valuable tool for public health institutions.

  18. Multiplex detection of protease activity with quantum dot nanosensors prepared by intein-mediated specific bioconjugation.

    Science.gov (United States)

    Xia, Zuyong; Xing, Yun; So, Min-Kyung; Koh, Ai Leen; Sinclair, Robert; Rao, Jianghong

    2008-11-15

    We report here a protease sensing nanoplatform based on semiconductor nanocrystals or quantum dots (QDs) and bioluminescence resonance energy transfer (QD-BRET) to detect the protease activity in complex biological samples. These nanosensors consist of bioluminescent proteins as the BRET donor, quantum dots as the BRET acceptor, and protease substrates sandwiched between the two as a sensing group. An intein-mediated conjugation strategy was developed for site-specific conjugation of proteins to QDs in preparing these QD nanosensors. In this traceless ligation, the intein itself is spliced out and excluded from the final conjugation product. With this method, we have synthesized a series of QD nanosensors for highly sensitive detection of an important class of protease matrix metalloproteinase (MMP) activity. We demonstrated that these nanosensors can detect the MMP activity in buffers and in mouse serum with the sensitivity to a few nanograms per milliliter and secreted proteases by tumor cells. The suitability of these nanosensors for a multiplex protease assay has also been shown.

  19. Multiplex-Touchdown PCR to Simultaneously Detect Cryptosporidium parvum, Giardia lamblia, and Cyclospora cayetanensis, the Major Causes of Traveler's Diarrhea.

    Science.gov (United States)

    Shin, Ji-Hun; Lee, Sang-Eun; Kim, Tong Soo; Ma, Da-Won; Chai, Jong-Yil; Shin, Eun-Hee

    2016-10-01

    This study aimed to develop a multiplex-touchdown PCR method to simultaneously detect 3 species of protozoan parasites, i.e., Cryptosporidium parvum, Giardia lamblia, and Cyclospora cayetanensis, the major causes of traveler's diarrhea and are resistant to standard antimicrobial treatments. The target genes included the Cryptosporidium oocyst wall protein for C. parvum, Glutamate dehydrogenase for G. lamblia, and 18S ribosomal RNA (18S rRNA) for C. cayetanensis. The sizes of the amplified fragments were 555, 188, and 400 bps, respectively. The multiplex-touchdown PCR protocol using a primer mixture simultaneously detected protozoa in human stools, and the amplified gene was detected in >1×103 oocysts for C. parvum, >1×104 cysts for G. lamblia, and >1 copy of the 18S rRNA gene for C. cayetanensis. Taken together, our protocol convincingly demonstrated the ability to simultaneously detect C. parvum, G. lamblia, and C. cayetanenesis in stool samples.

  20. Multiplex Lateral Flow Immunoassays Based on Amorphous Carbon Nanoparticles for Detecting Three Fusarium Mycotoxins in Maize.

    Science.gov (United States)

    Zhang, Xiya; Yu, Xuezhi; Wen, Kai; Li, Chenglong; Mujtaba Mari, Ghulam; Jiang, Haiyang; Shi, Weimin; Shen, Jianzhong; Wang, Zhanhui

    2017-09-13

    The detecting labels used for lateral flow immunoassays (LFAs) have been traditionally gold nanoparticles (GNPs) and, more recently, luminescent nanoparticles, such as quantum dots (QDs). However, these labels have low sensitivity and are costly, in particular, for trace detection of mycotoxins in cereals. Here, we provided a simple preparation procedure for amorphous carbon nanoparticles (ACNPs) and described multiplex LFAs employing ACNPs as labels (ACNP-LFAs) for detecting three Fusarium mycotoxins. The analytical performance of ACNPs in LFA was compared to GNPs and QDs using the same immunoreagents, except for the labels, allowing for their analytical characteristics to be objectively compared. The visual limit of detection for ACNP-LFAs in buffer was 8-fold better than GNPs and 2-fold better than QDs. Under optimized conditions, the quantitative limit of detection of ACNP-LFAs in maize was as low as 20 μg/kg for deoxynivalenol, 13 μg/kg for T-2 toxin, and 1 μg/kg for zearalenone. These measurements were much lower than the action level of these mycotoxins in maize. The accuracy and precision of the ACNP-LFAs were evaluated by analysis of spiked and incurred maize samples with recoveries of 84.6-109% and coefficients of variation below 13%. The results of ACNP-LFAs using naturally incurred maize samples showed good agreement with results from high-performance liquid chromatography-tandem mass spectrometry, indicating that ACNPs were more sensitive labels than and a promising alternative to GNPs used in LFAs for detecting mycotoxins in cereals.

  1. Direct simultaneous detection of 6 sexually transmitted pathogens from clinical specimens by multiplex polymerase chain reaction and auto-capillary electrophoresis.

    Science.gov (United States)

    Samra, Zmira; Rosenberg, Shoshana; Madar-Shapiro, Liora

    2011-05-01

    The availability of a reliable and user-friendly method to identify pathogens causing sexually transmitted diseases (STDs) is essential to reduce the complications and spread of infection. In this study, genital/urinary specimens from 113 patients with STDs were simultaneously tested for 6 pathogens using the automated Seeplex® (Seegene, Seoul, Korea) multiplex polymerase chain reaction (PCR)-based STD6B auto-capillary electrophoresis (ACE) system. The results were compared with conventional reference methods, including culture and PCR tests. The sensitivity of STD6B ACE was found to be 100% for Chlamydia trachomatis, Mycoplasma hominis, Mycoplasma genitalium, Neisseria gonorrhoeae, and Trichomonas vaginalis, and 98% for genital Ureoplasma (U. urealyticum and U. parvum). Specificity ranged from 97% to 100%. One pathogen was detected in 51 specimens, and 2 or more pathogens were detected in 24. In conclusion, the multiplex PCR and ACE system is highly sensitive and specific for the rapid, simultaneous detection of STD pathogens directly from a single specimen. Copyright © 2011 Elsevier Inc. All rights reserved.

  2. Rapid and accurate identification of Mycobacterium tuberculosis complex and common non-tuberculous mycobacteria by multiplex real-time PCR targeting different housekeeping genes.

    Science.gov (United States)

    Nasr Esfahani, Bahram; Rezaei Yazdi, Hadi; Moghim, Sharareh; Ghasemian Safaei, Hajieh; Zarkesh Esfahani, Hamid

    2012-11-01

    Rapid and accurate identification of mycobacteria isolates from primary culture is important due to timely and appropriate antibiotic therapy. Conventional methods for identification of Mycobacterium species based on biochemical tests needs several weeks and may remain inconclusive. In this study, a novel multiplex real-time PCR was developed for rapid identification of Mycobacterium genus, Mycobacterium tuberculosis complex (MTC) and the most common non-tuberculosis mycobacteria species including M. abscessus, M. fortuitum, M. avium complex, M. kansasii, and the M. gordonae in three reaction tubes but under same PCR condition. Genetic targets for primer designing included the 16S rDNA gene, the dnaJ gene, the gyrB gene and internal transcribed spacer (ITS). Multiplex real-time PCR was setup with reference Mycobacterium strains and was subsequently tested with 66 clinical isolates. Results of multiplex real-time PCR were analyzed with melting curves and melting temperature (T (m)) of Mycobacterium genus, MTC, and each of non-tuberculosis Mycobacterium species were determined. Multiplex real-time PCR results were compared with amplification and sequencing of 16S-23S rDNA ITS for identification of Mycobacterium species. Sensitivity and specificity of designed primers were each 100 % for MTC, M. abscessus, M. fortuitum, M. avium complex, M. kansasii, and M. gordonae. Sensitivity and specificity of designed primer for genus Mycobacterium was 96 and 100 %, respectively. According to the obtained results, we conclude that this multiplex real-time PCR with melting curve analysis and these novel primers can be used for rapid and accurate identification of genus Mycobacterium, MTC, and the most common non-tuberculosis Mycobacterium species.

  3. Multiplex PCR for the simultaneous identification and detection of Meloidogyne incognita, M. enterolobii, and M. javanica using DNA extracted directly from individual galls.

    Science.gov (United States)

    Hu, M X; Zhuo, K; Liao, J L

    2011-11-01

    Meloidogyne incognita, M. enterolobii, and M. javanica are the most widespread species of root-knot nematodes in South China, affecting many economically important crops, ornamental plants, and fruit trees. In this study, one pair of Meloidogyne universal primers was designed and three pairs of species-specific primers were employed successfully to rapidly detect and identify M. incognita, M. enterolobii, and M. javanica by multiplex polymerase chain reaction (PCR) using DNA extracted from individual galls. Multiplex PCR from all M. incognita, M. enterolobii, and M. javanica isolates generated two fragments of ≈500 and 1,000, 500 and 200, and 500 and 700 bp, respectively. The 500-bp fragment is the internal positive control fragment of rDNA 28S D2/D3 resulting from the use of the universal primers. Other Meloidogyne spp. included in this study generated only one fragment of ≈500 bp in size. Using this approach, M. incognita, M. enterolobii, and M. javanica were identified and detected using DNA extracted directly from individual galls containing the Meloidogyne spp. at various stages of their life cycle. Moreover, the percentage of positive PCR amplification increased with nematode development and detection was usually easy after the late stage of the second-stage juvenile. The protocol was applied to galls from naturally infested roots and the results were found to be fast, sensitive, robust, and accurate. This present study is the first to provide a definitive diagnostic tool for M. incognita, M. enterolobii, and M. javanica using DNA extracted directly from individual galls using a one-step multiplex PCR technique.

  4. Mobile Platform for Multiplexed Detection and Differentiation of Disease-Specific Nucleic Acid Sequences, Using Microfluidic Loop-Mediated Isothermal Amplification and Smartphone Detection.

    Science.gov (United States)

    Chen, Weili; Yu, Hojeong; Sun, Fu; Ornob, Akid; Brisbin, Ryan; Ganguli, Anurup; Vemuri, Vinay; Strzebonski, Piotr; Cui, Guangzhe; Allen, Karen J; Desai, Smit A; Lin, Weiran; Nash, David M; Hirschberg, David L; Brooks, Ian; Bashir, Rashid; Cunningham, Brian T

    2017-11-07

    New tools are needed to enable rapid detection, identification, and reporting of infectious viral and microbial pathogens in a wide variety of point-of-care applications that impact human and animal health. We report the design, construction, and characterization of a platform for multiplexed analysis of disease-specific DNA sequences that utilizes a smartphone camera as the sensor in conjunction with a hand-held "cradle" that interfaces the phone with a silicon-based microfluidic chip embedded within a credit-card-sized cartridge. Utilizing specific nucleic acid sequences for four equine respiratory pathogens as representative examples, we demonstrated the ability of the system to utilize a single 15 μL droplet of test sample to perform selective positive/negative determination of target sequences, including integrated experimental controls, in approximately 30 min. Our approach utilizes loop-mediated isothermal amplification (LAMP) reagents predeposited into distinct lanes of the microfluidic chip, which when exposed to target nucleic acid sequences from the test sample, generates fluorescent products that when excited by appropriately selected light emitting diodes (LEDs), are visualized and automatically analyzed by a software application running on the smartphone microprocessor. The system achieves detection limits comparable to those obtained by laboratory-based methods and instruments. Assay information is combined with the information from the cartridge and the patient to populate a cloud-based database for epidemiological reporting of test results.

  5. A multiplex real-time PCR panel assay for simultaneous detection and differentiation of 12 common swine viruses.

    Science.gov (United States)

    Shi, Xiju; Liu, Xuming; Wang, Qin; Das, Amaresh; Ma, Guiping; Xu, Lu; Sun, Qing; Peddireddi, Lalitha; Jia, Wei; Liu, Yanhua; Anderson, Gary; Bai, Jianfa; Shi, Jishu

    2016-10-01

    Mixed infection with different pathogens is common in swine production systems especially under intensive production conditions. Quick and accurate detection and differentiation of different pathogens are necessary for epidemiological surveillance, disease management and import and export controls. In this study, we developed and validated a panel of multiplex real-time PCR/RT-PCR assays composed of four subpanels, each detects three common swine pathogens. The panel detects 12 viruses or viral serotypes, namely, VSV-IN, VSV-NJ, SVDV, CSFV, ASFV, FMDV, PCV2, PPV, PRV, PRRSV-NA, PRRSV-EU and SIV. Correlation coefficients (R(2)) and PCR amplification efficiencies of all singular and triplex real-time PCR reactions are within the acceptable range. Comparison between singular and triplex real-time PCR assays of each subpanel indicates that there is no significant interference on assay sensitivities caused by multiplexing. Specificity tests on 226 target clinical samples or 4 viral strains and 91 non-target clinical samples revealed that the real-time PCR panel is 100% specific, and there is no cross amplification observed. The limit of detection of each triplex real-time PCR is less than 10 copies per reaction for DNA, and less than 16 copies per reaction for RNA viruses. The newly developed multiplex real-time PCR panel also detected different combinations of co-infections as confirmed by other means of detections. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. Sensitive Flow-through Immunoassay for Rapid Multiplex Determination of Cereal-borne Mycotoxins in Feed and Feed Ingredients.

    Science.gov (United States)

    Beloglazova, Natalia V; Graniczkowska, Kinga; Njumbe Ediage, Emmanuel; Averkieva, Olga; De Saeger, Sarah

    2017-08-23

    An easy-to-operate membrane-based flow-through test for multiplex screening of four mycotoxins (zearalenone, deoxynivalenol, aflatoxin B1, and ochratoxin A) in a variety of cereal-based feed ingredients and compound feeds, such as wheat, barley, soybean, wheat bran, rice, rice bran, maize, rapeseed meal, and sunflower meal, and various types of complete feed (duckling feed, swine feed, broiler feed, piglet feed) was developed and validated. First, the antibodies were evaluated by enzyme-linked immunosorbent assay and then employed in the membrane rapid test. The cutoff levels for zearalenone, deoxynivalenol, aflatoxin B1, and ochratoxin A were 50, 200, 1, and 10 μg/kg, respectively, based on European regulations and consumers' requirements. As sample pretreatment, consecutive steps of extraction, dilution, solid-phase extraction by addition of C18 sorbent, and final filtration of supernatant were followed. Both the sample preparation and the analysis procedure were simple, cost-effective, and easy to perform on-site in a nonlaboratory environment. The impact of sample processing on the result of the experiment was investigated supported by experimental design. The validation procedure was performed on the basis of Commission Regulation 2006/401/EC. The numbers of false-positive and false-negative outcomes were <5%, going along with the Commission Decision 2002/657/EC. Liquid chromatography-tandem mass spectrometry was performed as a confirmatory technique.

  7. Use of Multiplex PCR and PCR Restriction Enzyme Analysis for Detection and Exploration of the Variability in the Free-Living Amoeba Naegleria in the Environment

    Science.gov (United States)

    Pélandakis, Michel; Pernin, Pierre

    2002-01-01

    A multiplex PCR was developed to simultaneously detect Naegleria fowleri and other Naegleria species in the environment. Multiplex PCR was also capable of identifying N. fowleri isolates with internal transcribed spacers of different sizes. In addition, restriction fragment length polymorphism analysis of the PCR product distinguished the main thermophilic Naegleria species from the sampling sites. PMID:11916734

  8. A paper/polymer hybrid CD-like microfluidic SpinChip integrated with DNA-functionalized graphene oxide nanosensors for multiplex qLAMP detection.

    Science.gov (United States)

    Dou, Maowei; Sanjay, Sharma T; Dominguez, Delfina C; Zhan, Sihui; Li, XiuJun

    2017-10-03

    A paper/poly(methyl methacrylate) (PMMA) hybrid CD-like microfluidic SpinChip integrated with DNA probe-functionalized graphene oxide (GO) nanosensors was developed for multiplex quantitative LAMP detection (mqLAMP). This approach can simply and effectively address a major challenging problem of multiplexing in current LAMP methods.

  9. The Role of Multiplex Polymerase Chain Reaction in Detecting Etiological Causes of Bacterial Prostatitis Associated Benign Prostatic Hyperplasia

    Directory of Open Access Journals (Sweden)

    Bramastha Rosadi

    2015-01-01

    Full Text Available Background: Benign Prostatic Hyperplasia (BPH has been correlated with chronic prostatitis according recent study. Chronic pelvic pain is the chief complain of BPH followed by prostatitis. The gold standard of the etiological diagnosis is urine culture, but the negativity rate is still high. Multiplex polymerase chain reaction (PCR as a diagnostic tool in search of etiological causes could identify microorganism on DNA level. This research aims to find out the role of multiplex polymerase chain reaction as diagnostic tools on prostatitis patients. Material and Method: A total of 12 samples collected during the TURP procedure in Sanglah General Hospital Denpasar – Bali from February until May 2015. All of the samples has been diagnosed prostatitis clinically and perform urine culture test. The prostate specimen taken was sent to the Pathological anatomy for histopathology diagnostic and underwent multiplex PCR for etiologic diagnostic. Result: 12 samples have been declared as prostatitis based on histopathology examination, and then were analyzed using multiplex PCR. 10 samples were positive (6 were E. coli, 2 were C. trachomatis, the rest were N. gonorrhea and P. aeruginosa. The urine culture revealed 9 positive, within the result 6 were E. coli, and the others were P. aeruginosa, M. morganii and A. haemolyticus. Conclusion: In prostatitis patient, the etiological diagnostic was important. Multiplex PCR as diagnostic tools could detect the microorganism on a negative urine culture. The combination of the urine culture test and multiplex PCR revealed a better result on etiologic diagnosis which leads to a better management of the disease. 

  10. Rapid and not culture-dependent assay based on multiplex PCR-SSR analysis for monitoring inoculated yeast strains in industrial wine fermentations.

    Science.gov (United States)

    Cordero-Bueso, Gustavo; Rodríguez, María Esther; Garrido, Carlos; Cantoral, Jesús Manuel

    2017-01-01

    Wine industry needs a simple method for rapid diagnosis of the dominance of inoculated strains that could be performed routinely during the fermentation process. We present a suitable, high-throughput, and low-cost method to monitor rapidly the dominance of inoculated yeast strains in industrial fermentations of red and white wines using an activated carbon cleaning pretreatment, and a rapid DNA extraction method plus multiplex PCR-SSR analysis. We apply this technique directly to samples of fermenting wines without previously isolating yeast colonies. Results are obtained in a maximum time of 4.5 h.

  11. Sensitive Detection of Thirteen Bacterial Vaginosis-Associated Agents Using Multiplex Polymerase Chain Reaction.

    Science.gov (United States)

    Malaguti, Natália; Bahls, Larissa Danielle; Uchimura, Nelson Shozo; Gimenes, Fabrícia; Consolaro, Marcia Edilaine Lopes

    2015-01-01

    Bacterial vaginosis (BV) is characterized by a polymicrobial proliferation of anaerobic bacteria and depletion of lactobacilli, which are components of natural vaginal microbiota. Currently, there are limited conventional methods for BV diagnosis, and these methods are time-consuming, expensive, and rarely allow for the detection of more than one agent simultaneously. Therefore, we conceived and validated a multiplex polymerase chain reaction (M-PCR) assay for the simultaneous screening of thirteen bacterial vaginosis-associated agents (BV-AAs) related to symptomatic BV: Gardnerella vaginalis, Mobiluncus curtisii, Mobiluncus mulieris, Bacteroides fragilis, Mycoplasma hominis, Atopobium vaginae, Ureaplasma urealyticum, Megasphaera type I, Clostridia-like bacteria vaginosis-associated bacteria (BVABs) 1, 2, and 3, Sneathia sanguinegens, and Mycoplasma genitalium. The overall validation parameters of M-PCR compared to single PCR (sPCR) were extremely high, including agreement of 99.1% and sensitivity, specificity, and positive predictive values of 100.0%, negative predictive value of 97.0%, accuracy of 99.3%, and agreement with Nugent results of 100.0%. The prevalence of BV-AAs was very high (72.6%), and simultaneous agents were detected in 53.0%, which demonstrates the effectiveness of the M-PCR assay. Therefore, the M-PCR assay has great potential to impact BV diagnostic methods in vaginal samples and diminish associated complications in the near future.

  12. Sensitive Detection of Thirteen Bacterial Vaginosis-Associated Agents Using Multiplex Polymerase Chain Reaction

    Directory of Open Access Journals (Sweden)

    Natália Malaguti

    2015-01-01

    Full Text Available Bacterial vaginosis (BV is characterized by a polymicrobial proliferation of anaerobic bacteria and depletion of lactobacilli, which are components of natural vaginal microbiota. Currently, there are limited conventional methods for BV diagnosis, and these methods are time-consuming, expensive, and rarely allow for the detection of more than one agent simultaneously. Therefore, we conceived and validated a multiplex polymerase chain reaction (M-PCR assay for the simultaneous screening of thirteen bacterial vaginosis-associated agents (BV-AAs related to symptomatic BV: Gardnerella vaginalis, Mobiluncus curtisii, Mobiluncus mulieris, Bacteroides fragilis, Mycoplasma hominis, Atopobium vaginae, Ureaplasma urealyticum, Megasphaera type I, Clostridia-like bacteria vaginosis-associated bacteria (BVABs 1, 2, and 3, Sneathia sanguinegens, and Mycoplasma genitalium. The overall validation parameters of M-PCR compared to single PCR (sPCR were extremely high, including agreement of 99.1% and sensitivity, specificity, and positive predictive values of 100.0%, negative predictive value of 97.0%, accuracy of 99.3%, and agreement with Nugent results of 100.0%. The prevalence of BV-AAs was very high (72.6%, and simultaneous agents were detected in 53.0%, which demonstrates the effectiveness of the M-PCR assay. Therefore, the M-PCR assay has great potential to impact BV diagnostic methods in vaginal samples and diminish associated complications in the near future.

  13. Simultaneous detection of three fish rhabdoviruses using multiplex real-time quantitative RT-PCR assay.

    Science.gov (United States)

    Liu, Zongxiao; Teng, Yong; Liu, Hong; Jiang, Yulin; Xie, Xiayang; Li, Huifang; Lv, Jiangqiang; Gao, Longying; He, Junqiang; Shi, Xiujie; Tian, Feiyan; Yang, Jingshun; Xie, Congxin

    2008-04-01

    Spring viremia of carp virus (SVCV), infectious hematopoietic necrosis virus (IHNV) and viral hemorrhagic septicemia virus (VHSV) are three important fish rhabdoviruses, causing serious Office International des Epizooties (OIE) classified diseases in wild and farmed fish. Here, a new multiplex real-time quantitative RT-PCR (mqRT-PCR) assay was developed for simultaneous detection, identification and quantification of these three rhabdoviruses. The sets of primers and probes were targeted to conserved regions of glycoprotein (G) gene of SVCV, nucleoprotein (N) gene of IHNV and G gene of VHSV and used to amplify. The sensitivity, specificity and interference test of mqRT-PCR assay was analyzed. It was shown that the detection levels of 100 copies of SVCV, 220 copies of IHNV and 140 copies of VHSV were achieved, and there was no non-specific amplification and cross-reactivity using RNA of pike fry rhabdovirus (PFRV), infectious pancreatic necrosis virus (IPNV) and grass carp reovirus (GCRV). A total of 80 clinical fish samples were tested using the mqRT-PCR assay and the results were confirmed by antigen-capture ELISA and cell culture assay. This assay has the potential to be used for both research applications and diagnosis.

  14. A novel, multiplex, real-time PCR–based approach for the detection of the commonly occurring pathogenic fungi and bacteria

    Science.gov (United States)

    2013-01-01

    Background Polymerase chain reaction (PCR)-based techniques are widely used to identify fungal and bacterial infections. There have been numerous reports of different, new, real-time PCR-based pathogen identification methods although the clinical practicability of such techniques is not yet fully clarified. The present study focuses on a novel, multiplex, real-time PCR-based pathogen identification system developed for rapid differentiation of the commonly occurring bacterial and fungal causative pathogens of bloodstream infections. Results A multiplex, real-time PCR approach is introduced for the detection and differentiation of fungi, Gram-positive (G+) and Gram-negative (G-) bacteria. The Gram classification is performed with the specific fluorescence resonance energy transfer (FRET) probes recommended for LightCycler capillary real-time PCR. The novelty of our system is the use of a non-specific SYBR Green dye instead of labelled anchor probes or primers, to excite the acceptor dyes on the FRET probes. In conjunction with this, the use of an intercalating dye allows the detection of fungal amplicons. With the novel pathogen detection system, fungi, G + and G- bacteria in the same reaction tube can be differentiated within an hour after the DNA preparation via the melting temperatures of the amplicons and probes in the same tube. Conclusions This modified FRET technique is specific and more rapid than the gold-standard culture-based methods. The fact that fungi, G + and G- bacteria were successfully identified in the same tube within an hour after the DNA preparation permits rapid and early evidence-based management of bloodstream infections in clinical practice. PMID:24364823

  15. A novel, multiplex, real-time PCR-based approach for the detection of the commonly occurring pathogenic fungi and bacteria.

    Science.gov (United States)

    Horváth, Ádám; Pető, Zoltán; Urbán, Edit; Vágvölgyi, Csaba; Somogyvári, Ferenc

    2013-12-23

    Polymerase chain reaction (PCR)-based techniques are widely used to identify fungal and bacterial infections. There have been numerous reports of different, new, real-time PCR-based pathogen identification methods although the clinical practicability of such techniques is not yet fully clarified.The present study focuses on a novel, multiplex, real-time PCR-based pathogen identification system developed for rapid differentiation of the commonly occurring bacterial and fungal causative pathogens of bloodstream infections. A multiplex, real-time PCR approach is introduced for the detection and differentiation of fungi, Gram-positive (G+) and Gram-negative (G-) bacteria. The Gram classification is performed with the specific fluorescence resonance energy transfer (FRET) probes recommended for LightCycler capillary real-time PCR. The novelty of our system is the use of a non-specific SYBR Green dye instead of labelled anchor probes or primers, to excite the acceptor dyes on the FRET probes. In conjunction with this, the use of an intercalating dye allows the detection of fungal amplicons.With the novel pathogen detection system, fungi, G + and G- bacteria in the same reaction tube can be differentiated within an hour after the DNA preparation via the melting temperatures of the amplicons and probes in the same tube. This modified FRET technique is specific and more rapid than the gold-standard culture-based methods. The fact that fungi, G + and G- bacteria were successfully identified in the same tube within an hour after the DNA preparation permits rapid and early evidence-based management of bloodstream infections in clinical practice.

  16. Development of a multiplex polymerase chain reaction protocol for the simultaneous detection of Salmonella enterica serovar Typhi and Class 1 integron

    Directory of Open Access Journals (Sweden)

    Juthika Mandal

    2014-09-01

    Full Text Available Objective: To develop a multiplex polymerase chain reaction (PCR protocol for the simultaneous detection of Salmonella enterica serovar Typhi (S. Typhi and Class 1 integron, so as to aid rapid diagnosis of S. Typhi cases and help in the selection of treatment options based on the presence of the Class 1 integron that can carry resistance cassettes to a range of antibiotics. Methods: PCR for amplification of specific regions was done using fliC-d and intl primers and agarose gel electrophoresis was used for resolution of PCR products. Results: The fliC-d primer (S. Typhi specific amplified a 587 bp region and the intl primer (Class 1 integron specific amplified two bands approximately 500 and 550 bps. The developed method was specific for S. Typhi and did not amplify any products with Salmonella enterica serovar Typhimurium ATCC 14028, Salmonella enterica serovar Paratyphi and Escherichia coli O157:H7. Conclusions: The developed multiplex PCR protocol can be used for rapid diagnosis and aid in proper treatment strategies for patients infected with S. Typhi.

  17. Multiplex Minisequencing of the HBB Gene: A Rapid Strategy to Confirm the Most Frequent β-Thalassemia Mutations in the Tunisian Population.

    Science.gov (United States)

    Ben Charfeddine, Ilhem; Ben Lazreg, Taheni; M'sakni, Ahlem; Amara, Abdelbasset; Mlika, Adnène; Chaïeb, Anouar; Hlel, Khalid; Zouari, Noura; Zbidi, Faïza; Bouguila, Jihène; Soyah, Najla; Ayedi, Abdelkarim; Ben Hamouda, Hechmi; Abroug, Saoussen; Boughamoura, Lamia; Saad, Ali; Gribaa, Moez

    2015-01-01

    The β hemoglobinopathies [β-thalassemia (β-thal) and structural hemoglobin (Hb) variants such as Hb S (HBB: c.20A > T) and Hb E (HBB: c.79G > A)] are among the most common inherited diseases worldwide. In Tunisia, due to the high prevalence of consanguineous marriages, the recurrent risk of this disease is high. The average prevalence of hemoglobinopathies is 4.48%, reaching 12.50% in some focus regions. The molecular investigations on thalassemia contributed to establishing the spectrum of mutations in the Tunisian population. The total number of HBB gene mutations identified was 24. The two most frequent mutations, codon 39 (C > T) (HBB: c.118C > T) and IVS-I-110 (G > A) (HBB: c.93-21G > A) accounted for 70.0% of the total encountered β-thal cases. These two mutations together with IVS-I-2 (T > G) (HBB: c.92 + 2T > G) and the Hb S variant account for more than 90.0% of all HBB genetic variants in Tunisia. Thus, developing rapid, inexpensive and reliable mutation-specific molecular diagnostic assays targeting our Tunisian populations is our aim to facilitate routine detection of hemoglobinopathies. In this report, we describe the successful application of the multiplex minisequencing assay as an alternative strategy for genetic diagnosis of HBB gene disorders in Tunisia.

  18. Comparison between Saliva and Nasopharyngeal Swab Specimens for Detection of Respiratory Viruses by Multiplex Reverse Transcription-PCR.

    Science.gov (United States)

    Kim, Young-Gon; Yun, Seung Gyu; Kim, Min Young; Park, Kwisung; Cho, Chi Hyun; Yoon, Soo Young; Nam, Myung Hyun; Lee, Chang Kyu; Cho, Yun-Jung; Lim, Chae Seung

    2017-01-01

    Nasopharyngeal swabs (NPSs) are being widely used as specimens for multiplex real-time reverse transcription (RT)-PCR for respiratory virus detection. However, it remains unclear whether NPS specimens are optimal for all viruses targeted by multiplex RT-PCR. In addition, the procedure to obtain NPS specimens causes coughing in most patients, which possibly increases the risk of nosocomial spread of viruses. In this study, paired NPS and saliva specimens were collected from 236 adult male patients with suspected acute respiratory illnesses. Specimens were tested for 16 respiratory viruses by multiplex real-time RT-PCR. Among the specimens collected from the 236 patients, at least 1 respiratory virus was detected in 183 NPS specimens (77.5%) and 180 saliva specimens (76.3%). The rates of detection of respiratory viruses were comparable for NPS and saliva specimens (P = 0.766). Nine virus species and 349 viruses were isolated, 256 from NPS specimens and 273 from saliva specimens (P = 0.1574). Adenovirus was detected more frequently in saliva samples (P saliva samples was excluded by direct sequencing. In conclusion, neither of the sampling methods was consistently more sensitive than the other. We suggest that these cost-effective methods for detecting respiratory viruses in mixed NPS-saliva specimens might be valuable for future studies. Copyright © 2016 American Society for Microbiology.

  19. Comparison of three multiplex gastrointestinal platforms for the detection of gastroenteritis viruses.

    Science.gov (United States)

    Chhabra, Preeti; Gregoricus, Nicole; Weinberg, Geoffrey A; Halasa, Natasha; Chappell, James; Hassan, Ferdaus; Selvarangan, Rangaraj; Mijatovic-Rustempasic, Slavica; Ward, M Leanne; Bowen, Michael; Payne, Daniel C; Vinjé, Jan

    2017-10-01

    Viruses are major etiological agents of childhood gastroenteritis. In recent years, several molecular platforms for the detection of viral enteric pathogens have become available. We evaluated the performance of three multiplex platforms including Biofire's Gastrointestinal Panel (FilmArray), Luminex xTAG ® Gastrointestinal Pathogen Panel (GPP), and the TaqMan Array Card (TAC) for the detection of five gastroenteritis viruses using a coded panel of 300 archived stool samples. The FilmArray detected a virus in 199 (96.1%) and the TAC in 172 (83.1%) of the 207 samples (187 samples positive for a single virus and 20 samples positive for more than one virus) whereas the GPP detected a virus in 100 (78.7%) of the 127 (97 positive for one virus and three positive for more than one virus) samples. Overall the clinical accuracy was highest for the FilmArray (98%) followed by TAC (97.2%) and GPP (96.9%). The sensitivity of the FilmArray, GPP and TAC platforms was highest for rotavirus (100%, 95.8%, and 89.6%, respectively) and lowest for adenovirus type 40/41 (97.4%, 57.9% and 68.4%). The specificity of the three platforms ranged from 95.6% (rotavirus) to 99.6% (norovirus/sapovirus) for the FilmArray, 99.6% (norovirus) to 100% (rotavirus/adenovirus) for GPP, and 98.9% (astrovirus) to 100% (rotavirus/sapovirus) for TAC. The FilmArray demonstrated the best analytical performance followed by TAC. In recent years, the availability of multi-enteric molecular testing platforms has increased significantly and our data highlight the strengths and weaknesses of these platforms. Published by Elsevier B.V.

  20. Detection of small genomic imbalances using microarray-based multiplex amplifiable probe hybridization.

    Science.gov (United States)

    Patsalis, Philippos C; Kousoulidou, Ludmila; Männik, Katrin; Sismani, Carolina; Zilina, Olga; Parkel, Sven; Puusepp, Helen; Tõnisson, Neeme; Palta, Priit; Remm, Maido; Kurg, Ants

    2007-02-01

    Array-based genome-wide screening methods were recently introduced to clinical practice in order to detect small genomic imbalances that may cause severe genetic disorders. The continuous advancement of such methods plays an extremely important role in diagnostic genetics and medical genomics. We have modified and adapted the original multiplex amplifiable probe hybridization (MAPH) to a novel microarray format providing an important new diagnostic tool for detection of small size copy-number changes in any locus of human genome. Here, we describe the new array-MAPH diagnostic method and show proof of concept through fabrication, interrogation and validation of a human chromosome X-specific array. We have developed new bioinformatic tools and methodology for designing and producing amplifiable hybridization probes (200-600 bp) for array-MAPH. We designed 558 chromosome X-specific probes with median spacing 238 kb and 107 autosomal probes, which were spotted onto microarrays. DNA samples from normal individuals and patients with known and unknown chromosome X aberrations were analyzed for validation. Array-MAPH detected exactly the same deletions and duplications in blind studies, as well as other unknown small size deletions showing its accuracy and sensitivity. All results were confirmed by fluorescence in situ hybridization and probe-specific PCR. Array-MAPH is a new microarray-based diagnostic tool for the detection of small-scale copy-number changes in complex genomes, which may be useful for genotype-phenotype correlations, identification of new genes, studying genetic variation and provision of genetic services.

  1. New multiplex real-time PCR approach to detect gene mutations for spinal muscular atrophy.

    Science.gov (United States)

    Liu, Zhidai; Zhang, Penghui; He, Xiaoyan; Liu, Shan; Tang, Shi; Zhang, Rong; Wang, Xinbin; Tan, Junjie; Peng, Bin; Jiang, Li; Hong, Siqi; Zou, Lin

    2016-08-17

    Spinal muscular atrophy (SMA) is the most common autosomal recessive disease in children, and the diagnosis is complicated and difficult, especially at early stage. Early diagnosis of SMA is able to improve the outcome of SMA patients. In our study, Real-time PCR was developed to measure the gene mutation or deletion of key genes for SMA and to further analyse genotype-phenotype correlation. The multiple real-time PCR for detecting the mutations of survival of motor neuron (SMN), apoptosis inhibitory protein (NAIP) and general transcription factor IIH, polypeptide 2 gene (GTF2H2) was established and confirmed by DNA sequencing and multiplex ligation-dependent probe amplification (MLPA). The diagnosis and prognosis of 141 hospitalized children, 100 normal children and further 2000 cases of dry blood spot (DBS) samples were analysed by this multiple real-time PCR. The multiple real-time PCR was established and the accuracy of it to detect the mutations of SMN, NAIP and GTF2H2 was at least 98.8 % comparing with DNA sequencing and MLPA. Among 141 limb movement disorders children, 75 cases were SMA. 71 cases of SMA (94.67 %) were with SMN c.840 mutation, 9 cases (12 %) with NAIP deletion and 3 cases (4 %) with GTF2H2 deletion. The multiple real-time PCR was able to diagnose and predict the prognosis of SMA patients. Simultaneously, the real-time PCR was applied to detect trace DNA from DBS and able to make an early diagnosis of SMA. The clinical and molecular characteristics of SMA in Southwest of China were presented. Our work provides a novel way for detecting SMA in children by using real-time PCR and the potential usage in newborn screening for early diagnosis of SMA.

  2. Thermostabilization of indigenous multiplex polymerase chain reaction reagents for detection of enterotoxigenic Staphylococcus aureus.

    Science.gov (United States)

    Nagaraj, Sowmya; Ramlal, Shylaja; Kingston, Joseph; Batra, Harsh Vardhan

    2016-05-13

    Among DNA-based techniques, polymerase chain reaction (PCR) is the most widely accepted molecular tool for the detection of pathogens. However, the technique involves several reagents and multiple pipetting steps that often lead to error-prone results. Additionally, the reagents entail a cold-chain facility to maintain their stability during storage and transportation. The main aim of the present study was to simplify the utility of a pre-optimized multiplex PCR format that was developed to detect toxigenic strains of Staphylococcus aureus by providing stable, pre-mixed, and ready-to-use master mix in a lyophilized formulation. Master mix containing all reagents except the template was lyophilized in the presence of an excipient lyoprotectant to achieve long-term stability without altering the sensitivity, specificity and PCR performance. Bromophenol blue was also included in the master mix to reduce the risk of external contamination during gel loading. The stability of lyophilized master mix was analyzed at different temperatures. The PCR performance was also examined after exposure of master mix to notable temperature fluctuations during transportation. The shelf-life of lyophilized master mix was estimated to be 1.5 months at ambient temperature and 6 months at 4°C. Stability was unaffected by temperature fluctuations during transportation even in cold-chain-free conditions, thus reducing the cost required for cold storage. The sensitive, cost-effective, ready-to-use, and ambient temperature stable formulation could be implemented as a detection tool in food analysis and diagnostic laboratories and hospitals and for on-field application outside the laboratories, as well as for detection of toxigenic strains of S. aureus. Copyright © 2016. Published by Elsevier B.V.

  3. Epidemiology and Clinical Presentations of Respiratory Syncytial Virus Subgroups A and B Detected with Multiplex Real-Time PCR.

    Directory of Open Access Journals (Sweden)

    Wenkuan Liu

    Full Text Available Respiratory syncytial virus (RSV is one of the most important pathogenic infections of children and requires in-depth research worldwide, and especially in developing countries. We used a novel multiplex real-time PCR to test 5483 patients (≤ 14 years old hospitalized with respiratory illness in Guangzhou, China, over a 3-year period. Of these patients, 729 were positive for RSV-A (51.2%, 373/729 or RSV-B (48.8%, 356/729, but none was infected with both viruses. Two seasonal peaks in total RSV were detected at the changes from winter to spring and from summer to autumn. RSV-B was dominant in 2013 and RSV-A in 2015, whereas RSV-A and RSV-B cocirculated in 2014. The clinical presentations of 645 RSV-positive patients were analyzed. Bronchiolitis, dyspnea, coryza, vomiting, poor appetite, and diarrhea occurred more frequently in RSV-A-positive than RSV-B-positive patients, whereas chill, headache, myalgia, debility, and rash etc. were more frequent in RSV-B-positive than RSV-A-positive patients, suggesting specific clinical characteristics for RSV-A and RSV-B. Coinfectons with other pathogens were common and diverse. Bronchiolitis, fever (≥ 38°C, and poor appetite were more frequent in patients with single RSV infections than in coinfected patients, suggesting the key pathogenic activity of RSV. Analysis of the relationships between the comparative viral load and clinical presentations showed significant differences in bronchiolitis, fever (≥ 38°C, and rash etc. among patients with different viral loads. This study provides a novel rapid method for detecting RSV subgroups, and provides new insights into the epidemiology and clinical implications of RSV.

  4. Graphite-Based Nanocomposite Electrochemical Sensor for Multiplex Detection of Adenine, Guanine, Thymine, and Cytosine: A Biomedical Prospect for Studying DNA Damage.

    Science.gov (United States)

    Ng, Khan Loon; Khor, Sook Mei

    2017-09-19

    Guanine (G), adenine (A), thymine (T), and cytosine (C) are the four basic constituents of DNA. Studies on DNA composition have focused especially on DNA damage and genotoxicity. However, the development of a rapid, simple, and multiplex method for the simultaneous measurement of the four DNA bases remains a challenge. In this study, we describe a graphite-based nanocomposite electrode (Au-rGO/MWCNT/graphite) that uses a simple electro-co-deposition approach. We successfully applied the developed sensor for multiplex detection of G, A, T, and C, using square-wave voltammetry. The sensor was tested using real animal and plant DNA samples in which the hydrolysis of T and C could be achieved with 8 mol L-1 of acid. The electrochemical sensor exhibited excellent sensitivity (G = 178.8 nA/μg mL-1, A = 92.9 nA/μg mL-1, T = 1.4 nA/μg mL-1, and C = 15.1 9 nA/μg mL-1), low limit of detection (G, A = 0.5 μg mL-1; T, C = 1.0 μg mL-1), and high selectivity in the presence of common interfering factors from biological matrixes. The reliability of the established method was assessed by method validation and comparison with the ultraperformance liquid chromatography technique, and a correlation of 103.7% was achieved.

  5. Development of Multiplex-Mismatch Amplification Mutation-PCR Assay for Simultaneous Detection of Campylobacter jejuni and Mutation in gyrA Gene Related to Fluoroquinolone Resistance.

    Science.gov (United States)

    Cui, Mingquan; Wu, Chenbin; Zhang, Peng; Wu, Congming

    2016-11-01

    Campylobacter jejuni, a foodborne pathogen, is the major cause of enteritis in humans worldwide, however, its increasing resistance to fluoroquinolones reported recently is of a major concern. In the present study, multiplex-mismatch amplification mutation assay-polymerase chain reaction (MMAMA-PCR) was developed for the first time with the aim to quickly identify C. jejuni and to detect the single nucleotide mutation (C-257 to T) frequently observed in gyrA gene, associated with the acquisition of resistance to fluoroquinolones. In this assay, mismatch amplification mutation primers for the detection of gyrA mutation in C. jejuni were coupled with primers for the hip gene encoding for hippuricase and 16S rRNA gene of C. jejuni, respectively, in the multiplex PCR assay. The specificity and accuracy of this method were analyzed by the use of 78 C. jejuni strains with previously confirmed resistance phenotypes and the mutation (C-257 to T) in gyrA gene, as well as 107 clinical isolates of various bacterial species, including 29 C. jejuni isolates. This study indicates that MMAMA-PCR is a promising assay for the rapid identification of C. jejuni with a specific mutation in gyrA gene, responsible for the resistance to fluoroquinolones.

  6. Evaluating the use of dedicated swab for rapid antigen detection ...

    African Journals Online (AJOL)

    Background: Group A streptococcus (GAS) is the most common and fearful bacterial cause in pediatric acute pharyngitis due to its serious complications. Several generations of rapid antigen detection tests (RADTs) have been developed to facilitate rapid detection of GAS pharyngitis. We assessed the value of using a ...

  7. Rapid assessment of assignments using plagiarism detection software.

    Science.gov (United States)

    Bischoff, Whitney R; Abrego, Patricia C

    2011-01-01

    Faculty members most often use plagiarism detection software to detect portions of students' written work that have been copied and/or not attributed to their authors. The rise in plagiarism has led to a parallel rise in software products designed to detect plagiarism. Some of these products are configurable for rapid assessment and teaching, as well as for plagiarism detection.

  8. Lab on a chip for multiplexed immunoassays to detect bladder cancer using multifunctional dielectrophoretic manipulations.

    Science.gov (United States)

    Chuang, Cheng-Hsin; Wu, Ting-Feng; Chen, Cheng-Ho; Chang, Kai-Chieh; Ju, Jing-Wei; Huang, Yao-Wei; Van Nhan, Vo

    2015-07-21

    A multiplexed immunosensor has been developed for the detection of specific biomarkers Galectin-1 (Gal-1) and Lactate Dehydrogenase B (LDH-B) present in different grades of bladder cancer cell lysates. In order to immobilize nanoprobes with different antibodies on a single chip we employed three-step programmable dielectrophoretic manipulations for focusing, guiding and trapping to enhance the fluorescent response and reduce the interference between the two antibody arrays. The chip consisted of a patterned indium tin oxide (ITO) electrode for sensing and a middle fish bone shaped gold electrode for focusing and guiding. Using ITO electrodes for the sensing area can effectively eliminate the background noise of fluorescence response as compared to metal electrodes. It was also observed that the three step manipulation increased fluorescence response after immunosensing by about 4.6 times as compared to utilizing DEP for just trapping the nanoprobes. Two different-grade bladder cancer cell lysates (grade I: RT4 and grade III: T24) were individually analyzed for detecting the protein expression levels of Gal-1 and LDH-B. The fluorescence intensity observed for Gal-1 is higher than that of LDH-B in the T24 cell lysate; however the response observed in RT4 is higher for LDH-B as compared to Gal-1. Thus we can effectively identify the different grades of bladder cancer cells. In addition, the platform for DEP manipulation developed in this study can enable real time detection of multiple analytes on a single chip and provide more practical benefits for clinical diagnosis.

  9. Multi-analytic detection of antibiotic residues in honey using a multiplexing biochip assay.

    Science.gov (United States)

    Popa, Ionela Daniela; Schiriac, Elena Corina; Cuciureanu, Rodica

    2012-01-01

    Contamination of honey may occur through the common use of antibiotics and chemotherapeutics by certain beekeepers to cure or to prevent bacterial or other bee diseases. In order to protect human health the use of antimicrobials in apiculture is usually strictly regulated or banned. LC/MS/MS is currently regarded the tool of choice for analysis of drug residues in animal-derived food. Screening methods (immunoassays and biosensors) are also required and are being developed. The present paper reviews one of the most recent methods for analysis of drug residues in honey and also the abridged method validation according to the criteria set by Commission Decision 2002/657/EC for simultaneous determination of drug residues in honey samples using a test panel which screens for quinolones (QNL), ceftiofur (CEFT), thiamphenicol (TAF), streptomycin (STR), tylosin (TYL) and tetracyclines (TCN). A chemiluminescence--based biochip array sensing technique has been applied. Using a multiplex approach, metabolites of CEFT, TAF, QNL, STR, TCN and TYL were simultaneously detected in honey samples. The simultaneous immunoassays showed specificity for the target analytes. The limits of detection were between 1 microg/kg for TAF and 9 microg/kg for TCN. Good linearity was obtained, with determination coefficients higher than 0.980. The decision limit (CCalpha) and the capability of detection (CCbeta) were well below established Reference Point for Action (RPA). Method applicability was demonstrated through application to various kinds of honey. Biochip array technology allows simultaneous and selective multi-analytic measurement of antimicrobials in honey at levels well below established Reference Point for Action (RPA). The performance of biochip screening method was comparable to that achieved for the confirmatory LC/MS/MS method.

  10. Clinical Relevance of Pathogens Detected by Multiplex PCR in Blood of Very-Low-Birth Weight Infants with Suspected Sepsis - Multicentre Study of the German Neonatal Network.

    Science.gov (United States)

    Tröger, Birte; Härtel, Christoph; Buer, Jan; Dördelmann, Michael; Felderhoff-Müser, Ursula; Höhn, Thomas; Hepping, Nico; Hillebrand, Georg; Kribs, Angela; Marissen, Janina; Olbertz, Dirk; Rath, Peter-Michael; Schmidtke, Susanne; Siegel, Jens; Herting, Egbert; Göpel, Wolfgang; Steinmann, Joerg; Stein, Anja

    2016-01-01

    In the German Neonatal Network (GNN) 10% of very-low-birth weight infants (VLBWI) suffer from blood-culture confirmed sepsis, while 30% of VLBWI develop clinical sepsis. Diagnosis of sepsis is a difficult task leading to potential over-treatment with antibiotics. This study aims to investigate whether the results of blood multiplex-PCR (SeptiFast®) for common sepsis pathogens are relevant for clinical decision making when sepsis is suspected in VLBWI. We performed a prospective, multi-centre study within the GNN including 133 VLBWI with 214 episodes of suspected late onset sepsis (LOS). In patients with suspected sepsis a multiplex-PCR (LightCycler SeptiFast MGRADE-test®) was performed from 100 μl EDTA blood in addition to center-specific laboratory biomarkers. The attending neonatologist documented whether the PCR-result, which was available after 24 to 48 hrs, had an impact on the choice of antibiotic drugs and duration of therapy. PCR was positive in 110/214 episodes (51%) and blood culture (BC) was positive in 55 episodes (26%). Both methods yielded predominantly coagulase-negative staphylococci (CoNS) followed by Escherichia coli and Staphylococcus aureus. In 214 BC-PCR paired samples concordant results were documented in 126 episodes (59%; n = 32 were concordant pathogen positive results, n = 94 were negative in both methods). In 65 episodes (30%) we found positive PCR results but negative BCs, with CoNS being identified in 43 (66%) of these samples. Multiplex-PCR results influenced clinical decision making in 30% of episodes, specifically in 18% for the choice of antimicrobial therapy and in 22% for the duration of antimicrobial therapy. Multiplex-PCR results had a moderate impact on clinical management in about one third of LOS-episodes. The main advantage of multiplex-PCR was the rapid detection of pathogens from micro-volume blood samples. In VLBWI limitations include risk of contamination, lack of resistance testing and high costs. The high rate of

  11. One-step multiplex real time RT-PCR for the detection of bovine respiratory syncytial virus, bovine herpesvirus 1 and bovine parainfluenza virus 3

    Directory of Open Access Journals (Sweden)

    Thonur Leenadevi

    2012-03-01

    Full Text Available Abstract Background Detection of respiratory viruses in veterinary species has traditionally relied on virus detection by isolation or immunofluorescence and/or detection of circulating antibody using ELISA or serum neutralising antibody tests. Multiplex real time PCR is increasingly used to diagnose respiratory viruses in humans and has proved to be superior to traditional methods. Bovine respiratory disease (BRD is one of the most common causes of morbidity and mortality in housed cattle and virus infections can play a major role. We describe here a one step multiplex reverse transcriptase quantitative polymerase chain reaction (mRT-qPCR to detect the viruses commonly implicated in BRD. Results A mRT-qPCR assay was developed and optimised for the simultaneous detection of bovine respiratory syncytial virus (BRSV, bovine herpes virus type 1 (BoHV-1 and bovine parainfluenza virus type 3 (BPI3 i & ii nucleic acids in clinical samples from cattle. The assay targets the highly conserved glycoprotein B gene of BoHV-1, nucleocapsid gene of BRSV and nucleoprotein gene of BPI3. This mRT-qPCR assay was assessed for sensitivity, specificity and repeatability using in vitro transcribed RNA and recent field isolates. For clinical validation, 541 samples from clinically affected animals were tested and mRT-qPCR result compared to those obtained by conventional testing using virus isolation (VI and/or indirect fluorescent antibody test (IFAT. Conclusions The mRT-qPCR assay was rapid, highly repeatable, specific and had a sensitivity of 97% in detecting 102 copies of BRSV, BoHV-1 and BPI3 i & ii. This is the first mRT-qPCR developed to detect the three primary viral agents of BRD and the first multiplex designed using locked nucleic acid (LNA, minor groove binding (MGB and TaqMan probes in one reaction mix. This test was more sensitive than both VI and IFAT and can replace the aforesaid methods for virus detection during outbreaks of BRD.

  12. Listeria monocytogenes in spontaneous abortions in humans and its detection by multiplex PCR.

    Science.gov (United States)

    Kaur, S; Malik, S V S; Vaidya, V M; Barbuddhe, S B

    2007-11-01

    To assess the extent of Listeria monocytogenes in causation of human spontaneous abortions by isolation methods and PCR analysis for the presence of virulence-associated genes. A total of 305 samples comprising blood, urine, placental bits, faecal and vaginal swabs were collected from 61 patients with spontaneous abortions. Listeria spp. were isolated from 10 samples collected from nine (14.8%) patients. Confirmation of these isolates was based on biochemical tests, haemolysis on blood agar, CAMP test, phosphatidylinositol-specific phospholipase C (PI-PLC) assay followed by in vivo pathogenicity tests and multiplex PCR to detect virulence-associated genes (prfA, plcA, hlyA, actA and iap). Three isolates were confirmed as L. monocytogenes. Of these, two isolates turned out to be pathogenic and found to posses all five genes. However, the remaining two haemolytic L. monocytogenes isolates lacking the plcA gene and activity in the PI-PLC assay were found to be nonpathogenic by in vivo tests. The occurrence of pathogenic L. monocytogenes in cases of spontaneous abortions was 3.3%. It seems that the plcA gene and its expression have an important role as essential virulence determinants in pathogenic Listeria spp. The recovery of pathogenic L. monocytogenes isolates from cases of spontaneous abortion indicates the significance of listeric infection in pregnant women.

  13. DNAemia detection by multiplex PCR and biomarkers for infection in systemic inflammatory response syndrome patients.

    Directory of Open Access Journals (Sweden)

    Catherine Fitting

    Full Text Available Fast and reliable assays to precisely define the nature of the infectious agents causing sepsis are eagerly anticipated. New molecular biology techniques are now available to define the presence of bacterial or fungal DNA within the bloodstream of sepsis patients. We have used a new technique (VYOO® that allows the enrichment of microbial DNA before a multiplex polymerase chain reaction (PCR for pathogen detection provided by SIRS-Lab (Jena, Germany. We analyzed 72 sepsis patients and 14 non-infectious systemic inflammatory response syndrome (SIRS patients. Among the sepsis patients, 20 had a positive blood culture and 35 had a positive microbiology in other biological samples. Of these, 51.4% were positive using the VYOO® test. Among the sepsis patients with a negative microbiology and the non-infectious SIRS, 29.4% and 14.2% were positive with the VYOO® test, respectively. The concordance in bacterial identification between microbiology and the VYOO® test was 46.2%. This study demonstrates that these new technologies offer great hopes, but improvements are still needed.

  14. Multiplex PCR for the detection of toxigenic cyanobacteria in dietary supplements produced for human consumption.

    Science.gov (United States)

    Saker, Martin L; Welker, Martin; Vasconcelos, Vitor M

    2007-01-01

    The production of food supplements containing cyanobacteria is a growing worldwide industry. While there have been several reports of health benefits that can be gained from the consumption of these supplements, there have also been a growing number of studies showing the presence of toxins some of which (for example microcystins) are known to affect human health. In this paper, we report a multiplex polymerase chain reaction (PCR) technique that can be used to identify microcystin contamination in dietary supplements produced for human consumption. This method involves a PCR reaction containing three primer pairs, the first of which is used to amplify a 220-bp fragment of 16s rDNA specific to Microcystis, the most common microcystin-producing cyanobacterium. The second primer pair is used to amplify a 300-bp fragment of the mcyA gene, linked to microcystin biosynthesis in Anabaena, Microcystis, and Planktothrix. A third primer pair, used as a positive control, results in the amplification of a 650-bp fragment from the phycocyanin operon common to all cyanobacteria. This technique was found to be useful for detecting the presence of toxigenic Microcystis in all dietary supplements produced from the nontoxic cyanobacterium Aphanizomenon flos-aquae.

  15. Detection of virulence, antibiotic resistance and toxin (VAT) genes in Campylobacter species using newly developed multiplex PCR assays.

    Science.gov (United States)

    Laprade, Natacha; Cloutier, Michel; Lapen, David R; Topp, Edward; Wilkes, Graham; Villemur, Richard; Khan, Izhar U H

    2016-05-01

    Campylobacter species are one of the leading causes of bacterial gastroenteritis in humans worldwide. This twofold study was sought to: i) develop and optimize four single-tube multiplex PCR (mPCR) assays for the detection of six virulence (ciaB, dnaJ, flaA, flaB, pldA and racR), three toxin (cdtA, cdtB and cdtC) and one antibiotic resistance tet(O) genes in thermophilic Campylobacter spp. and ii) apply and evaluate the developed mPCR assays by testing 470 previously identified C. jejuni, C. coli and C. lari isolates from agricultural water. In each mPCR assay, a combination of two or three sets of primer pairs for virulence, antibiotic resistance and toxin (VAT) genes was used and optimized. Assay 1 was developed for the detection of dnaJ, racR and cdtC genes with expected amplification sizes of 720, 584 and 182bp. Assay 2 generated PCR amplicons for tet(O) and cdtA genes of 559 and 370bp. Assay 3 amplified cdtB ciaB, and pldA genes with PCR amplicon sizes of 620, 527 and 385bp. Assay 4 was optimized for flaA and flaB genes that generated PCR amplicons of 855 and 260bp. The primer pairs and optimized PCR protocols did not show interference and/or cross-amplification with each other and generated the expected size of amplification products for each target VAT gene for the C. jejuni ATCC 33291 reference strain. Overall, all ten target VAT genes were detected at a variable frequency in tested isolates of thermophilic Campylobacter spp. where cdtC, flaB, ciaB, cdtB, cdtA and pldA were commonly detected compared to the flaA, racR, dnaJ and tet(O) genes which were detected with less frequency. The developed mPCR assays are simple, rapid, reliable and sensitive tools for simultaneously assessing potential pathogenicity and antibiotic resistance profiling in thermophilic Campylobacter spp. The mPCR assays will be useful in diagnostic and analytical settings for routine screening of VAT characteristics of Campylobacter spp. as well as being applicable in epidemiological

  16. Rapid multiplex high resolution melting method to analyze inflammatory related SNPs in preterm birth

    Directory of Open Access Journals (Sweden)

    Pereyra Silvana

    2012-01-01

    Full Text Available Abstract Background Complex traits like cancer, diabetes, obesity or schizophrenia arise from an intricate interaction between genetic and environmental factors. Complex disorders often cluster in families without a clear-cut pattern of inheritance. Genomic wide association studies focus on the detection of tens or hundreds individual markers contributing to complex diseases. In order to test if a subset of single nucleotide polymorphisms (SNPs from candidate genes are associated to a condition of interest in a particular individual or group of people, new techniques are needed. High-resolution melting (HRM analysis is a new method in which polymerase chain reaction (PCR and mutations scanning are carried out simultaneously in a closed tube, making the procedure fast, inexpensive and easy. Preterm birth (PTB is considered a complex disease, where genetic and environmental factors interact to carry out the delivery of a newborn before 37 weeks of gestation. It is accepted that inflammation plays an important role in pregnancy and PTB. Methods Here, we used real time-PCR followed by HRM analysis to simultaneously identify several gene variations involved in inflammatory pathways on preterm labor. SNPs from TLR4, IL6, IL1 beta and IL12RB genes were analyzed in a case-control study. The results were confirmed either by sequencing or by PCR followed by restriction fragment length polymorphism. Results We were able to simultaneously recognize the variations of four genes with similar accuracy than other methods. In order to obtain non-overlapping melting temperatures, the key step in this strategy was primer design. Genotypic frequencies found for each SNP are in concordance with those previously described in similar populations. None of the studied SNPs were associated with PTB. Conclusions Several gene variations related to the same inflammatory pathway were screened through a new flexible, fast and non expensive method with the purpose of analyzing

  17. Comparison of two multiplex PCR assays for the detection of Listeria spp. and Listeria monocytogenes in biological samples

    OpenAIRE

    Budniak Sylwia; Kędrak-Jabłońska Agnieszka; Szczawińska Anna; Reksa Monika; Krupa Marek; Szulowski Krzysztof

    2016-01-01

    Introduction: The aim of the study was to optimise and compare two multiplex PCR assays for the detection of Listeria spp. and Listeria monocytogenes in biological samples including the liver, brain, and blood. Material and Methods: Three strains of L. monocytogenes and single strains of each of the species: L. ivanovii, L. innocua, L. grayi, L. welshimeri, and L. seeligeri were used. Additionally, five other species of bacterium were used to evaluate the specificity of the tests. Results: Sp...

  18. Presenting a rapid method for detection of Bacillus cereus, Listeria monocytogenes and Campylobacter jejuni in food samples

    Directory of Open Access Journals (Sweden)

    Ali Razei

    2017-08-01

    Full Text Available Objective(s: Listeria monocytogens, Bacillus cereus and Campylobacter jejuni are three toxin producing bacteria over the world, especially in Iran, and it is essential to find a certain, rapid procedure to identify these microorganisms. In this research, these bacteria were simultaneously detected by multiplex PCR technique in foods. Materials and Methods: The primary approval of bacterial strains was performed by biochemical tests. PCR primers were designed based on the nucleotide sequences of the NHEB/NHEC gene of B. cereus, the hly gene of L. monocytogenes and the C gene of C. jejuni. The specificity of Multiplex PCR method was determined using seven food poisoning bacteria including Salmonella typhi, Shigella dysentery, Yersinia pestis, Staphylococcus aureus, Clostridium perfringens, Clostridium botulinum and Vibrio cholerae. To confirm the reaction, DNA extraction was performed from 30 food samples (milk, and gene amplification was performed by PCR. The length of amplified fragments was 300 bp, 210 bp and 160 bpfor NHEB/NHEC, hly and C genes, respectively. Results: The detection limits of the PCR method were 5, 4 and 3 pg for L. monocytogenes, B. cereus and C. jejuni, respectively. Specifisity test showed that this reaction is spesific to these 3 bacteria. Conclusion: In this study, we  introduced a new multiplex PCR method for simultsnus detection of L. monocytogens, B. cereus and C. jejuni. These results can be used  for detection of other toxin producing bacteria in food.

  19. Multiplex detection of B-type natriuretic peptide, cardiac troponin I and C-reactive protein with photonic suspension array.

    Directory of Open Access Journals (Sweden)

    Wenbin Lu

    Full Text Available A novel photonic suspension array has been developed for multiplex immunoassay. The carriers of this array were silica colloidal crystal beads (SCCBs. The codes of these carriers have characteristic reflection peaks originating from their structural periodicity; therefore they do not suffer from fading, bleaching, quenching or chemical instability. In addition, the fluorescence background of SCCBs is negligible because no fluorescence materials or dyes are involved. With a sandwich method, the proposed suspension array was used for simultaneous multiplex detection of heart failure (HF and coronary heart disease (CAD biomarkers in one test tube. The results showed that the three biomarkers: cardiac troponin I (cTnI, C-reactive protein (CRP and B-type natriuretic peptide (BNP could be assayed in the ranges of 0.1-500 ng/ml, 1-500 mg/L and 0.02-50 ng/ml with detection limits of 0.01 ng/ml, 0.36 mg/L and 0.004 ng/ml at 3σ, respectively. There were no significant differences between the photonic suspension array and traditional parallel single-analyte test. This novel method demonstrated acceptable accuracy, high detection sensitivity and reproducibility and excellent storage stability. This technique provides a new strategy for low cost, automated, and simultaneous multiplex immunoassays of bio-markers.

  20. Assembly-line manipulation of droplets in microfluidic platform for fluorescence encoding and simultaneous multiplexed DNA detection.

    Science.gov (United States)

    Chen, Jinyang; Zhou, Guohua; Liu, Yufei; Ye, Tai; Xiang, Xia; Ji, Xinghu; He, Zhike

    2015-03-01

    In this article, a new mode of droplets manipulation is presented and applied for simultaneous multiplexed DNA detection. We call this droplets manipulation, "assembly-line manipulation of droplets (ALMD)". Firstly, multiple droplets containing the same target mixtures are generated in the microchannel, and then fused with later generated different droplets containing corresponding probes, respectively. Finally, all the fused droplets were fluorescence imaged on-line and real-time. The successful implementation of droplets fluorescence encoding based on ALMD shows the reproducibility and accuracy of this manipulation mode. As a proof-of-concept application, the simultaneous multiplexed DNA detection was carried out through the model of human immunodeficiency virus (HIV) gene sequence and variola virus (small pox, VV) gene sequence based on ALMD in the microfluidic system. It is proved that this method achieves simultaneous multiplexed DNA measurements with a significantly time-saving way and without different dye-labelled probes or complex operation procedures. In addition, it reveals the possibility of high-throughput biosensing with simple chip design and detection equipment. Copyright © 2014 Elsevier B.V. All rights reserved.

  1. Aqueous two-phase system patterning of detection antibody solutions for cross-reaction-free multiplex ELISA

    Science.gov (United States)

    Frampton, John P.; White, Joshua B.; Simon, Arlyne B.; Tsuei, Michael; Paczesny, Sophie; Takayama, Shuichi

    2014-05-01

    Accurate disease diagnosis, patient stratification and biomarker validation require the analysis of multiple biomarkers. This paper describes cross-reactivity-free multiplexing of enzyme-linked immunosorbent assays (ELISAs) using aqueous two-phase systems (ATPSs) to confine detection antibodies at specific locations in fully aqueous environments. Antibody cross-reactions are eliminated because the detection antibody solutions are co-localized only to corresponding surface-immobilized capture antibody spots. This multiplexing technique is validated using plasma samples from allogeneic bone marrow recipients. Patients with acute graft versus host disease (GVHD), a common and serious condition associated with allogeneic bone marrow transplantation, display higher mean concentrations for four multiplexed biomarkers (HGF, elafin, ST2 and TNFR1) relative to healthy donors and transplant patients without GVHD. The antibody co-localization capability of this technology is particularly useful when using inherently cross-reactive reagents such as polyclonal antibodies, although monoclonal antibody cross-reactivity can also be reduced. Because ATPS-ELISA adapts readily available antibody reagents, plate materials and detection instruments, it should be easily transferable into other research and clinical settings.

  2. Multiplex qPCR for reliable detection and differentiation of Burkholderia mallei and Burkholderia pseudomallei

    Directory of Open Access Journals (Sweden)

    Janse Ingmar

    2013-02-01

    Full Text Available Abstract Background Burkholderia mallei and B. pseudomallei are two closely related species of highly virulent bacteria that can be difficult to detect. Pathogenic Burkholderia are endemic in many regions worldwide and cases of infection, sometimes brought by travelers from unsuspected regions, also occur elsewhere. Rapid, sensitive methods for identification of B. mallei and B. pseudomallei are urgently needed in the interests of patient treatment and epidemiological surveillance. Methods Signature sequences for sensitive, specific detection of pathogenic Burkholderia based on published genomes were identified and a qPCR assay was designed and validated. Results A single-reaction quadruplex qPCR assay for the detection of pathogenic Burkholderia, which includes a marker for internal control of DNA extraction and amplification, was developed. The assay permits differentiation of B. mallei and B. pseudomallei strains, and probit analysis showed a very low detection limit. Use of a multicopy signature sequence permits detection of less than 1 genome equivalent per reaction. Conclusions The new assay permits rapid detection of pathogenic Burkholderia and combines enhanced sensitivity, species differentiation, and inclusion of an internal control for both DNA extraction and PCR amplification.

  3. Multiplex qPCR for reliable detection and differentiation of Burkholderia mallei and Burkholderia pseudomallei.

    Science.gov (United States)

    Janse, Ingmar; Hamidjaja, Raditijo A; Hendriks, Amber C A; van Rotterdam, Bart J

    2013-02-14

    Burkholderia mallei and B. pseudomallei are two closely related species of highly virulent bacteria that can be difficult to detect. Pathogenic Burkholderia are endemic in many regions worldwide and cases of infection, sometimes brought by travelers from unsuspected regions, also occur elsewhere. Rapid, sensitive methods for identification of B. mallei and B. pseudomallei are urgently needed in the interests of patient treatment and epidemiological surveillance. Signature sequences for sensitive, specific detection of pathogenic Burkholderia based on published genomes were identified and a qPCR assay was designed and validated. A single-reaction quadruplex qPCR assay for the detection of pathogenic Burkholderia, which includes a marker for internal control of DNA extraction and amplification, was developed. The assay permits differentiation of B. mallei and B. pseudomallei strains, and probit analysis showed a very low detection limit. Use of a multicopy signature sequence permits detection of less than 1 genome equivalent per reaction. The new assay permits rapid detection of pathogenic Burkholderia and combines enhanced sensitivity, species differentiation, and inclusion of an internal control for both DNA extraction and PCR amplification.

  4. Development of a Multiplex PCR Method to Detect Fungal Pathogens for Quarantine on Exported Cacti

    Directory of Open Access Journals (Sweden)

    Hyun ji Cho

    2016-02-01

    Full Text Available Major diseases in grafted cacti have been reported and Fusarium oxysporum, Bipolaris cactivora, Phytophthora spp. and Collectotrichum spp. are known as causal pathogens. These pathogens can lead to plant death after infection. Therefore, some European countries have quarantined imported cacti that are infected with specific fungal pathogens. Consequently, we developed PCR detection methods to identify four quarantined fungal pathogens and reduce export rejection rates of Korean grafted cacti. The pathogen specific primer sets F.oF-F.oR, B.CF-B.CR, P.nF-P.nR, and P.cF-P.CR were tested for F. oxysporum, B. cactivora, P. nicotinae, and P. cactorum, respectively. The F.oF-F.oR primer set was designed from the Fusarium ITS region; the B.CF-B.CR and P.nF-P.nR primers respectively from Bipolaris and Phytophthora ITS1; and the P.cF-P.CR primer set from the Ypt1protein gene region. The quarantine fungal pathogen primer pairs were amplified to the specific number of base pairs in each of the following fungal pathogens: 210-bp (F. oxysporum, 510-bp (B. cactivora, 313-bp (P. nicotinae, and 447-bp (P. cactorum. The detection limit for the mono- and multiplex PCR primer sets was 0.1 ng of template DNA under in vitro conditions. Therefore, each primer set successfully diagnosed contamination of quarantine pathogens in export grafted cacti. Consequently, our methodology is a viable tool to screen contamination of the fungal pathogen in exported grafted cacti.

  5. A multiplex real-time PCR for the detection and differentiation of Campylobacter phages.

    Science.gov (United States)

    Jäckel, Claudia; Hammerl, Jens A; Rau, Jörg; Hertwig, Stefan

    2017-01-01

    Campylobacter jejuni and C. coli are important food-borne pathogens that are widespread in animal husbandry. To combat Campylobacter along the food chain, the application of lytic phages has been shown to be a promising tool. Campylobacter phages are currently classified into three groups, of which group II and group III phages are the most common. Members of each group are closely related, whereas the two groups share only little DNA similarity. Moreover, while group III phages are specific for C. jejuni, group II phages additionally infect C. coli. Phage cocktails intended to be used for applications should be composed of various phages that differ in their host range and growth kinetics. The isolation of phages is generally performed by plaque assays. This approach has the limitation that phages are merely identified by their lytic activity on certain indicator strains and that relatively high numbers of phages must be present in a tested sample. Therefore, a more sensitive molecular detection system would be beneficial, which allows a pre-screening of samples and the quick detection and discrimination of group II and group III phages. New phages can then be isolated by use of indicator strains that may be different from those typically applied. On the basis of available Campylobacter phage genome sequences, we developed a multiplex PCR system for group II and group III phages selecting the tail tube gene and the gene for the base plate wedge, respectively, as target. Phages of both groups could be identified with primers deduced from the putative tail fiber gene. Efficient release of phage DNA from capsids was achieved by an extended heat treatment or digestion of phage particles with proteinase K/SDS yielding a detection limit of 1 pfu/ml. Individual detection of group II phages, group III phages and of both groups was studied with artificially contaminated chicken skin. To recover phages that had strongly adhered to the skin, stomaching was the most efficient

  6. Comparison and transfer testing of multiplex ligation detection methods for GM plants

    Directory of Open Access Journals (Sweden)

    Ujhelyi Gabriella

    2012-01-01

    Full Text Available Abstract Background With the increasing number of GMOs on the global market the maintenance of European GMO regulations is becoming more complex. For the analysis of a single food or feed sample it is necessary to assess the sample for the presence of many GMO-targets simultaneously at a sensitive level. Several methods have been published regarding DNA-based multidetection. Multiplex ligation detection methods have been described that use the same basic approach: i hybridisation and ligation of specific probes, ii amplification of the ligated probes and iii detection and identification of the amplified products. Despite they all have this same basis, the published ligation methods differ radically. The present study investigated with real-time PCR whether these different ligation methods have any influence on the performance of the probes. Sensitivity and the specificity of the padlock probes (PLPs with the ligation protocol with the best performance were also tested and the selected method was initially validated in a laboratory exchange study. Results Of the ligation protocols tested in this study, the best results were obtained with the PPLMD I and PPLMD II protocols and no consistent differences between these two protocols were observed. Both protocols are based on padlock probe ligation combined with microarray detection. Twenty PLPs were tested for specificity and the best probes were subjected to further evaluation. Up to 13 targets were detected specifically and simultaneously. During the interlaboratory exchange study similar results were achieved by the two participating institutes (NIB, Slovenia, and RIKILT, the Netherlands. Conclusions From the comparison of ligation protocols it can be concluded that two protocols perform equally well on the basis of the selected set of PLPs. Using the most ideal parameters the multiplicity of one of the methods was tested and 13 targets were successfully and specifically detected. In the

  7. Designing a biochip following multiplex polymerase chain reaction for the detection of Salmonella serovars Typhimurium, Enteritidis, Infantis, Hadar, and Virchow in poultry products

    Directory of Open Access Journals (Sweden)

    Yu-Cheng Chiang

    2018-01-01

    Full Text Available Salmonella-contaminated foods, especially poultry-derived foods (eggs, chicken meat, are the major source of salmonellosis. Not only in the European Union (EU, but also in the United States, Japan, and other countries, has salmonellosis been an issue of concern for food safety control agencies. In 2005, EU regulation 1003/2005 set a target for the control and reduction of five target Salmonella enterica serovars—S. Typhimurium, S. Enteritidis, S. Infantis, S. Hadar, and S. Virchow—in breeding flocks. Thus, a simple biochip for the rapid detection of any of these five Salmonella serovars in poultry products may be required. The objectives of this study were to design S. Virchow-specific primers and to develop a biochip for the simultaneous identification of all or any of these five Salmonella serovars in poultry and poultry products. Experimentally, we designed novel polymerase chain reaction (PCR primers for the specific detection of S. Virchow, S. Infantis, and S. Hadar. The specificity of all these primers and two known primer sets for S. Typhimurium and S. Enteritidis was then confirmed under the same PCR conditions using 57 target strains and 112 nontarget Salmonella strains as well as 103 non-Salmonella strains. Following multiplex PCR, strains of any of these five Salmonella serovars could be detected by a chromogenic biochip deployed with DNA probes specific to these five Salmonella serovars. In comparison with the multiplex PCR methods, the biochip assay could improve the detection limit of each of the Salmonella serovars from N×103 cfu/mL to N×102 cfu/mL sample in either the pure culture or the chicken meat samples. With an 8-hour enrichment step, the detection limit could reach up to N×100 cfu/mL.

  8. A multiplex nanoparticle-based bio-barcoded DNA sensor for the simultaneous detection of multiple pathogens.

    Science.gov (United States)

    Zhang, Deng; Huarng, Michael C; Alocilja, Evangelyn C

    2010-12-15

    A highly amplified, nanoparticle-based, bio-barcoded electrochemical biosensor for the simultaneous multiple detection of the protective antigen A (pagA) gene (accession number, M22589) of Bacillus anthracis and the insertion element (Iel) gene (accession number, Z83734) of Salmonella enteritidis is reported in this paper. The biosensor system is mainly composed of three nanoparticles: gold nanoparticles (AuNPs), magnetic nanoparticles (MNPs), and nanoparticle tracers (NTs, such as PbS and CdS). The AuNPs are coated with the first target-specific DNA probe (1pDNA), which can recognize one end of the target DNA sequence (tDNA), and many NT-terminated bio-barcode ssDNA (bDNA-NT), which act as signal reporter and amplifier. The MNPs are coated with the second target-specific DNA probe (2pDNA) that can recognize the other end of the target gene. After binding the nanoparticles with the target DNA, the following sandwich structure is formed: MNP-2pDNA/tDNA/1pDNA-AuNP-bDNA-NTs. A magnetic field is applied to separate the sandwich structure from the unreacted materials. Because the AuNPs have a large number of nanoparticle tracers per DNA probe binding event, there is substantial amplification. After the nanoparticle tracer is dissolved in 1M nitric acid, the NT(2+) ions are detected by square wave anodic stripping voltammetry (SWASV) on screen-printed carbon electrode (SPCE) chips. The results show that the detection limit of this multiplex bio-barcoded DNA sensor are 0.5 ng/mL of the insertion element (Iel) gene of S. enteritidis using CdS, and 50 pg/mL of the pagA gene of B. anthracis using PbS NTs. The nanoparticle-based bio-barcoded DNA sensor has potential application in rapid detection of multiple pathogenic agents in the same sample. Copyright © 2010 Elsevier B.V. All rights reserved.

  9. Differential detection of Entamoeba histolytica from Entamoeba dispar by parasitological and nested multiplex polymerase chain reaction methods

    Directory of Open Access Journals (Sweden)

    esmaiel fallah

    2014-02-01

    Full Text Available Introduction: Amebiasis is an intestinal illness caused by a one-celled parasite (amoeba called Entamoeba (E histolytica. E histolytica and E dispar are morphologicallyundistinguishable but have genetic and functional differences. E. histolytica is invasive andcause amoebiasis, but E dispar cause an asymptomatic colonization which does not need to bemedically treated. We have performed a nested multiplex Polymerase Chain Reaction (PCRtargeting small subunit rRNA (Ribosomal ribonucleic acid gene for differential detection of Ehistolytica and E dispar directly from stool samples. Methods: All the fecal samples collected without preservation and were screened for amebiccells by parasitological methods. Fecal samples that containing amebic cells were stored at -20ºC until DNA extraction. DNA extraction was down by using a DNA extraction kit. Thegenus specific primers were designed using nucleotide sequences of 18S-rRNA gene ofEntamoeba. Results: Thirty one (4.28% stool samples out of 724 samples were positive for E histolytica/E dispar. The nested multiplex PCR illustrated that the size of diagnostic fragments of PCR products was obviously different for two Entamoeba species, the specific product size for Ehistolytica and E dispar was 439 and 174 bp. The nested multiplex PCR was positive in 25 outof 31 stool specimens that 17 (54.8% samples were positive for E dispar and 8 (25.8%samples were positive for E histolytica. Conclusion: Nested multiplex PCR was useful for the specific detection of E histolytica and Edispar in stool samples. In current study we detected that E dispar was more prevalent in our study area.

  10. Rapid detection of Mycobacterium avium subsp. paratuberculosis ...

    African Journals Online (AJOL)

    Paratuberculosis, caused by Mycobacterium avium subsp. paratuberculosis, a suspect causative agent of Crohns disease in man, is an emerging disease of international proportions affecting all ruminants. Early stage detection of Mycobacterium avium subsp. paratuberculosis infection would accelerate progress in control ...

  11. RAPID DETECTION OF MICROBIAL CONTAMINATION IN GHANA ...

    African Journals Online (AJOL)

    2014-06-01

    Jun 1, 2014 ... pathogens in herbal medicines from Ghana. Methods: We employed different DNA extraction ... kits yielded significant amounts of DNA. PCR was able to detect pathogens present in the samples directly. ..... safety of dried spices and herbs from production and retail premises in the United Kingdom. Food.

  12. Multiplexed detection of protein cancer markers on Au/Ag-barcoded nanorods using fluorescent-conjugated polymers.

    Science.gov (United States)

    Zheng, Weiming; He, Lin

    2010-07-01

    Integration of fluorescent-conjugated polymers as detection moiety with metallic striped nanorods for multiplexed detection of clinically important cancer marker proteins in an immunoassay format was demonstrated in this report. Specifically, cationic conjugated polymers were introduced to protein complexes through electrostatic binding to negatively charged double-stranded DNA, which was tagged on detection antibodies prior to antigen recognition. The intense fluorescence emission of conjugated polymers resulted in highly sensitive detection of cancer marker proteins wherein an undiluted bovine serum sample as low as approximately 25 target molecules captured on each particle was detectable. Meanwhile, the use of polymer molecules as the detection probe did not obscure the optical pattern of underlying nanorods, i.e., the encoding capability of barcoded nanorods was preserved, which allowed simultaneous detection of three cancer marker proteins with good specificity.

  13. Multiplex enrichment quantitative PCR (ME-qPCR): a high-throughput, highly sensitive detection method for GMO identification.

    Science.gov (United States)

    Fu, Wei; Zhu, Pengyu; Wei, Shuang; Zhixin, Du; Wang, Chenguang; Wu, Xiyang; Li, Feiwu; Zhu, Shuifang

    2017-04-01

    Among all of the high-throughput detection methods, PCR-based methodologies are regarded as the most cost-efficient and feasible methodologies compared with the next-generation sequencing or ChIP-based methods. However, the PCR-based methods can only achieve multiplex detection up to 15-plex due to limitations imposed by the multiplex primer interactions. The detection throughput cannot meet the demands of high-throughput detection, such as SNP or gene expression analysis. Therefore, in our study, we have developed a new high-throughput PCR-based detection method, multiplex enrichment quantitative PCR (ME-qPCR), which is a combination of qPCR and nested PCR. The GMO content detection results in our study showed that ME-qPCR could achieve high-throughput detection up to 26-plex. Compared to the original qPCR, the Ct values of ME-qPCR were lower for the same group, which showed that ME-qPCR sensitivity is higher than the original qPCR. The absolute limit of detection for ME-qPCR could achieve levels as low as a single copy of the plant genome. Moreover, the specificity results showed that no cross-amplification occurred for irrelevant GMO events. After evaluation of all of the parameters, a practical evaluation was performed with different foods. The more stable amplification results, compared to qPCR, showed that ME-qPCR was suitable for GMO detection in foods. In conclusion, ME-qPCR achieved sensitive, high-throughput GMO detection in complex substrates, such as crops or food samples. In the future, ME-qPCR-based GMO content identification may positively impact SNP analysis or multiplex gene expression of food or agricultural samples. Graphical abstract For the first-step amplification, four primers (A, B, C, and D) have been added into the reaction volume. In this manner, four kinds of amplicons have been generated. All of these four amplicons could be regarded as the target of second-step PCR. For the second-step amplification, three parallels have been taken for

  14. Simultaneous detection of four garlic viruses by multiplex reverse transcription PCR and their distribution in Indian garlic accessions.

    Science.gov (United States)

    Majumder, S; Baranwal, V K

    2014-06-01

    Indian garlic is infected with Onion yellow dwarf virus (OYDV), Shallot latent virus (SLV), Garlic common latent virus (GarCLV) and allexiviruses. Identity and distribution of garlic viruses in various garlic accessions from different geographical regions of India were investigated. OYDV and allexiviruses were observed in all the garlic accessions, while SLV and GarCLV were observed only in a few accessions. A multiplex reverse transcription (RT)-PCR method was developed for the simultaneous detection and identification of OYDV, SLV, GarCLV and Allexivirus infecting garlic accessions in India. This multiplex protocol standardized in this study will be useful in indexing of garlic viruses and production of virus free seed material. Copyright © 2014 Elsevier B.V. All rights reserved.

  15. Evaluation of the Quantamatrix Multiplexed Assay Platform system for simultaneous detection of Mycobacterium tuberculosis and the rifampicin resistance gene using cultured mycobacteria.

    Science.gov (United States)

    Wang, Hye-Young; Uh, Young; Kim, Seoyong; Shim, Tae-Sun; Lee, Hyeyoung

    2017-08-01

    The differentiation of Mycobacterium tuberculosis complex (MTBC) from non-tuberculous mycobacteria (NTM) is of primary importance for infection control and the selection of anti-tuberculosis drugs. Up to date data on rifampicin (RIF)-resistant tuberculosis (TB) is essential for the early management of multidrug-resistant TB. The aim of this study was to evaluate the usefulness of a newly developed multiplexed, bead-based bioassay (Quantamatrix Multiplexed Assay Platform, QMAP) for the rapid differentiation of 23 Mycobacterium species including MTBC and RIF-resistant strains. A total of 314 clinical Mycobacterium isolates cultured from respiratory specimens were used in this study. The sensitivity and specificity of the QMAP system for Mycobacterium species were 100% (95% CI 99.15-100%, ptuberculosis (MTB) isolates, 196 were pan-susceptible and 27 were resistant to RIF according to QMAP results. All of the mutations in the RIF resistance-determining region detected by the QMAP system were confirmed by rpoB sequence analysis and a REBA MTB-Rifa reverse blot hybridization assay. The majority of the mutations (n=26, 96.3%), including those missing wild-type probe signals, were located in three codons (529-534, 524-529, and 514-520), and 17 (65.4%) of these mutations were detected by three mutation probes (531TTG, 526TAC, and 516GTC). The entire QMAP system assay takes about 3h to complete, while results from the culture-based conventional method can take up to 48-72h. Although improvements to the QMAP system are needed for direct respiratory specimens, it may be useful for rapid screening, not only to identify and accurately discriminate MTBC from NTM, but also to identify RIF-resistant MTB strains in positive culture samples. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  16. Development of multiplex loop mediated isothermal amplification (m-LAMP) label-based gold nanoparticles lateral flow dipstick biosensor for detection of pathogenic Leptospira.

    Science.gov (United States)

    Nurul Najian, A B; Engku Nur Syafirah, E A R; Ismail, Nabilah; Mohamed, Maizan; Yean, Chan Yean

    2016-01-15

    In recent years extensive numbers of molecular diagnostic methods have been developed to meet the need of point-of-care devices. Efforts have been made towards producing rapid, simple and inexpensive DNA tests, especially in the diagnostics field. We report on the development of a label-based lateral flow dipstick for the rapid and simple detection of multiplex loop-mediated isothermal amplification (m-LAMP) amplicons. A label-based m-LAMP lateral flow dipstick assay was developed for the simultaneous detection of target DNA template and a LAMP internal control. This biosensor operates through a label based system, in which probe-hybridization and the additional incubation step are eliminated. We demonstrated this m-LAMP assay by detecting pathogenic Leptospira, which causes the re-emerging disease Leptospirosis. The lateral flow dipstick was developed to detect of three targets, the LAMP target amplicon, the LAMP internal control amplicon and a chromatography control. Three lines appeared on the dipstick, indicating positive results for all representative pathogenic Leptospira species, whereas two lines appeared, indicating negative results, for other bacterial species. The specificity of this biosensor assay was 100% when it was tested with 13 representative pathogenic Leptospira species, 2 intermediate Leptospira species, 1 non-pathogenic Leptospira species and 28 other bacteria species. This study found that this DNA biosensor was able to detect DNA at concentrations as low as 3.95 × 10(-1) genomic equivalent ml(-1). An integrated m-LAMP and label-based lateral flow dipstick was successfully developed, promising simple and rapid visual detection in clinical diagnostics and serving as a point-of-care device. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. Detection and differentiation of Burkholderia pseudomallei, Burkholderia mallei and Burkholderia thailandensis by multiplex PCR.

    Science.gov (United States)

    Lee, May-Ann; Wang, Dongling; Yap, Eu Hian

    2005-03-01

    Burkholderia pseudomallei, a Gram-negative bacterium that causes melioidosis may be differentiated from closely related species of Burkholderia mallei that causes glanders and non-pathogenic species of Burkholderia thailandensis by multiplex PCR. The multiplex PCR consists of primers that flank a 10-bp repetitive element in B. pseudomallei and B. mallei amplifying PCR fragment of varying sizes between 400-700 bp, a unique sequence in B. thailandensis amplifying a PCR fragment of 308 bp and the metalloprotease gene amplifying a PCR fragment of 245 bp in B. pseudomallei and B. thailandensis. The multiplex PCR not only can differentiate the three Burkholderia species but can also be used for epidemiological typing of B. pseudomallei and B. mallei strains.

  18. Rapid Detection of Cellular Response to Biological Agents

    National Research Council Canada - National Science Library

    Williams, Bryan R

    2005-01-01

    Our program objective is to develop simple and rapid methods for detecting at a cellular level, individual responses to environmental stresses elaborated by exposure to infectious agents such as bacteria and viruses...

  19. Rapid Detection of Cellular Responses to Biological Agents

    National Research Council Canada - National Science Library

    Williams, Bryan

    2004-01-01

    Our program objective is to develop simple and rapid methods for detecting, at a cellular level, individual responses to environmental stresses elaborated by exposure to infectious agents such as bacteria and viruses...

  20. Rapid Detection of Cellular Responses to Biological Agents

    National Research Council Canada - National Science Library

    Williams, Bryan

    2003-01-01

    Our program objective is to develop simple and rapid methods for detecting, at a cellular level, individual responses to environmental stresses elaborated by exposure to infectious agents such as bacteria and viruses...

  1. ETV Tech Brief: Rapid Fungi and Bacteria Detection Technologies

    Science.gov (United States)

    Technical brief that summarizes the results for Mycometer, Inc. Mycometer®-test and Bactiquant®-test, which are rapid detection technologies for fungi and bacteria. The brief summarizes the results of the verification report and statement.

  2. Reliable, rapid and simple voltammetric detection of urea nitrate explosive.

    Science.gov (United States)

    Cagan, Avi; Lu, Donglai; Cizek, Karel; La Belle, Jeff; Wang, Joseph

    2008-05-01

    A highly selective and rapid electrochemical assay of the improvised explosive urea nitrate (UN) is reported. The method involves a short ( approximately 10 s) acid-catalyzed reaction of UN with 4-nitrotoluene (NT) followed by a rapid ( approximately 2 s) square-wave voltammetric (SWV) detection of the 2,4-dinitrotoluene (DNT) product. The new protocol offers great promise for a reliable field detection of UN, with significant advantages of speed, sensitivity, portability, simplicity, and cost.

  3. Rapid method for detection of salmonella in meat

    DEFF Research Database (Denmark)

    2016-01-01

    The present invention relates to a rapid method for the detection of Salmonella in meat as well as to a kit for performing said method. The method provides a time-to-result of less than 8 hours.......The present invention relates to a rapid method for the detection of Salmonella in meat as well as to a kit for performing said method. The method provides a time-to-result of less than 8 hours....

  4. Novel Multitarget Real-Time PCR Assay for Rapid Detection of Bordetella Species in Clinical Specimens ▿

    Science.gov (United States)

    Tatti, Kathleen M.; Sparks, Kansas N.; Boney, Kathryn O.; Tondella, Maria Lucia

    2011-01-01

    A novel multitarget real-time PCR (RT-PCR) assay for the rapid identification of Bordetella pertussis, B. parapertussis, and B. holmesii was developed using multicopy insertion sequences (ISs) in combination with the pertussis toxin subunit S1 (ptxS1) singleplex assay. The RT-PCR targets for the multiplex assay include IS481, commonly found in B. pertussis and B. holmesii; IS1001 of B. parapertussis; and the IS1001-like sequence of B. holmesii. Overall, 402 Bordetella species and 66 non-Bordetella species isolates were tested in the multitarget assay. Cross-reactivity was found only with 5 B. bronchiseptica isolates, which were positive with IS1001 of B. parapertussis. The lower limit of detection (LLOD) of the multiplex assay was similar to the LLOD of each target in an individual assay format, which was approximately 1 genomic equivalent per reaction for all targets. A total of 197 human clinical specimens obtained during cough-illness outbreak investigations were used to evaluate the multitarget RT-PCR assay. The multiplex assay results from 87 clinical specimens were compared to the individual RT-PCR assay and culture results. The multitarget assay is useful as a diagnostic tool to confirm B. pertussis infections and to rapidly identify other Bordetella species. In conclusion, the use of this multitarget RT-PCR approach increases specificity, while it decreases the amount of time, reagents, and specimen necessary for RT-PCRs used for accurate diagnosis of pertussis-like illness. PMID:21940464

  5. [Simultaneous detection of respiratory viruses and influenza A virus subtypes using multiplex PCR].

    Science.gov (United States)

    Ciçek, Candan; Bayram, Nuri; Anıl, Murat; Gülen, Figen; Pullukçu, Hüsnü; Saz, Eylem Ulaş; Telli, Canan; Cok, Gürsel

    2014-10-01

    This study was conducted to investigate the respiratory viruses and subtyping of influenza A virus when positive by multiplex PCR in patients with flu-like symptoms, after the pandemic caused by influenza A (H1N1)pdm09. Nasopharyngeal swab samples collected from 700 patients (313 female, 387 male; age range: 24 days-94 yrs, median age: 1 yr) between December 2010 - January 2013 with flu-like symptoms including fever, headache, sore throat, rhinitis, cough, myalgia as defined by the World Health Organization were included in the study. Nucleic acid extractions (Viral DNA/RNA Extraction Kit, iNtRON, South Korea) and cDNA synthesis (RevertAid First Strand cDNA Synthesis Kits, Fermentas, USA) were performed according to the manufacturer's protocol. Multiplex amplification of nucleic acids was performed using DPO (dual priming oligonucleotide) primers and RV5 ACE Screening Kit (Seegene, South Korea) in terms of the presence of influenza A (INF-A) virus, influenza B (INF-B) virus, respiratory syncytial virus (RSV), and the other respiratory viruses. PCR products were detected by automated polyacrylamide gel electrophoresis using Screen Tape multiple detection system. Specimens which were positive for viral nucleic acids have been further studied by using specific DPO primers, FluA ACE Subtyping and RV15 Screening (Seegene, South Korea) kits. Four INF-A virus subtypes [human H1 (hH1), human H3 (hH3), swine H1 (sH1), avian H5 (aH5)] and 11 other respiratory viruses [Adenovirus, parainfluenza virus (PIV) types 1-4, human bocavirus (HBoV), human metapneumovirus (HMPV), rhinovirus types A and B, human coronaviruses (HCoV) OC43, 229E/NL63] were investigated with those tests. In the study, 53.6% (375/700) of the patients were found to be infected with at least one virus and multiple respiratory virus infections were detected in 15.7% (59/375) of the positive cases, which were mostly (49/59, 83%) in pediatric patients. RSV and rhinovirus coinfections were the most prevalent (18

  6. Rapid Identification of Pathogens from Positive Blood Cultures by Multiplex PCR using the FilmArray System

    Science.gov (United States)

    Blaschke, Anne J.; Heyrend, Caroline; Byington, Carrie L.; Fisher, Mark A.; Barker, Elizabeth; Garrone, Nicholas F.; Thatcher, Stephanie A.; Pavia, Andrew T.; Barney, Trenda; Alger, Garrison D.; Daly, Judy A.; Ririe, Kirk M.; Ota, Irene; Poritz, Mark A.

    2012-01-01

    Sepsis is a leading cause of death. Rapid and accurate identification of pathogens and antimicrobial resistance directly from blood culture could improve patient outcomes. The FilmArray® (FA; Idaho Technology, Inc., Salt Lake City, UT) Blood Culture (BC) panel can identify > 25 pathogens and 4 antibiotic resistance genes from positive blood cultures in 1 hour. We compared a development version of the panel to conventional culture and susceptibility testing on 102 archived blood cultures from adults and children with bacteremia. Of 109 pathogens identified by culture, 95% were identified by FA. Among 111 prospectively collected blood cultures, the FA identified 84 of 92 pathogens (91%) covered by the panel. Among 25 Staphylococcus aureus and 21 Enterococcus species detected, FA identified all culture-proven MRSA and VRE. The FA BC panel is an accurate method for the rapid identification of pathogens and resistance genes from blood culture. PMID:22999332

  7. Early non-invasive detection of fetal Y chromosome sequences in maternal plasma using multiplex PCR.

    Science.gov (United States)

    Kolialexi, Aggeliki; Tounta, Georgia; Apostolou, Paraskevi; Vrettou, Christina; Papantoniou, Nikos; Kanavakis, Emmanuel; Antsaklis, Aris; Mavrou, Ariadni

    2012-03-01

    Clinical indications for fetal sex determination include risk of X-linked disorders, a family history of conditions associated with ambiguous development of the external genitalia, and some fetal ultrasound findings. It is usually performed in the first trimester from fetal material obtained through CVS and is associated with an approximately 1% risk of miscarriage. Ultrasound fetal sex determination is often performed after 11 weeks of gestation. This study aims to validate a reliable method for non-invasive prenatal diagnosis of fetal gender using maternal plasma cell-free fetal DNA (cffDNA) for fetal sex assessment in the first trimester of pregnancy and test its clinical utility in the diagnosis of potentially affected pregnancies in carriers of X-linked disorders. In the validation study, blood samples from 100 pregnant women at 6-11 weeks of gestation were analysed. In the clinical study, 17 pregnancies at risk of having an affected fetus were tested. 7 ml of maternal blood in EDTA were obtained and cffDNA was extracted using a commercially available kit. DNA was enzymatically digested using a methylation sensitive endonuclease (AciI) to remove maternal unmethylated sequences of the RASSF1A gene. A multiplex PCR was performed for the simultaneous amplification of target sequences of SRY and DYS14 from chromosome Y, along with RASSF1A and ACTB sequences. Amplification of these loci indicates fetal gender, confirms the presence of cffDNA and allows assessment of digestion efficiency. After establishing the appropriate experimental conditions, validation studies were successful in all 100 cases tested with no false negative or false positive results. Y chromosome-specific sequences were detected in 68 samples, and 32 cases were diagnosed as female based on the amplification of RASFF1A sequences only, in the absence of ACTB. In the clinical studies, fetal sex was correctly diagnosed in 16 pregnancies, and one case was reported as inconclusive. Fetal sex

  8. Multiplex Real-Time qPCR Assay for Simultaneous and Sensitive Detection of Phytoplasmas in Sesame Plants and Insect Vectors.

    Directory of Open Access Journals (Sweden)

    Cengiz Ikten

    Full Text Available Phyllody, a destructive and economically important disease worldwide caused by phytoplasma infections, is characterized by the abnormal development of floral structures into stunted leafy parts and contributes to serious losses in crop plants, including sesame (Sesamum indicum L.. Accurate identification, differentiation, and quantification of phyllody-causing phytoplasmas are essential for effective management of this plant disease and for selection of resistant sesame varieties. In this study, a diagnostic multiplex qPCR assay was developed using TaqMan® chemistry based on detection of the 16S ribosomal RNA gene of phytoplasmas and the 18S ribosomal gene of sesame. Phytoplasma and sesame specific primers and probes labeled with different fluorescent dyes were used for simultaneous amplification of 16SrII and 16SrIX phytoplasmas in a single tube. The multiplex real-time qPCR assay allowed accurate detection, differentiation, and quantification of 16SrII and 16SrIX groups in 109 sesame plant and 92 insect vector samples tested. The assay was found to have a detection sensitivity of 1.8 x 102 and 1.6 x 102 DNA copies for absolute quantification of 16SrII and 16SrIX group phytoplasmas, respectively. Relative quantification was effective and reliable for determination of phyllody phytoplasma DNA amounts normalized to sesame DNA in infected plant tissues. The development of this qPCR assay provides a method for the rapid measurement of infection loads to identify resistance levels of sesame genotypes against phyllody phytoplasma disease.

  9. Development and Assessment of a Single Tube Internally ControlledMultiplex PCR Assay to Detect Different Pathogenic Bacteria Involved inBlood Stream Infections

    Directory of Open Access Journals (Sweden)

    Mohammad Reza Arabestani

    2013-01-01

    Full Text Available Background: Bloodstream infections are associated with high morbidity and mortality. Delayed etiological diagnosis and inadequate antimicrobial therapy are associated with treatment failures. Objectives: This study describes the development and assessment of a new multiplex PCR that includes an Internal Control (IC for the assurance of the whole workflow from the extraction of the DNA until the revelation of the amplicons. Materials and Methods: A unique sequence was chosen for each pathogen and used for primer design. Primers for amplification of Enterobacteriaceae, Enterococcus spp, Staphylococcus spp, Acinetobacterbaumanii and IC were designed and tested for sensitivity and specificity on the basis of their standard strains. Results: The multiplex PCR showed a sensitivity ranging from 1 to 100 target copies per reaction or 50 to 100 colony forming unit (CFU per ml to the whole blood depending on the bacterial species. The specificity of this method was elevated and no false positive amplification was identified for 17 different species other than the target microorganisms. Moreover, the detection of the IC was observed in the concentration as low as 1 copy per reaction. The correct co-amplification of IC for each single bacterial species showed a correct whole workflow procedure starting from the extraction step. Conclusion: This new assay permits a rapid and accurate detection of some pathogenic microorganisms, that are among the most commonly detected ones in blood stream infections in Iran, with a simple and cost-effective method which includes the use of an internal control to validate the whole procedure thus avoiding false negative results.

  10. Multiplex Real-Time qPCR Assay for Simultaneous and Sensitive Detection of Phytoplasmas in Sesame Plants and Insect Vectors.

    Science.gov (United States)

    Ikten, Cengiz; Ustun, Rustem; Catal, Mursel; Yol, Engin; Uzun, Bulent

    2016-01-01

    Phyllody, a destructive and economically important disease worldwide caused by phytoplasma infections, is characterized by the abnormal development of floral structures into stunted leafy parts and contributes to serious losses in crop plants, including sesame (Sesamum indicum L.). Accurate identification, differentiation, and quantification of phyllody-causing phytoplasmas are essential for effective management of this plant disease and for selection of resistant sesame varieties. In this study, a diagnostic multiplex qPCR assay was developed using TaqMan® chemistry based on detection of the 16S ribosomal RNA gene of phytoplasmas and the 18S ribosomal gene of sesame. Phytoplasma and sesame specific primers and probes labeled with different fluorescent dyes were used for simultaneous amplification of 16SrII and 16SrIX phytoplasmas in a single tube. The multiplex real-time qPCR assay allowed accurate detection, differentiation, and quantification of 16SrII and 16SrIX groups in 109 sesame plant and 92 insect vector samples tested. The assay was found to have a detection sensitivity of 1.8 x 102 and 1.6 x 102 DNA copies for absolute quantification of 16SrII and 16SrIX group phytoplasmas, respectively. Relative quantification was effective and reliable for determination of phyllody phytoplasma DNA amounts normalized to sesame DNA in infected plant tissues. The development of this qPCR assay provides a method for the rapid measurement of infection loads to identify resistance levels of sesame genotypes against phyllody phytoplasma disease.

  11. Comparative study of colloidal gold and quantum dots as labels for multiplex screening tests for multi-mycotoxin detection.

    Science.gov (United States)

    Foubert, Astrid; Beloglazova, Natalia V; De Saeger, Sarah

    2017-02-22

    Quantum dots (QDs) and colloidal gold nanoparticles (CG) were evaluated as labels for multiplex lateral flow immunoassay (LFIA) for determination of mycotoxins deoxynivalenol (DON), zearalenone (ZEN) and T2/HT2-toxin (T2/HT2) in cereal matrices. Both developed assays were based on the same immunoreagents (except for the labels), therefore their analytical characteristics could be objectively compared. For both LFIAs antigens (DON-ovalbumin (OVA), ZEN-OVA and T2-OVA) and rabbit anti-mouse immunoglobulin were immobilized on a nitrocellulose membrane as three test lines and one control line, respectively. Depending on the LFIA, monoclonal antibodies (mAb) against DON, ZEN and T2 were conjugated with CdSeS/ZnS QDs or CG. T2 and HT2 were detected by one test line (T2-OVA) with an anti-T2 mAb which showed 110% cross-reactivity with HT2. Both tests were developed in accordance with the legal limits and were developed in such a way that they had the same cut-off limits of 1000 μg kg -1 , 80 μg kg -1 and 80 μg kg -1 for DON, ZEN and T2/HT2, respectively in order to allow a correct comparison. Applicability of these assays was demonstrated by analysis of naturally contaminated wheat samples. The results demonstrate that both the LFIAs can be used as rapid, cost-effective and convenient qualitative tool for on-site screening for simultaneous detection of DON, ZEN and HT2/T2 in wheat without special instrumentation. However, the QD-based LFIA consumed less immunoreagents and was more sensitive and economically beneficial. In addition, the results were easier to interpret, resulting in a lower false negative rate (<5%) which was in good agreement with Commission Decision 2002/657/EC regarding the performance of analytical methods intended for screening purposes. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. Real time quantitative amplification detection on a microarray: towards high multiplex quantitative PCR.

    NARCIS (Netherlands)

    Pierik, A.; Moamfa, M; van Zelst, M.; Clout, D.; Stapert, H.; Dijksman, Johan Frederik; Broer, D.; Wimberger-Friedl, R.

    2012-01-01

    Quantitative real-time polymerase chain reaction (qrtPCR) is widely used as a research and diagnostic tool. Notwithstanding its many powerful features, the method is limited in the degree of multiplexing to about 6 due to spectral overlap of the available fluorophores. A new method is presented that

  13. Comparison of two broadly multiplexed PCR systems for viral detection in clinical respiratory tract specimens from immunocompromised children.

    Science.gov (United States)

    Hayden, Randall T; Gu, Zhengming; Rodriguez, Alicia; Tanioka, Lisa; Ying, Claire; Morgenstern, Markus; Bankowski, Matthew J

    2012-04-01

    The detection of viral respiratory tract infections has evolved greatly with the development of PCR based commercial systems capable of simultaneously detecting a wide variety of pathogens. Evaluate the relative performance of two commercial broad range systems for the detection of viral agents in clinical respiratory tract specimens from immunocompromised children. A total of 176 patient samples were included in the analysis, representing only the first sample collected for each patient, and excluding failed reactions. Samples were de-identified and assayed in parallel using two different, broadly multiplexed PCR systems: ResPlex™ II Panel v2.0 (ResPlex), Qiagen, Hilden, Germany and FilmArray(®) Respiratory Panel (FilmArray), Idaho Technology Inc., Salt Lake City, UT. Method comparison was based upon pair-wise concordance of results according to patient age, viral target and number of targets detected. The two systems showed an overall concordance, by patient, of 83.8% (p=0.0001). FilmArray detected at least one target in 68.8% of samples, while ResPlex detected at least one target in 56.8%. ResPlex failed to detect 20.7% of FilmArray positives, and FilmArray failed to detect 4% of ResPlex positives. The relative performance of each system (including which system detected a higher number of positive samples) varied when stratified by target viral pathogen. Broadly multiplexed PCR is an effective means of detecting large numbers of clinically relevant respiratory viral pathogens. Copyright © 2011 Elsevier B.V. All rights reserved.

  14. Simultaneous detection of mastitis pathogens, Staphylococcus aureus, Streptococcus uberis, and Streptococcus agalactiae by multiplex real-time polymerase chain reaction.

    Science.gov (United States)

    Gillespie, B E; Oliver, S P

    2005-10-01

    The objective of this study was to develop a multiplex real-time polymerase chain reaction (PCR) method for simultaneous detection of Staphylococcus aureus, Streptococcus agalactiae, and Streptococcus uberis directly from milk. A genetic marker specific for Staph. aureus was used for primers and dual-labeled probe design. The target for Strep. agalactiae primers and dual-labeled probe was selected from the cfb gene encoding the Christie-Atkins-Munch-Petersen factor. The plasminogen activator gene was the target for primers and dual-labeled probe design for Strep. uberis. Quarter milk samples (n = 192) were analyzed by the multiplex real-time PCR assay and conventional microbiological methods. An additional 57 quarter milk samples were analyzed in a separate real-time PCR assay for Strep. agalactiae only. Using an overnight enrichment step, the real-time PCR technique correctly identified 96.4% of all quarter milk samples; 91.7% of Staph. aureus, 98.2% of Strep. agalactiae, and 100% of Strep. uberis. Results of conventional microbiological methods were used to determine the sensitivity and specificity of the multiplex real-time PCR procedure. The sensitivity of the procedure to correctly identify Staph. aureus, Strep. agalactiae, and Strep. uberis directly from milk was 95.5%, and the specificity was 99.6%. Results of this study indicate that the multiplex real-time PCR procedure has the potential to be a valuable diagnostic technique for simultaneous identification of Staph. aureus, Strep. agalactiae, and Strep. uberis directly from quarter milk samples.

  15. Use of a multiplex PCR to detect and identify Mycobacterium avium and M. intracellulare in blood culture fluids of AIDS patients.

    OpenAIRE

    Kulski, J. K.; Khinsoe, C; Pryce, T; Christiansen, K

    1995-01-01

    The presence of mycobacteria in blood culture fluids (BACTEC) of AIDS patients with positive growth indices (GIs, > 20 U) was investigated by using a multiplex PCR to detect and identify members of the genus Mycobacterium, M. avium, M. intracellulare, and M. tuberculosis. Three different methods of extracting mycobacterial DNA from blood culture fluid were compared for use with the multiplex PCR. Mycobacterial cells were pelleted from a small aliquot of blood culture fluid by centrifugation, ...

  16. Detection of Staphylococcus aureus enterotoxigenic strains in bovine raw milk by reversed passive latex agglutination and multiplex polymerase chain reaction

    Directory of Open Access Journals (Sweden)

    Asmaa Samy Mansour

    2017-08-01

    Full Text Available Aim: This review gives an outline of the assessment of enterotoxigenic Staphylococcus aureus tainting levels in raw milk from different sources in Egypt and characterization of enterotoxigenic strains utilizing a technique in light of PCR to identify genes coding for the production of staphylococcal enterotoxin (SE. The obtained data were compared with results from the application of the reversed passive latex. Materials and Methods: Multiplex PCR and reversed passive latex agglutination (RPLA were used. A total of 141 samples of raw milk (cow's milk=33, buffalo's milk=58, and bulk tank milk=50 were investigated for S. aureus contamination and tested for enterotoxin genes presence and toxin production. Results: S. aureus was detected in 23 (16.3% samples phenotypically and genotypically by amplification of nuc gene. The S. aureus isolates were investigated for SEs genes (sea to see by multiplex PCR and the toxin production by these isolates was screened by RPLA. SEs genes were detected in six isolates (26.1% molecularly; see was the most observed gene where detected in all isolates, two isolates harbored seb, and two isolates harbored sec. According to RPLA, three isolates produced SEB and SEC. Conclusion: The study revealed the widespread of S. aureus strains caring genes coding for toxins. The real significance of the presence of these strains or its toxins in raw milk and their possible impact a potential hazard for staphylococcal food poisoning by raw milk consumption. Therefore, detection of enterotoxigenic S. aureus strains in raw milk is necessary for consumer safety.

  17. Analytical Sensitivity Comparison between Singleplex Real-Time PCR and a Multiplex PCR Platform for Detecting Respiratory Viruses.

    Directory of Open Access Journals (Sweden)

    Jayme Parker

    Full Text Available Multiplex PCR methods are attractive to clinical laboratories wanting to broaden their detection of respiratory viral pathogens in clinical specimens. However, multiplexed assays must be well optimized to retain or improve upon the analytic sensitivity of their singleplex counterparts. In this experiment, the lower limit of detection (LOD of singleplex real-time PCR assays targeting respiratory viruses is compared to an equivalent panel on a multiplex PCR platform, the GenMark eSensor RVP. LODs were measured for each singleplex real-time PCR assay and expressed as the lowest copy number detected 95-100% of the time, depending on the assay. The GenMark eSensor RVP LODs were obtained by converting the TCID50/mL concentrations reported in the package insert to copies/μL using qPCR. Analytical sensitivity between the two methods varied from 1.2-1280.8 copies/μL (0.08-3.11 log differences for all 12 assays compared. Assays targeting influenza A/H3N2, influenza A/H1N1pdm09, influenza B, and human parainfluenza 1 and 2 were most comparable (1.2-8.4 copies/μL, <1 log difference. Largest differences in LOD were demonstrated for assays targeting adenovirus group E, respiratory syncytial virus subtype A, and a generic assay for all influenza A viruses regardless of subtype (319.4-1280.8 copies/μL, 2.50-3.11 log difference. The multiplex PCR platform, the GenMark eSensor RVP, demonstrated improved analytical sensitivity for detecting influenza A/H3 viruses, influenza B virus, human parainfluenza virus 2, and human rhinovirus (1.6-94.8 copies/μL, 0.20-1.98 logs. Broader detection of influenza A/H3 viruses was demonstrated by the GenMark eSensor RVP. The relationship between TCID50/mL concentrations and the corresponding copy number related to various ATCC cultures is also reported.

  18. Analytical Sensitivity Comparison between Singleplex Real-Time PCR and a Multiplex PCR Platform for Detecting Respiratory Viruses.

    Science.gov (United States)

    Parker, Jayme; Fowler, Nisha; Walmsley, Mary Louise; Schmidt, Terri; Scharrer, Jason; Kowaleski, James; Grimes, Teresa; Hoyos, Shanann; Chen, Jack

    2015-01-01

    Multiplex PCR methods are attractive to clinical laboratories wanting to broaden their detection of respiratory viral pathogens in clinical specimens. However, multiplexed assays must be well optimized to retain or improve upon the analytic sensitivity of their singleplex counterparts. In this experiment, the lower limit of detection (LOD) of singleplex real-time PCR assays targeting respiratory viruses is compared to an equivalent panel on a multiplex PCR platform, the GenMark eSensor RVP. LODs were measured for each singleplex real-time PCR assay and expressed as the lowest copy number detected 95-100% of the time, depending on the assay. The GenMark eSensor RVP LODs were obtained by converting the TCID50/mL concentrations reported in the package insert to copies/μL using qPCR. Analytical sensitivity between the two methods varied from 1.2-1280.8 copies/μL (0.08-3.11 log differences) for all 12 assays compared. Assays targeting influenza A/H3N2, influenza A/H1N1pdm09, influenza B, and human parainfluenza 1 and 2 were most comparable (1.2-8.4 copies/μL, <1 log difference). Largest differences in LOD were demonstrated for assays targeting adenovirus group E, respiratory syncytial virus subtype A, and a generic assay for all influenza A viruses regardless of subtype (319.4-1280.8 copies/μL, 2.50-3.11 log difference). The multiplex PCR platform, the GenMark eSensor RVP, demonstrated improved analytical sensitivity for detecting influenza A/H3 viruses, influenza B virus, human parainfluenza virus 2, and human rhinovirus (1.6-94.8 copies/μL, 0.20-1.98 logs). Broader detection of influenza A/H3 viruses was demonstrated by the GenMark eSensor RVP. The relationship between TCID50/mL concentrations and the corresponding copy number related to various ATCC cultures is also reported.

  19. A Multiplex RT-PCR Assay for S. aureus, L. monocytogenes, and Salmonella spp. Detection in Raw Milk with Pre-enrichment

    Directory of Open Access Journals (Sweden)

    Tian Ding

    2017-05-01

    Full Text Available This study firstly developed a multiplex real-time PCR (RT-PCR technique combined with a pre-enrichment step to simultaneously detect Staphylococcus aureus (S. aureus, Listeria monocytogenes (L. monocytogenes and Salmonella spp. in raw milk and the dairy farm environment (feces, soil, feed, water in one reaction. Brain heart infusion (BHI broth was selected for the enrichment step to increase the density of the target bacteria by using an incubation of 4 h before multiplex RT-PCR. The results showed that the detection limit of the multiplex real-time assay was approximately 102 CFU/mL for pure cultures and artificially contaminated milk without enrichment, while 12, 14, and 10 CFU/25 mL, respectively, for S. aureus, L. monocytogenes, and Salmonella spp. after pre-enrichment. The newly developed multiplex RT-PCR assay was applied to 46 dairy farm environmental samples and raw milk samples covering a wide variety of sample types. The results demonstrated that the multiplex RT-PCR assay coupled with the BHI enrichment broth was suitable for the simultaneous screening of S. aureus, L. monocytogenes, and Salmonella spp. in the pasture environment and in raw milk. The multiplex RT-PCR assay clearly and successfully shortened the total detection time and reduced labor compared to conventional culture-based methods for testing natural samples.

  20. Comparison of two multiplex methods for detection of respiratory viruses: FilmArray RP and xTAG RVP.

    Science.gov (United States)

    Rand, Kenneth H; Rampersaud, Howard; Houck, Herbert J

    2011-07-01

    We compared the FilmArray RP (Idaho Technology, Inc., Salt Lake City, UT) and the xTAG RVP (Luminex Corporation, Toronto, Canada) multiplex respiratory virus PCR methods for the detection of respiratory viruses in a set of 200 patient specimens frozen at -70 °C after standard viral culture and antigen detection methods were done. Both systems detected between 40 to 50% more viruses than traditional methods, primarily rhinoviruses and human metapneumovirus. The FilmArray RP detected significantly more total viruses either alone or as part of mixed infections than the xTAG RVP, as well as an additional 21.6% more respiratory syncytial viruses. The xTAG RVP requires 5 to 6 h with 2.5 to 3 h of hands-on time, while the FilmArray RP takes about an hour with 3 to 5 min of hands-on time, making it much easier to perform.

  1. Multiplex RT-PCR and indirect immunofluorescence assays for detection and subtyping of human influenza virus in Tunisia.

    Science.gov (United States)

    Ben M'hadheb, Manel; Harrabi, Myriam; Souii, Amira; Jrad-Battikh, Nadia; Gharbi, Jawhar

    2015-03-01

    Influenza viruses are negative stranded segmented RNA viruses belonging to Orthomyxoviridae family. They are classified into three types A, B, and C. Type A influenza viruses are classified into subtypes according to the antigenic characters of the surface glycoproteins: hemagglutinin (H) and neuraminidase (N). The aim of the present study is to develop a fast and reliable multiplex RT-PCR technique for detecting simultaneously the subtypes A/H1N1 and A/H3N2 of influenza virus. Our study included 398 patients (mean age 30.33 ± 19.92 years) with flu or flu-like syndromes, consulting physicians affiliated with collaborating teams. A multiplex RT-PCR detecting A/H1N1 and A/H3N2 influenza viruses and an examination by indirect immunofluorescence (IFI) were performed. In the optimized conditions, we diagnosed by IFI a viral infection in 90 patients (22.6 %): 85 cases of influenza type A, four cases of influenza type B, and only one case of coinfection with types A and B. An evaluation of the technique was performed on 19 clinical specimens positive in IFI, and we detected eight cases of A/H3N2, five cases of A/H1N1, one case of influenza virus type A which is not an H1N1 nor H3N2, and five negative cases. Multiplex RT-PCR is a sensitive technique allowing an effective and fast diagnosis of respiratory infections caused by influenza viruses in which the optimization often collides with problems of sensibility.

  2. Universal detection of phytoplasmas and Xylella spp. by TaqMan singleplex and multiplex real-time PCR with dual priming oligonucleotides.

    Directory of Open Access Journals (Sweden)

    Takao Ito

    Full Text Available Phytoplasmas and Xylella spp. are bacteria that cause many economically important plant diseases worldwide. TaqMan probe-based quantitative real-time polymerase chain reaction (qPCR assays have been utilized to universally detect phytoplasmas or Xylella fastidiosa. To develop a superior universal qPCR method, we used a dual priming oligonucleotide (DPO with two annealing sites as a reverse primer to target the well-conserved bacterial 16S rDNA. The new qPCR assays universally detected various species of phytoplasmas and subspecies of X. fastidiosa as well as Xylella taiwanensis, and generally showed superior threshold cycle values when amplifying specific or non-specific products compared to current universal qPCR assays. The proposed qPCR assays were integrated to develop a multiplex qPCR assay that simultaneously detected phytoplasmas, Xylella spp., and an internal plant DNA positive control within 1 hour. This assay could detect a minimum of ten bacterial cells and was compatible with crude extractions used in the rapid screening of various plants. The amplicons were of sufficient lengths to be directly sequenced for preliminary identification, and the primers could be used in universal conventional PCR assays. Additionally, reverse DPO primers can be utilized to improve other probe-based qPCR assays.

  3. Multiplex real-time PCR using SYBR® GreenER™ for the detection of DNA allergens in food.

    Science.gov (United States)

    Pafundo, Simona; Gullì, Mariolina; Marmiroli, Nelson

    2010-03-01

    We describe the development of a six-target real-time multiplex PCR assay with the SYBR® GreenER™ fluorescent dye, targeted to genes encoding for allergenic proteins commonly present in many processed food products (patent application pending). The assay was successfully trialled on reconstructed food matrices and on a range of commercial foodstuffs, and is proposed as a ready-to-use analytical tool for food manufacturers to detect the presence or confirm the absence of sequences encoding for important allergenic proteins of plant origin.

  4. The development of a GeXP-based multiplex reverse transcription-PCR assay for simultaneous detection of sixteen human respiratory virus types/subtypes

    Directory of Open Access Journals (Sweden)

    Li Jin

    2012-08-01

    Full Text Available Abstract Background Existing standard non-molecular diagnostic methods such as viral culture and immunofluorescent (DFA are time-consuming, labor intensive or limited sensitivity. Several multiplex molecular assays are costly. Therefore, there is a need for the development of a rapid and sensitive diagnosis of respiratory viral pathogens. Methods A GeXP-based multiplex RT-PCR assay (GeXP assay was developed to detect simultaneously sixteen different respiratory virus types/subtypes. Seventeen sets of chimeric primers were used to initiate the RT-PCR, and one pair of universal primers was used for the subsequent cycles of the RT-PCR. The specificity of the GeXP assay was examined with positive controls for each virus type/subtype. The sensitivity was evaluated by performing the assay on serial ten-fold dilutions of in vitro-transcribed RNA of all RNA viruses and the plasmids containing the Adv and HBoV target sequence. GeXP assay was further evaluated using 126 clinical specimens and compared with Luminex xTAG RVP Fast assay. Results The GeXP assay achieved a sensitivity of 20–200 copies for a single virus and 1000 copies when all of the 16 pre-mixed viral targets were present. Analyses of 126 clinical specimens using the GeXP assay demonstrated that GeXP assay and the RVP Fast assay were in complete agreement for 109/126 (88.51% of the specimens. GeXP assay was more sensitive than the RVP Fast assay for the detection of HRV and PIV3, and slightly less sensitive for the detection of HMPV, Adv, RSVB and HBoV. The whole process of the GeXP assay for the detection of 12 samples was completed within 2.5 hours. Conclusions In conclusion, the GeXP assay is a rapid, cost-effective, sensitive, specific and high throughput method for the detection of respiratory virus infections.

  5. Rapid virological diagnosis of central nervous system infections by use of a multiplex reverse transcription-PCR DNA microarray.

    Science.gov (United States)

    Leveque, Nicolas; Van Haecke, Adrien; Renois, Fanny; Boutolleau, David; Talmud, Deborah; Andreoletti, Laurent

    2011-11-01

    Viruses are the main etiological cause of central nervous system (CNS) infections. A rapid molecular diagnosis is recommended to improve the therapeutic management of patients. The aim of this study was to evaluate the performances of a DNA microarray, the Clart Entherpex kit (Genomica, Coslada, Spain), allowing the rapid and simultaneous detection of 9 DNA and RNA neurotropic viruses: herpes simplex virus 1 (HSV-1), HSV-2, varicella-zoster virus (VZV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), human herpesvirus 6 (HHV-6), HHV-7, HHV-8, and the human enteroviruses (HEVs). This evaluation was performed with 28 samples from the European proficiency panels (Quality Control for Molecular Diagnostics [QCMD]; Glasgow, Scotland) and then with 78 cerebrospinal fluid (CSF) specimens. The majority of the QCMD results obtained by the DNA microarray were similar to those recorded by the overall QCMD participants. The main discrepant results were observed for low concentrations of HSV-2 and HEVs. From the clinical samples, the kit detected 27 of the 28 herpesvirus CNS infections and all of the 30 HEV-positive CSF samples. No false-positive result was observed among the 20 virus-negative CSF samples. The clinical sensitivity, specificity, and negative and positive predictive values of the assay were 98.3, 100, 95.2, and 100%, respectively, when the results were compared to those of commercially available PCR assays. Interestingly, HHV-7 was detected in 11 (37%) of the 30 HEV-positive CSF samples from children suffering from aseptic meningitis causing significantly longer lengths of stay at the hospital than infection with HEVs alone (2.4 versus 1.4 days; P = 0.038). In conclusion, this preliminary study showed that this DNA microarray could be a valuable molecular diagnostic tool for single and mixed DNA and RNA virus infections of the CNS.

  6. Improved detection of deletions and duplications in the DMD gene using the multiplex ligation-dependent probe amplification (MLPA) method.

    Science.gov (United States)

    Sansović, Ivona; Barišić, Ingeborg; Dumić, Katja

    2013-04-01

    The multiplex ligation-dependent probe amplification (MLPA) assay is the most powerful tool in screening for deletions and duplications in the dystrophin gene in patients with Duchenne and Becker muscular dystrophy (DMD/BMD). The efficacy of the assay was validated by testing 20 unrelated male patients with DMD/BMD who had already been screened by multiplex PCR (mPCR). We detected two duplications that had been missed by mPCR. In one DMD patient showing an ambiguous MLPA result, a novel mutation (c.3808_3809insG) was identified. MLPA improved the mutation detection rate of mPCR by 15 %. The results of our study (1) confirmed MLPA to be the method of choice for detecting DMD gene rearrangements in DMD/BMD patients, (2) showed that ambiguous MLPA amplification products should be verified by other methods, and (3) indicated that the MLPA method could be used in screening even for small mutations located in the probe-binding regions.

  7. Real-time multiplex RT-PCR for the simultaneous detection of the five main grapevine viruses.

    Science.gov (United States)

    López-Fabuel, Irene; Wetzel, Thierry; Bertolini, Edson; Bassler, Alexandra; Vidal, Eduardo; Torres, Luis B; Yuste, Alberto; Olmos, Antonio

    2013-03-01

    A real-time multiplex RT-PCR has been developed for the simultaneous detection and identification of the major RNA viruses that infect grapevines (Grapevine fanleaf virus, Arabis mosaic virus, Grapevine leafroll-associated virus 1, Grapevine leafroll-associated virus 3 and Grapevine fleck virus). Serial dilutions of infected plant extracts were tested using the new method, and the results were compared with those obtained using a commercially available ELISA and real-time singleplex RT-PCR. The two real-time RT-PCR versions detected up to the same level of dilution and were at least 10,000 times more sensitive than the ELISA. In addition, 158 grapevine plants collected in a survey of the Protected Designation of Origin in Alicante, Spain were compared using the three methods. The results of the molecular methods were very similar, with only four discordant results, and both were able to detect many more infected plants than the ELISA. The high prevalence of Grapevine fleck virus, Grapevine leafroll-associated virus 3 and Grapevine fanleaf virus suggests that the main pathways of viral introduction are infected plant material that has escaped controls and/or uncontrolled traffic of propagating plant material. Real-time multiplex RT-PCR could be used to facilitate a better control of grapevine viruses. Copyright © 2012 Elsevier B.V. All rights reserved.

  8. Rapid Detection and Characterization of Emerging Foreign Animal Disease Pathogens

    Energy Technology Data Exchange (ETDEWEB)

    Jaing, C. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)

    2016-11-18

    To best safeguard human and animal health requires early detection and characterization of disease events. This must include effective surveillance for emerging infectious diseases. Both deliberate and natural outbreaks have enormous economic and public health impacts, and can present serious threats to national security. In this project, we developed novel next generation detection technologies to protect the agricultural economy and biosecurity. The first technology is a multiplexed assay to simultaneously detection 10 swine viral and bacterial pathogens. The second one is the Lawrence Livermore Microbial Detection Array (LLMDA) which can detect more than 10,000 microbial species including 4219 viruses, 5367 bacteria, 265 fungi, 117 protozoa and 293 archaea. We analyzed a series of swine clinical samples from past disease events to demonstrate the utility of the assays for faster and cheaper detection of emerging and foreign animal disease pathogens, and their utility as s routine diagnosis and surveillance tool. A second goal of the study is to better understand mechanisms of African swine fever virus (ASFV) infection in pigs to aid the development of countermeasures and diagnostics. There is no vaccine available for ASF. ASF outbreak is on the rise on several European countries. Though ASF is not currently in the U.S., a potential outbreak in the U.S. would be detrimental to the swine industry and the US agricultural economy. We pursued a genome-wide approach to characterize the pig immune responses after ASFV infection. We used RNA sequencing and bioinformatics methods to identify genes and pathways that are affected during ASF infection. We have identified a list of most differentially expressed genes that are in the immune response pathways.

  9. Rapid Molecular Detection of Multidrug-Resistant Tuberculosis by PCR-Nucleic Acid Lateral Flow Immunoassay

    Science.gov (United States)

    Kamphee, Hatairat; Chaiprasert, Angkana; Prammananan, Therdsak; Wiriyachaiporn, Natpapas; Kanchanatavee, Airin; Dharakul, Tararaj

    2015-01-01

    Several existing molecular tests for multidrug-resistant tuberculosis (MDR-TB) are limited by complexity and cost, hindering their widespread application. The objective of this proof of concept study was to develop a simple Nucleic Acid Lateral Flow (NALF) immunoassay as a potential diagnostic alternative, to complement conventional PCR, for the rapid molecular detection of MDR-TB. The NALF device was designed using antibodies for the indirect detection of labeled PCR amplification products. Multiplex PCR was optimized to permit the simultaneous detection of the drug resistant determining mutations in the 81-bp hot spot region of the rpoB gene (rifampicin resistance), while semi-nested PCR was optimized for the S315T mutation detection in the katG gene (isoniazid resistance). The amplification process additionally targeted a conserved region of the genes as Mycobacterium tuberculosis (Mtb) DNA control. The optimized conditions were validated with the H37Rv wild-type (WT) Mtb isolate and Mtb isolates with known mutations (MT) within the rpoB and katG genes. Results indicate the correct identification of WT (drug susceptible) and MT (drug resistant) Mtb isolates, with the least limit of detection (LOD) being 104 genomic copies per PCR reaction. NALF is a simple, rapid and low-cost device suitable for low resource settings where conventional PCR is already employed on a regular basis. Moreover, the use of antibody-based NALF to target primer-labels, without the requirement for DNA hybridization, renders the device generic, which could easily be adapted for the molecular diagnosis of other infectious and non-infectious diseases requiring nucleic acid detection. PMID:26355296

  10. Rapid Molecular Detection of Multidrug-Resistant Tuberculosis by PCR-Nucleic Acid Lateral Flow Immunoassay.

    Directory of Open Access Journals (Sweden)

    Hatairat Kamphee

    Full Text Available Several existing molecular tests for multidrug-resistant tuberculosis (MDR-TB are limited by complexity and cost, hindering their widespread application. The objective of this proof of concept study was to develop a simple Nucleic Acid Lateral Flow (NALF immunoassay as a potential diagnostic alternative, to complement conventional PCR, for the rapid molecular detection of MDR-TB. The NALF device was designed using antibodies for the indirect detection of labeled PCR amplification products. Multiplex PCR was optimized to permit the simultaneous detection of the drug resistant determining mutations in the 81-bp hot spot region of the rpoB gene (rifampicin resistance, while semi-nested PCR was optimized for the S315T mutation detection in the katG gene (isoniazid resistance. The amplification process additionally targeted a conserved region of the genes as Mycobacterium tuberculosis (Mtb DNA control. The optimized conditions were validated with the H37Rv wild-type (WT Mtb isolate and Mtb isolates with known mutations (MT within the rpoB and katG genes. Results indicate the correct identification of WT (drug susceptible and MT (drug resistant Mtb isolates, with the least limit of detection (LOD being 104 genomic copies per PCR reaction. NALF is a simple, rapid and low-cost device suitable for low resource settings where conventional PCR is already employed on a regular basis. Moreover, the use of antibody-based NALF to target primer-labels, without the requirement for DNA hybridization, renders the device generic, which could easily be adapted for the molecular diagnosis of other infectious and non-infectious diseases requiring nucleic acid detection.

  11. Multiplex Real-Time PCR for Detection of Staphylococcus aureus, mecA and Panton-Valentine Leukocidin (PVL) Genes from Selective Enrichments from Animals and Retail Meat

    Science.gov (United States)

    Velasco, Valeria; Sherwood, Julie S.; Rojas-García, Pedro P.; Logue, Catherine M.

    2014-01-01

    The aim of this study was to compare a real-time PCR assay, with a conventional culture/PCR method, to detect S. aureus, mecA and Panton-Valentine Leukocidin (PVL) genes in animals and retail meat, using a two-step selective enrichment protocol. A total of 234 samples were examined (77 animal nasal swabs, 112 retail raw meat, and 45 deli meat). The multiplex real-time PCR targeted the genes: nuc (identification of S. aureus), mecA (associated with methicillin resistance) and PVL (virulence factor), and the primary and secondary enrichment samples were assessed. The conventional culture/PCR method included the two-step selective enrichment, selective plating, biochemical testing, and multiplex PCR for confirmation. The conventional culture/PCR method recovered 95/234 positive S. aureus samples. Application of real-time PCR on samples following primary and secondary enrichment detected S. aureus in 111/234 and 120/234 samples respectively. For detection of S. aureus, the kappa statistic was 0.68–0.88 (from substantial to almost perfect agreement) and 0.29–0.77 (from fair to substantial agreement) for primary and secondary enrichments, using real-time PCR. For detection of mecA gene, the kappa statistic was 0–0.49 (from no agreement beyond that expected by chance to moderate agreement) for primary and secondary enrichment samples. Two pork samples were mecA gene positive by all methods. The real-time PCR assay detected the mecA gene in samples that were negative for S. aureus, but positive for Staphylococcus spp. The PVL gene was not detected in any sample by the conventional culture/PCR method or the real-time PCR assay. Among S. aureus isolated by conventional culture/PCR method, the sequence type ST398, and multi-drug resistant strains were found in animals and raw meat samples. The real-time PCR assay may be recommended as a rapid method for detection of S. aureus and the mecA gene, with further confirmation of methicillin-resistant S. aureus (MRSA) using

  12. Detection and characterization of recombinant DNA expressing vip3A-type insecticidal gene in GMOs--standard single, multiplex and construct-specific PCR assays.

    Science.gov (United States)

    Singh, Chandra K; Ojha, Abhishek; Bhatanagar, Raj K; Kachru, Devendra N

    2008-01-01

    Vegetative insecticidal protein (Vip), a unique class of insecticidal protein, is now part of transgenic plants for conferring resistance against lepidopteron pests. In order to address the imminent regulatory need for detection and labeling of vip3A carrying genetically modified (GM) products, we have developed a standard single PCR and a multiplex PCR assay. As far as we are aware, this is the first report on PCR-based detection of a vip3A-type gene (vip-s) in transgenic cotton and tobacco. Our assay involves amplification of a 284-bp region of the vip-s gene. This assay can possibly detect as many as 20 natural wild-type isolates bearing a vip3A-like gene and two synthetic genes of vip3A in transgenic plants. The limit of detection as established by our assay for GM trait (vip-s) is 0.1%. Spiking with nontarget DNA originating from diverse plant sources had no inhibitory effect on vip-s detection. Since autoclaving of vip-s bearing GM leaf samples showed no deterioration/interference in detection efficacy, the assay seems to be suitable for processed food products as well. The vip-s amplicon identity was reconfirmed by restriction endonuclease assay. The primer set for vip-s was equally effective in a multiplex PCR assay format (duplex, triplex and quadruplex), used in conjunction with the primer sets for the npt-II selectable marker gene, Cauliflower mosaic virus 35S promoter and nopaline synthetase terminator, enabling concurrent detection of the transgene, regulatory sequences and marker gene. Further, the entire transgene construct was amplified using the forward primer of the promoter and the reverse primer of the terminator. The resultant amplicon served as a template for nested PCR to confirm the construct integrity. The method is suitable for screening any vip3A-carrying GM plant and food. The availability of a reliable PCR assay method prior to commercial release of vip3A-based transgenic crops and food would facilitate rapid and efficient regulatory

  13. Nanomaterial-enabled Rapid Detection of Water Contaminants.

    Science.gov (United States)

    Mao, Shun; Chang, Jingbo; Zhou, Guihua; Chen, Junhong

    2015-10-28

    Water contaminants, e.g., inorganic chemicals and microorganisms, are critical metrics for water quality monitoring and have significant impacts on human health and plants/organisms living in water. The scope and focus of this review is nanomaterial-based optical, electronic, and electrochemical sensors for rapid detection of water contaminants, e.g., heavy metals, anions, and bacteria. These contaminants are commonly found in different water systems. The importance of water quality monitoring and control demands significant advancement in the detection of contaminants in water because current sensing technologies for water contaminants have limitations. The advantages of nanomaterial-based sensing technologies are highlighted and recent progress on nanomaterial-based sensors for rapid water contaminant detection is discussed. An outlook for future research into this rapidly growing field is also provided. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Rapid detection of EBOLA VP40 in microchip immunofiltration assay

    Science.gov (United States)

    Miethe, Peter; Gary, Dominik; Hlawatsch, Nadine; Gad, Anne-Marie

    2015-05-01

    In the spring of 2014, the Ebola virus (EBOV) strain Zaire caused a dramatic outbreak in several regions of West Africa. The RT-PCR and antigen capture diagnostic proved to be effective for detecting EBOV in blood and serum. In this paper, we present data of a rapid antigen capture test for the detection of VP40. The test was performed in a microfluidic chip for immunofiltration analysis. The chip integrates all necessary assay components. The analytical sensitivity of the rapid test was 8 ng/ml for recombinant VP40. In serum and whole blood samples spiked with virus culture material, the detection limit was 2.2 x 102 PFU/ml. The performance data of the rapid test (15 min) are comparable to that of the VP40 laboratory ELISA.

  15. Rapid identification of pathogens from positive blood cultures by multiplex polymerase chain reaction using the FilmArray system.

    Science.gov (United States)

    Blaschke, Anne J; Heyrend, Caroline; Byington, Carrie L; Fisher, Mark A; Barker, Elizabeth; Garrone, Nicholas F; Thatcher, Stephanie A; Pavia, Andrew T; Barney, Trenda; Alger, Garrison D; Daly, Judy A; Ririe, Kirk M; Ota, Irene; Poritz, Mark A

    2012-12-01

    Sepsis is a leading cause of death. Rapid and accurate identification of pathogens and antimicrobial resistance directly from blood culture could improve patient outcomes. The FilmArray® (FA; Idaho Technology, Salt Lake City, UT, USA) Blood Culture (BC) panel can identify >25 pathogens and 4 antibiotic resistance genes from positive blood cultures in 1 h. We compared a development version of the panel to conventional culture and susceptibility testing on 102 archived blood cultures from adults and children with bacteremia. Of 109 pathogens identified by culture, 95% were identified by FA. Among 111 prospectively collected blood cultures, the FA identified 84 (91%) of 92 pathogens covered by the panel. Among 25 Staphylococcus aureus and 21 Enterococcus species detected, FA identified all culture-proven methicillin-resistant S. aureus and vancomycin-resistant enterococci. The FA BC panel is an accurate method for the rapid identification of pathogens and resistance genes from blood culture. Copyright © 2012 Elsevier Inc. All rights reserved.

  16. Multiplex reverse transcription-polymerase chain reaction combined with on-chip electrophoresis as a rapid screening tool for candidate gene sets

    DEFF Research Database (Denmark)

    Wittig, Rainer; Salowsky, Rüdiger; Blaich, Stephanie

    2005-01-01

    Combining multiplex reverse transcription-polymerase chain reaction (mRT-PCR) with microfluidic amplicon analysis, we developed an assay for the rapid and reliable semiquantitative expression screening of 11 candidate genes for drug resistance in human malignant melanoma. The functionality...... of this approach was demonstrated by low interexperimental variations of amplicon quantities after endpoint analysis. When applied to RNA samples derived from drug-sensitive and -resistant melanoma cell lines, mRT-PCR delivered results qualitatively concordant with data obtained from Northern blot and array...... analyses. The screening of additional melanoma cell lines resulted in distinct expression patterns for ten candidate genes. Our approach reveals a rapid and easy-to-handle alternative for candidate gene set evaluation from limited amounts of RNA....

  17. Dual Recognition Element Lateral Flow Assay Toward Multiplex Strain Specific Influenza Virus Detection.

    Science.gov (United States)

    Le, Thao T; Chang, Pengxiang; Benton, Donald J; McCauley, John W; Iqbal, Munir; Cass, Anthony E G

    2017-06-20

    Different influenza virus strains have caused a number of recent outbreaks killing scores of people and causing significant losses in animal farming. Simple, rapid, sensitive, and specific detection of particular strains, such as a pandemic strain versus a previous seasonal influenza, plays a crucial role in the monitoring, controlling, and management of outbreaks. In this paper we describe a dual recognition element lateral flow assay (DRELFA) which pairs a nucleic acid aptamer with an antibody for use as a point-of-care platform which can detect particular strains of interest. The combination is used to overcome the individual limitations of antibodies' cross-reactivity and aptamers' slow binding kinetics. In the detection of influenza viruses, we show that DRELFA can discriminate a particular virus strain against others of the same subtype or common respiratory diseases while still exhibiting fast binding kinetic of the antibody-based lateral flow assay (LFA). The improvement in specificity that DRELFA exhibits is an advantage over the currently available antibody-based LFA systems for influenza viruses, which offer discrimination between influenza virus types and subtypes. Using quantitative real-time PCR (qRT-PCR), it showed that the DRELFA is very effective in localizing the analyte to the test line (consistently over 90%) and this is crucial for the sensitivity of the device. In addition, color intensities of the test lines showed a good correlation between the DRELFA and the qRT-PCR over a 50-fold concentration range. Finally, lateral flow strips with a streptavidin capture test line and an anti-antibody control line are universally applicable to specific detection of a wide range of different analytes.

  18. Usefulness of Multiplex Real-Time PCR for Simultaneous Pathogen Detection and Resistance Profiling of Staphylococcal Bacteremia

    Directory of Open Access Journals (Sweden)

    Yousun Chung

    2016-01-01

    Full Text Available Staphylococci are the leading cause of nosocomial blood stream infections. Fast and accurate identification of staphylococci and confirmation of their methicillin resistance are crucial for immediate treatment with effective antibiotics. A multiplex real-time PCR assay that targets mecA, femA specific for S. aureus, femA specific for S. epidermidis, 16S rRNA for universal bacteria, and 16S rRNA specific for staphylococci was developed and evaluated with 290 clinical blood culture samples containing Gram-positive cocci in clusters (GPCC. For the 262 blood cultures identified to the species level with the MicroScan WalkAway system (Siemens Healthcare Diagnostics, USA, the direct real-time PCR assay of positive blood cultures showed very good agreement for the categorization of staphylococci into methicillin-resistant S. aureus (MRSA, methicillin-susceptible S. aureus (MSSA, methicillin-resistant S. epidermidis (MRSE, methicillin-susceptible S. epidermidis (MSSE, methicillin-resistant non-S. epidermidis CoNS (MRCoNS, and methicillin-susceptible non-S. epidermidis CoNS (MSCoNS (κ=0.9313. The direct multiplex real-time PCR assay of positive blood cultures containing GPCC can provide essential information at the critical point of infection with a turnaround time of no more than 4 h. Further studies should evaluate the clinical outcome of using this rapid real-time PCR assay in glycopeptide antibiotic therapy in clinical settings.

  19. Usefulness of Multiplex Real-Time PCR for Simultaneous Pathogen Detection and Resistance Profiling of Staphylococcal Bacteremia.

    Science.gov (United States)

    Chung, Yousun; Kim, Taek Soo; Min, Young Gi; Hong, Yun Ji; Park, Jeong Su; Hwang, Sang Mee; Song, Kyoung-Ho; Kim, Eu Suk; Park, Kyoung Un; Kim, Hong Bin; Song, Junghan; Kim, Eui-Chong

    2016-01-01

    Staphylococci are the leading cause of nosocomial blood stream infections. Fast and accurate identification of staphylococci and confirmation of their methicillin resistance are crucial for immediate treatment with effective antibiotics. A multiplex real-time PCR assay that targets mecA, femA specific for S. aureus, femA specific for S. epidermidis, 16S rRNA for universal bacteria, and 16S rRNA specific for staphylococci was developed and evaluated with 290 clinical blood culture samples containing Gram-positive cocci in clusters (GPCC). For the 262 blood cultures identified to the species level with the MicroScan WalkAway system (Siemens Healthcare Diagnostics, USA), the direct real-time PCR assay of positive blood cultures showed very good agreement for the categorization of staphylococci into methicillin-resistant S. aureus (MRSA), methicillin-susceptible S. aureus (MSSA), methicillin-resistant S. epidermidis (MRSE), methicillin-susceptible S. epidermidis (MSSE), methicillin-resistant non-S. epidermidis CoNS (MRCoNS), and methicillin-susceptible non-S. epidermidis CoNS (MSCoNS) (κ = 0.9313). The direct multiplex real-time PCR assay of positive blood cultures containing GPCC can provide essential information at the critical point of infection with a turnaround time of no more than 4 h. Further studies should evaluate the clinical outcome of using this rapid real-time PCR assay in glycopeptide antibiotic therapy in clinical settings.

  20. Usefulness of Multiplex Real-Time PCR for Simultaneous Pathogen Detection and Resistance Profiling of Staphylococcal Bacteremia

    Science.gov (United States)

    Chung, Yousun; Kim, Taek Soo; Min, Young Gi; Hong, Yun Ji; Park, Jeong Su; Hwang, Sang Mee; Song, Kyoung-Ho; Kim, Eu Suk; Kim, Hong Bin; Song, Junghan; Kim, Eui-Chong

    2016-01-01

    Staphylococci are the leading cause of nosocomial blood stream infections. Fast and accurate identification of staphylococci and confirmation of their methicillin resistance are crucial for immediate treatment with effective antibiotics. A multiplex real-time PCR assay that targets mecA, femA specific for S. aureus, femA specific for S. epidermidis, 16S rRNA for universal bacteria, and 16S rRNA specific for staphylococci was developed and evaluated with 290 clinical blood culture samples containing Gram-positive cocci in clusters (GPCC). For the 262 blood cultures identified to the species level with the MicroScan WalkAway system (Siemens Healthcare Diagnostics, USA), the direct real-time PCR assay of positive blood cultures showed very good agreement for the categorization of staphylococci into methicillin-resistant S. aureus (MRSA), methicillin-susceptible S. aureus (MSSA), methicillin-resistant S. epidermidis (MRSE), methicillin-susceptible S. epidermidis (MSSE), methicillin-resistant non-S. epidermidis CoNS (MRCoNS), and methicillin-susceptible non-S. epidermidis CoNS (MSCoNS) (κ = 0.9313). The direct multiplex real-time PCR assay of positive blood cultures containing GPCC can provide essential information at the critical point of infection with a turnaround time of no more than 4 h. Further studies should evaluate the clinical outcome of using this rapid real-time PCR assay in glycopeptide antibiotic therapy in clinical settings. PMID:27403436

  1. FilmArray, an automated nested multiplex PCR system for multi-pathogen detection: development and application to respiratory tract infection.

    Directory of Open Access Journals (Sweden)

    Mark A Poritz

    Full Text Available The ideal clinical diagnostic system should deliver rapid, sensitive, specific and reproducible results while minimizing the requirements for specialized laboratory facilities and skilled technicians. We describe an integrated diagnostic platform, the "FilmArray", which fully automates the detection and identification of multiple organisms from a single sample in about one hour. An unprocessed biologic/clinical sample is subjected to nucleic acid purification, reverse transcription, a high-order nested multiplex polymerase chain reaction and amplicon melt curve analysis. Biochemical reactions are enclosed in a disposable pouch, minimizing the PCR contamination risk. FilmArray has the potential to detect greater than 100 different nucleic acid targets at one time. These features make the system well-suited for molecular detection of infectious agents. Validation of the FilmArray technology was achieved through development of a panel of assays capable of identifying 21 common viral and bacterial respiratory pathogens. Initial testing of the system using both cultured organisms and clinical nasal aspirates obtained from children demonstrated an analytical and clinical sensitivity and specificity comparable to existing diagnostic platforms. We demonstrate that automated identification of pathogens from their corresponding target amplicon(s can be accomplished by analysis of the DNA melting curve of the amplicon.

  2. FilmArray, an automated nested multiplex PCR system for multi-pathogen detection: development and application to respiratory tract infection.

    Science.gov (United States)

    Poritz, Mark A; Blaschke, Anne J; Byington, Carrie L; Meyers, Lindsay; Nilsson, Kody; Jones, David E; Thatcher, Stephanie A; Robbins, Thomas; Lingenfelter, Beth; Amiott, Elizabeth; Herbener, Amy; Daly, Judy; Dobrowolski, Steven F; Teng, David H-F; Ririe, Kirk M

    2011-01-01

    The ideal clinical diagnostic system should deliver rapid, sensitive, specific and reproducible results while minimizing the requirements for specialized laboratory facilities and skilled technicians. We describe an integrated diagnostic platform, the "FilmArray", which fully automates the detection and identification of multiple organisms from a single sample in about one hour. An unprocessed biologic/clinical sample is subjected to nucleic acid purification, reverse transcription, a high-order nested multiplex polymerase chain reaction and amplicon melt curve analysis. Biochemical reactions are enclosed in a disposable pouch, minimizing the PCR contamination risk. FilmArray has the potential to detect greater than 100 different nucleic acid targets at one time. These features make the system well-suited for molecular detection of infectious agents. Validation of the FilmArray technology was achieved through development of a panel of assays capable of identifying 21 common viral and bacterial respiratory pathogens. Initial testing of the system using both cultured organisms and clinical nasal aspirates obtained from children demonstrated an analytical and clinical sensitivity and specificity comparable to existing diagnostic platforms. We demonstrate that automated identification of pathogens from their corresponding target amplicon(s) can be accomplished by analysis of the DNA melting curve of the amplicon.

  3. Time-multiplexed open-path TDLAS spectrometer for dynamic, sampling-free, interstitial H2 18O and H2 16O vapor detection in ice clouds

    Science.gov (United States)

    Kühnreich, B.; Wagner, S.; Habig, J. C.; Möhler, O.; Saathoff, H.; Ebert, V.

    2015-04-01

    An advanced in situ diode laser hygrometer for simultaneous, sampling-free detection of interstitial H2 16O and H2 18O vapor was developed and tested in the aerosol interaction and dynamics in atmosphere (AIDA) cloud chamber during dynamic cloud formation processes. The spectrometer to measure isotope-resolved water vapor concentrations comprises two rapidly time-multiplexed DFB lasers near 1.4 and 2.7 µm and an open-path White cell with 227-m absorption path length and 4-m mirror separation. A dynamic water concentration range from 2.6 ppb to 87 ppm for H2 16O and 87 ppt to 3.6 ppm for H2 18O could be achieved and was used to enable a fast and direct detection of dynamic isotope ratio changes during ice cloud formation in the AIDA chamber at temperatures between 190 and 230 K. Relative changes in the H2 18O/H2 16O isotope ratio of 1 % could be detected and resolved with a signal-to-noise ratio of 7. This converts to an isotope ratio resolution limit of 0.15 % at 1-s time resolution.

  4. A multiplex RT-PCR for simultaneous detection and identification of five viruses and two viroids infecting chrysanthemum.

    Science.gov (United States)

    Zhao, Xiting; Liu, Xingliang; Ge, Beibei; Li, Mingjun; Hong, Bo

    2015-05-01

    Pathogens causing significant economic losses in chrysanthemum include tomato aspermy virus (TAV), chrysanthemum virus B (CVB), cucumber mosaic virus (CMV), tobacco mosaic virus (TMV), potato virus Y (PVY), chrysanthemum stunt viroid (CSVd) and chrysanthemum chlorotic mottle viroid (CChMVd). A multiplex reverse transcription polymerase chain reaction (RT-PCR) method, using specific primer sets for each virus or viroid, was developed for simultaneous detection and differentiation of TAV, CVB, CMV, TMV, PVY, CChMVd, and CSVd. The RT-PCR method was validated by testing chrysanthemum samples collected from different regions of China. In this study, CVB, TAV, TMV, PVY, CSVd, CMV, and CChMVd were detected, respectively, in 24.7 %, 17.5 %, 4.4 %, 4.4 %, 2.9 %, 2.5 %, and 1.5 % of the samples tested. These results indicate that CVB and TAV (24.7 % and 17.5 %) are common, whereas CMV, TMV, CChMVd, CSVd, and PVY (all below 5 %) are less frequently encountered. This new multiplex RT-PCR method has potential to be used routinely in large-scale virus and viroid surveys.

  5. Rapid Detection, Characterization, and Enumeration of Foodborne Pathogens

    DEFF Research Database (Denmark)

    This book provides a new focus on the rapid detection of food–borne pathogens, employing food production chains as a starting point instead of a specific method or various detection technologies. This reference is organized by production chains or contamination scenarios, and offers a unique...... in implementation. It also provides guidelines for faster, more user–friendly, and cost–effective enumeration of pathogens....

  6. 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

  7. A rapid ultrasound particle agglutination method for HIV antibody detection: Comparison with conventional rapid HIV tests.

    Science.gov (United States)

    Bystryak, Simon; Ossina, Natalya

    2017-11-01

    We present the results of the feasibility and preliminary studies on analytical performance of a rapid test for detection of human immunodeficiency virus (HIV) antibodies in human serum or plasma that is an important advance in detecting HIV infection. Current methods for rapid testing of antibodies against HIV are qualitative and exhibit poor sensitivity (limit of detection). In this paper, we describe an ultrasound particle agglutination (UPA) method that leads to a significant increase of the sensitivity of conventional latex agglutination tests for HIV antibody detection in human serum or plasma. The UPA method is based on the use of: 1) a dual mode ultrasound, wherein a first single-frequency mode is used to accelerate the latex agglutination process, and then a second swept-frequency mode of sonication is used to disintegrate non-specifically bound aggregates; and 2) a numerical assessment of results of the agglutination process. The numerical assessment is carried out by optical detection and analysis of moving patterns in the resonator cell during the swept-frequency mode. The single-step UPA method is rapid and more sensitive than the three commercial rapid HIV test kits analyzed in the study: analytical sensitivity of the new UPA method was found to be 510-, 115-, and 80-fold higher than that for Capillus™, Multispot™ and Uni-Gold™ Recombigen HIV antibody rapid test kits, respectively. The newly developed UPA method opens up additional possibilities for detection of a number of clinically significant markers in point-of-care settings. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Trichomonas spp. in pigeons: detection by OSOM Trichomonas Rapid Test.

    Science.gov (United States)

    Valek, Petr; Kunca, Tomas; Langrova, Iva; Hartlova, Helena; Brozova, Adela; Jankovska, Ivana; Kudrnacova, Marie; Sloup, Vladislav

    2013-12-01

    The efficacy of the OSOM Trichomonas Rapid Test (developed for rapid diagnosis of human Trichomonas vaginalis) in detection of Trichomonas spp. in pigeons (Columba livia) was investigated. Two oral cavity swabs were taken from 50 farm pigeons. Cultivation in Diamond Trichomonas medium was used as a reference method. According to a morphological determination, Trichomonas gallinae was the only protozoan found; however, no further molecular analysis was conducted. The OSOM Trichomonas test was positive in 39 oral swabs. In comparison with the cultivation method three samples were identified as false negative and one as false positive. Test specificity and sensitivity were established as 93% and 90%, respectively. Using Cohen's Kappa, the concordance between the two testing methods was found to be strong (kappa = 0.7506, 95% CI = 0.5162-0.9850). The OSOM Trichomonas test is not able to distinguish between Trichomonas species; however, results suggest that the test is suitable for the rapid detection of Trichomonas spp. infection in pigeons.

  9. Palm kernel agar: An alternative culture medium for rapid detection ...

    African Journals Online (AJOL)

    Palm kernel agar: An alternative culture medium for rapid detection of aflatoxins in agricultural commodities. ... a pink background and blue or blue green fluorescence of palm kernel agar Under long wave UV light (366nm) as against the white background of DCA, which often interferes with fluorescence with corresponding ...

  10. Evaluation of a direct colorimetric assay for rapid detection of ...

    African Journals Online (AJOL)

    Yemane Berhane

    bromide (MTT) for a rapid detection of rifampicin resistance. Methods: Sputum was inoculated directly into 7H9 .... a loopful of the corresponding broth on nutrient agar and incubating it at 370C for 24 hours before performing the .... and Training in Tropical Diseases (TDR). This study was part of the MSc thesis of DW at Addis ...

  11. How well does physician selection of microbiologic tests identify Clostridium difficile and other pathogens in paediatric diarrhoea? Insights using multiplex PCR-based detection.

    Science.gov (United States)

    Stockmann, C; Rogatcheva, M; Harrel, B; Vaughn, M; Crisp, R; Poritz, M; Thatcher, S; Korgenski, E K; Barney, T; Daly, J; Pavia, A T

    2015-02-01

    The objective of this study was to compare the aetiologic yield of standard-of-care microbiologic testing ordered by physicians with that of a multiplex PCR platform. Stool specimens obtained from children and young adults with gastrointestinal illness were evaluated by standard laboratory methods and a developmental version of the FilmArray Gastrointestinal (GI) Diagnostic System (FilmArray GI Panel), a rapid multiplex PCR platform that detects 23 bacterial, viral and protozoal agents. Results were classified according to the microbiologic tests requested by the treating physician. A median of three (range 1-10) microbiologic tests were performed by the clinical laboratory during 378 unique diarrhoeal episodes. A potential aetiologic agent was identified in 46% of stool specimens by standard laboratory methods and in 65% of specimens tested using the FilmArray GI Panel (p FilmArray GI Panel. Notably, 11 (12%) cases of norovirus were identified among children who only had testing for Clostridium difficile ordered. Among those who had C. difficile testing ordered in combination with other tests, an additional pathogen was identified in 57% of stool specimens with the FilmArray GI Panel. For patients who had no C. difficile testing performed, the FilmArray GI Panel identified a pathogen in 63% of cases, including C. difficile in 8%. Physician-specified laboratory testing may miss important diarrhoeal pathogens. Additionally, standard laboratory testing is likely to underestimate co-infections with multiple infectious diarrhoeagenic agents. Copyright © 2014 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

  12. Poly(m-phenylenediamine nanospheres and nanorods: selective synthesis and their application for multiplex nucleic acid detection.

    Directory of Open Access Journals (Sweden)

    Yingwei Zhang

    Full Text Available In this paper, we demonstrate for the first time that poly(m-phenylenediamine (PMPD nanospheres and nanorods can be selectively synthesized via chemical oxidation polymerization of m-phenylenediamine (MPD monomers using ammonium persulfate (APS as an oxidant at room temperature. It suggests that the pH value plays a critical role in controlling the the morphology of the nanostructures and fast polymerization rate favors the anisotropic growth of PMPD under homogeneous nucleation condition. We further demonstrate that such PMPD nanostructures can be used as an effective fluorescent sensing platform for multiplex nucleic acid detection. A detection limit as low as 50 pM and a high selectivity down to single-base mismatch could be achieved. The fluorescence quenching is attributed to photoinduced electron transfer from nitrogen atom in PMPD to excited fluorophore. Most importantly, the successful use of this sensing platform in human blood serum system is also demonstrated.

  13. Comparison of the fast track diagnostics respiratory 21 and Seegene Allplex multiplex polymerase chain reaction assays for the detection of respiratory viruses.

    Science.gov (United States)

    Barratt, Kevin; Anderson, Trevor P; Fahey, Jennifer A; Jennings, Lance C; Werno, Anja M; Murdoch, David R

    2017-04-01

    Real-time multiplex PCR assays are increasingly used for respiratory virus detection, and offer automated analysis in a closed tube system, but they have the disadvantage of low-throughput due to multiplexing limitations. In this study, the established fast-track respiratory 21 assay (FTD) (fast-track diagnostics, Junglinster Luxembourg) was compared to the new Seegene Allplex assay (Seegene) (Seegene Inc. Seoul, Korea) which offers greater multiplexing as multiple targets can be detected in each fluorescence channel. The Seegene Allplex assay is quicker to perform than previous Seegene respiratory multiplex assays. The assays were evaluated using 199 mostly upper respiratory tract samples. A respiratory pathogen was found in 127/199 (63.8%) of samples by the FTD assay and 123/199 (61.8%) using the Seegene assay. Kappa agreement was between 0.87 and 1 for all targets except human bocavirus and adenovirus. Although the performance of the assays were similar, the Seegene assay had the advantage of simultaneous detection of two gene targets for each of the common Influenza A subtypes, improved throughput of 30 samples per run and automated result analysis. The FTD assay could only test 17 samples per run but validation for use on several different real-time thermal cyclers made it easier to integrate into an existing laboratory system. Both assays were cost effective compared to in-house multiplex PCR respiratory virus screening.

  14. Multiplex polymerase chain reaction for the detection of high-risk-human papillomavirus types in formalin-fixed paraffin-embedded cervical tissues

    Directory of Open Access Journals (Sweden)

    Mini P Singh

    2017-01-01

    Full Text Available Detecting high-risk-human papillomavirus (HPV types has become an integral part of the cervical cancer screening programmes. This study aimed to develop a multiplex polymerase chain reaction (PCR for identification of HPV types 16 and 18 along with the beta globin gene in formalin-fixed and paraffin-embedded cervical biopsy specimens. A total of 59 samples from patients with cervical abnormalities were tested. HPV 16 positivity was 50% in cervical cancers and 52.9% in cervical intraepithelial neoplasia. Our multiplex PCR protocol can be used as a simple and cost-effective tool for high-risk-HPV detection in cervical cancer screening programmes.

  15. Multiplex polymerase chain reaction for the detection of high-risk-human papillomavirus types in formalin-fixed paraffin-embedded cervical tissues.

    Science.gov (United States)

    Singh, Mini P; Gupta, Nalini; Deepak, T; Kumar, Archit; Ratho, Radha Kanta

    2017-01-01

    Detecting high-risk-human papillomavirus (HPV) types has become an integral part of the cervical cancer screening programmes. This study aimed to develop a multiplex polymerase chain reaction (PCR) for identification of HPV types 16 and 18 along with the beta globin gene in formalin-fixed and paraffin-embedded cervical biopsy specimens. A total of 59 samples from patients with cervical abnormalities were tested. HPV 16 positivity was 50% in cervical cancers and 52.9% in cervical intraepithelial neoplasia. Our multiplex PCR protocol can be used as a simple and cost-effective tool for high-risk-HPV detection in cervical cancer screening programmes.

  16. Evaluation of a rapid single multiplex microsatellite-based assay for use in forensic genetic investigations in dogs.

    Science.gov (United States)

    Clark, Leigh Anne; Famula, Thomas R; Murphy, Keith E

    2004-10-01

    To develop a set of microsatellite markers, composed of a minimal number of these markers, suitable for use in forensic genetic investigations in dogs. Blood, tissue, or buccal epithelial cells from 364 dogs of 85 breeds and mixed breeds and 19 animals from related species in the family Canidae. 61 tetranucleotide microsatellite markers were characterized on the basis of number and size of alleles, ease of genotyping, chromosomal location, and ability to be coamplified. The range in allele size, number of alleles, total heterozygosity, and fixation index for each marker were determined by use of genotype data from 383 dogs and related species. Polymorphism information content was calculated for several breeds of dogs. 7 microsatellite markers could be coamplified. These markers were labeled with fluorescent dyes, multiplexed into a single reaction, and optimized for resolution in a commercial genetic analyzer. The multiplex set was used to identify sires for 2 mixed litters. The test was not species specific; genotype information collected for wolves, coyotes, jackals, New Guinea singing dogs, and an African wild dog could not distinguish between these species. This set of 7 microsatellite markers is useful in forensic applications (ie, identification of dogs and determination of parentage) in closely related animals and is applicable to a wide range of species belonging to the family Canidae.

  17. Development of a rapid multiplex SSR genotyping method to study populations of the fungal plant pathogen Zymoseptoria tritici.

    Science.gov (United States)

    Gautier, Angélique; Marcel, Thierry C; Confais, Johann; Crane, Charles; Kema, Gert; Suffert, Frédéric; Walker, Anne-Sophie

    2014-06-18

    Zymoseptoria tritici is a hemibiotrophic ascomycete fungus causing leaf blotch of wheat that often decreases yield severely. Populations of the fungus are known to be highly diverse and poorly differentiated from each other. However, a genotyping tool is needed to address further questions in large collections of isolates, regarding regional population structure, adaptation to anthropogenic selective pressures, and dynamics of the recently discovered accessory chromosomes. This procedure is limited by costly and time-consuming simplex PCR genotyping. Recent development of genomic approaches and of larger sets of SSRs enabled the optimization of microsatellite multiplexing. We report here a reliable protocol to amplify 24 SSRs organized in three multiplex panels, and covering all Z. tritici chromosomes. We also propose an automatic allele assignment procedure, which allows scoring alleles in a repeatable manner across studies and laboratories. All together, these tools enabled us to characterize local and worldwide populations and to calculate diversity indexes consistent with results reported in the literature. This easy-to-use, accurate, repeatable, economical, and faster technical strategy can provide useful genetic information for evolutionary inferences concerning Z. tritici populations. Moreover, it will facilitate the comparison of studies from different scientific groups.

  18. Multiplex PCR for the detection of Mycoplasma fermentans, M. hominis and M. penetrans in cell cultures and blood samples of patients with chronic fatigue syndrome.

    Science.gov (United States)

    Choppa, P C; Vojdani, A; Tagle, C; Andrin, R; Magtoto, L

    1998-10-01

    A multiplex polymerase chain reaction (PCR) was initially developed to detect the presence of mycoplasma genus DNA sequences in cell cultures and to differentiate between three human pathogenic mycoplasma species simultaneously. The assay in turn, proved to be a useful tool for the detection of mycoplasma infection in human DNA samples. One set of oligonucleotide primers which are specific for a highly conserved region among all members of the genus mycoplasma along with three other primer sets which are specific for Mycoplasma fermentans, Mycoplasma hominis andMycoplasma penetrans species were used in this assay. The sensitivity of detection was determined by infecting peripheral blood mononuclear cells (PBMC) of healthy individuals with known bacterial copy numbers from each species, extracting the DNA, and subjecting 1 microgram of DNA from each sample to 40 cycles of amplification. By using agarose gel electrophoresis the detection level was determined to be 7, 7, 9 and 15 mycoplasma cells per microgram of human genomic DNA for M. genus,M. fermentans, M. hominis and M. penetrans, respectively. The assay was applied to DNA extracted from the PBMCs of individuals suffering from chronic fatigue syndrome (CFS) (n=100) as determined by the Center for Disease Control (CDC) criteria, and compared to healthy individuals (n=100). The percentage of M. genus infection was found to be 52% in CFS patients and only 15% in healthy individuals. Mycoplasma fermentans, M. hominis andM. penetrans were detected in 32, 9 and 6% of the CFS patients while they were detected in 8, 3 and 2% of the healthy control subjects, respectively. This assay provides a rapid and cost efficient procedure to screen either cell cultures or clinical samples for the presence of three potentially pathogenic species of mycoplasma with a high level of sensitivity and specificity. Copyright 1998 Academic Press.

  19. Radiometric method for the rapid detection of Leptospira organisms

    Energy Technology Data Exchange (ETDEWEB)

    Manca, N.; Verardi, R.; Colombrita, D.; Ravizzola, G.; Savoldi, E.; Turano, A.

    1986-02-01

    A rapid and sensitive radiometric method for detection of Leptospira interrogans serovar pomona and Leptospira interrogans serovar copenhageni is described. Stuart's medium and Middlebrook TB (12A) medium supplemented with bovine serum albumin, catalase, and casein hydrolysate and labeled with /sup 14/C-fatty acids were used. The radioactivity was measured in a BACTEC 460. With this system, Leptospira organisms were detected in human blood in 2 to 5 days, a notably shorter time period than that required for the majority of detection techniques.

  20. Detection of Escherichia coli Enteropathogens by Multiplex Polymerase Chain Reaction from Children's Diarrheal Stools in Two Caribbean–Colombian Cities

    Science.gov (United States)

    Arzuza, Octavio; Urbina, Delfina; Bai, Jing; Guerra, Julio; Montes, Oscar; Puello, Marta; Mendoza, Ketty; Castro, Gregorio Y.

    2010-01-01

    Abstract Acute diarrheal disease is a leading cause of childhood morbidity and mortality in the developing world and Escherichia coli intestinal pathogens are important causative agents. Information on the epidemiology of E. coli intestinal pathogens and their association with diarrheal disease is limited because no diagnostic testing is available in countries with limited resources. To evaluate the prevalence of E. coli intestinal pathogens in a Caribbean–Colombian region, E. coli clinical isolates from children with diarrhea were analyzed by a recently reported two-reaction multiplex polymerase chain reaction (Gomez-Duarte et al., Diagn Microbiol Infect Dis 2009;63:1–9). The phylogenetic group from all E. coli isolates was also typed by a single-reaction multiplex polymerase chain reaction. We found that among 139 E. coli strains analyzed, 20 (14.4%) corresponded to E. coli diarrheagenic pathotypes. Enterotoxigenic, shiga-toxin–producing, enteroaggregative, diffuse adherent, and enteropathogenic E. coli pathotypes were detected, and most of them belonged to the phylogenetic groups A and B1, known to be associated with intestinal pathogens. This is the first report on the molecular characterization of E. coli diarrheogenic isolates in Colombia and the first report on the potential role of E. coli in childhood diarrhea in this geographic area. PMID:19839760

  1. Detection of Escherichia coli enteropathogens by multiplex polymerase chain reaction from children's diarrheal stools in two Caribbean-Colombian cities.

    Science.gov (United States)

    Gómez-Duarte, Oscar G; Arzuza, Octavio; Urbina, Delfina; Bai, Jing; Guerra, Julio; Montes, Oscar; Puello, Marta; Mendoza, Ketty; Castro, Gregorio Y

    2010-02-01

    Acute diarrheal disease is a leading cause of childhood morbidity and mortality in the developing world and Escherichia coli intestinal pathogens are important causative agents. Information on the epidemiology of E. coli intestinal pathogens and their association with diarrheal disease is limited because no diagnostic testing is available in countries with limited resources. To evaluate the prevalence of E. coli intestinal pathogens in a Caribbean-Colombian region, E. coli clinical isolates from children with diarrhea were analyzed by a recently reported two-reaction multiplex polymerase chain reaction (Gomez-Duarte et al., Diagn Microbiol Infect Dis 2009;63:1-9). The phylogenetic group from all E. coli isolates was also typed by a single-reaction multiplex polymerase chain reaction. We found that among 139 E. coli strains analyzed, 20 (14.4%) corresponded to E. coli diarrheagenic pathotypes. Enterotoxigenic, shiga-toxin-producing, enteroaggregative, diffuse adherent, and enteropathogenic E. coli pathotypes were detected, and most of them belonged to the phylogenetic groups A and B1, known to be associated with intestinal pathogens. This is the first report on the molecular characterization of E. coli diarrheogenic isolates in Colombia and the first report on the potential role of E. coli in childhood diarrhea in this geographic area.

  2. Simultaneous detection of cow and buffalo species in milk from China, India, and Pakistan using multiplex real-time PCR.

    Science.gov (United States)

    Cottenet, G; Blancpain, C; Golay, P-A

    2011-08-01

    Asian countries are major producers of cow and buffalo milk. For quality and authenticity purposes, a multiplex real-time PCR assay was developed to specifically and simultaneously detect DNA from these 2 bovine species. Targeting the cytochrome b gene of mitochondrial DNA, common PCR primers amplified a 105-bp fragment, and 2 fluorescent probes specific to either cow or buffalo were designed for their identification. Specificity was successfully tested on 6 other species, including sheep and goat, and sensitivity reached 1% of cow DNA in buffalo DNA and vice versa. As an evaluation, the method was tested using 119 freeze-dried Asian milk samples from regional industrial milk facilities. Although these samples did not cover the entire Asian zone, the multiplex assay indicated that approximately 20% of the samples (mainly from India) showed high levels of cross-contamination of cow milk by buffalo milk, and vice versa. Fast, sensitive, and straightforward, this method is fit-for-purpose for the authenticity control of Asian milk. Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  3. Detection of selected antibiotic resistance genes using multiplex PCR assay in mastitis pathogens in the Czech Republic

    Directory of Open Access Journals (Sweden)

    Vladimir Pyatov

    2017-01-01

    Full Text Available The aim of this research was to develop multiplex polymerase chain reaction assays for the detection of aminoglycoside (strA, strB, sulphonamide (sulI, sulII, tetracycline (tetA, tetB, tetK, tetM, tetO, macrolide and lincosamide (msrA, ermA, ermB, ermC, mefA/E genes of resistance in mastitis pathogens (Escherichia coli, Staphylococcus aureus, Streptococcus uberis, Streptococcus agalactiae and Streptococcus dysgalactiae. Applying the established assays, we investigated the distribution of antibiotic resistance genes in the above mentioned species isolated from milk samples in the Czech Republic. Each assay consisted of seven pairs of primers. Six of them amplified fragments of antibiotic resistance genes and one pair a fragment of a species specific gene. Polymerase chain reaction conditions were optimized to amplify seven gene fragments simultaneously in one reaction. In total, 249 isolates were used, among which 111 were positive for E. coli, 52 for S. aureus and 86 for Streptococcus spp. The majority (60.2% of bacteria carried at least one antibiotic resistance gene and 44.6% were multidrug-resistant. The designed multiplex polymerase chain reaction assays may be applied as diagnostic method to replace or complement standard techniques of antibiotic susceptibility testing in the mentioned pathogens.

  4. An FPGA-based rapid wheezing detection system.

    Science.gov (United States)

    Lin, Bor-Shing; Yen, Tian-Shiue

    2014-01-29

    Wheezing is often treated as a crucial indicator in the diagnosis of obstructive pulmonary diseases. A rapid wheezing detection system may help physicians to monitor patients over the long-term. In this study, a portable wheezing detection system based on a field-programmable gate array (FPGA) is proposed. This system accelerates wheezing detection, and can be used as either a single-process system, or as an integrated part of another biomedical signal detection system. The system segments sound signals into 2-second units. A short-time Fourier transform was used to determine the relationship between the time and frequency components of wheezing sound data. A spectrogram was processed using 2D bilateral filtering, edge detection, multithreshold image segmentation, morphological image processing, and image labeling, to extract wheezing features according to computerized respiratory sound analysis (CORSA) standards. These features were then used to train the support vector machine (SVM) and build the classification models. The trained model was used to analyze sound data to detect wheezing. The system runs on a Xilinx Virtex-6 FPGA ML605 platform. The experimental results revealed that the system offered excellent wheezing recognition performance (0.912). The detection process can be used with a clock frequency of 51.97 MHz, and is able to perform rapid wheezing classification.

  5. Assessment of a novel multiplex real-time PCR assay for the detection of the CBPP agent Mycoplasma mycoides subsp. mycoides SC through experimental infection in cattle

    Directory of Open Access Journals (Sweden)

    Tomaso Herbert

    2011-08-01

    Full Text Available Abstract Background Mycoplasma mycoides subsp. mycoides SC is the pathogenic agent of contagious bovine pleuropneumonia (CBPP, the most important disease of cattle in Africa causing significant economic losses. The re-emergence of CBPP in Europe in the 1980s and 1990s illustrates that it is still a threat also to countries that have successfully eradicated the disease in the past. Nowadays, probe-based real-time PCR techniques are among the most advanced tools for a reliable identification and a sensitive detection of many pathogens, but only few protocols have been published so far for CBPP diagnosis. Therefore we developed a novel TaqMan®-based real-time PCR assay comprising the amplification of two independent targets (MSC_0136 and MSC_1046 and an internal exogenous amplification control in a multiplex reaction and evaluated its diagnostic performance with clinical samples. Results The assays detected 49 MmmSC strains from diverse temporal and geographical origin, but did not amplify DNA from 82 isolates of 20 non-target species confirming a specificity of 100%. The detection limit was determined to be 10 fg DNA per reaction for the MSC_0136 assay and 100 fg per reaction for the MSC_1046 assay corresponding to 8 and 80 genome equivalents, respectively. The diagnostic performance of the assay was evaluated with clinical samples from 19 experimentally infected cattle and from 20 cattle without CBPP and compared to those of cultivation and a conventional PCR protocol. The two rt-PCR tests proved to be the most sensitive methods and identified all 19 infected animals. The different sample types used were not equally suitable for MmmSC detection. While 94.7% of lung samples from the infected cohort were positively tested in the MSC_0136 assay, only 81% of pulmonal lymph nodes, 31% of mediastinal lymph nodes and 25% of pleural fluid samples gave a positive result. Conclusions The developed multiplex rt-PCR assay is recommended as an efficient tool

  6. Rapid detection and identification of human hookworm infections through high resolution melting (HRM analysis.

    Directory of Open Access Journals (Sweden)

    Romano Ngui

    Full Text Available BACKGROUND: Hookworm infections are still endemic in low and middle income tropical countries with greater impact on the socioeconomic and public health of the bottom billion of the world's poorest people. In this study, a real-time polymerase chain reaction (PCR coupled with high resolution melting-curve (HRM analysis was evaluated for an accurate, rapid and sensitive tool for species identification focusing on the five human hookworm species. METHODS: Real-time PCR coupled with HRM analysis targeting the second internal transcribed spacer (ITS-2 of nuclear ribosomal DNA as the genetic marker was used to identify and distinguish hookworm species in human samples. Unique and distinct characteristics of HRM patterns were produced for each of the five hookworm species. The melting curves were characterized by peaks of 79.24±0.05°C and 83.00±0.04°C for Necator americanus, 79.12±0.10°C for Ancylostoma duodenale, 79.40±0.10°C for Ancylostoma ceylanicum, 79.63±0.05°C for Ancylostoma caninum and 79.70±0.14°C for Ancylostoma braziliense. An evaluation of the method's sensitivity and specificity revealed that this assay was able to detect as low as 0.01 ng/µl hookworm DNA and amplification was only recorded for hookworm positive samples. CONCLUSION: The HRM assay developed in this study is a rapid and straightforward method for the diagnosis, identification and discrimination of five human hookworms. This assay is simple compared to other probe-based genotyping methods as it does not require multiplexing, DNA sequencing or post-PCR processing. Therefore, this method offers a new alternative for rapid detection of human hookworm species.

  7. Rapid detection and identification of human hookworm infections through high resolution melting (HRM) analysis.

    Science.gov (United States)

    Ngui, Romano; Lim, Yvonne A L; Chua, Kek Heng

    2012-01-01

    Hookworm infections are still endemic in low and middle income tropical countries with greater impact on the socioeconomic and public health of the bottom billion of the world's poorest people. In this study, a real-time polymerase chain reaction (PCR) coupled with high resolution melting-curve (HRM) analysis was evaluated for an accurate, rapid and sensitive tool for species identification focusing on the five human hookworm species. Real-time PCR coupled with HRM analysis targeting the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA as the genetic marker was used to identify and distinguish hookworm species in human samples. Unique and distinct characteristics of HRM patterns were produced for each of the five hookworm species. The melting curves were characterized by peaks of 79.24±0.05°C and 83.00±0.04°C for Necator americanus, 79.12±0.10°C for Ancylostoma duodenale, 79.40±0.10°C for Ancylostoma ceylanicum, 79.63±0.05°C for Ancylostoma caninum and 79.70±0.14°C for Ancylostoma braziliense. An evaluation of the method's sensitivity and specificity revealed that this assay was able to detect as low as 0.01 ng/µl hookworm DNA and amplification was only recorded for hookworm positive samples. The HRM assay developed in this study is a rapid and straightforward method for the diagnosis, identification and discrimination of five human hookworms. This assay is simple compared to other probe-based genotyping methods as it does not require multiplexing, DNA sequencing or post-PCR processing. Therefore, this method offers a new alternative for rapid detection of human hookworm species.

  8. Evaluation of multiplex tandem real-time PCR for detection of Cryptosporidium spp., Dientamoeba fragilis, Entamoeba histolytica, and Giardia intestinalis in clinical stool samples.

    Science.gov (United States)

    Stark, D; Al-Qassab, S E; Barratt, J L N; Stanley, K; Roberts, T; Marriott, D; Harkness, J; Ellis, J T

    2011-01-01

    The aim of this study was to describe the first development and evaluation of a multiplex tandem PCR (MT-PCR) assay for the detection and identification of 4 common pathogenic protozoan parasites, Cryptosporidium spp., Dientamoeba fragilis, Entamoeba histolytica, and Giardia intestinalis, from human clinical samples. A total of 472 fecal samples submitted to the Department of Microbiology at St. Vincent's Hospital were included in the study. The MT-PCR assay was compared to four real-time PCR (RT-PCR) assays and microscopy by a traditional modified iron hematoxylin stain. The MT-PCR detected 28 G. intestinalis, 26 D. fragilis, 11 E. histolytica, and 9 Cryptosporidium sp. isolates. Detection and identification of the fecal protozoa by MT-PCR demonstrated 100% correlation with the RT-PCR results, and compared to RT-PCR, MT-PCR exhibited 100% sensitivity and specificity, while traditional microscopy of stained fixed fecal smears exhibited sensitivities and specificities of 56% and 100% for Cryptosporidium spp., 38% and 99% for D. fragilis, 47% and 97% for E. histolytica, and 50% and 100% for G. intestinalis. No cross-reactivity was detected in 100 stool samples containing various other bacterial, viral, and protozoan species. The MT-PCR assay was able to provide rapid, sensitive, and specific simultaneous detection and identification of the four most important diarrhea-causing protozoan parasites that infect humans. This study also highlights the lack of sensitivity demonstrated by microscopy, and thus, molecular methods such as MT-PCR must be considered the diagnostic methods of choice for enteric protozoan parasites.

  9. Evaluation of Multiplex Tandem Real-Time PCR for Detection of Cryptosporidium spp., Dientamoeba fragilis, Entamoeba histolytica, and Giardia intestinalis in Clinical Stool Samples ▿

    Science.gov (United States)

    Stark, D.; Al-Qassab, S. E.; Barratt, J. L. N.; Stanley, K.; Roberts, T.; Marriott, D.; Harkness, J.; Ellis, J. T.

    2011-01-01

    The aim of this study was to describe the first development and evaluation of a multiplex tandem PCR (MT-PCR) assay for the detection and identification of 4 common pathogenic protozoan parasites, Cryptosporidium spp., Dientamoeba fragilis, Entamoeba histolytica, and Giardia intestinalis, from human clinical samples. A total of 472 fecal samples submitted to the Department of Microbiology at St. Vincent's Hospital were included in the study. The MT-PCR assay was compared to four real-time PCR (RT-PCR) assays and microscopy by a traditional modified iron hematoxylin stain. The MT-PCR detected 28 G. intestinalis, 26 D. fragilis, 11 E. histolytica, and 9 Cryptosporidium sp. isolates. Detection and identification of the fecal protozoa by MT-PCR demonstrated 100% correlation with the RT-PCR results, and compared to RT-PCR, MT-PCR exhibited 100% sensitivity and specificity, while traditional microscopy of stained fixed fecal smears exhibited sensitivities and specificities of 56% and 100% for Cryptosporidium spp., 38% and 99% for D. fragilis, 47% and 97% for E. histolytica, and 50% and 100% for G. intestinalis. No cross-reactivity was detected in 100 stool samples containing various other bacterial, viral, and protozoan species. The MT-PCR assay was able to provide rapid, sensitive, and specific simultaneous detection and identification of the four most important diarrhea-causing protozoan parasites that infect humans. This study also highlights the lack of sensitivity demonstrated by microscopy, and thus, molecular methods such as MT-PCR must be considered the diagnostic methods of choice for enteric protozoan parasites. PMID:21048004

  10. Rapid Generation of Multiplexed Cell Cocultures Using Acoustic Droplet Ejection Followed by Aqueous Two-Phase Exclusion Patterning

    Science.gov (United States)

    Fang, Yu; Frampton, John P.; Raghavan, Shreya; Sabahi-Kaviani, Rahman; Luker, Gary

    2012-01-01

    The development of tools for patterning cocultures of cells is a fundamental interest among cell biologists and tissue engineers. Although a variety of systems exist for micropatterning cells, the methods used to generate cell micropatterns are often cumbersome and difficult to adapt for tissue engineering purposes. This study combines acoustic droplet ejection and aqueous two-phase system exclusion patterning to introduce a method for patterning cocultures of cells in multiplexed arrays. This new method uses focused acoustic radiation pressure to eject discrete droplets of uniform size from the surface of a dextran solution containing cells. The size of droplets is controlled by adjusting ultrasound parameters, such as pulse, duration, and amplitude. The ejected dextran droplets are captured on a cell culture substrate that is manipulated by a computer-controlled 3D positioning system according to predesigned patterns. Polyethylene glycol solution containing an additional cell type is then added to the culture dish to produce a two-phase system capable of depositing different types of cells around the initial pattern of cells. We demonstrate that our method can produce patterns of islands or lines with two or more cell types. Further, we demonstrate that patterns can be multiplexed for studies involving combinations of multiple cell types. This method offers a tool to transfer cell-containing samples in a contact-free, nozzle-less manner, avoiding sample cross-contamination. It can be used to pattern cell cocultures without complicated fabrication of culture substrates. These capabilities were used to examine the response of cancer cells to the presence of a ligand (CXCL12) secreted from surrounding cocultured cells. PMID:22356298

  11. Detection of pathogenic strains by multiplex PCR and antimicrobial sensitivity of Escherichia coli isolated from piglets

    OpenAIRE

    Macêdo, N.R.; Menezes,C.P.L.; Lage, A.P.; Ristow,L.E.; Reis, A.; Guedes,R.M.C.

    2007-01-01

    Avaliou-se a freqüência dos genes de fímbrias (K88, K99, 987P, F18 e F41) e toxinas (LT, Stb, StaP e Stx2e) de cepas de E. coli isoladas de leitões com diarréia usando a técnica de PCR multiplex com primers específicos para esses genes, e estudou-se o padrão de sensibilidade das cepas patogênicas pelo método de difusão em disco ao florfenicol, ceftiofur sódico, colistina, fosfomicina, neomicina, norfloxacina, sulfa + trimetoprim, doxiciclina, tetraciclina e lincomicina. Foram utilizadas 144 a...

  12. Multiplexed optical pathogen detection with lab-on-a-chip devices.

    Science.gov (United States)

    Schulze, Holger; Giraud, Gerard; Crain, Jason; Bachmann, Till T

    2009-04-01

    Infectious diseases are still a main cause of human morbidity and mortality. Advanced diagnostics is considered to be a key driver to improve the respective therapeutic outcome. The main factors influencing the impact of diagnostics include: assay speed, availability, information content, in-vitro diagnostics and cost, for which molecular assays are providing the most promising opportunities. Miniaturisation and integration of assay steps into lab-on-a-chip devices has been described as an appropriate way to speed up assay time and make assays available onsite at competitive costs. As meaningful assays for infectious diseases need to include a whole range of clinical relevant information about the pathogen, multiplexed functionality is often required for which optical transduction is particularly well suited. The aim of this review is to assess existing developments in this field and to give an outlook on future requirements and solutions. (c) 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Multiplex ligation-dependent probe amplification detects DCX gene deletions in band heterotopia.

    Science.gov (United States)

    Mei, D; Parrini, E; Pasqualetti, M; Tortorella, G; Franzoni, E; Giussani, U; Marini, C; Migliarini, S; Guerrini, R

    2007-02-06

    Subcortical band heterotopia (SBH, or double cortex syndrome) is a neuronal migration disorder consisting of heterotopic bands of gray matter located between the cortex and the ventricular surface, with or without concomitant pachygyria. Most cases show diffuse or anteriorly predominant (A>P) migration abnormality. All familial and 53% to 84% of sporadic cases with diffuse or A>P SBH harbor a mutation of the DCX gene, leaving the genetic causes unexplained, and genetic counseling problematic, in the remaining patients. Our purpose was to verify the extent to which exonic deletions or duplications of the DCX gene would account for sporadic SBH with A>P gradient but normal gene sequencing. We identified 23 patients (22 women, 1 man) with sporadic, diffuse, or anteriorly predominant SBH. After sequencing the DCX gene and finding mutations in 12 (11 women, 1 man), we used multiplex ligation-dependent probe amplification (MLPA) to search for whole-exon deletions or duplications in the 11 remaining women. We used semiquantitative fluorescent multiplex PCR (SQF-PCR) and Southern blot to confirm MLPA findings. MLPA assay uncovered two deletions encompassing exons 3 to 5, and one involving exon 6, in 3 of 11 women (27%) and raised the percentage of DCX mutations from 52% to 65% in our series. SQF-PCR performed in all three women and Southern blot analysis performed in two confirmed the deletions. MLPA uncovers large genomic deletions of the DCX gene in a subset of patients with SBH in whom no mutations are found after gene sequencing. Deletions of DCX are an underascertained cause of SBH.

  14. A multiplex protein-free lateral flow assay for detection of microRNAs based on unmodified molecular beacons.

    Science.gov (United States)

    Javani, Atefeh; Javadi-Zarnaghi, Fatemeh; Rasaee, Mohammad Javad

    2017-11-15

    Lateral flow assays (LFAs) have promising potentials for point-of-care applications. Recently, many LFAs have been reported that are based on hybridization of oligonucleotide strands. Mostly, biotinylated capture DNAs are immobilized on the surface of a nitrocellulose membrane via streptavidin interactions. During the assay, stable colorful complexes get formed that are visible by naked eyes. Here, we present an inexpensive and unique design of LFA that applies unmodified oligonucleotides at capture lines. The presented LFA do not utilize streptavidin or any other affinity protein. We employ structural switch of molecular beacons (MB) in combination with base stacking hybridization (BSH) phenomenon. The unique design of the reported LFA provided high selectivity for target oligonucleotides. We validated potential applications of the system for detection of DNA mimics of two microRNAs in multiplex assays. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. Rapid identification of ST131 Escherichia coli by a novel multiplex real-time allelic discrimination assay.

    Science.gov (United States)

    François, Patrice; Bonetti, Eve-Julie; Fankhauser, Carolina; Baud, Damien; Cherkaoui, Abdessalam; Schrenzel, Jacques; Harbarth, Stephan

    2017-09-01

    Escherichia coli sequence type 131 is increasingly described in severe hospital infections. We developed a rapid real-time allelic discrimination assay for the rapid identification of E. coli ST131 isolates. This rapid assay represents an affordable alternative to sequence-based strategies before completing characterization of potentially highly virulent isolates of E. coli. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Individual differences in detecting rapidly presented fearful faces.

    Directory of Open Access Journals (Sweden)

    Dandan Zhang

    Full Text Available Rapid detection of evolutionarily relevant threats (e.g., fearful faces is important for human survival. The ability to rapidly detect fearful faces exhibits high variability across individuals. The present study aimed to investigate the relationship between behavioral detection ability and brain activity, using both event-related potential (ERP and event-related oscillation (ERO measurements. Faces with fearful or neutral facial expressions were presented for 17 ms or 200 ms in a backward masking paradigm. Forty-two participants were required to discriminate facial expressions of the masked faces. The behavioral sensitivity index d' showed that the detection ability to rapidly presented and masked fearful faces varied across participants. The ANOVA analyses showed that the facial expression, hemisphere, and presentation duration affected the grand-mean ERP (N1, P1, and N170 and ERO (below 20 Hz and lasted from 100 ms to 250 ms post-stimulus, mainly in theta band brain activity. More importantly, the overall detection ability of 42 subjects was significantly correlated with the emotion effect (i.e., fearful vs. neutral on ERP (r = 0.403 and ERO (r = 0.552 measurements. A higher d' value was corresponding to a larger size of the emotional effect (i.e., fearful--neutral of N170 amplitude and a larger size of the emotional effect of the specific ERO spectral power at the right hemisphere. The present results suggested a close link between behavioral detection ability and the N170 amplitude as well as the ERO spectral power below 20 Hz in individuals. The emotional effect size between fearful and neutral faces in brain activity may reflect the level of conscious awareness of fearful faces.

  17. Rapid Visual Tests: Fast and Reliable Detection of Ochratoxin A

    Directory of Open Access Journals (Sweden)

    Miguel Lopez-Ferber

    2010-08-01

    Full Text Available This paper reviews the early detection strategies that have been employed for the rapid monitoring of ochratoxin A (OTA contamination of food. OTA, a mycotoxin mainly produced by some Aspergillus and Penicillium species, is found in cereals, coffee, wine, pork and grapes. To minimize the entry of this mycotoxin into the food chain, rapid diagnostic tools are required. To this end, the potential use of lateral flow devices has also been developed. In this study, we analyze the robustness of test strips using published methods for colorimetric detection. Different test formats are discussed, and challenges in the development of lateral flow devices for on-site determination of OTA, with requirements such as robustness, speed, and cost-effectiveness, are discussed.

  18. Rapid and Sensitive Detection of BLAD in Cattle Population

    Directory of Open Access Journals (Sweden)

    Daniela Elena Ilie

    2014-05-01

    Full Text Available Bovine leukocyte adhesion deficiency (BLAD is an autosomal recessive disorder with negative impact on dairy cattle breeding. The molecular basis of BLAD is a single point mutation (A→G, resulting in a single amino acid substitution (aspartic acid → glycine at amino acid 128 in the adhesion molecule CD18. The object of this study was to establish a fast and sensitive molecular genotyping assay to detect BLAD carriers using high-resolution melting (HRM curve analysis. We tested animals with known genotypes for BLAD that were previously confirmed by PCR-RFLP method, and then examined the sensitivity of mutation detection using PCR followed by HRM curve analysis. BLAD carriers were readily detectable using HRM assay. Thus, the PCR-HRM genotyping method is a rapid, easily interpretable, reliable and cost-effective assay for BLAD mutant allele detection. This assay can be useful in cattle genotyping and genetic selection.

  19. Rapid detection of methanol in artisanal alcoholic beverages

    Science.gov (United States)

    de Goes, R. E.; Muller, M.; Fabris, J. L.

    2015-09-01

    In the industry of artisanal beverages, uncontrolled production processes may result in contaminated products with methanol, leading to risks for consumers. Owing to the similar odor of methanol and ethanol, as well as their common transparency, the distinction between them is a difficult task. Contamination may also occur deliberately due to the lower price of methanol when compared to ethanol. This paper describes a spectroscopic method for methanol detection in beverages based on Raman scattering and Principal Component Analysis. Associated with a refractometric assessment of the alcohol content, the method may be applied in field for a rapid detection of methanol presence.

  20. Amperometric immunosensor for rapid detection of Mycobacterium tuberculosis

    Science.gov (United States)

    Hiraiwa, Morgan; Kim, Jong-Hoon; Lee, Hyun-Boo; Inoue, Shinnosuke; Becker, Annie L.; Weigel, Kris M.; Cangelosi, Gerard A.; Lee, Kyong-Hoon; Chung, Jae-Hyun

    2015-05-01

    Tuberculosis (TB) has been a major public health problem, which can be better controlled by using accurate and rapid diagnosis in low-resource settings. A simple, portable, and sensitive detection method is required for point-of-care (POC) settings. This paper studies an amperometric biosensor using a microtip immunoassay for a rapid and low-cost detection of Mycobacterium tuberculosis (MTB) in sputum. MTB in sputum is specifically captured on the functionalized microtip surface and detected by electric current. According to the numerical study, the current signal on the microtip surface is linearly changed with increasing immersion depth. Using a reference microtip, the immersion depth is compensated for a sensing microtip. On the microtip surface, target bacteria are concentrated and organized by a coffee-ring effect, which amplifies the electric current. To enhance the signal-to-noise ratio, both the sample processing and rinsing steps are presented with the use of deionized water as a medium for the amperometric measurement. When applied to cultured MTB cells spiked into human sputum, the detection limit was 100 CFU mL-1, comparable to a more labor-intensive fluorescence detection method reported previously.

  1. [Rapid detection of Shigella dysenteriae by PCR assay].

    Science.gov (United States)

    Chen, Hongyuan; Zhong, Qingping; Wang, Li; Sun, Yuanming

    2010-09-01

    Based on the invasive plasmid antigen H gene (ipaH) of S. dysenteriae, one pair of specific primers was designed for PCR assays in this study. The concentrations of dNTP, Mg2+ and primer, dosage of Taq DNA polymerase, annealing temperature and circulating parameter in the PCR amplification system were optimized. In this way, a rapid and stable method of PCR assay for the detection of S. dysenteriae was established. The specificity and sensitivity of PCR were also analyzed. The detection limits of pure culture and genomic DNA in the PCR assay were 1.06 x 10(2) cfu/ml and 106.34 pg/PCR system, respectively. The detection limit for S. dysenteriae in artificially contaminated food samples was 3.21 x 10(4) cfu/ml. These results indicated that the PCR method for S. dysenteriae detection was simple, rapid, high in specificity and sensitivity and suitable for the detection of pathogens in foods caused by Shigella dysenteriae.

  2. Development of a Multiplex PCR Method for Detection of the Genes Encoding 16S rRNA, Coagulase, Methicillin Resistance and Enterotoxins in Staphylococcus aureus

    Science.gov (United States)

    A multiplex PCR method was developed for simultaneous detection of the genes encoding methicillin resistance (mecA), staphylococcal enterotoxins A, B and C (sea, seb and sec), coagulase (coa) and 16S rRNA. The primers for amplification of the 16S rRNA gene were specific for Staphylococcus spp., and ...

  3. Multiplex detection of IgG and IgM to Rift Valley fever virus nucleoprotein, nonstructural proteins, and glycoprotein in ovine and bovine

    Science.gov (United States)

    A multiplex fluorescence microsphere immunoassay (FMIA) was used to detect bovine and ovine IgM and IgG antibodies to several Rift Valley fever virus (RVFV) proteins, including the major surface glycoprotein, Gn; the nonstructural proteins, NSs and NSm; and the nucleoprotein, N. Target antigens were...

  4. Reconfigurable intensity modulation and direct detection optical transceivers for variable-rate wavelength-division-multiplexing passive optical networks utilizing digital signal processing-based symbol mapper

    Science.gov (United States)

    Zhang, Zhiguo; Zhang, Bingbing; Chen, Yanxu; Chen, Xue

    2017-01-01

    Variable-rate intensity modulation and direct detection-based optical transceivers with software-controllable reconfigurability and transmission performance adaptability are experimentally demonstrated, utilizing M-QAM symbol mapping implemented in MATLAB® programs. A frequency division multiplexing-based symbol demapping and wavelength management method is proposed for the symbol demapper and tunable laser management used in colorless optical network unit.

  5. Multiplex PCR assays for simultaneous detection of six major serotypes and two virulence-associated phenotypes of Streptococcus suis in tonsillar specimens from pigs

    NARCIS (Netherlands)

    Wisselink, H.J.; Joosten, J.J.; Smith, H.E.

    2002-01-01

    Multiplex PCR assays for the detection and identification of various Streptococcus suis strains in tonsillar specimens from pigs were developed and evaluated. In two separate reactions, five distinct DNA targets were amplified. Three targets, based on the S. suis capsular polysaccharide (cps) genes

  6. Development of a multiplex TaqMan real-time PCR assay for the detection of Chlamydia psittaci and Chlamydia pneumoniae in human clinical specimens.

    Science.gov (United States)

    Wolff, Bernard J; Morrison, Shatavia S; Winchell, Jonas M

    2017-11-27

    Diagnosis of Chlamydia psittaci and Chlamydia pneumoniae infections has traditionally relied on serological assays. We developed a multiplex real-time PCR assay for detection of C. psittaci, C. pneumoniae and an internal control. Results of this assay demonstrated 100% concordance compared to results of previously tested human clinical specimens. Published by Elsevier Inc.

  7. Detection of Mycobacterium chelonae, Mycobacterium abscessus Group, and Mycobacterium fortuitum Complex by a Multiplex Real-Time PCR Directly from Clinical Samples Using the BD MAX System.

    Science.gov (United States)

    Rocchetti, Talita T; Silbert, Suzane; Gostnell, Alicia; Kubasek, Carly; Campos Pignatari, Antonio C; Widen, Raymond

    2017-03-01

    A new multiplex PCR test was designed to detect Mycobacterium chelonae, Mycobacterium abscessus group, and Mycobacterium fortuitum complex on the BD MAX System. A total of 197 clinical samples previously submitted for mycobacterial culture were tested using the new protocol. Samples were first treated with proteinase K, and then each sample was inoculated into the BD MAX Sample Buffer Tube. Extraction and multiplex PCR were performed by the BD MAX System, using the BD MAX ExK TNA-3 extraction kit and BD TNA Master Mix, along with specific in-house designed primers and probes for each target. The limit of detection of each target, as well as specificity, was evaluated. Of 197 clinical samples included in this study, 133 were positive and 60 were negative for mycobacteria by culture, and another 4 negative samples were spiked with M. chelonae ATCC 35752. The new multiplex PCR on the BD MAX had 97% concordant results with culture for M. abscessus group detection, 99% for M. chelonae, and 100% for M. fortuitum complex. The new multiplex PCR test performed on the BD MAX System proved to be a sensitive and specific test to detect M. chelonae, M. abscessus group, and M. fortuitum complex by real-time PCR on an automated sample-in results-out platform. Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

  8. Simultaneous detection of virulence factors from a colony in diarrheagenic Escherichia coli by a multiplex PCR assay with Alexa Fluor-labeled primers.

    Science.gov (United States)

    Kuwayama, Masaru; Shigemoto, Naoki; Oohara, Sachiko; Tanizawa, Yukie; Yamada, Hiroko; Takeda, Yoshihiro; Matsuo, Takeshi; Fukuda, Shinji

    2011-07-01

    We have developed simultaneous detection of eight genes associated with the five categories of diarrheagenic Escherichia coli by the multiplex PCR assay with Alexa Fluor-labeled primers. This assay can easily distinguish eight genes based on the size and color of amplified products without gel staining. Copyright © 2011 Elsevier B.V. All rights reserved.

  9. Simple, multiplexed, PCR-based barcoding of DNA enables sensitive mutation detection in liquid biopsies using sequencing.

    Science.gov (United States)

    Ståhlberg, Anders; Krzyzanowski, Paul M; Jackson, Jennifer B; Egyud, Matthew; Stein, Lincoln; Godfrey, Tony E

    2016-06-20

    Detection of cell-free DNA in liquid biopsies offers great potential for use in non-invasive prenatal testing and as a cancer biomarker. Fetal and tumor DNA fractions however can be extremely low in these samples and ultra-sensitive methods are required for their detection. Here, we report an extremely simple and fast method for introduction of barcodes into DNA libraries made from 5 ng of DNA. Barcoded adapter primers are designed with an oligonucleotide hairpin structure to protect the molecular barcodes during the first rounds of polymerase chain reaction (PCR) and prevent them from participating in mis-priming events. Our approach enables high-level multiplexing and next-generation sequencing library construction with flexible library content. We show that uniform libraries of 1-, 5-, 13- and 31-plex can be generated. Utilizing the barcodes to generate consensus reads for each original DNA molecule reduces background sequencing noise and allows detection of variant alleles below 0.1% frequency in clonal cell line DNA and in cell-free plasma DNA. Thus, our approach bridges the gap between the highly sensitive but specific capabilities of digital PCR, which only allows a limited number of variants to be analyzed, with the broad target capability of next-generation sequencing which traditionally lacks the sensitivity to detect rare variants. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  10. Prenatal BACs-on-Beads™ : a new technology for rapid detection of aneuploidies and microdeletions in prenatal diagnosis.

    Science.gov (United States)

    Vialard, F; Simoni, G; Aboura, A; De Toffol, S; Molina Gomes, D; Marcato, L; Serero, S; Clement, P; Bouhanna, P; Rouleau, E; Grimi, B; Selva, J; Gaetani, E; Maggi, F; Joseph, A; Benzacken, B; Grati, F R

    2011-05-01

    Molecular cytogenetic techniques on uncultured prenatal samples are the sole tests applied in some countries in cases with advanced maternal age (AMA) or increased risk after prenatal screening. Moreover, there is a trend to perform invasive prenatal diagnosis (PD) during the first trimester before ultrasound manifestations, so new rapid and reliable assays are necessary to investigate microdeletions not detectable with the conventional karyotype. We report the validation study of the prenatal bacterial artificial chromosomes-on-Beads™ (BoBs™ ; CE-IVD), a bead-based multiplex assay detecting chromosomes 13, 18, 21, X/Y aneuploidies and nine microdeletion regions having an overall detection rate of 1/1700. We retrospectively studied 408 selected samples and prospectively tested 212 consecutive samples ascertained for conventional karyotyping. We did not find false-positive results. Triploidies were not detected. Maternal cell contamination of male samples up to 90% was unmasked inspecting gonosome profiles. Mosaic conditions at 20 to 30% were revealed. Failures were due to low amount of DNA. Prenatal BoBs™ is a robust technology for the investigation of fetuses with normal karyotype with or without sonographic abnormalities. Running