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Sample records for reaction pcr assay

  1. Monitoring Acidophilic Microbes with Real-Time Polymerase Chain Reaction (PCR) Assays

    Energy Technology Data Exchange (ETDEWEB)

    Frank F. Roberto

    2008-08-01

    Many techniques that are used to characterize and monitor microbial populations associated with sulfide mineral bioleaching require the cultivation of the organisms on solid or liquid media. Chemolithotrophic species, such as Acidithiobacillus ferrooxidans and Leptospirillum ferrooxidans, or thermophilic chemolithotrophs, such as Acidianus brierleyi and Sulfolobus solfataricus can grow quite slowly, requiring weeks to complete efforts to identify and quantify these microbes associated with bioleach samples. Real-time PCR (polymerase chain reaction) assays in which DNA targets are amplified in the presence of fluorescent oligonucleotide primers, allowing the monitoring and quantification of the amplification reactions as they progress, provide a means of rapidly detecting the presence of microbial species of interest, and their relative abundance in a sample. This presentation will describe the design and use of such assays to monitor acidophilic microbes in the environment and in bioleaching operations. These assays provide results within 2-3 hours, and can detect less than 100 individual microbial cells.

  2. A new polymerase chain reaction (PCR) assay for the trinucleotide repeat that is unstable and expanded on Huntington's disease chromosomes.

    Science.gov (United States)

    Warner, J P; Barron, L H; Brock, D J

    1993-06-01

    The Huntington's Disease (HD) Collaborative Research Group has recently published the sequence of a new cDNA, IT15, containing a polymorphic trinucleotide (CAG)n repeat that is expanded and unstable on HD chromosomes. There is a correlation between the repeat size and the age of onset of symptoms. The suggested polymerase chain reaction (PCR) assay of the (CAG)n repeat requires unusual reaction components and primer concentrations and the use of 5% polyacrylamide sequencing gels to resolve the amplification products. We present a simple PCR assay that produces a smaller product using standard reaction conditions. This gives better resolution of the (CAG)n expansion observed on HD chromosomes by acrylamide gel electrophoresis and allows sufficient product to be obtained to perform assays using agarose gels. This will allow diagnostic labs to do rapid and accurate presymptomatic testing of HD in high risk families.

  3. Translation of a laboratory-validated equine herpesvirus-1 specific real-time PCR assay into an insulated isothermal polymerase chain reaction (iiPCR) assay for point-of-need diagnosis using POCKIT™ nucleic acid analyzer.

    Science.gov (United States)

    Balasuriya, Udeni B R; Lee, Pei-Yu Alison; Tsai, Yun-Long; Tsai, Chuan-Fu; Shen, Yu-Han; Chang, Hsiao-Fen Grace; Skillman, Ashley; Wang, Hwa-Tang Thomas; Pronost, Stéphane; Zhang, Yan

    2017-03-01

    Equine herpesvirus myeloencephalopathy (EHM), a major problem for the equine industry in the United States, is caused by equine herpesvirus-1 (EHV-1). In addition, EHV-1 is associated with upper respiratory disease, abortion, and chorioretinal lesions in horses. Here we describe the development and evaluation of an inexpensive, user-friendly insulated isothermal PCR (iiPCR) method targeting open reading 30 (ORF30) to detect both neuropathogenic and non-neuropathogenic strains on the field-deployable POCKIT™ device for point-of-need detection of EHV-1. The analytical sensitivity of the EHV-1 iiPCR assay was 13 genome equivalents per reaction. The assay did not cross react with ten non-target equine viral pathogens. Performance of the EHV-1 iiPCR assay was compared to two previously described real-time PCR (qPCR) assays in two laboratories by using 104 archived clinical samples. All 53 qPCR-positive and 46 of the 51 qPCR-negative samples tested positive and negative, respectively, by the iiPCR. The agreement between the two assays was 95.19% (confidence interval 90.48-99.90%) with a kappa value of 0.90. In conclusion, the newly developed EHV-1 iiPCR assay is robust to provide specificity and sensitivity comparable to qPCR assays for the detection of EHV-1 nucleic acid in clinical specimens.

  4. Application of a real time Polymerase Chain Reaction (PCR) assay for the early diagnosis of human leptospirosis in Sri Lanka.

    Science.gov (United States)

    Denipitiya, D T H; Chandrasekharan, N V; Abeyewickreme, W; Hartskeerl, C M; Hartskeerl, R A; Jiffrey, A M; Hapugoda, M D

    2016-11-01

    Leptospirosis has a major impact on health in Sri Lanka but is probably grossly under-recognized due to difficulties in clinical diagnosis and lack of diagnostic laboratory services. The objective of this study was to establish and evaluate a SYBR Green-based real-time Polymerase Chain Reaction (rt-PCR) assay for early, rapid and definitive laboratory diagnosis of leptospirosis in Sri Lanka. The rt-PCR assay was established and analytical specificity and sensitivity were determined using reference DNA samples. Evaluation of the assay for diagnosis of clinical samples was performed using two panels of serum samples obtained from 111 clinically suspected adult patients. Patients were confirmed as leptospirosis (n = 65) and non-leptospirosis (n = 30) by the Patoc - MAT. Other 16 samples gave ambiguous results. The analytical sensitivity of the rt-PCR was approximately 60 genome copies and no cross-reactivity was observed with saprophytic Leptospira spp. and other pathogenic microorganisms. Based on confirmation with Patoc-MAT on paired samples this corresponds to a diagnostic sensitivity and specificity of 67.7% (44/65) and 90.0% (27/30), respectively. This study showed that rt-PCR has the potential to facilitate rapid and definitive diagnosis of leptospirosis during early phase of infection in Sri Lanka. Copyright © 2016 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

  5. Detection of Eperythrozoon wenyoni by PCR assay

    Institute of Scientific and Technical Information of China (English)

    Jian WANG; Yutao ZHU; Jianhua QIN; Fumei ZHANG; Yuelan ZHAO

    2009-01-01

    The objective of this research was to develop a detection method for Eperythrozoon wenyoni infection using polymerase chain reaction (PCR) assay technique. A pair of primers was designed and synthesized according to the conservative sequence 16S rRNA. The PCR assay was performed with the primers. A 985-bp fragment was amplified by using PCR. The amplified fragments with the expected size were identified by EcoR I restriction digestion. The crossing-reaction, specific-reaction and duplicate-reaction indicated that the PCR method is a specific, sensitive, fast and effective method for diagnosing E. Wenyoni infection at group level.

  6. Simultaneous detection of five enteric viruses associated with gastroenteritis by use of a PCR assay: a single real-time multiplex reaction and its clinical application.

    Science.gov (United States)

    Jiang, Yixiang; Fang, Lin; Shi, Xiaolu; Zhang, Hailong; Li, Yinghui; Lin, Yiman; Qiu, Yaqun; Chen, Qingliang; Li, Hui; Zhou, Li; Hu, Qinghua

    2014-04-01

    We developed a highly sensitive reverse transcription and multiplex real-time PCR (rtPCR) assay that can identify five viruses, including six genogroups, in a single reaction: norovirus genogroups I and II; sapovirus genogroups I, II, IV, and V; human rotavirus A; adenovirus serotypes 40 and 41; and human astrovirus. In comparison to monoplex rtPCR assays, the sensitivities and specificities of the multiplex rtPCR ranged from 75% to 100% and from 99% to 100%, respectively, evaluated on 812 clinical stool specimens.

  7. Quantitative polymerase chain reaction (PCR) assays for a bacterial thiaminase I gene and the thiaminase-producing bacterium Paenibacillus thiaminolyticus.

    Science.gov (United States)

    Richter, C.A.; Wright-Osment, Maureen K.; Zajicek, J.L.; Honeyfield, D.C.; Tillitt, D.E.

    2009-01-01

    The thiaminase I enzyme produced by the gram-positive bacterium Paenibacillus thiaminolyticus isolated from the viscera of Lake Michigan alewives Alosa pseudoharengus is currently the only defined source of the thiaminase activity linked to thiamine (vitamin B1) deficiency in early mortality syndrome (EMS) in the larvae of Great Lakes salmonines. Diets of alewife or isolated strains of P. thiaminolyticus mixed in a semipurified diet and fed to lake trout Salvelinus namaycush have been shown to produce EMS in fry. We utilized quantitative polymerase chain reaction (Q-PCR) to aid in studies of the sources of P. thiaminolyticus and thiaminase I. Quantitative PCR assays were established to detect the thiaminase I gene of P. thiaminolyticus, the 16S rRNA gene from most species of bacteria, and the 16S rRNA gene specifically from P. thiaminolyticus and a few closely related taxa. The Q-PCR assays are linear over at least six orders of magnitude and can detect the thiaminase I gene of P. thiaminolyticus from as few as 1,000 P. thiaminolyticus cells/g of sample or the Paenibacillus 16S rRNA gene from as few as 100 P. thiaminolyticus cells/g of sample. The initial results from alewife viscera samples with high thiaminase activity yielded unexpectedly low densities of P. thiaminolyticus cells; Paenibacillus thiaminolyticus was detectable in 2 of 6 alewife viscera tested at densities on the order of 100 cells/g out of 100,000,000 total bacterial cells/g. The low numbers of P. thiaminolyticus detected suggest that alewives contain additional non-P. thiaminolyticus sources of thiaminase activity.

  8. Use of a high resolution melt real-time polymerase chain reaction (PCR) assay for the environmental monitoring of Vibrio cholerae

    CSIR Research Space (South Africa)

    Le Rouw, Wouter J

    2011-10-01

    Full Text Available A real-time polymerase chain reaction (PCR) assay utilizing high resolution melt (HRM) curve analysis was developed and tested for the monitoring of Vibrio cholerae in water samples. The assay utilized previously published primers that are specific...

  9. Loop mediated isothermal amplification assay using hydroxy naphthol blue, conventional polymerase chain reaction and real-time PCR in the diagnosis of intraocular tuberculosis.

    Science.gov (United States)

    Balne, P K; Basu, S; Rath, S; Barik, M R; Sharma, S

    2015-01-01

    This study is a comparative evaluation (Chi-square test) of a closed tube loop mediated isothermal amplification assay using hydroxy naphthol blue dye (HNB-LAMP), real-time polymerase chain reaction (PCR) and conventional PCR in the diagnosis of intraocular tuberculosis. Considering clinical presentation as the gold standard in 33 patients, the sensitivity of HNB-LAMP assay (75.8%) was higher (not significant, P value 0.2) than conventional PCR (57.6%) and lower than real-time PCR (90.9%). Specificity was 100% by all three methods. No amplification was observed in negative controls (n = 20) by all three methods. The cost of the HNB-LAMP assay was Rs. 500.00 and it does not require thermocycler, therefore, it can be used as an alternative to conventional PCR in resource-poor settings.

  10. Loop mediated isothermal amplification assay using hydroxy naphthol blue, conventional polymerase chain reaction and real-time PCR in the diagnosis of intraocular tuberculosis

    Directory of Open Access Journals (Sweden)

    P K Balne

    2015-01-01

    Full Text Available This study is a comparative evaluation (Chi-square test of a closed tube loop mediated isothermal amplification assay using hydroxy naphthol blue dye (HNB-LAMP, real-time polymerase chain reaction (PCR and conventional PCR in the diagnosis of intraocular tuberculosis. Considering clinical presentation as the gold standard in 33 patients, the sensitivity of HNB-LAMP assay (75.8% was higher (not significant, P value 0.2 than conventional PCR (57.6% and lower than real-time PCR (90.9%. Specificity was 100% by all three methods. No amplification was observed in negative controls (n = 20 by all three methods. The cost of the HNB-LAMP assay was Rs. 500.00 and it does not require thermocycler, therefore, it can be used as an alternative to conventional PCR in resource-poor settings.

  11. Development of a multiplex PCR-ligase detection reaction assay for diagnosis of infection by the four parasite species causing malaria in humans.

    Science.gov (United States)

    McNamara, David T; Thomson, Jodi M; Kasehagen, Laurin J; Zimmerman, Peter A

    2004-06-01

    The diagnosis of infections caused by Plasmodium species is critical for understanding the nature of malarial disease, treatment efficacy, malaria control, and public health. The demands of field-based epidemiological studies of malaria will require faster and more sensitive diagnostic methods as new antimalarial drugs and vaccines are explored. We have developed a multiplex PCR-ligase detection reaction (LDR) assay that allows the simultaneous diagnosis of infection by all four parasite species causing malaria in humans. This assay exhibits sensitivity and specificity equal to those of other PCR-based assays, identifying all four human malaria parasite species at levels of parasitemias equal to 1 parasitized erythrocyte/microl of blood. The multiplex PCR-LDR assay goes beyond other PCR-based assays by reducing technical procedures and by detecting intraindividual differences in species-specific levels of parasitemia. Application of the multiplex PCR-LDR assay will provide the sensitivity and specificity expected of PCR-based diagnostic assays and will contribute new insight regarding relationships between the human malaria parasite species and the human host in future epidemiological studies.

  12. Giardia and Cryptosporidium spp. dissemination during wastewater treatment and comparative detection via immunofluorescence assay (IFA), nested polymerase chain reaction (nested PCR) and loop mediated isothermal amplification (LAMP).

    Science.gov (United States)

    Gallas-Lindemann, Carmen; Sotiriadou, Isaia; Plutzer, Judit; Noack, Michael J; Mahmoudi, Mohammad Reza; Karanis, Panagiotis

    2016-06-01

    Environmental water samples from the Lower Rhine area in Germany were investigated via immunofluorescence assays (IFAs), nested polymerase chain reaction (nested PCR) and loop-mediated isothermal amplification (LAMP) to detect the presence of Giardia spp. (n=185) and Cryptosporidium spp. (n=227). The samples were concentrated through filtration or flocculation, and oocysts were purified via centrifugation through a sucrose density gradient. For all samples, IFA was performed first, followed by DNA extraction for the nested PCR and LAMP assays. Giardia cysts were detected in 105 samples (56.8%) by IFA, 62 samples (33.5%) by nested PCR and 79 samples (42.7%) by LAMP. Cryptosporidium spp. were detected in 69 samples (30.4%) by IFA, 95 samples (41.9%) by nested PCR and 99 samples (43.6%) by LAMP. According to these results, the three detection methods are complementary for monitoring Giardia and Cryptosporidium in environmental waters.

  13. A novel polymerase chain reaction (PCR based assay for authentication of cell lines or tissues from human, pig and chicken origin

    Directory of Open Access Journals (Sweden)

    MARIO GORENJAK

    2012-01-01

    Full Text Available A polymerase chain reaction based assay was developed for authentication of cell lines or tissues from human, pig and chicken origin. Specificity was achieved by species specific primer design targeting the mitochondrial D-loop sequence. Amplicon sizes were 114 bp, 169 bp and 645-648 bp for chicken, human and pig derived cell lines, respectively. Primers were tested for species specificity and non-specificity between haplogroups of the same organisms using BLAST tool and subsequently for cross amplification DNA extracted from human, chicken and pig venous blood as a positive control. Primers were also amplifying specific products in DNA extracted from individual cell line in both functional cell models and intentionally mixed cell lines consisting functional cell models. The PCR assay developed in this study represents a low-cost species specific end-point PCR based assay of the mitochondrial D-loop for the authentication of the cell line origin.

  14. A modified molecular beacons-based multiplex real-time PCR assay for simultaneous detection of eight foodborne pathogens in a single reaction and its application.

    Science.gov (United States)

    Hu, Qinghua; Lyu, Dongyue; Shi, Xiaolu; Jiang, Yixiang; Lin, Yiman; Li, Yinghui; Qiu, Yaqun; He, Lianhua; Zhang, Ran; Li, Qingge

    2014-03-01

    Foodborne disease outbreaks are often caused by one of the major pathogens. Early identification of the causal pathogen is crucial for disease control and prevention. We describe a real-time polymerase chain reaction (rtPCR) assay that can identify, in a single reaction, up to eight common foodborne bacterial pathogens, including Salmonella enterica subsp. enterica, Listeria monocytogenes, Escherichia coli O157, Vibrio parahaemolyticus, V. vulnificus, Campylobacter jejuni, Enterobacter sakazakii, and Shigella spp. This multiplex rtPCR assay takes advantage of modified molecular beacons and the multicolor combinational probe coding strategy to discriminate each pathogen and the homo-tag assisted non-dimer (HAND) system to prevent dimer formation. The detection limits of the assay ranged from 1.3×10(3) colony-forming units (CFU)/g stool (L. monocytogenes) to 1.6×10(4) CFU/g stool (Shigella spp.). The target genes were 100% specific as assessed on 986 reference strains covering 41 species since no cross-reactions were observed. The assay was applied to the detection of foodborne pathogens in 11,167 clinical samples and the results were compared with culture methods for further validation. The sensitivity and specificity of the rtPCR were 100% and 99%, respectively. When performed in a 96-well rtPCR system, more than 90 samples could be analyzed within 3 h. Given the high accuracy, sensitivity, specificity, and short turn-around time, the established assay could be used for the rapid and reliable identification of the causative pathogens responsible for a certain foodborne disease outbreak and rapid screening of these major foodborne pathogens in laboratory-based surveillance of outpatient clinical samples or even food samples.

  15. Quantitation of viable Coxiella burnetii in milk using an integrated cell culture-polymerase chain reaction (ICC-PCR) assay.

    Science.gov (United States)

    Stewart, Diana; Shieh, Y-Carol; Tortorello, Mary; Kukreja, Ankush; Shazer, Arlette; Schlesser, Joseph

    2015-11-01

    The obligate intracellular pathogen Coxiella burnetii has long been considered the most heat resistant pathogen in raw milk, making it the reference pathogen for determining pasteurisation conditions for milk products. New milk formulations and novel non-thermal processes require validation of effectiveness which requires a more practical method for analysis than using the currently used animal model for assessing Coxiella survival. Also, there is an interest in better characterising thermal inactivation of Coxiella in various milk formulations. To avoid the use of the guinea pig model for evaluating Coxiella survival, an Integrated Cell Culture-PCR (ICC-PCR) method was developed for determining Coxiella viability in milk. Vero cell cultures were directly infected from Coxiella-contaminated milk in duplicate 24-well plates. Viability of the Coxiella in milk was shown by a ≥ 0.5 log genome equivalent (ge)/ml increase in the quantity of IS111a gene from the baseline post-infection (day 0) level after 9-11 d propagation. Coxiella in skim, 2%, and whole milk, and half and half successfully infected Vero cells and increased in number by at least 2 logs using a 48-h infection period followed by 9-d propagation time. As few as 125 Coxiella ge/ml in whole milk was shown to infect and propagate at least 2 logs in the optimised ICC-PCR assay, though variable confirmation of propagation was shown for as low as 25 Coxiella ge/ml. Applicability of the ICC-PCR method was further proven in an MPN format to quantitate the number of viable Coxiella remaining in whole milk after 60 °C thermal treatment at 0, 20, 40, 60 and 90 min.

  16. Development of a TaqMan Probe-Based Insulated Isothermal Polymerase Chain Reaction (iiPCR) Assay for Detection of Fusarium oxysporum f. sp. cubense Race 4

    Science.gov (United States)

    Lin, Yi-Jia; Chang, Tsai-De; Hong, Li-Ling; Chen, Tzu-Yu; Chang, Pi-Fang Linda

    2016-01-01

    This study developed a novel and inexpensive detection method based on a TaqMan probe-based insulated isothermal polymerase chain reaction (iiPCR) method for the rapid detection of Panama disease caused by Fusarium oxysporum f. sp. cubense (Foc) race 4, which is currently among the most serious fungal vascular diseases worldwide. By using the portable POCKIT™ device with the novel primer set iiFoc-1/iiFoc-2, the Foc race 4 iiPCR assay (including DNA amplification and signal monitoring) could be completed within one hour. The developed Foc race 4 iiPCR assay is thus a user-friendly and efficient platform designed specifically for the detection of Foc race 4. The detection limit of this optimized Foc iiPCR system was estimated to be 1 copy of the target standard DNA as well as 1 fg of the Foc genomic DNA. This approach can serve as a rapid detection method for in planta detection of Foc race 4 in field-infected banana. It was concluded that this molecular detection procedure based on iiPCR has good potential for use as an efficient detection method. PMID:27448242

  17. Development of a TaqMan Probe-Based Insulated Isothermal Polymerase Chain Reaction (iiPCR Assay for Detection of Fusarium oxysporum f. sp. cubense Race 4.

    Directory of Open Access Journals (Sweden)

    Ying-Hong Lin

    Full Text Available This study developed a novel and inexpensive detection method based on a TaqMan probe-based insulated isothermal polymerase chain reaction (iiPCR method for the rapid detection of Panama disease caused by Fusarium oxysporum f. sp. cubense (Foc race 4, which is currently among the most serious fungal vascular diseases worldwide. By using the portable POCKIT™ device with the novel primer set iiFoc-1/iiFoc-2, the Foc race 4 iiPCR assay (including DNA amplification and signal monitoring could be completed within one hour. The developed Foc race 4 iiPCR assay is thus a user-friendly and efficient platform designed specifically for the detection of Foc race 4. The detection limit of this optimized Foc iiPCR system was estimated to be 1 copy of the target standard DNA as well as 1 fg of the Foc genomic DNA. This approach can serve as a rapid detection method for in planta detection of Foc race 4 in field-infected banana. It was concluded that this molecular detection procedure based on iiPCR has good potential for use as an efficient detection method.

  18. Droplet digital polymerase chain reaction (ddPCR) assays integrated with an internal control for quantification of bovine, porcine, chicken and turkey species in food and feed

    National Research Council Canada - National Science Library

    Hanan R Shehata; Jiping Li; Shu Chen; Helen Redda; Shumei Cheng; Nicole Tabujara; Honghong Li; Keith Warriner; Robert Hanner

    2017-01-01

    .... Reliable techniques are needed to monitor these issues. Droplet Digital PCR (ddPCR) assays were developed and evaluated for detection and quantification of bovine, porcine, chicken and turkey DNA in food and feed samples...

  19. Interlaboratory comparison of three microbial source tracking quantitative polymerase chain reaction (qPCR) assays from fecal-source and environmental samples

    Science.gov (United States)

    Stelzer, Erin A.; Strickler, Kriston M.; Schill, William B.

    2012-01-01

    During summer and early fall 2010, 15 river samples and 6 fecal-source samples were collected in West Virginia. These samples were analyzed by three laboratories for three microbial source tracking (MST) markers: AllBac, a general fecal indicator; BacHum, a human-associated fecal indicator; and BoBac, a ruminant-associated fecal indicator. MST markers were analyzed by means of the quantitative polymerase chain reaction (qPCR) method. The aim was to assess interlaboratory precision when the three laboratories used the same MST marker and shared deoxyribonucleic acid (DNA) extracts of the samples, but different equipment, reagents, and analyst experience levels. The term assay refers to both the markers and the procedure differences listed above. Interlaboratory precision was best for all three MST assays when using the geometric mean absolute relative percent difference (ARPD) and Friedman's statistical test as a measure of interlaboratory precision. Adjustment factors (one for each MST assay) were calculated using results from fecal-source samples analyzed by all three laboratories and applied retrospectively to sample concentrations to account for differences in qPCR results among labs using different standards and procedures. Following the application of adjustment factors to qPCR results, ARPDs were lower; however, statistically significant differences between labs were still observed for the BacHum and BoBac assays. This was a small study and two of the MST assays had 52 percent of samples with concentrations at or below the limit of accurate quantification; hence, more testing could be done to determine if the adjustment factors would work better if the majority of sample concentrations were above the quantification limit.

  20. Development of a multiplex real-time PCR assay for the detection of Bordetella pertussis and Bordetella parapertussis in a single tube reaction.

    Science.gov (United States)

    Arbefeville, Sophie; Levi, Michael H; Ferrieri, Patricia

    2014-02-01

    Pertussis is an infectious respiratory disease caused by the fastidious bacterium Bordetella pertussis, which may infect unvaccinated, previously vaccinated children, and adults in whom immunity has waned. Infants are at a particular risk for severe disease and complications. Bordetella parapertussis may cause a similar illness, however the symptoms are less severe and of shorter duration. Pertussis is a highly contagious disease and early diagnosis is essential. Studies have shown that PCR is 2-4 times more likely than culture to detect Bordetella pertussis. We developed a multiplex, real-time PCR assay using analyte-specific reagent (ASR) primers and probes dispensed in a convenient lyophilized bead format that targeted the multi-copy insertion sequences IS481 and IS1001 of B. pertussis and B. parapertussis, respectively. These specific ASRs were used in conjunction with Cepheid Smartmix. Included in the ASRs is a competitive internal control to evaluate the performance of the PCR reaction. After DNA extraction, amplification and detection were done on the Smart Cycler System, which performs integrated amplification and detection automatically in a single step. Specificity of the assay was confirmed using multiple distinct bacterial strains. Sensitivity of the assay and extraction efficiency were evaluated on DNA isolated from pure bacterial cultures and on spiked respiratory specimens. We also spiked different swab types and transport media to evaluate for interfering substances. To assess accuracy, we studied different patient specimen types received from two outside laboratories that used similar or different methods to detect B. pertussis and B. parapertussis. The sensitivity and the specificity of the assay for B. pertussis were 90% and 96%, respectively, and for B. parapertussis 71% (only 7 positive specimens were available for testing) and 100%, respectively. Our assay was found to be a valid method for the simultaneous detection of B. pertussis and B

  1. Polymerase chain reaction (PCR assay for rapid diagnosis and its role in prevention of human Brucellosis in Punjab, India

    Directory of Open Access Journals (Sweden)

    Moti Yohannes Gemechu

    2011-01-01

    Conclusions: Early-case reporting is possible by rapid tests like PCR. Thus, PCR is a promising diagnostic tool for routine investigation and surveillance of brucellosis which is the key element for management of prevention and control programmes. But patient condition before testing, optimal clinical specimen, sample volume used, simple and efficient DNA extraction protocol are the points of concern for PCR to be used as a routine test in clinical laboratory practice.

  2. "Use of Random Amplified Polymorphic DNA Polymerase Chain Reaction (RAPD-PCR and ITS2 PCR assays for differentiation of populations and putative sibling species of Anopheles fluviatilis (Diptera: Culicidae in Iran"

    Directory of Open Access Journals (Sweden)

    SR Naddaf Dezfouli

    2002-09-01

    Full Text Available Anopheles fluviatilis complex is known to be a vector of malaria in Iran. Since mosquitoes of this species cover a wide geographical range in Iran, they might have evolved into different separated populations. Random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR assay was used to differentiate geographic populations of this species. DNA was extracted from individual mosquitoes from 8 localities in 4 south and southeast provinces and amplified in PCR reactions using 18 single primers of arbitrary nucleotide sequence. Results of RAPD-PCR showed that Kazeroun populations could simply be differentiated from other populations using a diagnostic fragment amplified with primer UBC-306. But other populations could not be differentiated either visually or by means of statistical analysis. Moreover ITS2 fragments of some selected specimens were amplified using a pair of universal primer and sequenced as a key standard for detection of putative sibling species. Sequence analysis of the ITS2 fragments revealed a very high (100% homology among the populations. These findings are crucial in epidemiological studies concerning relatedness of geographic populations and vector movement in the region. Results of RAPD-PCR and ITS2 analysis suggest that this taxon in Iran comprises of only one species with a low genetic variation among geographic populations.

  3. Evaluation of a real-time polymerase chain reaction (PCR) assay for detection of anisakis simplex parasite as a food-borne allergen source in seafood products.

    Science.gov (United States)

    Lopez, Itziar; Pardo, Miguel Angel

    2010-02-10

    Anisakis simplex has been recognized as an important cause of disease in humans and as a food-borne allergen source. Actually, this food-borne parasite was recently identified as an emerging food safety risk. An A. simplex -specific primer-probe system based on a real-time polymerase chain reaction (PCR) detection assay has been successfully optimized and validated with seafood samples. In addition, a DNA extraction procedure has been optimized to detect the presence of the nematode in food samples. The assay is a very reliable, specific, and sensitive methodology to detect the presence of traces of this parasite in seafood products, including highly processed samples. As a result, 13 sequences of cytochrome c oxidase II gene were obtained and scrutinized to calculate intra- and interspecific variabilities of 0 and 35-67%, respectively. Finally, an efficiency of 2.07 +/- 0.14 of the assay was calculated, and a limit of detection of 40 ppm parasite in 25 g of sample was also optimized. Actually, the presence of this parasite in several seafood products has been demonstrated, enforcing the necessity of a design for a good manufacturing practice protocol for the processing industry to minimize the presence of this parasite as a food-borne allergen source in seafood products.

  4. A simple real-time polymerase chain reaction (PCR)-based assay for authentication of the Chinese Panax ginseng cultivar Damaya from a local ginseng population.

    Science.gov (United States)

    Wang, H; Wang, J; Li, G

    2016-06-27

    Panax ginseng is one of the most important medicinal plants in the Orient. Owing to its increasing demand in the world market, cultivated ginseng has become the main source of medicinal material. Among the Chinese ginseng cultivars, Damaya commands higher prices and is grown in significant proportions among the local ginseng population. Due to the lack of rapid and accurate authentication methods, Damaya is distributed among different cultivars in the local ginseng population in China. Here, we identified a unique, Damaya-specific single nucleotide polymorphism (SNP) site present in the second intron of mitochondrial cytochrome c oxidase subunit 2 (cox2). Based on this SNP, a Damaya cultivar-specific primer was designed and an allele-specific polymerase chain reaction (PCR) was optimized for the effective molecular authentication of Damaya. We designed a method by combining a simple DNA isolation method with real-time allele-specific PCR using SYBR Green I fluorescent dye, and proved its efficacy in clearly discriminated Damaya cultivar from other Chinese ginseng cultivars according to the allelic discrimination analysis. Hence, this study provides a simple and rapid assay for the differentiation and conservation of Damaya from the local Chinese ginseng population.

  5. Development and evaluation of a reverse transcription-insulated isothermal polymerase chain reaction (RT-iiPCR) assay for detection of equine arteritis virus in equine semen and tissue samples using the POCKIT™ system.

    Science.gov (United States)

    Carossino, Mariano; Lee, Pei-Yu A; Nam, Bora; Skillman, Ashley; Shuck, Kathleen M; Timoney, Peter J; Tsai, Yun-Long; Ma, Li-Juan; Chang, Hsiao-Fen G; Wang, Hwa-Tang T; Balasuriya, Udeni B R

    2016-08-01

    Equine arteritis virus (EAV) is the causative agent of equine viral arteritis (EVA), a respiratory and reproductive disease of horses. Most importantly, EAV induces abortion in pregnant mares and can establish persistent infection in up to 10-70% of the infected stallions, which will continue to shed the virus in their semen. The objective of this study was to develop and evaluate a reverse transcription insulated isothermal polymerase chain reaction (RT-iiPCR) for the detection of EAV in semen and tissue samples. The newly developed assay had a limit of detection of 10 RNA copies and a 10-fold higher sensitivity than a previously described real-time RT-PCR (RT-qPCR). Evaluation of 125 semen samples revealed a sensitivity and specificity of 98.46% and 100.00%, respectively for the RT-qPCR assay, and 100.00% and 98.33%, respectively for the RT-iiPCR assay. Both assays had the same accuracy (99.2%, k=0.98) compared to virus isolation. Corresponding values derived from testing various tissue samples (n=122) collected from aborted fetuses, foals, and EAV carrier stallions are as follows: relative sensitivity, specificity, and accuracy of 88.14%, 96.83%, and 92.62% (k=0.85), respectively for the RT-qPCR assay, and 98.31%, 92.06%, and 95.08% (k=0.90), respectively for the RT-iiPCR assay. These results indicate that RT-iiPCR is a sensitive, specific, and a robust test enabling detection of EAV in semen and tissue samples with very considerable accuracy. Even though the RT-qPCR assay showed a sensitivity and specificity equal to virus isolation for semen samples, its diagnostic performance was somewhat limited for tissue samples. Thus, this new RT-iiPCR could be considered as an alternative tool in the implementation of EAV control and prevention strategies.

  6. Development and Validation of a HPV-32 Specific PCR Assay

    Directory of Open Access Journals (Sweden)

    Leigh Janet

    2009-06-01

    Full Text Available Abstract Background Human Papillomavirus-32 (HPV-32 has traditionally been associated with focal-epithelial-hyperplasia (FEH. It is also present in 58% of oral warts of HIV-positive individuals whose prevalence is increasing. Current methods for the detection of HPV-32 are labor-intensive and insensitive so the goal of this work was to develop a highly sensitive and easy to use specific polymerase chain reaction (PCR assay. Materials and methods An HPV-32 L1 specific PCR assay was developed and optimized. The sensitivity and specificity was compared to previous assays utilized for detection (PGMY and MY09/11 PCR with dot blot hybridization using cloned HPV-32 L1, the closely related HPV-42 L1 as well as clinical samples (oral swabs and fluids from 89 HIV-positive subjects. Results The HPV-32 specific PCR assay showed improved sensitivity to 5 copies of HPV-32 as compared to the PGMY PCR, MY09/11 PCR and dot blot which had a limit of detection of approximately 3,000 copies. Using the HPV-32 dot blot hybridization assay as the gold standard, the HPV-32 specific PCR assay has a sensitivity of 95.8% and 88.9% by sample and subject, respectively, and specificity was 87.8% and 58.8% by sample and subject, respectively. The low sensitivity is due to the HPV-32 specific PCR assays ability to detect more HPV-32 positive samples and may be the new gold standard. Conclusion Due to the ease, sensitivity, and specificity the HPV-32 specific PCR assay is superior to previous assays and is ideal for detection of HPV-32 in large cohorts. This assay provides an excellent tool to study the natural history of HPV-32 infection and the development of oral warts.

  7. Evaluation of a broad range real-time polymerase chain reaction (RT-PCR) assay for the diagnosis of septic synovitis in horses.

    Science.gov (United States)

    Elmas, Colette R; Koenig, Judith B; Bienzle, Dorothee; Cribb, Nicola C; Cernicchiaro, Natalia; Coté, Nathalie M; Weese, J Scott

    2013-07-01

    Septic synovitis is a potentially debilitating and life-threatening disorder in horses. We hypothesized that a universal bacterial real-time PCR (RT-PCR) assay would have improved sensitivity and decreased turn-around time for detection of bacteria in synovial fluid (SF) samples. Forty-eight SF samples were collected from 36 horses that presented to two referral institutions with suspected septic synovitis. Universal RT-PCR, bacterial culture and SF analysis were performed on all samples, and an interpretation on the sample being septic or not was derived by three board certified specialists from the history, clinical assessment and SF characteristics. RT-PCR results were compared to a composite standard comprised of positive culture and interpretation by all three specialists of samples as "septic". For 41 of 48 samples (85%), culture and RT-PCR results were concordant. Compared to the composite standard, 83% of samples were correctly classified by RT-PCR (turn-around time of approximately 4 hours). Relative sensitivity and specificity of RT-PCR were 87% and 72% respectively, and 56% and 86% for culture. Hence, universal RT-PCR was a rapid and highly sensitive test, which may accelerate diagnosis and improve outcome for horses with septic synovitis.

  8. Effects of Holding Time, Storage, and the Preservation of Samples on Sample Integrity for the Detection of Fecal Indicator Bacteria by Quantitative Polymerase Chain Reaction (qPCR)-based assays.

    Science.gov (United States)

    The purpose of this project was to answer questions related to storage of samples to be analyzed by the quantitative polymerase chain reaction (qPCR)-based assays for fecal indicator bacteria. The project was divided into two parts. The first part was to determine if filters th...

  9. The development of a real-time reverse transcription-polymerase chain reaction (rRT-PCR) assay using TaqMan technology for the pan detection of bluetongue virus (BTV).

    Science.gov (United States)

    Mulholland, Catherine; McMenamy, Michael J; Hoffmann, Bernd; Earley, Bernadette; Markey, Bryan; Cassidy, Joseph; Allan, Gordon; Welsh, Michael D; McKillen, John

    2017-07-01

    Bluetongue virus (BTV) is an infectious, non-contagious viral disease of domestic and wild ruminants that is transmitted by adult females of certain Culicoides species. Since 2006, several serotypes including BTV-1, 2, 4, 6, 8, 9 and 16, have spread from the Mediterranean basin into Northern Europe for the first time. BTV-8 in particular, caused a major epidemic in northern Europe. As a result, it is evident that most European countries are at risk of BTV infection. The objective of this study was to develop and validate a real-time reverse transcriptase-polymerase chain reaction (rRT-PCR) assay based on TaqMan technology for the detection of representative strains of all BTV serotypes. Primers and probes were based on genome segment 10 of the virus, the NS3 gene. The assay was tested for sensitivity, and specificity. The analytical sensitivity of the rRT-PCR assay was 200 copies of RNA per reaction. The assay did not amplify the closely related orbivirus epizootic hemorrhagic disease virus (EHDV) but successfully detected all BTV reference strains including clinical samples from animals experimentally infected with BTV-8. This real time RT-PCR assay offers a sensitive, specific and rapid alternative assay for the pan detection of BTV that could be used as part of a panel of diagnostic assays for the detection of all serotypes of BTV. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

  10. Multiplex PCR Assay for Identification of Human Diarrheagenic Escherichia coli

    OpenAIRE

    Toma, Claudia; Lu, Yan; Higa, Naomi; Nakasone, Noboru; Isabel CHINEN; Baschkier, Ariela; Rivas, Marta; Iwanaga, Masaaki

    2003-01-01

    A multiplex PCR assay for the identification of human diarrheagenic Escherichia coli was developed. The targets selected for each category were eae for enteropathogenic E. coli, stx for Shiga toxin-producing E. coli, elt and est for enterotoxigenic E. coli, ipaH for enteroinvasive E. coli, and aggR for enteroaggregative E. coli. This assay allowed the categorization of a diarrheagenic E. coli strain in a single reaction tube.

  11. Multiplex PCR Assay for Identification of Human Diarrheagenic Escherichia coli

    OpenAIRE

    2003-01-01

    A multiplex PCR assay for the identification of human diarrheagenic Escherichia coli was developed. The targets selected for each category were eae for enteropathogenic E. coli, stx for Shiga toxin-producing E. coli, elt and est for enterotoxigenic E. coli, ipaH for enteroinvasive E. coli, and aggR for enteroaggregative E. coli. This assay allowed the categorization of a diarrheagenic E. coli strain in a single reaction tube.

  12. Comparison of Droplet Digital PCR and Quantitative PCR Assays for Quantitative Detection of Xanthomonas citri Subsp. citri.

    Directory of Open Access Journals (Sweden)

    Yun Zhao

    Full Text Available Droplet digital polymerase chain reaction (ddPCR is a novel molecular biology technique providing absolute quantification of target nucleic acids without the need for an external calibrator. Despite its emerging applications in medical diagnosis, there are few reports of its use for the detection of plant pathogens. This work was designed to assess the diagnosis potential of the ddPCR for absolute quantitative detection of Xanthomonas citri subsp. citri, a quarantine plant pathogenic bacterium that causes citrus bacterial canker in susceptible Citrus species. We transferred an established quantitative PCR (qPCR assay for citrus bacterial canker diagnosis directly to the ddPCR format and compared the performance of the two methods. The qPCR assay has a broader dynamic range compared to the ddPCR assay and the ddPCR assay has a significantly higher degree of sensitivity compared to the qPCR assay. The influence of PCR inhibitors can be reduced considerably in the ddPCR assay because the collection of end-point fluorescent signals and the counting of binomial events (positive or negative droplets are associated with a Poisson algorithm. The ddPCR assay also shows lower coefficient of variation compared to the qPCR assay especially in low target concentration. The linear association of the measurements by ddPCR and qPCR assays is strong (Pearson correlation = 0.8633; P<0.001. Receiver operating characteristic analysis indicates the ddPCR methodology is a more robust approach for diagnosis of citrus bacterial canker. In summary, the results demonstrated that the ddPCR assay has the potential for the quantitative detection of X. citri subsp. citri with high precision and accuracy as compared with the results from qPCR assay. Further studies are required to evaluate and validate the value of ddPCR technology in the diagnosis of plant disease and quarantine applications.

  13. An optimized one-tube, semi-nested PCR assay for Paracoccidioides brasiliensis detection

    Directory of Open Access Journals (Sweden)

    Amanda de Faveri Pitz

    2013-12-01

    Full Text Available Introduction Herein, we report a one-tube, semi-nested-polymerase chain reaction (OTsn-PCR assay for the detection of Paracoccidioides brasiliensis. Methods We developed the OTsn-PCR assay for the detection of P. brasiliensis in clinical specimens and compared it with other PCR methods. Results The OTsn-PCR assay was positive for all clinical samples, and the detection limit was better or equivalent to the other nested or semi-nested PCR methods for P. brasiliensis detection. Conclusions The OTsn-PCR assay described in this paper has a detection limit similar to other reactions for the molecular detection of P. brasiliensis, but this approach is faster and less prone to contamination than other conventional nested or semi-nested PCR assays.

  14. Practical Prediction of Ten Common Streptococcus pneumoniae Serotypes/Serogroups in One PCR Reaction by Multiplex Ligation-Dependent Probe Amplification and Melting Curve (MLPA-MC Assay in Shenzhen, China.

    Directory of Open Access Journals (Sweden)

    Lijuan Wu

    Full Text Available Streptococcus pneumoniae has more than 95 distinct serotypes described to date. However, only certain serotypes are more likely to cause pneumococcal diseases. Thus serotype surveillance is important for vaccine formula design as well as in post-vaccine serotype shift monitor. The goal of this study was to develop a practical screening assay for ten Shenzhen China common pneumococcal serotypes/serogroups in one molecular reaction.A molecular assay, based on multiplex ligation-dependent probe amplification (MLPA and melting curve (MC analysis, was developed in an integrated approach (MLPA-MC for the detection of ten capsular serotypes/serogroups 4, 6 (6A/6B/6C/6D, 9V/9A, 14, 15F/15A, 15B/15C, 18 (18F/18A/18B/18C, 19F, 19A and 23F. We designed serotype/serogroup-specific MLPA probes and fluorescent detection probes to discriminate the different serotypes/serogroups in one molecular reaction. The three steps of MLPA-MC assay are continuous reactions in one well detected by LightCycler 480. A total of 210 S. pneumoniae isolates from our local Maternity and Child Health Hospital were randomly chosen to evaluate the assay against published multiplex PCR assays.Our results showed that 198 (94.3% of S. pneumoniae isolates were type-able by our assays and the results were in complete concordance with the published multiplex PCRs. Using the MLPA-MC assay, 96 S. pneumoniae isolates could be typed within 3 hours with limited hands-on time. This serotype/serogroup-screening assay can be easily modified or extended by modification of the serotype/serogroup-specific MLPA probes combinations according to the needs of different laboratories.We recommend use of this assay as a starting point for screening serotype/serogroup frequencies. There is a need for this assay to be combined with other molecular typing assays, like published serotype specific PCRs, or even the Quellung reaction for serotype confirmation.

  15. Development and validation of a real time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay for investigation of wild poliovirus type 1-South Asian (SOAS) strain reintroduced into Israel, 2013 to 2014.

    Science.gov (United States)

    Hindiyeh, M Y; Moran-Gilad, J; Manor, Y; Ram, D; Shulman, L M; Sofer, D; Mendelson, E

    2014-02-20

    In February 2013, wild poliovirus type 1 (WPV1) was reintroduced into southern Israel and resulted in continuous silent circulation in the highly immune population. As a part of the public health emergency response, a novel real time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay was developed, to allow for the sensitive and specific detection of the circulatingWPV1-South Asian (SOAS) strain. Specific primers and probes derived from the VP-1 region were designed, based on sequenced sewage isolates, and used to simultaneously amplify this WPV1-SOAS sequence together with bacteriophage MS-2 as internal control. High titre WPV1-SOAS stock virus was used for assay optimisation and 50 processed sewage samples collected from southern Israel and tested by reference culture based methods were used for analytical validation of the assay’s performance. The limit of detection of the multiplex qRT-PCR (SOAS/MS-2) assay was 0.1 plaque-forming unit (pfu)/reaction (20 pfu/mL) for WPV1-SOAS RNA with 100% sensitivity, specificity, positive and negative predictive values when compared to the culture based method. The turnaround time was rapid, providing results for environmental samples within 24 to 48 hours from completion of sewage processing, instead of five to seven days by culture-based analysis. Direct sewage testing by qRT-PCR assay proved to be a useful tool for rapid detection and environmental surveillance of WPV1-SOAS circulating strain during emergency response. Application of the approach for detection of WPV1-SOAS in stool samples obtained during acute flaccid paralysis (AFP) surveillance or field surveys should be further evaluated.

  16. Immuno-PCR assay for sensitive detection of proteins in real time

    Science.gov (United States)

    The immuno-PCR (IPCR) assay combines the versatility and robustness of immunoassays with the exponential signal amplification power of the polymerase chain reaction (PCR). Typically, IPCR allows a 10–1,000-fold increase in sensitivity over the analogous enzyme-linked immunosorbent assay (ELISA). Thi...

  17. The polymerase chain reaction (PCR): general methods.

    Science.gov (United States)

    Waters, Daniel L E; Shapter, Frances M

    2014-01-01

    The polymerase chain reaction (PCR) converts very low quantities of DNA into very high quantities and is the foundation of many specialized techniques of molecular biology. PCR utilizes components of the cellular machinery of mitotic cell division in vitro which respond predictably to user inputs. This chapter introduces the principles of PCR and discusses practical considerations from target sequence definition through to optimization and application.

  18. A novel nested multiplex polymerase chain reaction (PCR assay for differential detection of Entamoeba histolytica, E. moshkovskii and E. dispar DNA in stool samples

    Directory of Open Access Journals (Sweden)

    Parija Subhash C

    2007-05-01

    Full Text Available Abstract Background E. histolytica, a pathogenic amoeba, is indistinguishable in its cyst and trophozoite stages from those of non-pathogenic E. moshkovskii and E. dispar by light microscopy. We have developed a nested multiplex PCR targeting a 16S-like rRNA gene for differential detection of all the three morphologically similar forms of E. histolytica, E. moshkovskii and E. dispar simultaneously in stool samples. Results The species specific product size for E. histolytica, E. moshkovskii and E. dispar was 439, 553 and 174 bp respectively, which was clearly different for all the three Entamoeba species. The nested multiplex PCR showed a sensitivity of 94% and specificity of 100% for the demonstration of E. histolytica, E. moshkovskii and E. dispar DNA in stool samples. The PCR was positive for E. histolytica, E. moshkovskii and E. dispar in a total of 190 out of 202 stool specimens (94% sensitive that were positive for E. histolytica/E. dispar/E. moshkovskii by examination of stool by microscopy and/or culture. All the 35 negative control stool samples that were negative for E. histolytica/E. dispar/E. moshkovskii by microscopy and culture were also found negative by the nested multiplex PCR (100% specific. The result from the study shows that only 34.6% of the patient stool samples that were positive for E. histolytica/E. dispar/E. moshkovskii by examination of stool by microscopy and/or culture, were actually positive for pathogenic E. histolytica and the remaining majority of the stool samples were positive for non-pathogenic E. dispar or E. moshkovskii as demonstrated by the use of nested multiplex PCR. Conclusion The present study reports a new nested multiplex PCR strategy for species specific detection and differentiation of E. histolytica, E. dispar and E. moshkovskii DNA in stool specimens. The test is highly specific, sensitive and also rapid, providing the results within 12 hours of receiving stool specimens.

  19. Multiplex real-time PCR assay for Legionella species.

    Science.gov (United States)

    Kim, Seung Min; Jeong, Yoojung; Sohn, Jang Wook; Kim, Min Ja

    2015-12-01

    Legionella pneumophila serogroup 1 (sg1) accounts for the majority of infections in humans, but other Legionella species are also associated with human disease. In this study, a new SYBR Green I-based multiplex real-time PCR assay in a single reaction was developed to allow the rapid detection and differentiation of Legionella species by targeting specific gene sequences. Candidate target genes were selected, and primer sets were designed by referring to comparative genomic hybridization data of Legionella species. The Legionella species-specific groES primer set successfully detected all 30 Legionella strains tested. The xcpX and rfbA primers specifically detected L. pneumophila sg1-15 and L. pneumophila sg1, respectively. In addition, this assay was validated by testing clinical samples and isolates. In conclusion, this novel multiplex real-time PCR assay might be a useful diagnostic tool for the rapid detection and differentiation of Legionella species in both clinical and epidemiological studies.

  20. [Usefulness of a real-time quantitative polymerase-chain reaction (PCR) assay for the diagnosis of congenital and postnatal cytomegalovirus infection].

    Science.gov (United States)

    Reina, J; Weber, I; Riera, E; Busquets, M; Morales, C

    2014-05-01

    Cytomegalovirus (CMV) is the main virus causing congenital and postnatal infections in the pediatric population. The aim of this study is to evaluate the usefulness of a quantitative real-time PCR in the diagnosis of these infections using urine as a single sample. We studied all the urine samples of newborns (PCR (PCRc), and quantitative real-time PCR (PCRq). We analyzed 332 urine samples (270 to rule out congenital infection and 62 postnatal infections). Of the first, 22 were positive in the PCRq, 19 in the PCRc, and 17 in the culture. PCRq had a sensitivity of 100%, on comparing the culture with the rest of the techniques. Using the PCRq as a reference method, culture had a sensitivity of 77.2%, and PCRc 86.3%. In cases of postnatal infection, PCRq detected 16 positive urines, the PCRq 12, and the cell culture 10. The urines showed viral loads ranging from 2,178 to 116,641 copies/ml. The genomic amplification technique PCRq in real time was more sensitive than the other techniques evaluated. This technique should be considered as a reference (gold standard), leaving the cell culture as a second diagnostic level. The low cost and the automation of PCRq would enable the screening for CMV infection in large neonatal and postnatal populations. Copyright © 2013 Asociación Española de Pediatría. Published by Elsevier Espana. All rights reserved.

  1. DEVELOPMENT OF HOMOLOGOUS VIRAL INTERNAL CONTROLS FOR USE IN RT-PCR ASSAYS OF WATERBORNE ENTERIC VIRUSES

    Science.gov (United States)

    Enteric viruses often contaminate water sources causing frequent outbreaks of gastroenteritis. Reverse transcription-polymerase chain reaction (RT-PCR) assays are commonly used for detection of human enteric viruses in environmental and drinking water samples. RT-PCR provides ...

  2. DEVELOPMENT OF HOMOLOGOUS VIRAL INTERNAL CONTROLS FOR USE IN RT-PCR ASSAYS OF WATERBORNE ENTERIC VIRUSES

    Science.gov (United States)

    Enteric viruses often contaminate water sources causing frequent outbreaks of gastroenteritis. Reverse transcription-polymerase chain reaction (RT-PCR) assays are commonly used for detection of human enteric viruses in environmental and drinking water samples. RT-PCR provides ...

  3. Development of a real-time SYBR Green PCR assay for the rapid detection of Dermatophilus congolensis.

    Science.gov (United States)

    García, Alfredo; Martínez, Remigio; Benitez-Medina, José Manuel; Risco, David; Garcia, Waldo Luis; Rey, Joaquín; Alonso, Juan Manuel; Hermoso de Mendoza, Javier

    2013-01-01

    Methods such as real time (RT)-PCR have not been developed for the rapid detection and diagnosis of Dermatophilus (D.) congolensis infection. In the present study, a D. congolensis-specific SYBR Green RT-PCR assay was evaluated. The detection limit of the RT-PCR assay was 1 pg of DNA per PCR reaction. No cross-reaction with nucleic acids extracted from Pseudomonas aeruginosa, Mycobacterium tuberculosis, Staphylococcus aureus, or Austwickia chelonae was observed. Finally, the RT-PCR assay was used to evaluate clinical samples collected from naturally infected animals with D. congolensis. The results showed that this assay is a fast and reliable method for diagnosing dermatophilosis.

  4. Detection of bovine herpesvirus 4 glycoprotein B and thymidine kinase DNA by PCR assays in bovine milk

    NARCIS (Netherlands)

    Wellenberg, G.J.; Verstraten, E.; Belak, S.; Verschuren, S.B.E.; Rijsewijk, F.A.M.; Peshev, R.; Oirschot, van J.T.

    2001-01-01

    A polymerase chain reaction (PCR) assay was developed to detect bovine herpesvirus 4 (BHV4) glycoprotein B (gB) DNA, and a nested-PCR assay was modified for the detection of BHV4 thymidine kinase (TK) DNA in bovine milk samples. To identify false-negative PCR results, internal control templates were

  5. Detection of bovine herpesvirus 4 glycoprotein B and thymidine kinase DNA by PCR assays in bovine milk

    NARCIS (Netherlands)

    Wellenberg, G.J.; Verstraten, E.; Belak, S.; Verschuren, S.B.E.; Rijsewijk, F.A.M.; Peshev, R.; Oirschot, van J.T.

    2001-01-01

    A polymerase chain reaction (PCR) assay was developed to detect bovine herpesvirus 4 (BHV4) glycoprotein B (gB) DNA, and a nested-PCR assay was modified for the detection of BHV4 thymidine kinase (TK) DNA in bovine milk samples. To identify false-negative PCR results, internal control templates were

  6. Real-Time PCR (RT-PCR) Assays for Burkholderia mallei and B. pseudomallei

    Science.gov (United States)

    2005-10-01

    1 Real - time PCR (RT-PCR) Assays for Burkholderia mallei and B. pseudomallei Vipin K. Rastogi1, Tu-chen Cheng1, Lisa Collins1 and Jennifer Bagley2 1...A 3. DATES COVERED - 4. TITLE AND SUBTITLE Real - time PCR (RT-PCR) Assays for Burkholderia mallei and B.pseudomallei 5a. CONTRACT NUMBER 5b...risk. There is currently no real - time PCR assay for detection of both of these pathogens. Primers and probes corresponding to specific genomic regions

  7. Detection of Hepatitis B Virus DNA by Duplex Scorpion Primer-based PCR Assay

    Institute of Scientific and Technical Information of China (English)

    KONG De-Ming孔德明; SHEN Han-Xi沈含熙; MI Huai-Feng宓怀风

    2004-01-01

    The application of a new fiuorogenic probe-based PCR assay (PCR duplex scorpion primer assay) to the detection of Hepatitis B virus (HBV) DNA in human sera was described. Duplex scorpion primer is a modified variant of duplex Amplifluor, and the incorporation of a PCR stopper between probe and primer sequences improve the detection specificity and sensitivity. Combined with PCR amplification, this probe can give unambiguous positive results for the reactions initiated with more than 20 HBV molecules. In addition, the particular unimolecular probing mechanism of this probe makes the use of short target-specific probe sequence possible, which will render this probe applicable in some specific systems.

  8. Development of a real-time PCR assay for quantification of Citrobacter rodentium.

    Science.gov (United States)

    Sagaidak, Sofia; Taibi, Amel; Wen, Bijun; Comelli, Elena M

    2016-07-01

    Molecular tools to quantify Citrobacter rodentium are not available. We developed a quantitative PCR assay targeting the espB gene. This assay is specific, has a linearity range of about 6.7×10(1) to 6.7×10(6)cells/PCR reaction (92% efficiency) and a detection limit of about 10(4)cells/g wet feces.

  9. Development of a PCR assay suitable for Campylobacter spp. mass screening programs in broiler production

    DEFF Research Database (Denmark)

    Bang, Dang Duong; Pedersen, Karl; Madsen, Mogens

    2001-01-01

    culture techniques since 1998. However, using conventional culture methods is time consuming and laborious, and therefore a Polymerase Chain Reaction (PCR) Campylobacter detection assay suitable for mass screening of cloacal swab samples from broilers was developed. By comparing the PCR detection...... with conventional culture methods, significantly more samples were found positive for Campylobacter with the PCR method. The PCR method is rapid, sensitive and suitable for mass screening for Campylobacter in poultry. Using this PCR method Campylobacter can be detected within 15 h. Notably, the method can...

  10. Development of a PCR ELISA assay for the identification of Campylobacter jejuni and Campylobacter coli.

    Science.gov (United States)

    Sails, A D; Fox, A J; Bolton, F J; Wareing, D R; Greenway, D L; Borrow, R

    2001-10-01

    A polymerase chain reaction (PCR) assay was developed based on a solution-hybridization colorimetric end-point detection format (PCR ELISA) for the identification of Campylobacter jejuni and Campylobacter coli. PCR primers were designed to target a gene sequence with species-specific motifs. Five biotin-labelled probes targeted to the species-specific motifs were investigated for the detection of digoxygenin-labelled PCR products from C. jejuni and C. coli using the PCR ELISA format. Two probes were identified, one which reacts with both the C. jejuni and C. coli target sequences (probe CC2) and one probe which reacts with the C. jejuni target sequence only (probe CJ2). The specificity of the assay with the CJ2 and CC2 probes was investigated with a range of Campylobacter spp., Arcobacter spp., Helicobacter spp. and a range of unrelated organisms. The PCR ELISA assay and probes were demonstrated to be specific for C. jejuni and C. coli. The sensitivity of the PCR ELISA assay was demonstrated to be 10-100-fold more sensitive than a gel-based PCR method using the same primers. This PCR ELISA assay is sensitive, specific and significantly reduces the time needed for the identification of C. jejuni and C. coli and has the potential to facilitate early detection of these important gastro-intestinal pathogens. Copyright 2001 Academic Press.

  11. Calibrated user-friendly reverse transcriptase-PCR assay

    DEFF Research Database (Denmark)

    Bor, M V; Sørensen, B S; Rammer, P

    1998-01-01

    We report a competitive reverse transcriptase-PCR (RT-PCR) assay and a calibrated user-friendly RT-PCR assay (CURT-PCR) for epidermal growth factor receptor (EGFR) mRNA. A calibrator was prepared from isolated rat liver RNA, and the amount of EGFR mRNA was determined by competitive RT-PCR. In CUR...... and to accept or reject the results according to existing rules for quality assurance....... and the corresponding RNA internal standard. Competitive RT-PCR and CURT-PCR were used for rat liver samples from 21 different animals. Comparable results were obtained by the two methods. The imprecision of the CURT-PCR method was 8% (n = 20), and the imprecision of the traditional competitive RT-PCR was 16% (n = 17......). We conclude that the CURT-PCR method developed is suitable for routine applications such as quantitation of EGFR expression in tumor biopsies. The imprecision is relatively low. Furthermore, the use of a calibration curve makes it possible to analyze a large number of samples in one analytical run...

  12. Comparison of the performance in detection of HPV infections between the high-risk HPV genotyping real time PCR and the PCR-reverse dot blot assays.

    Science.gov (United States)

    Zhang, Lahong; Dai, Yibei; Chen, Jiahuan; Hong, Liquan; Liu, Yuhua; Ke, Qiang; Chen, Yiwen; Cai, Chengsong; Liu, Xia; Chen, Zhaojun

    2017-08-29

    A new multiplex real-time PCR assay, the high-risk HPV genotyping real time PCR assay (HR HPV RT-PCR), has been developed to detect 15 high-risk HPV types with respective viral loads. In this report, a total of 684 cervical specimens from women diagnosed with vaginitis were assessed by the HR HPV RT-PCR and the PCR reaction and reverse dot blot (PCR-RDB) assays, using a PCR-sequencing method as a reference standard. A total coincidence of 97.7% between the HR HPV RT PCR and the PCR-RDB assays was determined with a Kappa value of 0.953. The HR HPV RT PCR assay had sensitivity, specificity, and concordance rates (accuracy) of 99.7%, 99.7%, and 99.7%, respectively, as confirmed by PCR-sequencing, while the PCR-RDB assay had respective rates of 98.8%, 97.1%, and 98.0%. The overall rate of HPV infection, determined by PCR-sequencing, in women diagnosed with vaginitis was 49.85%, including 36.26% of single infection and 13.6% of multiple infections. The most common infections among the 15 high-risk HPV types in women diagnosed with vaginitis were HPV-52, HPV-16, and HPV-58, with a total detection rate of 10.23%, 7.75%, and 5.85%, respectively. We conclude that the HR HPV RT PCR assay exhibits better clinical performance than the PCR-RDB assay, and is an ideal alternative method for HPV genotyping. In addition, the HR HPV RT PCR assay provides HPV DNA viral loads, and could serve as a quantitative marker in the diagnosis and treatment of single and multiple HPV infections. © 2017 Wiley Periodicals, Inc.

  13. A Highly Sensitive Telomerase Activity Assay that Eliminates False-Negative Results Caused by PCR Inhibitors

    Directory of Open Access Journals (Sweden)

    Hidenobu Yaku

    2013-09-01

    Full Text Available An assay for telomerase activity based on asymmetric polymerase chain reaction (A-PCR on magnetic beads (MBs and subsequent application of cycling probe technology (CPT is described. In this assay, the telomerase reaction products are immobilized on MBs, which are then washed to remove PCR inhibitors that are commonly found in clinical samples. The guanine-rich sequences (5'-(TTAGGGn-3' of the telomerase reaction products are then preferentially amplified by A-PCR, and the amplified products are subsequently detected via CPT, where a probe RNA with a fluorophore at the 5' end and a quencher at the 3' end is hydrolyzed by RNase H in the presence of the target DNA. The catalyst-mediated cleavage of the probe RNA enhances fluorescence from the 5' end of the probe. The assay allowed us to successfully detect HeLa cells selectively over normal human dermal fibroblast (NHDF cells. Importantly, this selectivity produced identical results with regard to detection of HeLa cells in the absence and presence of excess NHDF cells; therefore, this assay can be used for practical clinical applications. The lower limit of detection for HeLa cells was 50 cells, which is lower than that achieved with a conventional telomeric repeat amplification protocol assay. Our assay also eliminated false-negative results caused by PCR inhibitors. Furthermore, we show that this assay is appropriate for screening among G-quadruplex ligands to find those that inhibit telomerase activity.

  14. Rapid electrochemiluminescence assays of polymerase chain reaction products.

    Science.gov (United States)

    Kenten, J H; Casadei, J; Link, J; Lupold, S; Willey, J; Powell, M; Rees, A; Massey, R

    1991-09-01

    We demonstrate the first use of an electrochemiluminescent (ECL) label, [4-(N-succimidyloxycarbonylpropyl)-4'-methyl-2,2'- bipyridine]ruthenium(II) dihexafluorophosphate (Origen label; IGEN Inc.), in DNA probe assays. This label allows rapid (less than 25 min) quantification and detection of polymerase chain reaction (PCR)-amplified products from oncogenes, viruses, and cloned genes. For the PCR, we used labeled oligonucleotide primers complementary to human papiloma virus and the Ha-ras oncogene. These samples were followed by ECL analysis or hybridization with specific, Origen-labeled oligonucleotide probes. These studies demonstrate the speed, specificity, and effectiveness of the new ECL labels, compared with 32P, for nucleic acid probe applications. We describe formats involving conventional methodologies and a new format that requires no wash step, allowing simple and rapid sample analysis. These rapid assays also reduce PCR contamination, by requiring less sample handling. Improvements in ECL detectability are currently under investigation for use in DNA probe assays without amplification.

  15. Specific PCR and real-time PCR assays for detection and quantitation of 'Candidatus Phytoplasma phoenicium'.

    Science.gov (United States)

    Jawhari, Maan; Abrahamian, Peter; Sater, Ali Abdel; Sobh, Hana; Tawidian, Patil; Abou-Jawdah, Yusuf

    2015-02-01

    Almond witches' broom (AlmWB) is a fast-spreading lethal disease of almond, peach and nectarine associated with 'Candidatus Phytoplasma phoenicium'. The development of PCR and quantitative real-time PCR (qPCR) assays for the sensitive and specific detection of the phytoplasma is of prime importance for early detection of 'Ca. P. phoenicium' and for epidemiological studies. The developed qPCR assay herein uses a TaqMan(®) probe labeled with Black Hole Quencher Plus. The specificity of the PCR and that of the qPCR detection protocols were tested on 17 phytoplasma isolates belonging to 11 phytoplasma 16S rRNA groups, on samples of almond, peach, nectarine, native plants and insects infected or uninfected with the phytoplasma. The developed assays showed high specificity against 'Ca. P. phoenicium' and no cross-reactivity against any other phytoplasma, plant or insect tested. The sensitivity of the developed PCR and qPCR assays was similar to the conventional nested PCR protocol using universal primers. The qPCR assay was further validated by quantitating AlmWB phytoplasma in different hosts, plant parts and potential insect vectors. The highest titers of 'Ca. P. phoenicium' were detected in the phloem tissues of stems and roots of almond and nectarine trees, where they averaged from 10(5) to 10(6) genomic units per nanogram of host DNA (GU/ng of DNA). The newly developed PCR and qPCR protocols are reliable, specific and sensitive methods that are easily applicable to high-throughput diagnosis of AlmWB in plants and insects and can be used for surveys of potential vectors and alternative hosts.

  16. Fast detection of Noroviruses using a real-time PCR assay and automated sample preparation

    Directory of Open Access Journals (Sweden)

    Schmid Michael

    2004-06-01

    Full Text Available Abstract Background Noroviruses (NoV have become one of the most commonly reported causative agents of large outbreaks of non-bacterial acute gastroenteritis worldwide as well as sporadic gastroenteritis in the community. Currently, reverse transcriptase polymerase chain reaction (RT-PCR assays have been implemented in NoV diagnosis, but improvements that simplify and standardize sample preparation, amplification, and detection will be further needed. The combination of automated sample preparation and real-time PCR offers such refinements. Methods We have designed a new real-time RT-PCR assay on the LightCycler (LC with SYBR Green detection and melting curve analysis (Tm to detect NoV RNA in patient stool samples. The performance of the real-time PCR assay was compared with that obtained in parallel with a commercially available enzyme immunoassay (ELISA for antigen detection by testing a panel of 52 stool samples. Additionally, in a collaborative study with the Baden-Wuerttemberg State Health office, Stuttgart (Germany the real-time PCR results were blindly assessed using a previously well-established nested PCR (nPCR as the reference method, since PCR-based techniques are now considered as the "gold standard" for NoV detection in stool specimens. Results Analysis of 52 clinical stool samples by real-time PCR yielded results that were consistent with reference nPCR results, while marked differences between the two PCR-based methods and antigen ELISA were observed. Our results indicate that PCR-based procedures are more sensitive and specific than antigen ELISA for detecting NoV in stool specimens. Conclusions The combination of automated sample preparation and real-time PCR provided reliable diagnostic results in less time than conventional RT-PCR assays. These benefits make it a valuable tool for routine laboratory practice especially in terms of rapid and appropriate outbreak-control measures in health-care facilities and other settings.

  17. Performance of MycAssay Aspergillus DNA real-time PCR assay compared with the galactomannan detection assay for the diagnosis of invasive aspergillosis from serum samples.

    Science.gov (United States)

    Danylo, Alexis; Courtemanche, Chantal; Pelletier, René; Boudreault, Alexandre A

    2014-08-01

    Invasive aspergillosis (IA) is a major problem in the immunocompromised population, and its diagnosis is difficult due to the low sensitivity of available tests. Detection of Aspergillus nucleic acid by polymerase chain reaction (PCR) in serum samples is a promising diagnostic tool; however, use of multiple "in-house" methods precludes standardization. The first commercial PCR assay, MycAssay Aspergillus (Myconostica, Ltd), became available recently, and its performance in the diagnosis of IA was evaluated and compared with the galactomannan (GM) assay. Serum samples obtained from patients with hematological cancer were tested retrospectively with MycAssay Aspergillus PCR. Per-episode and per-test analyses were undertaken with 146 sera from 35 hematological patients. Sixteen patients had proven or probable IA and 19 had possible or no IA. In per-episode analysis, MycAssay Aspergillus had a sensitivity of 43.8% (95% confidence interval [CI], 19.8%-70.1%) and a specificity of 63.2% (95% CI, 38.4%-83.7%) for IA diagnosis. In per-test analyses, MycAssay Aspergillus had a lower specificity than the GM assay (83.3% vs. 93.1%, P = 0.04). The addition of PCR to routine clinical practice would have permitted the diagnosis of one additional probable IA in our cohort. Use of PCR instead of GM assay would have delayed the diagnosis in two cases. Aspergillus DNA detection by PCR with serum specimens using MycAssay showed a lower specificity than the GM assay and was associated with a low sensitivity for IA diagnosis. More studies are needed to determine the exact role of MycAssay in IA diagnosis in patients with hematological malignancy.

  18. Differential susceptibility of PCR reactions to inhibitors: an important and unrecognised phenomenon

    Directory of Open Access Journals (Sweden)

    Miller Robert F

    2008-08-01

    Full Text Available Abstract Background PCR inhibition by nucleic acid extracts is a well known yet poorly described phenomenon. Inhibition assessment generally depends on the assumption that inhibitors affect all PCR reactions to the same extent; i.e. that the reaction of interest and the control reaction are equally susceptible to inhibition. To test this assumption we performed inhibition assessment on DNA extracts from human urine samples, fresh urine and EDTA using different PCR reactions. Results When copurified inhibitors were assessed using two different PCR reactions one reaction appeared to be inhibited whilst the other was not. Further experiments using various concentrations of unextracted urine to inhibit six different PCR reactions revealed that susceptibility to inhibition was highly variable between reactions. Similar results were obtained using EDTA as the PCR inhibitor. We could find no obvious explanation why one reaction should be more susceptible to inhibition than another, although a possible association with amplicon GC content was noted. Conclusion These findings have serious implications for all PCR-based gene expression studies, including the relatively new PCR array method, and for both qualitative and quantitative PCR-based molecular diagnostic assays, suggesting that careful consideration should be given to inhibition compatibility when conducting PCR analyses. We have demonstrated unequivocally that it is not safe to assume that different PCR reactions are equally susceptible to inhibition by substances co-purified in nucleic acid extracts.

  19. The development of a qualitative real-time RT-PCR assay for the detection of hepatitis C virus

    NARCIS (Netherlands)

    Clancy, A.; Crowley, B.; Niesters, H.; Herra, C.

    2008-01-01

    Real-time polymerase chain reaction (PCR) represents a favourable option for the detection of hepatitis C virus (HCV). A real-time reverse transcriptase PCR (RT-PCR) assay was developed as a qualitative diagnostic screening method for the detection of HCV using the ABI PRISM 7500 Sequence Detection

  20. The development of a qualitative real-time RT-PCR assay for the detection of hepatitis C virus

    NARCIS (Netherlands)

    Clancy, A.; Crowley, B.; Niesters, H.; Herra, C.

    2008-01-01

    Real-time polymerase chain reaction (PCR) represents a favourable option for the detection of hepatitis C virus (HCV). A real-time reverse transcriptase PCR (RT-PCR) assay was developed as a qualitative diagnostic screening method for the detection of HCV using the ABI PRISM 7500 Sequence Detection

  1. Calibration-free assays on standard real-time PCR devices

    Science.gov (United States)

    Debski, Pawel R.; Gewartowski, Kamil; Bajer, Seweryn; Garstecki, Piotr

    2017-03-01

    Quantitative Polymerase Chain Reaction (qPCR) is one of central techniques in molecular biology and important tool in medical diagnostics. While being a golden standard qPCR techniques depend on reference measurements and are susceptible to large errors caused by even small changes of reaction efficiency or conditions that are typically not marked by decreased precision. Digital PCR (dPCR) technologies should alleviate the need for calibration by providing absolute quantitation using binary (yes/no) signals from partitions provided that the basic assumption of amplification a single target molecule into a positive signal is met. Still, the access to digital techniques is limited because they require new instruments. We show an analog-digital method that can be executed on standard (real-time) qPCR devices. It benefits from real-time readout, providing calibration-free assessment. The method combines advantages of qPCR and dPCR and bypasses their drawbacks. The protocols provide for small simplified partitioning that can be fitted within standard well plate format. We demonstrate that with the use of synergistic assay design standard qPCR devices are capable of absolute quantitation when normal qPCR protocols fail to provide accurate estimates. We list practical recipes how to design assays for required parameters, and how to analyze signals to estimate concentration.

  2. Gold nanoparticle-based RT-PCR and real-time quantitative RT-PCR assays for detection of Japanese encephalitis virus

    Science.gov (United States)

    Huang, Su-Hua; Yang, Tsuey-Ching; Tsai, Ming-Hong; Tsai, I.-Shou; Lu, Huang-Chih; Chuang, Pei-Hsin; Wan, Lei; Lin, Ying-Ju; Lai, Chih-Ho; Lin, Cheng-Wen

    2008-10-01

    Virus isolation and antibody detection are routinely used for diagnosis of Japanese encephalitis virus (JEV) infection, but the low level of transient viremia in some JE patients makes JEV isolation from clinical and surveillance samples very difficult. We describe the use of gold nanoparticle-based RT-PCR and real-time quantitative RT-PCR assays for detection of JEV from its RNA genome. We tested the effect of gold nanoparticles on four different PCR systems, including conventional PCR, reverse-transcription PCR (RT-PCR), and SYBR green real-time PCR and RT-PCR assays for diagnosis in the acute phase of JEV infection. Gold nanoparticles increased the amplification yield of the PCR product and shortened the PCR time compared to the conventional reaction. In addition, nanogold-based real-time RT-PCR showed a linear relationship between Ct and template amount using ten-fold dilutions of JEV. The nanogold-based RT-PCR and real-time quantitative RT-PCR assays were able to detect low levels (1-10 000 copies) of the JEV RNA genomes extracted from culture medium or whole blood, providing early diagnostic tools for the detection of low-level viremia in the acute-phase infection. The assays described here were simple, sensitive, and rapid approaches for detection and quantitation of JEV in tissue cultured samples as well as clinical samples.

  3. Gold nanoparticle-based RT-PCR and real-time quantitative RT-PCR assays for detection of Japanese encephalitis virus

    Energy Technology Data Exchange (ETDEWEB)

    Huang, S-H; Tsai, M-H; Lin, C-W [Department of Biotechnology, College of Health Science, Asia University, Wufeng, Taichung, Taiwan (China); Yang, T-C; Chuang, P-H [Department of Medical Laboratory Science and Biotechnology, China Medical University, Taichung, Taiwan (China); Tsai, I-S; Lu, H-C [Nanotechnology Research Center, Feng Chia University, Taichung, Taiwan (China); Wan Lei; Lin, Y-J [Department of Medical Genetics and Medical Research, China Medical University Hospital, Taichung, Taiwan (China); Lai, C-H [Department of Microbiology and Immunology, China Medical University, Taichung, Taiwan (China)], E-mail: cwlin@mail.cmu.edu.tw

    2008-10-08

    Virus isolation and antibody detection are routinely used for diagnosis of Japanese encephalitis virus (JEV) infection, but the low level of transient viremia in some JE patients makes JEV isolation from clinical and surveillance samples very difficult. We describe the use of gold nanoparticle-based RT-PCR and real-time quantitative RT-PCR assays for detection of JEV from its RNA genome. We tested the effect of gold nanoparticles on four different PCR systems, including conventional PCR, reverse-transcription PCR (RT-PCR), and SYBR green real-time PCR and RT-PCR assays for diagnosis in the acute phase of JEV infection. Gold nanoparticles increased the amplification yield of the PCR product and shortened the PCR time compared to the conventional reaction. In addition, nanogold-based real-time RT-PCR showed a linear relationship between Ct and template amount using ten-fold dilutions of JEV. The nanogold-based RT-PCR and real-time quantitative RT-PCR assays were able to detect low levels (1-10 000 copies) of the JEV RNA genomes extracted from culture medium or whole blood, providing early diagnostic tools for the detection of low-level viremia in the acute-phase infection. The assays described here were simple, sensitive, and rapid approaches for detection and quantitation of JEV in tissue cultured samples as well as clinical samples.

  4. Final Report Nucleic Acid System - Hybrid PCR and Multiplex Assay Project Phase 2

    Energy Technology Data Exchange (ETDEWEB)

    Koopman, R P; Langlois, R G; Nasarabadi, S; Benett, W J; Colston, B W; Johnson, D C; Brown, S B; Stratton, P L; Milanovich, F P

    2002-04-17

    This report covers phase 2 (year 2) of the Nucleic Acid System--Hybrid PCR and Multiplex Assay project. The objective of the project is to reduce to practice the detection and identification of biological warfare pathogens by the nucleic acid recognition technique of PCR (polymerase chain reaction) in a multiplex mode using flow cytometry. The Hybrid instrument consists of a flow-through PCR module capable of handling a multiplexed PCR assay, a hybridizing module capable of hybridizing multiplexed PCR amplicons and beads, and a flow cytometer module for bead-based identification, all controlled by a single computer. Multiplex immunoassay using bead-based Luminex flow cytometry is available, allowing rapid screening for many agents. PCR is highly specific and complements and verifies immunoassay. It can also be multiplexed and detection provided using the bead-based Luminex flow cytometer. This approach allows full access to the speed and 100-fold multiplex capability of flow cytometry for rapid screening as well as the accuracy and specificity of PCR. This project has two principal activities: (1) Design, build and test a prototype hybrid PCR/flow cytometer with the basic capabilities for rapid, broad spectrum detection and identification, and (2) Develop and evaluate multiplex flow analysis assay protocols and reagents for the simultaneous detection of PCR products. This project requires not only building operationally functional instrumentation but also developing the chemical assays for detection of priority pathogens. This involves development and evaluation of multiplex flow analysis assay protocols and reagents for the simultaneous detection of PCR products.

  5. Automated 5 ' nuclease PCR assay for identification of Salmonella enterica

    DEFF Research Database (Denmark)

    Hoorfar, Jeffrey; Ahrens, Peter; Rådström, P.

    2000-01-01

    A simple and ready-to-go test based on a 5' nuclease (TaqMan) PCR technique was developed for identification of presumptive Salmonella enterica isolates. The results were compared with those of conventional methods. The TaqMan assay was evaluated for its ability to accurately detect 210 S. enterica...... isolates, including 100 problematic "rough" isolates. An internal positive control was designed to use the same Salmonella primers for amplification of a spiked nonrelevant template (116 bp) in the sample tube. The PCR test correctly identified all the Salmonella strains by resulting in positive end...... Salmonella strains tested resulted in positive FAM and TET signals. In addition, it was found that the complete PCR mixture, predispensed in microwell plates, could be stored for up to 3 months at -20 degrees C, Thus, the diagnostic TaqMan assay developed can be a useful and simple alternative method...

  6. A pentaplex PCR assay for detection and characterization of Vibrio vulnificus and Vibrio parahaemolyticus isolates.

    Science.gov (United States)

    Bhattacharyya, N; Hou, A

    2013-09-01

    Vibrio parahaemolyticus and Vibrio vulnificus are the leading causes of seafood-related illnesses and also can cause wound infections. These bacteria often co-exist in marine and estuarine environments. However, there have been no reported protocols that can detect and characterize (i.e. pathogenic or nonpathogenic) them in a single PCR. In this study, we developed a pPCR assay with a combination of two species-specific and three pathogenic-specific PCR primers to simultaneously detect virulent (viuB in V. vulnificus and tdh/trh in V. parahaemolyticus) and nonvirulent (vvhA in V. vulnificus and tlh in V. parahaemolyticus) markers of the two species in bacterial isolates. The assay was validated by three methods. First, the pPCR was used to characterize 300 bacterial isolates consisting of seven reference strains and 293 environmental strains isolated from the Gulf of Mexico water. Results were compared with characterizations based on single-gene PCR amplifications and previously published multiplex PCR protocols. Second, 51 isolates characterized with the pPCR were analysed by 16S rRNA sequencing to confirm any false-negative/positive reaction. Finally, the effectiveness of the assay for heterogeneous bacterial samples was validated. The pPCR correctly characterized isolates from the Gulf with an efficiency of 96·6-98·7%.

  7. Simultaneous Detection of Three Arboviruses Using a Triplex RT-PCR Enzyme Hybridization Assay

    Institute of Scientific and Technical Information of China (English)

    Dan Dong; Shi-hong Fu; Li-hua Wang; Zhi Lv; Tai-yuan Li; Guo-dong Liang

    2012-01-01

    Arboviruses represent a serious problem to public health and agriculture worldwide.Fast,accurate identification of the viral agents of arbovirus-associated disease is essential for epidemiological surveillance and laboratory investigation.We developed a cost-effective,rapid,and highly sensitive one-step "triplex RT-PCR enzyme hybridization"assay for simultaneous detections of Japanese Encephallitis virus (JEV,Flaviviridae)Getah virus (GETV,Togaviridae),and Tahyna virus (TAHV,Bunyaviridae) using three pairs of primers to amplify three target sequences in one RT-PCR reaction.The analytical sensitivity of this assay was 1 PFU/mL for JEV,10PFU/mL for GETV,and 10 PFU/mL for TAHV.This assay is significantly more rapid and less expensive than the traditional serological detection and single RT-PCR reaction methods.When “triplex RT-PCR enzyme hybridization” was applied to 29 cerebrospinal fluid(CSF)samples that were JEV-positive by normal RT-PCR assay,all samples were strongly positive for JEV,but negative for GETV and TAHV,demonstrating a good sensitivity,specificity,and performance at CSF specimen detection.

  8. Development of a real-time SYBR Green PCR assay for the rapid detection of Dermatophilus congolensis

    Science.gov (United States)

    Martínez, Remigio; Benitez-Medina, José Manuel; Risco, David; García, Waldo Luis; Rey, Joaquín; Alonso, Juan Manuel; de Mendoza, Javier Hermoso

    2013-01-01

    Methods such as real time (RT)-PCR have not been developed for the rapid detection and diagnosis of Dermatophilus (D.) congolensis infection. In the present study, a D. congolensis-specific SYBR Green RT-PCR assay was evaluated. The detection limit of the RT-PCR assay was 1 pg of DNA per PCR reaction. No cross-reaction with nucleic acids extracted from Pseudomonas aeruginosa, Mycobacterium tuberculosis, Staphylococcus aureus, or Austwickia chelonae was observed. Finally, the RT-PCR assay was used to evaluate clinical samples collected from naturally infected animals with D. congolensis. The results showed that this assay is a fast and reliable method for diagnosing dermatophilosis. PMID:23820221

  9. Novel PCR assay for determining the genetic sex of mice.

    Science.gov (United States)

    McFarlane, L; Truong, V; Palmer, J S; Wilhelm, D

    2013-01-01

    A number of studies require the determination of the genetic sex of mouse embryos before sexual differentiation and/or of mutant mice that display partial or complete sex reversal. The majority of current methods for sexing by PCR involve multiplexing of 2 primer pairs. We have developed a novel sexing PCR using a single primer pair that amplifies fragments from the X and the Y chromosome with a clear size difference between the respective amplicons. This assay provides a rapid and reliable method to identify the genetic sex of mice across different mouse strains.

  10. Polymerase chain reaction assay for avian polyomavirus.

    OpenAIRE

    Phalen, D.N.; Wilson, V G; Graham, D L

    1991-01-01

    A polymerase chain reaction assay was developed for detection of budgerigar fledgling disease virus (BFDV). The assay used a single set of primers complementary to sequences located in the putative coding region for the BFDV VP1 gene. The observed amplification product had the expected size of 550 bp and was confirmed to derive from BFDV DNA by its restriction digestion pattern. This assay was specific for BFDV and highly sensitive, being able to detect as few as 20 copies of the virus. By us...

  11. Techniques for minimizing the effects of PCR inhibitors in the chytridiomycosis assay.

    Science.gov (United States)

    Kosch, T A; Summers, K

    2013-03-01

    Chytridiomycosis is an amphibian disease of global conservation concern that is caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd). Since the discovery of Bd in 1998, several methods have been used for detection of Bd; among these polymerase chain reaction (PCR) from skin swabs is accepted as the best method due to its noninvasiveness, high sensitivity and ease of use. However, PCR is not without problems - to be successful, this technique is dependent upon the presence of nondegraded DNA template and reaction contents that are free from inhibitors. Here, we report on an investigation of several techniques aimed at improving the reliability of the Bd PCR assay by minimizing the effects of humic acid (HA), a potent PCR inhibitor. We compared the effectiveness of four DNA extraction kits (DNeasy, QIAamp DNA Stool, PowerLyzer Power Soil and PrepMan Ultra) and four PCR methods (Amplitaq Gold, bovine serum albumin, PowerClean DNA Clean-up and inhibitor resistant Taq Polymerase). The results of this and previous studies indicate that chytridiomycosis studies that use PCR methods for disease detection may be significantly underestimating the occurrence of Bd. Our results suggest that to minimize the inhibitory effects of HA, DNeasy should be used for sample DNA extraction and Amplitaq Gold with bovine serum albumin should be used for the Bd PCR assay. We also outline protocols tested, show the results of our methods comparisons and discuss the pros and cons of each method.

  12. A Trio of Human Molecular Genetics PCR Assays

    Science.gov (United States)

    Reinking, Jeffrey L.; Waldo, Jennifer T.; Dinsmore, Jannett

    2013-01-01

    This laboratory exercise demonstrates three different analytical forms of the polymerase chain reaction (PCR) that allow students to genotype themselves at four different loci. Here, we present protocols to allow students to a) genotype a non-coding polymorphic Variable Number of Tandem Repeat (VNTR) locus on human chromosome 5 using conventional…

  13. A Trio of Human Molecular Genetics PCR Assays

    Science.gov (United States)

    Reinking, Jeffrey L.; Waldo, Jennifer T.; Dinsmore, Jannett

    2013-01-01

    This laboratory exercise demonstrates three different analytical forms of the polymerase chain reaction (PCR) that allow students to genotype themselves at four different loci. Here, we present protocols to allow students to a) genotype a non-coding polymorphic Variable Number of Tandem Repeat (VNTR) locus on human chromosome 5 using conventional…

  14. A quantitative real-time RT-PCR assay for mature C. albicans biofilms

    Directory of Open Access Journals (Sweden)

    Dongari-Bagtzoglou Anna

    2011-05-01

    Full Text Available Abstract Background Fungal biofilms are more resistant to anti-fungal drugs than organisms in planktonic form. Traditionally, susceptibility of biofilms to anti-fungal agents has been measured using the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl-2H-tetrazolium-5-carboxyanilide (XTT assay, which measures the ability of metabolically active cells to convert tetrazolium dyes into colored formazan derivatives. However, this assay has limitations when applied to high C. albicans cell densities because substrate concentration and solubility are limiting factors in the reaction. Because mature biofilms are composed of high cell density populations we sought to develop a quantitative real-time RT-PCR assay (qRT-PCR that could accurately assess mature biofilm changes in response to a wide variety of anti-fungal agents, including host immune cells. Results The XTT and qRT-PCR assays were in good agreement when biofilm changes were measured in planktonic cultures or in early biofilms which contain lower cell densities. However, the real-time qRT-PCR assay could also accurately quantify small-medium size changes in mature biofilms caused by mechanical biomass reduction, antifungal drugs or immune effector cells, that were not accurately quantifiable with the XTT assay. Conclusions We conclude that the qRT-PCR assay is more accurate than the XTT assay when measuring small-medium size effects of anti-fungal agents against mature biofilms. This assay is also more appropriate when mature biofilm susceptibility to anti-fungal agents is tested on complex biological surfaces, such as organotypic cultures.

  15. Ultrasensitive Detection of RNA and DNA Viruses Simultaneously Using Duplex UNDP-PCR Assay

    Science.gov (United States)

    Wang, Zengguo; Zhang, Xiujuan; Zhao, Xiaomin; Du, Qian; Chang, Lingling; Tong, Dewen

    2015-01-01

    Mixed infection of multiple viruses is common in modern intensive pig rearing. However, there are no methods available to detect DNA and RNA viruses in the same reaction system in preclinical level. In this study, we aimed to develop a duplex ultrasensitive nanoparticle DNA probe-based PCR assay (duplex UNDP-PCR) that was able to simultaneously detect DNA and RNA viruses in the same reaction system. PCV2 and TGEV are selected as representatives of the two different types of viruses. PCV2 DNA and TGEV RNA were simultaneously released from the serum sample by boiling with lysis buffer, then magnetic beads and gold nanoparticles coated with single and/or duplex specific probes for TGEV and PCV2 were added to form a sandwich-like complex with nucleic acids released from viruses. After magnetic separation, DNA barcodes specific for PCV2 and TGEV were eluted using DTT and characterized by specific PCR assay for specific DNA barcodes subsequently. The duplex UNDP-PCR showed similar sensitivity as that of single UNDP-PCR and was able to detect 20 copies each of PCV2 and TGEV in the serum, showing approximately 250-fold more sensitivity than conventional duplex PCR/RT-PCR assays. No cross-reaction was observed with other viruses. The positive detection rate of single MMPs- and duplex MMPs-based duplex UNDP-PCR was identical, with 29.6% for PCV2, 9.3% for TGEV and 3.7% for PCV2 and TGEV mixed infection. This duplex UNDP-PCR assay could detect TGEV (RNA virus) and PCV2 (DNA virus) from large-scale serum samples simultaneously without the need for DNA/RNA extraction, purification and reverse transcription of RNA, and showed a significantly increased positive detection rate for PCV2 (29%) and TGEV (11.7%) preclinical infection than conventional duplex PCR/RT-PCR. Therefore, the established duplex UNDP-PCR is a rapid and economical detection method, exhibiting high sensitivity, specificity and reproducibility. PMID:26544710

  16. Ultrasensitive Detection of RNA and DNA Viruses Simultaneously Using Duplex UNDP-PCR Assay.

    Directory of Open Access Journals (Sweden)

    Yong Huang

    Full Text Available Mixed infection of multiple viruses is common in modern intensive pig rearing. However, there are no methods available to detect DNA and RNA viruses in the same reaction system in preclinical level. In this study, we aimed to develop a duplex ultrasensitive nanoparticle DNA probe-based PCR assay (duplex UNDP-PCR that was able to simultaneously detect DNA and RNA viruses in the same reaction system. PCV2 and TGEV are selected as representatives of the two different types of viruses. PCV2 DNA and TGEV RNA were simultaneously released from the serum sample by boiling with lysis buffer, then magnetic beads and gold nanoparticles coated with single and/or duplex specific probes for TGEV and PCV2 were added to form a sandwich-like complex with nucleic acids released from viruses. After magnetic separation, DNA barcodes specific for PCV2 and TGEV were eluted using DTT and characterized by specific PCR assay for specific DNA barcodes subsequently. The duplex UNDP-PCR showed similar sensitivity as that of single UNDP-PCR and was able to detect 20 copies each of PCV2 and TGEV in the serum, showing approximately 250-fold more sensitivity than conventional duplex PCR/RT-PCR assays. No cross-reaction was observed with other viruses. The positive detection rate of single MMPs- and duplex MMPs-based duplex UNDP-PCR was identical, with 29.6% for PCV2, 9.3% for TGEV and 3.7% for PCV2 and TGEV mixed infection. This duplex UNDP-PCR assay could detect TGEV (RNA virus and PCV2 (DNA virus from large-scale serum samples simultaneously without the need for DNA/RNA extraction, purification and reverse transcription of RNA, and showed a significantly increased positive detection rate for PCV2 (29% and TGEV (11.7% preclinical infection than conventional duplex PCR/RT-PCR. Therefore, the established duplex UNDP-PCR is a rapid and economical detection method, exhibiting high sensitivity, specificity and reproducibility.

  17. Quantitative CrAssphage PCR Assays for Human Fecal ...

    Science.gov (United States)

    Environmental waters are monitored for fecal pollution to protect public health and water resources. Traditionally, general fecal indicator bacteria are used; however, they cannot distinguish human fecal waste from pollution from other animals. Recently, a novel bacteriophage, crAssphage, was discovered by metagenomic data mining and reported to be abundant in and closely associated with human fecal waste. To confirm bioinformatic predictions, 384 primer sets were designed along the length of the crAssphage genome. Based upon initial screening, two novel crAssphage qPCR assays (CPQ_056 and CPQ_064) were designed and evaluated in reference fecal samples and water matrices. The assays exhibited high specificities (98.6%) when tested against a large animal fecal reference library and were highly abundant in raw sewage and sewage impacted water samples. In addition, CPQ_056 and CPQ_064 assay performance was compared to HF183/BacR287 and HumM2 methods in paired experiments. Findings confirm viral crAssphage qPCR assays perform at a similar level to well established bacterial human-associated fecal source identification technologies. These new viral based assays could become important water quality management and research tools. To inform the public.

  18. Development and Application of Nested PCR Assay for Detection of Dairy Cattle-Derived Cyclospora sp.

    Institute of Scientific and Technical Information of China (English)

    XIAO Shu-min; LI Guo-qing; LI Wei-hua; ZHOU Rong-qiong; YANG Jian-wei

    2007-01-01

    To develop a nested PCR assay for the detection of cattle-derived Cyclospora sp.,two pairs of primers were designed on the basis of the cattle-derived Cyclospora sp.sequences.These primers selectively amplified a 168-bp DNA fragment of the 18S rRNA gene of cattle-derived Cyclospora sp.With these primers,a nested PCR assay for the detection of cattlederived Cyclospora sp.was developed.The nested PCR assay was specific and there is no cross-reaction with other parasites,such as Eimeria spp.,Cryptosporidium spp.,Giardia sp.,Toxoplasma sp.,Trichuris sp.and cattle ciliate.The assay was able to detect as low as 2.85 x 10-2 fg of the control positive DNA.The results of the detection of clinical samples indicated that the assay coincided with microscopic examination.The results show that the nested PCR assay will be an effective tool for the detection of Cyclospora sp.in cattle feces.

  19. Detection of Aspergillus flavus and A. fumigatus in Bronchoalveolar Lavage Specimens of Hematopoietic Stem Cell Transplants and Hematological Malignancies Patients by Real-Time Polymerase Chain Reaction, Nested PCR and Mycological Assays.

    Science.gov (United States)

    Zarrinfar, Hossein; Mirhendi, Hossein; Fata, Abdolmajid; Khodadadi, Hossein; Kordbacheh, Parivash

    2015-01-01

    Pulmonary aspergillosis (PA) is one of the most serious complications in immunocompromised patients, in particular among hematopoietic stem cell transplants (HSCT) and patients with hematological malignancies. The current study aimed to evaluate the incidence of PA and utility of molecular methods in HSCT and patients with hematological malignancies, four methods including direct examination, culture, nested polymerase chain reaction (PCR) and real-time PCR were performed on bronchoalveolar lavage (BAL) specimens in Tehran, Iran. During 16 months, 46 BAL specimens were obtained from individuals with allogeneic HSCT (n = 18) and patients with hematological malignancies (n = 28). Direct wet mounts with 20% potassium hydroxide (KOH) and culture on mycological media were performed. The molecular detection of Aspergillus fumigatus and A. flavus was done by amplifying the conserved sequences of internal transcribed spacer 1 (ITS1) ribosomal DNA by nested-PCR and the β-tubulin gene by TaqMan real-time PCR. Seven (15.2%) out of 46 specimens were positive in direct examination and showed branched septate hyphae; 11 (23.9%) had positive culture including eight (72.7%) A. flavus and three (27.3%) A. fumigatus; 22 (47.8%) had positive nested-PCR and eight (17.4%) had positive real-time PCR. The incidence of invasive pulmonary aspergillosis (IPA) in these patients included proven IPA in 1 (2.2%), probable IPA in 10 (21.7%), possible IPA in 19 (41.3%) and not IPA in 16 cases (34.8%). The incidence of IPA in allogeneic HSCT and patients with hematological malignancies was relatively high and A. flavus was the most common cause of PA. As molecular methods had higher sensitivity, it may be useful as screening methods in HSCT and patients with hematological malignancies, or to determine when empirical antifungal therapy can be withheld.

  20. Development of a real-time PCR assay for detection and quantification of Anaplasma ovis infection.

    Science.gov (United States)

    Chi, Q; Liu, Z; Li, Y; Yang, J; Chen, Z; Yue, C; Luo, J; Yin, H

    2013-11-01

    Anaplasma ovis is a tick-borne intra-erythrocytic rickettsial pathogen of small ruminants. Real-time PCR possesses merits of rapidity, accuracy, reliability, automation and ease of standardization, but has not been used for detection of A. ovis, to the best of our knowledge. In this study, a real-time PCR assay was developed for detection and quantification of A. ovis. Species-specific primers and TaqMan probe were designed based on the gltA gene. No cross-reactions were observed with Anaplasma marginale, Anaplasma bovis, Anaplasma phagocytophilum, Borrelia burgdorferi s. l., Chlamydia psittaci, Mycoplasma mycoides, Theileria luwenshuni and Babesia sp. Xinjiang isolate. Analytic sensitivity results revealed that real-time PCR could detect as few as 10 copies of the gltA gene. The performance of real-time PCR was assessed by testing 254 blood samples from goats and comparing with the results from conventional PCR. This demonstrated that the real-time PCR assay was significantly more sensitive than conventional PCR. Our results indicated that real-time PCR is a useful approach for detecting A. ovis infections and has potential as an alternative tool for ecological and epidemiological surveillance of ovine anaplasmosis. © 2013 Blackwell Verlag GmbH.

  1. Real-time PCR assay for rapid qualitative and quantitative detection of Entamoeba histolytica.

    Science.gov (United States)

    Orosz, Erika; Perkátai, Katalin; Kapusinszky, Beatrix; Farkas, Agnes; Kucsera, István

    2012-12-01

    Simple real-time PCR assay with one set of primer and probe for rapid, sensitive qualitative and quantitative detection of Entamoeba histolytica has been used. Consensus sequences were used to amplify a species-specific region of the 16S rRNA gene, and fluorescence resonance energy transfer hybridization probes were used for detection in a LightCycler platform (Roche). The anchor probe sequence was designed to be a perfect match for the 16S rRNA gene of Entamoeba species, while the acceptor probe sequence was designed for Entamoeba histolytica, which allowed differentiation. The performed characteristics of the real-time PCR assay were compared with ELISA antigen and microscopical detection from 77 samples of individuals with suspected clinical diagnosis of imported E. histolytica infection. Stool and liver abscess pus samples were examined with analytical sensitivity of 5 parasites per PCR reaction. The melting curve means Tms (standard deviation) in clinical isolates were 54°C. The real-time assay was 100% sensitive and specific for differentiation of Entamoeba histolytica, compared with conventional ELISA or microscopy. This real-time PCR assay with melting curve analysis is rapid, and specific for the detection and differentiation of Entamoeba histolytica. The suitability for routine use of this assay in clinical diagnostic laboratories is discussed.

  2. Temperature Switch PCR (TSP: Robust assay design for reliable amplification and genotyping of SNPs

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    Mather Diane E

    2009-12-01

    Full Text Available Abstract Background Many research and diagnostic applications rely upon the assay of individual single nucleotide polymorphisms (SNPs. Thus, methods to improve the speed and efficiency for single-marker SNP genotyping are highly desirable. Here, we describe the method of temperature-switch PCR (TSP, a biphasic four-primer PCR system with a universal primer design that permits amplification of the target locus in the first phase of thermal cycling before switching to the detection of the alleles. TSP can simplify assay design for a range of commonly used single-marker SNP genotyping methods, and reduce the requirement for individual assay optimization and operator expertise in the deployment of SNP assays. Results We demonstrate the utility of TSP for the rapid construction of robust and convenient endpoint SNP genotyping assays based on allele-specific PCR and high resolution melt analysis by generating a total of 11,232 data points. The TSP assays were performed under standardised reaction conditions, requiring minimal optimization of individual assays. High genotyping accuracy was verified by 100% concordance of TSP genotypes in a blinded study with an independent genotyping method. Conclusion Theoretically, TSP can be directly incorporated into the design of assays for most current single-marker SNP genotyping methods. TSP provides several technological advances for single-marker SNP genotyping including simplified assay design and development, increased assay specificity and genotyping accuracy, and opportunities for assay automation. By reducing the requirement for operator expertise, TSP provides opportunities to deploy a wider range of single-marker SNP genotyping methods in the laboratory. TSP has broad applications and can be deployed in any animal and plant species.

  3. Detection limits and cost comparisons of human- and gull-associated conventional and quantitative PCR assays in artificial and environmental waters

    Science.gov (United States)

    Riedel, Timothy E.; Zimmer-Faust, Amity G.; Thulsiraj, Vanessa; Madi, Tania; Hanley, Kaitlyn T.; Ebentier, Darcy L.; Byappanahalli, Muruleedhara N.; Layton, Blythe; Raith, Meredith; Boehm, Alexandria B.; Griffith, John F.; Holden, Patricia A.; Shanks, Orin C.; Weisberg, Stephen B.; Jay, Jennifer A.

    2014-01-01

    Some molecular methods for tracking fecal pollution in environmental waters have both PCR and quantitative PCR (qPCR) assays available for use. To assist managers in deciding whether to implement newer qPCR techniques in routine monitoring programs, we compared detection limits (LODs) and costs of PCR and qPCR assays with identical targets that are relevant to beach water quality assessment. For human-associated assays targeting Bacteroidales HF183 genetic marker, qPCR LODs were 70 times lower and there was no effect of target matrix (artificial freshwater, environmental creek water, and environmental marine water) on PCR or qPCR LODs. The PCR startup and annual costs were the lowest, while the per reaction cost was 62% lower than the Taqman based qPCR and 180% higher than the SYBR based qPCR. For gull-associated assays, there was no significant difference between PCR and qPCR LODs, target matrix did not effect PCR or qPCR LODs, and PCR startup, annual, and per reaction costs were lower. Upgrading to qPCR involves greater startup and annual costs, but this increase may be justified in the case of the human-associated assays with lower detection limits and reduced cost per sample.

  4. Molecular surveillance of true nontypeable Haemophilus influenzae: an evaluation of PCR screening assays.

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    Michael J Binks

    Full Text Available BACKGROUND: Unambiguous identification of nontypeable Haemophilus influenzae (NTHi is not possible by conventional microbiology. Molecular characterisation of phenotypically defined NTHi isolates suggests that up to 40% are Haemophilus haemolyticus (Hh; however, the genetic similarity of NTHi and Hh limits the power of simple molecular techniques such as PCR for species discrimination. METHODOLOGY/PRINCIPAL FINDINGS: Here we assess the ability of previously published and novel PCR-based assays to identify true NTHi. Sixty phenotypic NTHi isolates, classified by a dual 16S rRNA gene PCR algorithm as NTHi (n = 22, Hh (n = 27 or equivocal (n = 11, were further characterised by sequencing of the 16S rRNA and recA genes then interrogated by PCR-based assays targeting the omp P2, omp P6, lgtC, hpd, 16S rRNA, fucK and iga genes. The sequencing data and PCR results were used to define NTHi for this study. Two hpd real time PCR assays (hpd#1 and hpd#3 and the conventional iga PCR assay were equally efficient at differentiating study-defined NTHi from Hh, each with a receiver operator characteristic curve area of 0.90 [0.83; 0.98]. The hpd#1 and hpd#3 assays were completely specific against a panel of common respiratory bacteria, unlike the iga PCR, and the hpd#3 assay was able to detect below 10 copies per reaction. CONCLUSIONS/SIGNIFICANCE: Our data suggest an evolutionary continuum between NTHi and Hh and therefore no single gene target could completely differentiate NTHi from Hh. The hpd#3 real time PCR assay proved to be the superior method for discrimination of NTHi from closely related Haemophilus species with the added potential for quantification of H. influenzae directly from specimens. We suggest the hpd#3 assay would be suitable for routine NTHi surveillance and to assess the impact of antibiotics and vaccines, on H. influenzae carriage rates, carriage density, and disease.

  5. Comparison of two real-time PCR assays for the detection of malaria parasites from hemolytic blood samples - Short communication.

    Science.gov (United States)

    Hagen, Ralf Matthias; Hinz, Rebecca; Tannich, Egbert; Frickmann, Hagen

    2015-06-01

    We compared the performance of an in-house and a commercial malaria polymerase chain reaction (PCR) assay using freeze-thawed hemolytic blood samples. A total of 116 freeze-thawed ethylenediamine tetraacetic acid (EDTA) blood samples of patients with suspicion of malaria were analyzed by an in-house as well as by a commercially available real-time PCR. Concordant malaria negative PCR results were reported for 39 samples and malaria-positive PCR results for 67 samples. The in-house assay further detected one case of Plasmodium falciparum infection, which was negative in the commercial assay as well as five cases of P. falciparum malaria and three cases of Plasmodium vivax malaria, which showed sample inhibition in the commercial assay. The commercial malaria assay was positive in spite of a negative in-house PCR result in one case. In all concordant results, cycle threshold values of P. falciparum-positive samples were lower in the commercial PCR than in the in-house assay. Although Ct values of the commercial PCR kit suggest higher sensitivity in case of concordant results, it is prone to inhibition if it is applied to hemolytic freeze-thawed blood samples. The number of misidentifications was, however, identical for both real-time PCR assays.

  6. Detection of Genitourinary Tract Chlamydia trachomatis Infection In Urine specimens by PCR Assay

    Institute of Scientific and Technical Information of China (English)

    李洪霞; 温泉; 夏迎华; 张林

    2001-01-01

    Objective: To compare the sensitivity and specificity of the cervical/urethral swabs with voided urine specimens for the detection of genitourinary tract infection with Chlamydia trachomatis and determine whether urine specimens can replace the cervical/urethral swabs in detection of C. trachomatis. Methods: The matched cervical/urethral swabs and voided urine specimens were collected from 569 patients of STD clinics.Polymerase chain reaction (PCR) assay specific for C. trachomatis plasmid DNA and rapid antigen testing (Clear view assay) was used to detect C. trachomatis. Standard criteria that defined """"true"""" positive included: 1) positive PCR results both in cervical/urethral swab and voided urine specimen or 2) positive voided urine results both by PCR assay and clear view test or 3)positive results in both PCR assay of cervical/urethral swab and clear view test of voided urine. For statistical analysis, the chi-square test was used. Results: The prevalence of C. trachomatis in patients with symptoms was 12.1% (28/231) in women and 10.4%(10/96) in men, with no significant difference between them (x2=0.21,P>0.05). The prevalence of C. trachomatis in patients with no symptoms was 11.0% (11/100) in women and 15.5% (22/142) in men, with a significant difference existing between them. (x2=4.0, P0.05) existed between PCR testing of swabs (sensitivity 87.3 %; specificity 99.2 %) and PCR testing of urine (sensitivity 88.7%; specificity 98.8%). As for clear view assay, sensitivity was 60.6% and specificity was 100%. Conclusions: PCR assay is superior to clear view in detecting C. trachomatis. Although both PCR testing of swabs and PCR testing of urine specimens both have high sensitivity and specificity, urine specimen testing is more cost-effective, practical and noninvasive. Thus urine specimens can take the place of the swabs in PCR testing for chlamydia.

  7. A pentaplex PCR assay for the detection and differentiation of Shigella species.

    Science.gov (United States)

    Ojha, Suvash Chandra; Yean Yean, Chan; Ismail, Asma; Singh, Kirnpal-Kaur Banga

    2013-01-01

    The magnitude of shigellosis in developing countries is largely unknown because an affordable detection method is not available. Current laboratory diagnosis of Shigella spp. is laborious and time consuming and has low sensitivity. Hence, in the present study, a molecular-based diagnostic assay which amplifies simultaneously four specific genes to identify invC for Shigella genus, rfc for S. flexneri, wbgZ for S. sonnei, and rfpB for S. dysenteriae, as well as one internal control (ompA) gene, was developed in a single reaction to detect and differentiate Shigella spp. Validation with 120 Shigella strains and 37 non-Shigella strains yielded 100% specificity. The sensitivity of the PCR was 100 pg of genomic DNA, 5.4 × 10(4) CFU/ml, or approximately 120 CFU per reaction mixture of bacteria. The sensitivity of the pentaplex PCR assay was further improved following preincubation of the stool samples in gram-negative broth. A preliminary study with 30 diarrhoeal specimens resulted in no cross-reaction with other non-Shigella strains tested. We conclude that the developed pentaplex PCR assay is robust and can provide information about the four target genes that are essential for the identification of the Shigella genus and the three Shigella species responsible for the majority of shigellosis cases.

  8. A Pentaplex PCR Assay for the Detection and Differentiation of Shigella Species

    Science.gov (United States)

    Ojha, Suvash Chandra; Yean Yean, Chan; Ismail, Asma; Banga Singh, Kirnpal-Kaur

    2013-01-01

    The magnitude of shigellosis in developing countries is largely unknown because an affordable detection method is not available. Current laboratory diagnosis of Shigella spp. is laborious and time consuming and has low sensitivity. Hence, in the present study, a molecular-based diagnostic assay which amplifies simultaneously four specific genes to identify invC for Shigella genus, rfc for S. flexneri, wbgZ for S. sonnei, and rfpB for S. dysenteriae, as well as one internal control (ompA) gene, was developed in a single reaction to detect and differentiate Shigella spp. Validation with 120 Shigella strains and 37 non-Shigella strains yielded 100% specificity. The sensitivity of the PCR was 100 pg of genomic DNA, 5.4 × 104 CFU/ml, or approximately 120 CFU per reaction mixture of bacteria. The sensitivity of the pentaplex PCR assay was further improved following preincubation of the stool samples in Gram-negative broth. A preliminary study with 30 diarrhoeal specimens resulted in no cross-reaction with other non-Shigella strains tested. We conclude that the developed pentaplex PCR assay is robust and can provide information about the four target genes that are essential for the identification of the Shigella genus and the three Shigella species responsible for the majority of shigellosis cases. PMID:23509722

  9. A Pentaplex PCR Assay for the Detection and Differentiation of Shigella Species

    Directory of Open Access Journals (Sweden)

    Suvash Chandra Ojha

    2013-01-01

    Full Text Available The magnitude of shigellosis in developing countries is largely unknown because an affordable detection method is not available. Current laboratory diagnosis of Shigella spp. is laborious and time consuming and has low sensitivity. Hence, in the present study, a molecular-based diagnostic assay which amplifies simultaneously four specific genes to identify invC for Shigella genus, rfc for S. flexneri, wbgZ for S. sonnei, and rfpB for S. dysenteriae, as well as one internal control (ompA gene, was developed in a single reaction to detect and differentiate Shigella spp. Validation with 120 Shigella strains and 37 non-Shigella strains yielded 100% specificity. The sensitivity of the PCR was 100 pg of genomic DNA, 5.4 × 104 CFU/ml, or approximately 120 CFU per reaction mixture of bacteria. The sensitivity of the pentaplex PCR assay was further improved following preincubation of the stool samples in Gram-negative broth. A preliminary study with 30 diarrhoeal specimens resulted in no cross-reaction with other non-Shigella strains tested. We conclude that the developed pentaplex PCR assay is robust and can provide information about the four target genes that are essential for the identification of the Shigella genus and the three Shigella species responsible for the majority of shigellosis cases.

  10. Development of Quantitative Real-time PCR Assays for Different Clades of “Candidatus Accumulibacter”

    Science.gov (United States)

    Zhang, An Ni; Mao, Yanping; Zhang, Tong

    2016-05-01

    We designed novel quantitative real-time polymerase chain reaction (qPCR) primers for the polyphosphate kinase 1 (ppk1) gene, targeting eight individual “Candidatus Accumulibacter” (referred to as Accumulibacter) clades. An evaluation of primer sets was conducted regarding the coverage, specificity, and PCR efficiency. (i) All primer sets were designed to cover all available sequences of the target clade. (ii) The phylogenetic analysis of the sequences retrieved from the qPCR products by each primer set demonstrated a high level of specificity. (iii) All calibration curves presented high PCR efficiencies in the range of 85–112% (R2 = 0.962–0.998). In addition, the possible interference of non-target amplicons was individually examined using the qPCR assay for 13 Accumulibacter clades, which were either undetected or showed negligible detection. With the primers designed by other research groups, a highly selective and sensitive qPCR-based method was developed to quantify all Accumulibacter clades, with the exception of Clade IE, in one assay, which enables more comprehensive insights into the community dynamics. The applicability to environmental samples was demonstrated by profiling the Accumulibacter clades in activated sludge samples of nine full-scale wastewater treatment plants.

  11. Quantitative multiplex real-time PCR assay for shrimp allergen: comparison of commercial master mixes and PCR platforms in rapid cycling.

    Science.gov (United States)

    Eischeid, Anne C; Kasko, Sasha M

    2015-01-01

    Real-time PCR has been used widely in numerous fields. In food safety, it has been applied to detection of microbes and other contaminants, including food allergens. Interest in rapid (fast) cycling real-time PCR has grown because it yields results in less time than does conventional cycling. However, fast cycling can adversely affect assay performance. Here we report on tests of commercial master mixes specifically designed for fast real-time PCR using a shrimp allergen assay we previously developed and validated. The objective of this work was to determine whether specialized commercial master mixes lead to improved assay performance in rapid cycling. Real-time PCR assays were carried out using four different master mixes and two different rapid cycling protocols. Results indicated that specialized master mixes did yield quality results. In many cases, linear ranges spanned up to 7 orders of magnitude, R(2) values were at least 0.95, and reaction efficiencies were within or near the optimal range of 90 to 110%. In the faster of the two rapid cycling protocols tested, assay performance and PCR amplification were markedly better for the shorter PCR product. In conclusion, specialized commercial master mixes were effective as part of rapid cycling protocols, but conventional cycling as used in our previous work is more reliable for the shrimp assay tested.

  12. Polymerase chain reaction assay for avian polyomavirus.

    Science.gov (United States)

    Phalen, D N; Wilson, V G; Graham, D L

    1991-05-01

    A polymerase chain reaction assay was developed for detection of budgerigar fledgling disease virus (BFDV). The assay used a single set of primers complementary to sequences located in the putative coding region for the BFDV VP1 gene. The observed amplification product had the expected size of 550 bp and was confirmed to derive from BFDV DNA by its restriction digestion pattern. This assay was specific for BFDV and highly sensitive, being able to detect as few as 20 copies of the virus. By using the polymerase chain reaction, BFDV was detected in adult, nestling, and embryo budgerigar (Melopsitticus undulatus) tissue DNAs and in sera from adult and nestling budgerigars. These results suggest the possibility of persistent infections in adult birds and lend further support to previously described evidence of possible in ovo transmission. BFDV was also detected in chicken embryo fibroblast cell cultures and chicken eggs inoculated with the virus. A 550-bp product with identical restriction enzyme sites was amplified from a suspected polyomavirus isolated from a peach-faced lovebird (Agapornis pesonata) and from tissue DNA from a Hahn's macaw (Ara nobilis) and a sun conure (Aratinga solstitialis) with histological lesions suggestive of polyomavirus infection. These fragments also hybridized with a BFDV-derived probe, proving that they were derived from a polyomavirus very similar, if not identical, to BFDV.

  13. Cutaneous and visceral leishmaniasis co-infection in dogs from Rio de Janeiro, Brazil: evaluation by specific PCR and RFLP-PCR assays

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    Marize Quinhones Pires

    2014-04-01

    Full Text Available Introduction During a diagnostic evaluation of canine visceral leishmaniasis (VL, two of seventeen dogs were found to be co-infected by Leishmania (Viannia braziliensis and Leishmania (Leishmania chagasi. Methods Specific polymerase chain reaction (PCR and restriction fragment length polymorphism-PCR (RFLP-PCR assays were performed. Results PCR assays for Leishmania subgenus identification followed by RFLP-PCR analysis in biopsies from cutaneous lesions and the spleen confirmed the presence of Leishmania (Viannia braziliensis and Leishmania (Leishmania chagasi in those fragments. Conclusions This report reinforces the importance of using serological and molecular techniques in the epidemiological surveillance of canine populations in endemic areas in which both diseases are known to co-exist. In such cases, a reassessment of the control measures is required.

  14. A polymerase chain reaction assay for ascosporic inoculum of Sclerotinia species

    Science.gov (United States)

    A PCR assay was developed which amplified a 170-bp fragment of the intergenic spacer region of Sclerotinia sclerotiorum, the cause of white mould. Sensitivity was 10 S. sclerotiorum ascospores per DNA extraction (0.2 ascospores per PCR reaction). The presence of soil did not affect sensitivity a...

  15. Application of three duplex real-time PCR assays for simultaneous detection of human seasonal and avian influenza viruses.

    Science.gov (United States)

    Stefańska, Ilona; Dzieciatkowski, Tomasz; Brydak, Lidia B; Romanowska, Magdalena

    2013-08-01

    This study was performed to develop real-time PCR (qPCR) for detection of human seasonal and avian influenza viruses in duplex format. First duplex qPCR detects haemagglutinin (HA) gene of influenza virus A(H1N1)pdm09 and HA gene of influenza virus A(H3N2), the second reaction detects neuraminidase (NA) gene of influenza virus A(H3N2) and NA gene of influenza virus A(H1N1)pdm09 and A(H5N1), and the third reaction detects HA gene of influenza A(H5N1) and nonstructural protein gene of influenza B virus. Primers and probes were designed using multiple alignments of target gene sequences of different reference strains. Assays were optimised for identical thermocycling conditions. Their specificity was confirmed by conventional PCR and monoplex qPCR with nucleic acids isolated from different influenza viruses and other respiratory pathogens. Plasmid constructs with a fragment of specific gene were used to assess sensitivity of the assay. The limit of detection ranged from 27 to 96 cDNA copies/reaction. Clinical specimens (n = 107) have been tested using new assays, immunofluorescence and monoplex qRT-PCR. It has been shown that developed assays have been capable of rapid and accurate simultaneous detection and differentiation of influenza viruses. They are more sensitive than immunofluorescence and at least as sensitive as monoplex qRT-PCR.

  16. Evaluation of real-time RT-PCR assays for detection and quantification of norovirus genogroups I and II.

    Science.gov (United States)

    Rupprom, Kitwadee; Chavalitshewinkoon-Petmitr, Porntip; Diraphat, Pornphan; Kittigul, Leera

    2017-02-20

    Noroviruses are the leading cause of acute gastroenteritis in humans. Real-time reverse transcription-polymerase chain reaction (real-time RT-PCR) is a promising molecular method for the detection of noroviruses. In this study, the performance of three TaqMan real-time RT-PCR assays was assessed, which were one commercially available real-time RT-PCR kit (assay A: Norovirus Real Time RT-PCR kit) and two in-house real-time RT-PCR assays (assay B: LightCycler RNA Master Hybprobe and assay C: RealTime ready RNA Virus Master). Assays A and B showed higher sensitivity than assay C for norovirus GI, while they all had the same sensitivity (10(3) DNA copies/mL) for GII DNA standard controls. Assay B had the highest efficiency for both genogroups. No cross-reactivity was observed among GI and GII noroviruses, rotavirus, hepatitis A virus, and poliovirus. The detection rates of these assays in GI and GII norovirus-positive fecal samples were not significantly different. However, the mean quantification cycle (Cq) value of assay B for GII was lower than assays A and C with statistical significance (P-value, 0.000). All three real-time RT-PCR assays could detect a variety of noroviruses including GI.2, GII.2, GII.3, GII.4, GII.6, GII.12, GII.17, and GII.21. This study suggests assay B as a suitable assay for the detection and quantification of noroviruses GI and GII due to good analytical sensitivity and higher performance to amplify norovirus on DNA standard controls and clinical samples.

  17. 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.

  18. A quantitative PCR (TaqMan assay for pathogenic Leptospira spp

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    Symonds Meegan L

    2002-07-01

    Full Text Available Abstract Background Leptospirosis is an emerging infectious disease. The differential diagnosis of leptospirosis is difficult due to the varied and often "flu like" symptoms which may result in a missed or delayed diagnosis. There are over 230 known serovars in the genus Leptospira. Confirmatory serological diagnosis of leptospirosis is usually made using the microscopic agglutination test (MAT which relies on the use of live cultures as the source of antigen, often performed using a panel of antigens representative of local serovars. Other techniques, such as the enzyme linked immunosorbent assay (ELISA and slide agglutination test (SAT, can detect different classes of antibody but may be subject to false positive reactions and require confirmation of these results by the MAT. Methods The polymerase chain reaction (PCR has been used to detect a large number of microorganisms, including those of clinical significance. The sensitivity of PCR often precludes the need for isolation and culture, thus making it ideal for the rapid detection of organisms involved in acute infections. We employed real-time (quantitative PCR using TaqMan chemistry to detect leptospires in clinical and environmental samples. Results and Conclusions The PCR assay can be applied to either blood or urine samples and does not rely on the isolation and culture of the organism. Capability exists for automation and high throughput testing in a clinical laboratory. It is specific for Leptospira and may discriminate pathogenic and non-pathogenic species. The limit of detection is as low as two cells.

  19. Real-time multiplex PCR assay for detection of Yersinia pestis and Yersinia pseudotuberculosis.

    Science.gov (United States)

    Matero, Pirjo; Pasanen, Tanja; Laukkanen, Riikka; Tissari, Päivi; Tarkka, Eveliina; Vaara, Martti; Skurnik, Mikael

    2009-01-01

    A multiplex real-time polymerase chain reaction (PCR) assay was developed for the detection of Yersinia pestis and Yersinia pseudotuberculosis. The assay includes four primer pairs, two of which are specific for Y. pestis, one for Y. pestis and Y. pseudotuberculosis and one for bacteriophage lambda; the latter was used as an internal amplification control. The Y. pestis-specific target genes in the assay were ypo2088, a gene coding for a putative methyltransferase, and the pla gene coding for the plasminogen activator. In addition, the wzz gene was used as a target to specifically identify both Y. pestis and the closely related Y. pseudotuberculosis group. The primer and probe sets described for the different genes can be used either in single or in multiplex PCR assays because the individual probes were designed with different fluorochromes. The assays were found to be both sensitive and specific; the lower limit of the detection was 10-100 fg of extracted Y. pestis or Y. pseudotuberculosis total DNA. The sensitivity of the tetraplex assay was determined to be 1 cfu for the ypo2088 and pla probe labelled with FAM and JOE fluorescent dyes, respectively.

  20. Surface effects on PCR reactions in multichip microfluidic platforms.

    Science.gov (United States)

    Panaro, Nicholas J; Lou, Xing Jian; Fortina, Paolo; Kricka, Larry J; Wilding, Peter

    2004-03-01

    We evaluated the compatibility of several common plastics, commercially available plastic tubing and disposable syringes which might be useful in the construction of microfluidic platforms with respect to the polymerase chain reaction (PCR). A simple and inexpensive plastic test module was constructed in order to evaluate some of the construction plastics. We also investigated the effect of addition of PEG 8000 to PCR reaction mixtures on the compatibility of materials. These studies identified several common plastics, plastic tubing, and disposable syringes which were compatible with the PCR reaction.

  1. Single rapid TaqMan fluorogenic probe based PCR assay that detects all four dengue serotypes.

    Science.gov (United States)

    Warrilow, David; Northill, Judith A; Pyke, Alyssa; Smith, Greg A

    2002-04-01

    Public health laboratories require rapid diagnosis of dengue outbreaks for application of measures such as vector control. We have developed a rapid single fluorogenic probe-based polymerase chain reaction assay for the detection of all four dengue serotypes (FUDRT-PCR). The method employs primers and probe that are complementary to the evolutionarily conserved 3' untranslated region of the dengue genome. The assay detected viral RNA of strains of all four dengue serotypes but not of the flaviviruses Japanese encephalitis virus, Murray Valley encephalitis virus, Kunjin, Stratford, West Nile, Alfuy or Yellow fever. When compared to an existing nested-PCR assay for the detection of dengue on clinical samples, FUDRT-PCR detected dengue 1 (100%, n=14), dengue 2 (85%, n=13), dengue 3 (64%, n=14) and dengue 4 (100%, n=3) with the indicated sensitivities. FUDRT-PCR enables diagnosis of acute dengue infection in four hours from sample receipt. In addition, a single-test procedure should result in a reduction in the number of tests performed with considerable cost savings for diagnostic laboratories.

  2. Quantitative polymerase chain reaction (PCR) for detection of aquatic animal pathogens in a diagnostic laboratory setting

    Science.gov (United States)

    Purcell, Maureen K.; Getchell, Rodman G.; McClure, Carol A.; Weber, S.E.; Garver, Kyle A.

    2011-01-01

    Real-time, or quantitative, polymerase chain reaction (qPCR) is quickly supplanting other molecular methods for detecting the nucleic acids of human and other animal pathogens owing to the speed and robustness of the technology. As the aquatic animal health community moves toward implementing national diagnostic testing schemes, it will need to evaluate how qPCR technology should be employed. This review outlines the basic principles of qPCR technology, considerations for assay development, standards and controls, assay performance, diagnostic validation, implementation in the diagnostic laboratory, and quality assurance and control measures. These factors are fundamental for ensuring the validity of qPCR assay results obtained in the diagnostic laboratory setting.

  3. Development of a versatile and stable internal control system for RT-qPCR assays.

    Science.gov (United States)

    Felder, Eva; Wölfel, Roman

    2014-11-01

    RT-qPCR, an established method for the detection of RNA viruses, requires internal RNA controls for the correct interpretation of PCR results. Robust and versatile RT-PCR controls can be achieved for example by packaging RNA into a virus-derived protein shell. In this study a MS2-based internal control system was developed, that allows stable and universal packing of different RNAs into non-infectious, non-lytic MS2-based viral like particles (VLPs). Two competitive internal controls for a hantavirus assay and a Crimean-Congo Hemorrhagic Fever Virus (CCHFV) assay were cloned for the expression of VLPs. The expression of VLPs containing the RNA of interest could be induced with arabinose in Escherichia coli. The VLPs proved to be temperature resistant and could be frozen and thawed several times without degradation. Distinction of IC RNA from the target RNA was facilitated by a clear shift in the melting temperature or by specific hybridization signals. Furthermore, target and IC PCR amplification could be easily distinguished by their size in gel-electrophoretic analyses. Limits of detection were determined, demonstrating that the application of the IC did not reduce the sensitivity of the target RT-qPCR reactions. The system can be adapted to nearly any required sequence, resulting in a highly flexible method with broad range applications.

  4. Validation of an ultrasensitive digital droplet PCR assay for HIV-2 plasma RNA quantification.

    Science.gov (United States)

    Ruelle, Jean; Yfantis, Vasilieios; Duquenne, Armelle; Goubau, Patrick

    2014-01-01

    Low or undetectable plasma viral load (VL) using current qPCR assays is common for HIV-2 patients. Digital PCR is an emerging technology enabling more precision and reproducibility than qPCR at low DNA/RNA copy numbers. Available data related to digital droplet PCR (ddPCR, Bio-Rad) underscore issues linked to the threshold definition of positivity, coupled to the specificity of low copy results (1). A RT-PCR protocol was set up using the One-Step RT-ddPCR Kit for Probes on the QX200 platform (Bio-Rad, Hercules, CA) in an accredited environment (ISO15189:2012 norm). Parameters tested were in line with the digital MIQE guidelines (2). Inter-run coefficient of variation (CV) was established using synthetic RNA controls diluted in HIV-negative plasma. The ddPCR assay was compared to a qRT-PCR previously used in routine (LOQ 50 cop/mL (3)) using 46 clinical samples and the NIBSC international HIV-2 RNA standard. The optimal PCR efficiency and the best separation between positive and negative droplets were obtained with a mixture containing 0.5 mM manganese acetate, 700 nM primers and 250 nM of the 5'FAM-probe. Using a manual threshold to define positivity, 7.74% of negative controls (n=168) were scored as positive due to one positive droplet. The presence of two positive droplets or more was not observed for negative controls. Serial dilutions of a positive control showed excellent linearity (R2=0.999) and enabled us to define a limit of quantification of two positives droplets, which corresponds to 0.14 copies/μL in the reaction mixture and to seven copies per mL of plasma. The inter-run coefficient of variation was 3.37% at a mean value of 4,468 cop/mL, 19.59% at 416 cop/mL and 32.28% at 8 cop/mL. The NIBSC standard of 1,000 IU was quantified 1,400 copies by ddPCR and close to 5,000 copies by qPCR (delta log superior to 0.5). Among 46 clinical samples, 22 were undetectable with both qPCR and ddPCR, 12 were detected with both methods (respective means of 10,612 and 2

  5. Validation of an ultrasensitive digital droplet PCR assay for HIV-2 plasma RNA quantification

    Directory of Open Access Journals (Sweden)

    Jean Ruelle

    2014-11-01

    Full Text Available Introduction: Low or undetectable plasma viral load (VL using current qPCR assays is common for HIV-2 patients. Digital PCR is an emerging technology enabling more precision and reproducibility than qPCR at low DNA/RNA copy numbers. Available data related to digital droplet PCR (ddPCR, Bio-Rad underscore issues linked to the threshold definition of positivity, coupled to the specificity of low copy results (1. Materials and Methods: A RT-PCR protocol was set up using the One-Step RT-ddPCR Kit for Probes on the QX200 platform (Bio-Rad, Hercules, CA in an accredited environment (ISO15189:2012 norm. Parameters tested were in line with the digital MIQE guidelines (2. Inter-run coefficient of variation (CV was established using synthetic RNA controls diluted in HIV-negative plasma. The ddPCR assay was compared to a qRT-PCR previously used in routine (LOQ 50 cop/mL (3 using 46 clinical samples and the NIBSC international HIV-2 RNA standard. Results: The optimal PCR efficiency and the best separation between positive and negative droplets were obtained with a mixture containing 0.5 mM manganese acetate, 700 nM primers and 250 nM of the 5’FAM-probe. Using a manual threshold to define positivity, 7.74% of negative controls (n=168 were scored as positive due to one positive droplet. The presence of two positive droplets or more was not observed for negative controls. Serial dilutions of a positive control showed excellent linearity (R2=0.999 and enabled us to define a limit of quantification of two positives droplets, which corresponds to 0.14 copies/μL in the reaction mixture and to seven copies per mL of plasma. The inter-run coefficient of variation was 3.37% at a mean value of 4,468 cop/mL, 19.59% at 416 cop/mL and 32.28% at 8 cop/mL. The NIBSC standard of 1,000 IU was quantified 1,400 copies by ddPCR and close to 5,000 copies by qPCR (delta log superior to 0.5. Among 46 clinical samples, 22 were undetectable with both qPCR and ddPCR, 12 were

  6. Development of a multiplex PCR assay for detection and discrimination of Theileria annulata and Theileria sergenti in cattle.

    Science.gov (United States)

    Junlong, Liu; Li, Youquan; Liu, Aihong; Guan, Guiquan; Xie, Junren; Yin, Hong; Luo, Jianxun

    2015-07-01

    Aim to construct a simple and efficient diagnostic assay for Theileria annulata and Theileria sergenti, a multiplex polymerase chain reaction (PCR) method was developed in this study. Following the alignment of the related sequences, two primer sets were designed specific targeting on T. annulata cytochrome b (COB) gene and T. sergenti internal transcribed spacer (ITS) sequences. It was found that the designed primers could react in one PCR system and generating amplifications of 818 and 393 base pair for T. sergenti and T. annulata, respectively. The standard genomic DNA of both species Theileria was serial tenfold diluted for testing the sensitivity, while specificity test confirmed both primer sets have no cross-reaction with other Theileria and Babesia species. In addition, 378 field samples were used for evaluation of the utility of the multiplex PCR assay for detection of the pathogens infection. The detection results were compared with the other two published PCR methods which targeting on T. annulata COB gene and T. sergenti major piroplasm surface protein (MPSP) gene, respectively. The developed multiplex PCR assay has similar efficient detection with COB and MPSP PCR, which indicates this multiplex PCR may be a valuable assay for the epidemiological studies for T. annulata and T. sergenti.

  7. 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

    be performed. The SNPforID consortium (www.snpforid.org) was established in 2003 with the principal goal of developing a SNP-based system of DNA analysis that would have comparable discrimination power and ease of use to those of existing short tandem repeat (STR) based techniques. Here, we describe a strategy...... 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...... a multiple injection approach for DNA sequencers that can effectively detect all the SNPs amplified in a single electrophoretic run. We present SNP data for 700 unrelated individuals from 9 populations...

  8. Comparison of the conventional multiplex RT-PCR, real time RT-PCR and Luminex xTAG® RVP fast assay for the detection of respiratory viruses.

    Science.gov (United States)

    Choudhary, Manohar L; Anand, Siddharth P; Tikhe, Shamal A; Walimbe, Atul M; Potdar, Varsha A; Chadha, Mandeep S; Mishra, Akhilesh C

    2016-01-01

    Detection of respiratory viruses using polymerase chain reaction (PCR) is sensitive, specific and cost effective, having huge potential for patient management. In this study, the performance of an in-house developed conventional multiplex RT-PCR (mRT-PCR), real time RT-PCR (rtRT-PCR) and Luminex xTAG(®) RVP fast assay (Luminex Diagnostics, Toronto, Canada) for the detection of respiratory viruses was compared. A total 310 respiratory clinical specimens predominantly from pediatric patients, referred for diagnosis of influenza A/H1N1pdm09 from August 2009 to March 2011 were tested to determine performance characteristic of the three methods. A total 193 (62.2%) samples were detected positive for one or more viruses by mRT-PCR, 175 (56.4%) samples by real time monoplex RT-PCR, and 138 (44.5%) samples by xTAG(®) RVP fast assay. The overall sensitivity of mRT-PCR was 96.9% (95% CI: 93.5, 98.8), rtRT-PCR 87.9% (95% CI: 82.5, 92.1) and xTAG(®) RVP fast was 68.3% (95% CI: 61.4, 74.6). Rhinovirus was detected most commonly followed by respiratory syncytial virus group B and influenza A/H1N1pdm09. The monoplex real time RT-PCR and in-house developed mRT-PCR are more sensitive, specific and cost effective than the xTAG(®) RVP fast assay.

  9. Real-time immuno-PCR assay for detecting PCBs in soil samples.

    Science.gov (United States)

    Chen, Han-Yu; Zhuang, Hui-Sheng

    2009-06-01

    A fast and robust assay, based on immuno-polymerase chain reaction (IPCR) techniques, was developed for the detection of polychlorinated biphenyls (PCBs) in soil samples. Real-time IPCR (rt-IPCR) is a powerful technique that combines enzyme-linked immunosorbent assay (ELISA) with the specificity and sensitivity of PCR. In our assay, indirect ELISAs based on immobilization of PCB37 hapten-ovalbumin conjugates was used for evaluation of the immune response. The effect of optimal reagent concentrations to reduce background fluorescence was also investigated. Using the optimized assay, the linear sensitivity range of the assay covered more than six orders of magnitude, and the minimum detection limits reached 5 fg ml(-1) antigen. Rt-IPCR was tested for its cross-reactivity profiles using four selected congeners and four Aroclor products. The assays were highly specific for congeners but less specific for Aroclor1242. We took four soil samples to validate the method, and the results were confirmed by gas chromatography/mass spectrometry (GC/MS). The rt-IPCR results for soil samples correlated well with the concentrations of PCBs obtained by GC/MS (r = 0.99, n = 6). These data indicate that this highly specific, sensitive, and robust assay can be modified for detecting PCB compounds in the environment.

  10. Detection of Nicotiana DNA in Tobacco Products Using a Novel Multiplex Real-Time PCR Assay.

    Science.gov (United States)

    Korchinski, Katie L; Land, Adrian D; Craft, David L; Brzezinski, Jennifer L

    2016-07-01

    Establishing that a product contains tobacco is a requirement for the U.S. Food and Drug Administration's regulation and/or prosecution of tobacco products. Therefore, a multiplex real-time PCR method was designed to determine if Nicotiana (tobacco) DNA is present in tobacco products. The PCR method simultaneously amplifies a 73 bp fragment of the cytochrome P450 monoxygenase CYP82E4 gene and 66 bp fragment in the nia-1 gene for nitrate reductase, which are detected using dual-labeled TaqMan probes. The assay is capable of detecting approximately 7.8 pg purified tobacco DNA, with a similar sensitivity for either gene target while incorporating an internal positive control (IPC). DNA was extracted from prepared tobacco products-including chewing tobacco, pipe tobacco, and snuff-or from the cut fill (no wrapper) of cigarettes and cigars. Of the 13 products analyzed, 12 were positive for both tobacco-specific markers and the IPC. DNA was also extracted from the fill of five varieties of herbal cigarettes, which were negative for both tobacco-specific gene targets and positive for the IPC. Our method expands on current assays by introducing a multiplex reaction, targeting two sequences in two different genes of interest, incorporating an IPC into the reaction, and lowering the LOD and LOQ while increasing the efficiency of the PCR.

  11. Rapid PCR-based assay for Sclerotinia sclerotiorum detection on soybean seeds

    Directory of Open Access Journals (Sweden)

    Edilaine Mauricia Gelinski Grabicoski

    2015-02-01

    Full Text Available Caused by Sclerotinia sclerotiorum, white mold is an important seed-transmitted disease of soybean (Glycine max. Incubation-based methods available for the detection and quantification of seed-borne inoculum such as the blotter test, paper roll and Neon-S assay are time-consuming, laborious, and not always sensitive. In this study, we developed and evaluated a molecular assay for the detection of S. sclerotiorum in soybean seeds using a species-specific PCR (polymerase chain reaction primer set and seed soaking (without DNA extraction for up to 72 h. The PCR products were amplified in all the samples infected with the pathogen, but not in the other samples of plant material or the other seed-borne fungi DNA. The minimum amount of DNA detected was 10 pg, or one artificially infested seed in a 400-seed sample (0.25 % fungal incidence and one naturally infected seed in a 300-seed sample (0.33 % incidence. The PCR-based assay was rapid (< 9 h, did not require DNA extraction and was very sensitive.

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

    OpenAIRE

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

    2014-01-01

    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 amplif...

  13. Detection of four important Eimeria species by multiplex PCR in a single assay.

    Science.gov (United States)

    You, Myung-Jo

    2014-06-01

    The oocysts of some of the recognized species of chicken coccidiosis are difficult to distinguish morphologically. Diagnostic laboratories are increasingly utilizing DNA-based technologies for the specific identification of Eimeria species. This study reports a multiplex polymerase chain reaction (PCR) assay based on internal transcribed spacer-1 (ITS-1) for the simultaneous diagnosis of the Eimeria tenella, Eimeria acervulina, Eimeria maxima, and Eimeria necatrix species, which infect domestic fowl. Primer pairs specific to each species were designed in order to generate a ladder of amplification products ranging from 20 to 25 bp, and a common optimum annealing temperature for these species was determined to be 52.5 °C. Sensitivity tests were performed for each species, showing a detection threshold of 1-5 pg. All the species were amplified homogeneously, and a homogenous band ladder was observed, indicating that the assay permitted the simultaneous detection of all the species in a single-tube reaction. In the phylogenic study, there was a clear species clustering, which was irrespective of geographical location, for all the ITS-1 sequences used. This multiplex PCR assay represents a rapid and potential cost-effective diagnostic method for the detection of some key Eimeria species that infect domestic fowl.

  14. Multiplex PCR Assay for Identifi cation and Differentiation of Campylobacter jejuni and Campylobacter coli Isolates.

    Science.gov (United States)

    Pavlova, Maria R; Dobreva, Elina G; Ivanova, Katucha I; Asseva, Galina D; Ivanov, Ivan N; Petrov, Peter K; Velev, Valeri R; Tomova, Ivelina I; Tiholova, Maida M; Kantardjiev, Todor V

    2016-01-01

    Campylobacter spp. are important causative agents of gastrointestinal infections in humans. The most frequently isolated strains of this bacterial genus are Campylobacter jejuni and Campylobacter coli. To date, genetic methods for bacterial identification have not been used in Bulgaria. We optimized the multiplex PSR assay to identify Campylobacter spp. and differentiate C. jejuni from C. coli in clinical isolates. We also compared this method with the routinely used biochemical methods. To identify Campylobacter spp. and discriminate C. coli from C. jejuni in clinical isolates using multiplex PCR assay. Between February 2014 and January 2015 we studied 93 stool samples taken from patients with diarrheal syndrome and identified 40 species of Campylobacter spp. in them. The clinical material was cultured in microaerophilic atmosphere, the isolated strains being biochemically diff erentiated (hydrolysis of sodium hippurate for C. jejuni, and hydrolysis of indoxyl acetate for C. coli). DNA was isolated from the strains using QiaAmp MiniKit (QIAGEN, Germany). Twenty strains were tested with multiplex PCR for the presence of these genes: cadF, characteristic for Campylobacter spp., hipO for C. jejuni and asp for C. coli. The biochemical tests identified 16 strains of C. jejuni, 3 strains of C. coli, and 1 strain of C. upsaliensis. After the multiplex PCR assay the capillary gel electrophoresis confirmed 16 strains of C. jejuni, 2 strains of C. coli and 2 strains of Campylobacter spp. - because of the presence of the gene cadF. C. jejuni has the gene hipO, and it is possible that this gene may not be expressed in the biochemical differentiation yielding a negative reaction as a result. In comparison, we can conclude that the genetic differentiation is a more accurate method than the biochemical tests. The multiplex PCR assay is a fast, accurate method for identifi cation of Campylobacter spp. which makes it quite necessary in the clinical diagnostic practice.

  15. Development of a multiplex reverse transcription-PCR assay for simultaneous detection of garlic viruses

    Institute of Scientific and Technical Information of China (English)

    HU Xin-xi; LEI Yan; WANG Pei; TANG Lin-fei; HE Chang-zheng; SONG Yong; XIONG Xing-yao; NIE Xian-zhou

    2015-01-01

    A preliminary screening for garlic viruses in garlic plants in Hunan, China, using existing monoplex (simplex) reverse tran-scription-polymerase chain reaction (RT-PCR) procedures detected four viruses/virus groups. These viruses/virus groups were Onion yel ow dwarf virus (OYDV), Leek yel ow stripe virus (LYSV), Shal ot latent virus (SLV) and al exiviruses (e.g., garlic viruses A, B, C, D, E, X). Sequence analysis of the projected al exivirus amplicons revealed the al exivirus in the infected garlic plants was Garlic virus D (GarV-D), which shared 92–97%sequence identities with various isolates from the world. A multiplex RT-PCR (mRT-PCR) was therefore developed to simultaneously detect and differentiate the four viruses/virus groups. To achieve this, four primer pairs targeting al exiviruses, OYDV, LYSV and SLV were designed. The anticipated amplicon sizes are 183 bp (al exiviruses), 265 bp (OYDV), 404 bp (LYSV) and 592 bp (SLV), respectively. Al primer pairs produced virus-speciifc fragments in both simplex and multiplex formats, thus conifrming the efifcacy of the newly developed mRT-PCR for detection of these viruses. The mRT-PCR further was evaluated by applying it to garlic plant samples col ected in two geographic locations in Hunan. Al exiviruses, OYDV, LYSV and SLV were detected in 50.9, 40.3, 28.3 and 58.5%of leaf samples, respectively;and mixed infections with two or more viruses accounted for 54%of the garlic samples. The results obtained by mRT-PCR were conifrmed by simplex RT-PCR assays. In conclusion, this newly devel-oped mRT-PCR provides a rapid, sensitive and reliable method for the detection and identiifcation of major garlic viruses.

  16. Optimized PCR assay for detection of white spot syndrome virus (WSSV).

    Science.gov (United States)

    Nunan, Linda M; Lightner, Donald V

    2011-01-01

    A rapid PCR assay for detection of white spot syndrome virus (WSSV) was developed based on the nested PCR procedure described by Lo et al. (1996) and outlined as the recommended PCR diagnostic assay in the Manual of Diagnostic Tests for Aquatic Animals published by the Office of International Epizootics (OIE, 2009). The optimized procedure incorporated the second step primers used in the nested WSSV PCR. By adjusting the annealing temperature and shortening the cycling times, this modified assay is substantially faster and as sensitive as the recommended OIE protocol. The modified PCR test was compared directly to the two-step nested PCR protocol and a modified nested procedure. The sensitivity of the published assay was determined by template dilutions of semi-purified WSSV virions that had been quantitated using real-time PCR for detection of WSSV. Various isolates were tested using the modified procedure, to ensure that the assay was able to detect WSSV from different geographical locations.

  17. Primer design for PCR reactions in forensic biology.

    Science.gov (United States)

    Elkins, Kelly M

    2015-01-01

    The polymerase chain reaction (PCR) is a popular method to copy DNA in vitro. Its invention revolutionized fields ranging from clinical medicine to anthropology, molecular biology, and forensic biology. The method employs one of many available heat-stable DNA polymerases in a reaction that is repeated many times in situ. The DNA polymerase reads a template DNA strand and using the components of the reaction mix, catalyzes the addition of free 2'-deoxynucleotide triphosphate nitrogenous bases to short segment of DNA that forms a complement with the template via Watson-Crick base pairing. This short segment of DNA is referred to as a PCR primer and it is essential to the success of the reaction. The most widely used application of PCR in forensic labs is the amplification of short tandem repeat (STR) loci used in DNA typing. The STRs are routinely evaluated in concert with 16 or more reactions, a multiplex, run in one test tube simultaneously. In a multiplex, it is essential that the primers work specifically and accurately on the intended reactions without hindering the other reactions. The primers, which are very specific, also can be used to probe single nucleotide polymorphisms (SNPs) in a DNA sequence of interest by single base extension. Primers are often designed using one of many available automated software packages. Here the process of manually designing PCR primers for forensic biology using no-cost software is described.

  18. Development of a non invasion real-time PCR assay for the quantitation of chicken parvovirus in fecal swabs

    Science.gov (United States)

    The present study describes the development of a real time Taqman polymerase chain reaction (PCR) assay using a fluorescent labeled probe for the detection and quantitation of chicken parvovirus (ChPV) in feces. The primers and probes were designed based on the nucleotide sequence of the non struct...

  19. Edesign: Primer and Enhanced Internal Probe Design Tool for Quantitative PCR Experiments and Genotyping Assays.

    Directory of Open Access Journals (Sweden)

    Yasumasa Kimura

    Full Text Available Analytical PCR experiments preferably use internal probes for monitoring the amplification reaction and specific detection of the amplicon. Such internal probes have to be designed in close context with the amplification primers, and may require additional considerations for the detection of genetic variations. Here we describe Edesign, a new online and stand-alone tool for designing sets of PCR primers together with an internal probe for conducting quantitative real-time PCR (qPCR and genotypic experiments. Edesign can be used for selecting standard DNA oligonucleotides like for instance TaqMan probes, but has been further extended with new functions and enhanced design features for Eprobes. Eprobes, with their single thiazole orange-labelled nucleotide, allow for highly sensitive genotypic assays because of their higher DNA binding affinity as compared to standard DNA oligonucleotides. Using new thermodynamic parameters, Edesign considers unique features of Eprobes during primer and probe design for establishing qPCR experiments and genotyping by melting curve analysis. Additional functions in Edesign allow probe design for effective discrimination between wild-type sequences and genetic variations either using standard DNA oligonucleotides or Eprobes. Edesign can be freely accessed online at http://www.dnaform.com/edesign2/, and the source code is available for download.

  20. Detection of mandarin in orange juice by single-nucleotide polymorphism qPCR assay.

    Science.gov (United States)

    Aldeguer, Miriam; López-Andreo, María; Gabaldón, José A; Puyet, Antonio

    2014-02-15

    A dual-probe real time PCR (qPCR) DNA-based analysis was devised for the identification of mandarin in orange juice. A single nucleotide polymorphism at the trnL-trnF intergenic region of the chloroplast chromosome was confirmed in nine orange (Citrus sinensis) and thirteen commercial varieties of mandarin, including Citrus reticulata and Citrus unshiu species and a mandarin × tangelo hybrid. Two short minor-groove binding fluorescent probes targeting the polymorphic sequence were used in the dual-probe qPCR, which allowed the detection of both species in single-tube reactions. The similarity of PCR efficiencies allowed a simple estimation of the ratio mandarin/orange in the juice samples, which correlated to the measured difference of threshold cycle values for both probes. The limit of detection of the assay was 5% of mandarin in orange juice, both when the juice was freshly prepared (not from concentrate) or reconstituted from concentrate, which would allow the detection of fraudulently added mandarin juice. The possible use of the dual-probe system for quantitative measurements was also tested on fruit juice mixtures. qPCR data obtained from samples containing equal amounts of mandarin and orange juice revealed that the mandarin target copy number was approximately 2.6-fold higher than in orange juice. The use of a matrix-adapted control as calibrator to compensate the resulting C(T) bias allowed accurate quantitative measurements to be obtained.

  1. Edesign: Primer and Enhanced Internal Probe Design Tool for Quantitative PCR Experiments and Genotyping Assays.

    Science.gov (United States)

    Kimura, Yasumasa; Soma, Takahiro; Kasahara, Naoko; Delobel, Diane; Hanami, Takeshi; Tanaka, Yuki; de Hoon, Michiel J L; Hayashizaki, Yoshihide; Usui, Kengo; Harbers, Matthias

    2016-01-01

    Analytical PCR experiments preferably use internal probes for monitoring the amplification reaction and specific detection of the amplicon. Such internal probes have to be designed in close context with the amplification primers, and may require additional considerations for the detection of genetic variations. Here we describe Edesign, a new online and stand-alone tool for designing sets of PCR primers together with an internal probe for conducting quantitative real-time PCR (qPCR) and genotypic experiments. Edesign can be used for selecting standard DNA oligonucleotides like for instance TaqMan probes, but has been further extended with new functions and enhanced design features for Eprobes. Eprobes, with their single thiazole orange-labelled nucleotide, allow for highly sensitive genotypic assays because of their higher DNA binding affinity as compared to standard DNA oligonucleotides. Using new thermodynamic parameters, Edesign considers unique features of Eprobes during primer and probe design for establishing qPCR experiments and genotyping by melting curve analysis. Additional functions in Edesign allow probe design for effective discrimination between wild-type sequences and genetic variations either using standard DNA oligonucleotides or Eprobes. Edesign can be freely accessed online at http://www.dnaform.com/edesign2/, and the source code is available for download.

  2. Development of A PCR-ELISA Assay for the Detection of Campylobacter jejuni%空肠弯曲杆菌PCR-ELISA检测方法的建立

    Institute of Scientific and Technical Information of China (English)

    唐梦君; 周生; 张小燕; 唐修君; 蒲俊华; 高玉时

    2013-01-01

    A polymerase chain reaction (PCR) assay was developed based on a solution-hybridization colorimetric end-point detection format (PCR-ELISA) for the detection of C.jejuni.PCR primers were designed to target gyrA gene.Digoxygenin-labelled probes were investigated for the detection of biotin-labelled PCR products from C.jejuni using the PCR-ELISA format.The specificity of the assay was investigated.The results showed that only expected fragments of C.jejuni strains were successfully amplified,whereas the Escherichia coli,Salmonella,L.rnonocytogenes and a range of unrelated organisms were negative.The PCR-ELISA assay and probes were demonstrated to be specific for C.jejuni.The detection threshold value is 2 fg.The sensitivity of the PCR-ELISA assay was demonstrated to be 10-fold more sensitive than a gel-based PCR method using the same primers.The detection limit of feces simulated samples was 50 cfu/mL.Results of detecting C.jejuni in one hundred samples in feces in chicken showed that positive rate was 69% by PCR-ELISA methods whereas it was 60% by PCR.This PCR-ELISA assay is sensitive,specific and has the potential to apply in the field of risk assessment of food-borne pathogens and of the large-scale detection researches.%针对空肠弯曲杆菌(Campylobacter jejuni)旋转酶基因(gyrA gene)设计特异性引物和探针,将生物素和地高辛分别标记上游引物5'端和核酸探针3 '端,并对反应条件进行优化,建立空肠弯曲杆菌PCR-ELISA检测方法.结果表明:该方法能特异的检测空肠弯曲杆菌基因组DNA,检测阈值为2fg,敏感性是常规PCR的10倍.对模拟泄殖腔样本进行检测,检测限为50 cfu/mL.对100份临床样品进行检测,PCR和PCR-ELISA方法阳性检出率分别为60%和69%.

  3. 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.

  4. Segmenting fluid effect on PCR reactions in microfluidic platforms.

    Science.gov (United States)

    Walsh, E J; King, C; Grimes, R; Gonzalez, A

    2005-12-01

    This paper evaluates the compatibility of segmenting fluids for two phase flow applications in biomedical microdevices. The evaluated fluids are chosen due to the variations in fluid properties and cost, while also reflecting their use in the recent literature. These segmenting fluids are examined to determine their compatibility with the Polymerase Chain Reaction (PCR), through controlled experiments. The results are the first to provide a quantitative measure of segmenting fluid compatibility with PCR.

  5. Evaluation of single and double-locus real-time PCR assays for methicillin-resistant Staphylococcus aureus (MRSA surveillance

    Directory of Open Access Journals (Sweden)

    Arielly Haya

    2010-04-01

    Full Text Available Abstract Background Methicillin-resistant Staphylococcus aureus (MRSA is a human pathogen, representing an infection control challenge. Conventional MRSA screening takes up to three days, therefore development of rapid detection is essential. Real time-PCR (rt-PCR is the fastest method fulfilling this task. All currently published or commercially available rt-PCR MRSA assays relay on single or double-locus detection. Double-locus assays are based on simultaneous detection of mecA gene and a S. aureus-specific gene. Such assays cannot be applied on clinical samples, which often contain both coagulase-negative staphylococci (CoNS and S. aureus, either of which can carry mecA. Single-locus assays are based on detection of the staphylococcal cassette chromosome mec (SCCmec element and the S. aureus-specific orfX gene, assuming that it is equivalent to mecA detection. Findings Parallel evaluation of several published single and double-locus rt-PCR MRSA assays of 150 pure culture strains, followed by analysis of 460 swab-derived clinical samples which included standard identification, susceptibility testing, followed by PCR detection of staphylococcal suspected isolates and in-PCR mixed bacterial populations analysis indicated the following findings. Pure cultures analysis indicated that one of the single-locus assay had very high prevalence of false positives (Positive predictive value = 77.8% and was excluded from further analysis. Analysis of 460 swab-derived samples indicated that the second single-locus assay misidentified 16 out of 219 MRSA's and 13 out of 90 methicillin-sensitive S. aureus's (MSSA were misidentified as MRSA's. The double-locus detection assay misidentified 55 out of 90 MSSA's. 46 MSSA containing samples were misidentified as MRSA and 9 as other than S. aureus ending with low positive predicted value ( Conclusion The results indicate that high prevalence of false-positive and false-negative reactions occurs in such assays.

  6. Rapid and sensitive detection of Feline immunodeficiency virus using an insulated isothermal PCR-based assay with a point-of-need PCR detection platform.

    Science.gov (United States)

    Wilkes, Rebecca Penrose; Kania, Stephen A; Tsai, Yun-Long; Lee, Pei-Yu Alison; Chang, Hsiu-Hui; Ma, Li-Juan; Chang, Hsiao-Fen Grace; Wang, Hwa-Tang Thomas

    2015-07-01

    Feline immunodeficiency virus (FIV) is an important infectious agent of cats. Clinical syndromes resulting from FIV infection include immunodeficiency, opportunistic infections, and neoplasia. In our study, a 5' long terminal repeat/gag region-based reverse transcription insulated isothermal polymerase chain reaction (RT-iiPCR) was developed to amplify all known FIV strains to facilitate point-of-need FIV diagnosis. The RT-iiPCR method was applied in a point-of-need PCR detection platform--a field-deployable device capable of generating automatically interpreted RT-iiPCR results from nucleic acids within 1 hr. Limit of detection 95% of FIV RT-iiPCR was calculated to be 95 copies standard in vitro transcription RNA per reaction. Endpoint dilution studies with serial dilutions of an ATCC FIV type strain showed that the sensitivity of lyophilized FIV RT-iiPCR reagent was comparable to that of a reference nested PCR. The established reaction did not amplify any nontargeted feline pathogens, including Felid herpesvirus 1, feline coronavirus, Feline calicivirus, Feline leukemia virus, Mycoplasma haemofelis, and Chlamydophila felis. Based on analysis of 76 clinical samples (including blood and bone marrow) with the FIV RT-iiPCR, test sensitivity was 97.78% (44/45), specificity was 100.00% (31/31), and agreement was 98.65% (75/76), determined against a reference nested-PCR assay. A kappa value of 0.97 indicated excellent correlation between these 2 methods. The lyophilized FIV RT-iiPCR reagent, deployed on a user-friendly portable device, has potential utility for rapid and easy point-of-need detection of FIV in cats.

  7. Validation of a PCR Assay for Chlamydophila abortus rRNA gene detection in a murine model

    Directory of Open Access Journals (Sweden)

    Francielle Gibson da Silva-Zacarias

    2009-11-01

    Full Text Available Chlamydophila abortus (C. abortus is associated with reproductive problems in cattle, sheep, and goats. Diagnosis of C. abortus using embryonated chicken eggs or immortalized cell lines has a very low sensitivity. Polymerase chain reaction (PCR assays have been used to detect C. abortus infection in clinical specimens and organ fragments, such as placenta, fetal organs, vaginal secretions, and semen. The aim of this study was to develop a PCR assay for the amplification of an 856-bp fragment of the rRNA gene of the Chlamydiaceae family. The PCR assay was evaluated using organs from 15 mice experimentally infected with the S26/3 reference strain of C. abortus. The results of the rRNA PCR were compared to the results from another PCR system (Omp2 PCR that has been previously described for the Omp2 (outer major protein gene from the Chlamydiaceae family. From the 15 C. abortus-inoculated mice, 13 (K=0.84, standard error =0.20 tested positive using the rRNA PCR assay and 9 (K=0.55, standard error=0.18 tested positive using the Omp2 PCR assay. The detection limit, measured using inclusion-forming units (IFU, for C. abortus with the rRNA PCR (1.05 IFU was 100-fold lower than for the Omp2 PCR (105 IFU. The higher sensitivity of the rRNA PCR, as compared to the previously described PCR assay, and the specificity of the assay, demonstrated using different pathogenic microorganisms of the bovine reproductive system, suggest that the new PCR assay developed in this study can be used for the molecular diagnosis of C. abortus in abortion and other reproductive failures in bovines, caprines, and ovines.Chlamydophila abortus (C. abortus é frequentemente associada a distúrbios reprodutivos em bovinos, ovinos e caprinos. Para o diagnóstico, os métodos de cultivo em ovo embrionado de galinha e em células de linhagem contínua apresentam baixa sensibilidade. A reação em cadeia da polimerase (PCR tem sido utilizada em placenta, órgãos fetais, secre

  8. Assessing the performance capabilities of LRE-based assays for absolute quantitative real-time PCR.

    Directory of Open Access Journals (Sweden)

    Robert G Rutledge

    Full Text Available BACKGROUND: Linear regression of efficiency or LRE introduced a new paradigm for conducting absolute quantification, which does not require standard curves, can generate absolute accuracies of +/-25% and has single molecule sensitivity. Derived from adapting the classic Boltzmann sigmoidal function to PCR, target quantity is calculated directly from the fluorescence readings within the central region of an amplification profile, generating 4-8 determinations from each amplification reaction. FINDINGS: Based on generating a linear representation of PCR amplification, the highly visual nature of LRE analysis is illustrated by varying reaction volume and amplification efficiency, which also demonstrates how LRE can be used to model PCR. Examining the dynamic range of LRE further demonstrates that quantitative accuracy can be maintained down to a single target molecule, and that target quantification below ten molecules conforms to that predicted by Poisson distribution. Essential to the universality of optical calibration, the fluorescence intensity generated by SYBR Green I (FU/bp is shown to be independent of GC content and amplicon size, further verifying that absolute scale can be established using a single quantitative standard. Two high-performance lambda amplicons are also introduced that in addition to producing highly precise optical calibrations, can be used as benchmarks for performance testing. The utility of limiting dilution assay for conducting platform-independent absolute quantification is also discussed, along with the utility of defining assay performance in terms of absolute accuracy. CONCLUSIONS: Founded on the ability to exploit lambda gDNA as a universal quantitative standard, LRE provides the ability to conduct absolute quantification using few resources beyond those needed for sample preparation and amplification. Combined with the quantitative and quality control capabilities of LRE, this kinetic-based approach has the

  9. DEVELOPMENT OF SEMI-QUANTITATIVE PCR ASSAYS FOR THE DETECTION AND ENUMERATION OF GAMBIERDISCUS SPECIES (GONYAULACALES, DINOPHYCEAE)(1).

    Science.gov (United States)

    Vandersea, Mark W; Kibler, Steven R; Holland, William C; Tester, Patricia A; Schultz, Thomas F; Faust, Maria A; Holmes, Michael J; Chinain, Mirelle; Wayne Litaker, R

    2012-08-01

    Ciguatera fish poisoning (CFP) is a serious health problem in tropical regions and is caused by the bioaccumulation of lipophilic toxins produced by dinoflagellates in the genus Gambierdiscus. Gambierdiscus species are morphologically similar and are difficult to distinguish from one another even when using scanning electron microscopy. Improved identification and detection methods that are sensitive and rapid are needed to identify toxic species and investigate potential distribution and abundance patterns in relation to incidences of CFP. This study presents the first species-specific, semi-quantitative polymerase chain reaction (qPCR) assays that can be used to address these questions. These assays are specific for five Gambierdiscus species and one undescribed ribotype. The assays utilized a SYBR green format and targeted unique sequences found within the SSU, ITS, and the D1/D3 LSU ribosomal domains. Standard curves were constructed using known concentrations of cultured cells and 10-fold serial dilutions of rDNA PCR amplicons containing the target sequence for each specific assay. Assay sensitivity and accuracy were tested using DNA extracts purified from known concentrations of multiple Gambierdiscus species. The qPCR assays were used to assess Gambierdiscus species diversity and abundance in samples collected from nearshore areas adjacent to Ft. Pierce and Jupiter, Florida USA. The results indicated that the practical limit of detection for each assay was 10 cells per sample. Most interestingly, the qPCR analysis revealed that as many as four species of Gambierdiscus were present in a single macrophyte sample.

  10. Development and validation of a real-time quantitative PCR assay for rapid identification of Bacillus anthracis in environmental samples.

    Science.gov (United States)

    Irenge, Léonid M; Durant, Jean-François; Tomaso, Herbert; Pilo, Paola; Olsen, Jaran S; Ramisse, Vincent; Mahillon, Jacques; Gala, Jean-Luc

    2010-11-01

    A real-time polymerase chain reaction (PCR) assay was developed for rapid identification of Bacillus anthracis in environmental samples. These samples often harbor Bacillus cereus bacteria closely related to B. anthracis, which may hinder its specific identification by resulting in false positive signals. The assay consists of two duplex real-time PCR: the first PCR allows amplification of a sequence specific of the B. cereus group (B. anthracis, B. cereus, Bacillus thuringiensis, Bacillus weihenstephanensis, Bacillus pseudomycoides, and Bacillus mycoides) within the phosphoenolpyruvate/sugar phosphotransferase system I gene and a B. anthracis specific single nucleotide polymorphism within the adenylosuccinate synthetase gene. The second real-time PCR assay targets the lethal factor gene from virulence plasmid pXO1 and the capsule synthesis gene from virulence plasmid pXO2. Specificity of the assay is enhanced by the use of minor groove binding probes and/or locked nucleic acids probes. The assay was validated on 304 bacterial strains including 37 B. anthracis, 67 B. cereus group, 54 strains of non-cereus group Bacillus, and 146 Gram-positive and Gram-negative bacteria strains. The assay was performed on various environmental samples spiked with B. anthracis or B. cereus spores. The assay allowed an accurate identification of B. anthracis in environmental samples. This study provides a rapid and reliable method for improving rapid identification of B. anthracis in field operational conditions.

  11. Tracking the Invasion of Small Numbers of Cells in Paper-Based Assays with Quantitative PCR.

    Science.gov (United States)

    Truong, Andrew S; Lochbaum, Christian A; Boyce, Matthew W; Lockett, Matthew R

    2015-11-17

    Paper-based scaffolds are an attractive material for culturing mammalian cells in a three-dimensional environment. There are a number of previously published studies, which utilize these scaffolds to generate models of aortic valves, cardiac ischemia and reperfusion, and solid tumors. These models have largely relied on fluorescence imaging and microscopy to quantify cells in the scaffolds. We present here a polymerase chain reaction (PCR)-based method, capable of quantifying multiple cell types in a single culture with the aid of DNA barcodes: unique sequences of DNA introduced to the genome of individual cells or cell types through lentiviral transduction. PCR-based methods are highly specific and are amenable to high-throughput and multiplexed analyses. To validate this method, we engineered two different breast cancer lines to constitutively express either a green or red fluorescent protein. These cells lines allowed us to directly compare the ability of fluorescence imaging (of the fluorescent proteins) and qPCR (of the unique DNA sequences of the fluorescent proteins) to quantify known numbers of cells in the paper based-scaffolds. We also used both methods to quantify the distribution of these breast cell lines in homotypic and heterotypic invasion assays. In the paper-based invasion assays, a single sheet of paper containing cells suspended in a hydrogel was sandwiched between sheets of paper containing only hydrogel. The stack was incubated, and the cells invaded the adjacent layers. The individual sheets of the invasion assay were then destacked and the number of cells in each layer quantified. Our results show both methods can accurately detect cell populations of greater than 500 cells. The qPCR method can repeatedly and accurately detect as few as 50 cells, allowing small populations of highly invasive cells to be detected and differentiated from other cell types.

  12. Polymerase chain reaction-based assays for the diagnosis of human brucellosis.

    Science.gov (United States)

    Wang, Ying; Wang, Zhanli; Zhang, Yaxian; Bai, Liyun; Zhao, Yue; Liu, Chunfang; Ma, An; Yu, Hui

    2014-08-01

    Polymerase chain reaction (PCR) is an in vitro technique for the nucleic acid amplification, which is commonly used to diagnose infectious diseases. The use of PCR for pathogens detection, genotyping and quantification has some advantages, such as high sensitivity, high specificity, reproducibility and technical ease. Brucellosis is a common zoonosis caused by Brucella spp., which still remains as a major health problem in many developing countries around the world. The direct culture and immunohistochemistry can be used for detecting infection with Brucella spp. However, PCR has the potential to address limitations of these methods. PCR are now one of the most useful assays for the diagnosis in human brucellosis. The aim of this review was to summarize the main PCR techniques and their applications for diagnosis and follow-up of patients with brucellosis. Moreover, advantages or limitation of the different PCR methods as well as the evaluation of PCR results for treatment and follow-up of human brucellosis were also discussed.

  13. LUX real-time PCR assay for the detection of porcine circovirus type 2.

    Science.gov (United States)

    Vilcek, Stefan; Vlasakova, Michaela; Jackova, Anna

    2010-05-01

    Light Upon eXtension real-time PCR (LUX real-time PCR) assay was developed for the detection of porcine circovirus type 2 (PCV2). The primers flanking a 114 bp fragment were selected from ORF1. The optimized assay could detect 20 viral copies of pBluescript SK+ plasmid containing inserted PCV2 DNA. The dynamic range of quantitative analysis covered a 7-order interval ranging from 20 to 2 x 10(8) genome equivalents per assay with the best results in the range from 2 x 10(2) to 2 x 10(7) viral copies. The LUX real-time PCR assay had a high specificity since it detected PCV2 but not PCV1, CSFV, PRRSV or negative samples. There was good agreement between the LUX real-time PCR and the conventional PCR when lymph nodes from PCV2 infected animals were tested. A comparison of the LUX real-time PCR with the TaqMan PCR and SYBR Green PCR indicated that the amount of viral copies determined using linear calibration curve differed from assay to assay but not more than an order. LUX real-time PCR, similar to the TaqMan PCR, was more specific for generation of fluorogenic signal than SYBR Green PCR.

  14. Comparison of a PCR assay in whole blood and serum specimens for canine brucellosis diagnosis.

    Science.gov (United States)

    Keid, L B; Soares, R M; Vasconcellos, S A; Salgado, V R; Megid, J; Richtzenhain, L J

    2010-07-17

    The performance of a serum PCR assay was compared with that of a blood PCR assay for the diagnosis of canine brucellosis caused by Brucella canis in 72 dogs. The dogs were classified into three groups (infected, non-infected and suspected brucellosis) according to the results of blood culture and serological tests. The sensitivities of blood PCR and serum PCR were, respectively, 97.14 per cent and 25.71 per cent. The specificities of both were 100 per cent. In the group of dogs with suspected brucellosis, three were positive by blood PCR and none was positive by serum PCR. Serum PCR showed little value for the direct diagnosis of canine brucellosis as the assay had low diagnostic sensitivity and fewer positive dogs were detected by this test than by blood culture, blood PCR, rapid slide agglutination test (RSAT) and RSAT with 2-mercaptoethanol.

  15. Real time TaqMan RT-PCR assay for the detection of Cucumber green mottle mosaic virus.

    Science.gov (United States)

    Hongyun, Chen; Wenjun, Zhao; Qinsheng, Gu; Qing, Chen; Shiming, Lin; Shuifang, Zhu

    2008-05-01

    A real time reverse-transcription polymerase chain reaction (RT-PCR) was developed for efficient detection of Cucumber green mottle mosaic virus (CGMMV). The method was designed to use a duo-primer system with a TaqMan probe targeting the conserved sequence in 3' noncoding region (NCR) of CGMMV to detect isolates of this virus collected in China. The sensitivity of the real time RT-PCR assay was 0.13 pg of total RNA or 50 molecules of RNA transcripts. This level of sensitivity indicated that the one step real time RT-PCR developed in the present study could be used for routine testing assays. The real time RT-PCR method could assist in the implementation of quarantine measures for prevention and control of the disease caused by CGMMV.

  16. Comparison of kDNA PCR-hybridization assay with three PCR methods for canines visceral Leishmaniasis diagnosis

    Energy Technology Data Exchange (ETDEWEB)

    Pilatti, Marcia M.; Andrade, Antero S.R. [Centro de Desenvolvimento da Tecnologia Nuclear (CDTN/CNEN-MG), Belo Horizonte, MG (Brazil)], e-mail: marciapilatti@yahoo.com.br, e-mail: antero@cdtn.br; Ferreira, Sidney A. [Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG (Brazil). Dept. de Parasitologia], e-mail: saninoalmeida@gmail.com

    2009-07-01

    The sensitivity of the kDNA PCR-Hybridization assay, which uses radioactive DNA probes (labeled with {sup 32}P), was compared with three conventional PCR methods used for canine visceral leishmaniasis diagnosis. All PCR methods had two steps: a first amplification followed by hybridization or by a new amplification (nested or semi nested). Two methods (kDNA PCR-Hybridization and kDNA snPCR) used primers addressed to kinetoplast minicircles and the other two methods to the coding (LnPCR) and intergenic noncoding regions (ITS-1 nPCR) of the ribosomal rRNA genes. The comparison was accomplished in two groups of 23 infected dogs using samples collected by the conjunctival swab procedure. In the Group 1 the DNA was extracted from cotton swabs by phenol-chloroform and in Group 2 by boiling. The most efficient PCR methods in the Group 1 were those based on kDNA targets. The kDNA PCR-Hybridization was able to detect parasites in 22/23 dogs (95.6%) and in 40/46 samples (86.9%). The kDNA snPCR was positive for 21/23 dogs (91.3%) and for 40/46 samples (86.9%). The positivities of the kDNA based methods were significantly higher than the positivities verified for the methods based on ribosomal rRNA genes (p<0.05). In the Group 2 the kDNA PCR- Hybridization showed a better performance detecting parasites in 18/23 dogs (78.3%) and in 31/46 samples (67.4%), significantly higher than the other three methods (p<0.05). The higher sensitivity of the minicircle kDNA based assays reported by others was confirmed in this study and kDNA PCR-Hybridization showed the best sensitivity among the assays evaluated. (author)

  17. Detection of Staphylococcus aureus in Dairy Products by Polymerase Chain Reaction Assay

    Institute of Scientific and Technical Information of China (English)

    YANG Yang; SU Xu-dong; YUAN Yao-wu; KANG Chun-yu; LI Ying-jun; ZHANG wei; ZHONG Xiao-ying

    2007-01-01

    A polymerase chain reaction (PCR) assay was employed for direct detection of Staphylococcus aureus without enrichment in dairy products. A solvent extraction procedure was successfully modified for the extraction of Staphylococcus aureus DNA from artificially contaminated whole milk, skim milk, and cheese. A primer targeting the thermostable nuclease gene (nuc) was used in the PCR. A DNA fragment of 279 bp was amplified. The PCR product was confirmed by DNA sequencing. In this study, the PCR, GB- 4789.10-94, Perifilm RSA.Count Plate, and Baird-Parker + RPF Agar were compared.The sensitivity of the PCR was 10 CFU mL-1 of whole milk, skim milk, and 55 CFU g-1 of cheese. The developed methodology allowed for detection of Staphylococcus aureus in dairy products in less than 6 h. The time taken for the development of this PCR assay was 12-24 h, less than the time taken by the general PCR assay using the enrichment method, and the coincidence rate of this developed PCR was 94.3%, the sensitivity was 100%. It was a rapid, sensitive, and effective method for PCR to detect Staphylococcus aureus in milk and milk products.

  18. Multiplex real-time PCR assay for detection and classification of Klebsiella pneumoniae carbapenemase gene (bla KPC) variants.

    Science.gov (United States)

    Chen, Liang; Mediavilla, José R; Endimiani, Andrea; Rosenthal, Marnie E; Zhao, Yanan; Bonomo, Robert A; Kreiswirth, Barry N

    2011-02-01

    Carbapenem resistance mediated by plasmid-borne Klebsiella pneumoniae carbapenemases (KPC) is an emerging problem of significant clinical importance in Gram-negative bacteria. Multiple KPC gene variants (bla(KPC)) have been reported, with KPC-2 (bla(KPC-2)) and KPC-3 (bla(KPC-3)) associated with epidemic outbreaks in New York City and various international settings. Here, we describe the development of a multiplex real-time PCR assay using molecular beacons (MB-PCR) for rapid and accurate identification of bla(KPC) variants. The assay consists of six molecular beacons and two oligonucleotide primer pairs, allowing for detection and classification of all currently described bla(KPC) variants (bla(KPC-2) to bla(KPC-11)). The MB-PCR detection limit was 5 to 40 DNA copies per reaction and 4 CFU per reaction using laboratory-prepared samples. The MB-PCR probes were highly specific for each bla(KPC) variant, and cross-reactivity was not observed using DNA isolated from several bacterial species. A total of 457 clinical Gram-negative isolates were successfully characterized by our MB-PCR assay, with bla(KPC-3) and bla(KPC-2) identified as the most common types in the New York/New Jersey metropolitan region. The MB-PCR assay described herein is rapid, sensitive, and specific and should be useful for understanding the ongoing evolution of carbapenem resistance in Gram-negative bacteria. As novel bla(KPC) variants continue to emerge, the MB-PCR assay can be modified in response to epidemiologic developments.

  19. Comparison of quantitative PCR assays for Escherichia coli targeting ribosomal RNA and single copy genes

    Science.gov (United States)

    Aims: Compare specificity and sensitivity of quantitative PCR (qPCR) assays targeting single and multi-copy gene regions of Escherichia coli. Methods and Results: A previously reported assay targeting the uidA gene (uidA405) was used as the basis for comparing the taxono...

  20. Diagnostic efficacy of a real time-PCR assay for Chlamydia trachomatis infection in infertile women in north India

    Directory of Open Access Journals (Sweden)

    Benu Dhawan

    2014-01-01

    Full Text Available Background & objectives: Little is known about the prevalence of Chlamydia trachomatis infection in Indian women with infertility. To improve the diagnosis of C. trachomatis infection in developing countries, there is an urgent need to establish cost-effective molecular test with high sensitivity and specificity. This study was conducted to determine the diagnostic utility of a real time-PCR assay for detention of C. trachomatis infection in infertile women attending an infertility clinic in north India. The in house real time-PCR assay was also compared with a commercial real-time PCR based detection system. Methods: Endocervical swabs, collected from 200 infertile women were tested for C. trachomatis by three different PCR assays viz. in-house real time-PCR targeting the cryptic plasmid using published primers, along with omp1 gene and cryptic plasmid based conventional PCR assays. Specimens were also subjected to direct fluorescence assay (DFA and enzyme immunoassay (EIA Performance of in-house real time-PCR was compared with that of COBAS Taqman C. trachomatis Test, version 2.0 on all in-house real time-PCR positive sample and 30 consecutive negative samples. Results: C. trachomatis infection was found in 13.5 per cent (27/200 infertile women by in-house real time-PCR, 11.5 per cent (23/200 by cryptic plasmid and/or omp1 gene based conventional PCR, 9 per cent (18/200 by DFA and 6.5 per cent (7/200 by EIA. The in-house real time-PCR exhibited a sensitivity and specificity of 100 per cent, considering COBAS Taqman CT Test as the gold standard. The negative and positive predictive values of the in-house real time-PCR were 100 per cent. The in-house real time-PCR could detect as low as 10 copies of C. trachomatis DNA per reaction. Interpretation & conclusions: In-house real time-PCR targeting the cryptic plasmid of C. trachomatis exhibited an excellent sensitivity and specificity similar to that of COBAS Taqman CT Test, v2.0 for detection of C

  1. A multiplex real-time polymerase chain reaction assay to diagnose Epiphyas postvittana (Lepidoptera: Tortricidae).

    Science.gov (United States)

    Barr, N B; Ledezma, L A; Farris, R E; Epstein, M E; Gilligan, T M

    2011-10-01

    A molecular assay for diagnosis of light brown apple moth, Epiphyas postvittana (Walker) (Lepidoptera: Tortricidae), in North America is reported. The assay multiplexes two TaqMan real-time polymerase chain reaction (RT-PCR) probe systems that are designed to target DNA segments of the internal transcribed spacer region 2 (ITS2) and 18S rRNA gene. The RT-PCR probe designed for the 18S target recognizes a DNA sequence conserved in all of the moths included in the study and functions as a control in the assay. The second probe recognizes a segment of the ITS2 specifically found in E. postvittana and not found in the other moths included in the study, i.e., this segment is not conserved. Inclusion of the two markers in a single multiplex reaction did not affect assay performance. The assay was tested against 637 moths representing > 90 taxa in 15 tribes in all three subfamilies in the Tortricidae. The assay generated no false negatives based on analysis of 355 E. postvittana collected from California, Hawaii, England, New Zealand, and Australia. Analysis of a data set including 282 moths representing 41 genera generated no false positives. Only three inconclusive results were generated from the 637 samples. Spike experiments demonstrated that DNA contamination in the assay can affect samples differently. Contaminated samples analyzed with the ITS2 RT-PCR assay and DNA barcode methodology by using the cytochrome oxidase I gene can generate contradictory diagnoses.

  2. Development of a polymerase chain reaction assay for the detection of pseudorabies virus in clinical samples

    OpenAIRE

    Lester J Pérez; Heidy Díaz de Arce

    2009-01-01

    Aujeszky's disease, also known as pseudorabies causes severe economic losses in swine industry and affects the pig husbandry all over the world. The conventional diagnostic procedure is time-consuming and false-negative results may occur in submissions from latently infected animals. The development, optimization and evaluation of a polymerase chain reaction (PCR) assay are presented for the diagnosis of pseudorabies infection. This assay was based on the amplification of a highly conserved v...

  3. Buoyancy-Driven Polymerase Chain Reaction (PCR) Devices

    Energy Technology Data Exchange (ETDEWEB)

    Ness, K D; Wheeler, E K; Benett, W; Stratton, P; Christian, A; Chen, A; Ortega, J; Weisgraber, T H; Goodson, K E

    2004-09-28

    Polymerase chain reaction (PCR) facilitates DNA detection by significantly increasing the concentration of specific DNA segments. A new class of PCR instruments uses a buoyancy-driven re-circulating flow to thermally cycle the DNA sample and benefits from reduced cycle times, low sample volumes, a miniaturized format, and low power consumption. This paper analyzes a specific buoyancy PCR device in a micro-channel ''race-track'' geometry to determine key parameters about PCR cycle times and other figures of merit as functions of device dimensions. The 1-D model balances the buoyancy driving force with frictional losses. A hydrostatic pressure imbalance concept is used between the left and right sides of the fluid loop to calculate the buoyancy driving force. Velocity and temperature distributions within the channels are determined from two-dimensional analysis of the channel section, with developing region effects included empirically through scaled values of the local Nusselt number. Good agreement between four independent verification steps validate the 1-D simulation approach: (1) analytical expressions for the thermal entrance length are compared against, (2) comparison with a full 3-D finite element simulation, (3) comparison with an experimental flow field characterization, and (4) calculation of the minimum PCR runtime required to get a positive PCR signal from the buoyancy-driven PCR device. The 1-D approach closely models an actual buoyancy-driven PCR device and can further be used as a rapid design tool to simulate buoyancy PCR flows and perform detailed design optimizations studies.

  4. Comparison of PCR-ELISA and LightCycler real-time PCR assays for detecting Salmonella spp. in milk and meat samples

    DEFF Research Database (Denmark)

    Perelle, Sylvie; Dilasser, Françoise; Malorny, Burkhard

    2004-01-01

    , minced beef and raw milk, and 92 naturally-contaminated milk and meat samples. When using either PCR-ELISA or LC-PCR assays, only Salmonella strains were detected. PCR-ELISA and LC-PCR assays gave with pure Salmonella cultures the same detection limit level of 10(3) CFU/ml, which corresponds respectively...

  5. Simultaneous Detection of CDC Category "A" DNA and RNA Bioterrorism Agents by Use of Multiplex PCR & RT-PCR Enzyme Hybridization Assays

    Directory of Open Access Journals (Sweden)

    Kelly J. Henrickson

    2009-10-01

    Full Text Available Assays to simultaneously detect multiple potential agents of bioterrorism are limited. Two multiplex PCR and RT-PCR enzyme hybridization assays (mPCR-EHA, mRT-PCR-EHA were developed to simultaneously detect many of the CDC category “A” bioterrorism agents. The “Bio T” DNA assay was developed to detect: Variola major (VM, Bacillus anthracis (BA, Yersinia pestis (YP, Francisella tularensis (FT and Varicella zoster virus (VZV. The “Bio T” RNA assay (mRT-PCR-EHA was developed to detect: Ebola virus (Ebola, Lassa fever virus (Lassa, Rift Valley fever (RVF, Hantavirus Sin Nombre species (HSN and dengue virus (serotypes 1-4. Sensitivity and specificity of the 2 assays were tested by using genomic DNA, recombinant plasmid positive controls, RNA transcripts controls, surrogate (spiked clinical samples and common respiratory pathogens. The analytical sensitivity (limit of detection (LOD of the DNA asssay for genomic DNA was 1×100~1×102 copies/mL for BA, FT and YP. The LOD for VZV whole organism was 1×10-2 TCID50/mL. The LOD for recombinant controls ranged from 1×102~1×103copies/mL for BA, FT, YP and VM. The RNA assay demonstrated LOD for RNA transcript controls of 1×104~1×106 copies/mL without extraction and 1×105~1×106 copies/mL with extraction for Ebola, RVF, Lassa and HSN. The LOD for dengue whole organisms was ~1×10-4 dilution for dengue 1 and 2, 1×104 LD50/mL and 1×102 LD50/mL for dengue 3 and 4. The LOD without extraction for recombinant plasmid DNA controls was ~1×103 copies/mL (1.5 input copies/reaction for Ebola, RVF, Lassa and HSN. No cross-reactivity of primers and probes used in both assays was detected with common respiratory pathogens or between targeted analytes. Clinical sensitivity was estimated using 264 surrogate clinical samples tested with the BioT DNA assay and 549 samples tested with the BioT RNA assay. The clinical specificity is 99.6% and 99.8% for BioT DNA assay and BioT RNA assay, respectively. The

  6. Development and application of multiplex PCR assays for detection of virus-induced respiratory disease complex in dogs

    Science.gov (United States)

    PIEWBANG, Chutchai; RUNGSIPIPAT, Anudep; POOVORAWAN, Yong; TECHANGAMSUWAN, Somporn

    2016-01-01

    Canine infectious respiratory disease complex (CIRDC) viruses have been detected in dogs with respiratory illness. Canine influenza virus (CIV), canine parainfluenza virus (CPIV), canine distemper virus (CDV), canine respiratory coronavirus (CRCoV), canine adenovirus type 2 (CAdV-2) and canine herpesvirus 1 (CaHV-1), are all associated with the CIRDC. To allow diagnosis, two conventional multiplex polymerase chain reactions (PCR) were developed to simultaneously identify four RNA and two DNA viruses associated with CIRDC. The two multiplex PCR assays were then validated on 102 respiratory samples collected from 51 dogs with respiratory illness by sensitivity and specificity determination in comparison to conventional simplex PCR and a rapid three-antigen test kit. All six viruses were detected in either individual or multiple infections. The developed multiplex PCR assays had a >87% sensitivity and 100% specificity compared to their simplex counterpart. Compared to the three-antigen test kit, the multiplex PCR assays yielded 100% sensitivity and more than 83% specificity for detection of CAdV-2 and CDV, but not for CIV. Therefore, the developed multiplex PCR modalities were able to simultaneously diagnose a panel of CIRDC viruses and facilitated specimen collection through being suitable for use of nasal or oral samples. PMID:27628592

  7. Development and validation of a Myxoma virus real-time polymerase chain reaction assay.

    Science.gov (United States)

    Albini, Sarah; Sigrist, Brigitte; Güttinger, Regula; Schelling, Claude; Hoop, Richard K; Vögtlin, Andrea

    2012-01-01

    To aid in the rapid diagnosis of myxomatosis in rabbits, a real-time polymerase chain reaction (PCR) for the specific detection of Myxoma virus is described. Primers and probe were designed to amplify a 147-bp fragment within the Serp2 gene. The assay was able to detect 23 copies of a synthesized oligo indicating a reliable sensitivity. In addition, the real-time PCR did not detect the Rabbit fibroma virus used in myxomatosis vaccines. The novel PCR was shown to be able to detect Myxoma virus in fresh and paraffin-embedded rabbit tissues originating from myxomatosis cases from various regions in Switzerland.

  8. Development and validation of a Myxoma virus real-time polymerase chain reaction assay

    OpenAIRE

    Albini, S; Sigrist, B; Guttinger, R; Schelling, C; Hoop, R K; Vogtlin, A

    2012-01-01

    To aid in the rapid diagnosis of myxomatosis in rabbits, a real-time polymerase chain reaction (PCR) for the specific detection of Myxoma virus is described. Primers and probe were designed to amplify a 147-bp fragment within the Serp2 gene. The assay was able to detect 23 copies of a synthesized oligo indicating a reliable sensitivity. In addition, the real-time PCR did not detect the Rabbit fibroma virus used in myxomatosis vaccines. The novel PCR was shown to be able to detect Myxoma virus...

  9. A SYBR Green RT-PCR assay in single tube to detect human and bovine noroviruses and control for inhibition

    Directory of Open Access Journals (Sweden)

    Saegerman Claude

    2008-08-01

    Full Text Available Abstract Background Noroviruses are single-stranded RNA viruses belonging to the family Caliciviridae. They are a major cause of epidemic and sporadic gastroenteritis in humans and clinical signs and lesions of gastroenteritis were reported in bovines. Due to their genetic proximity, potential zoonotic transmission or animal reservoir can be hypothesized for noroviruses. RT-PCR has become the "gold standard" for the detection of noroviruses in faecal and environmental samples. With such samples, the control for inhibition of the reaction during amplification and detection is crucial to avoid false negative results, which might otherwise not be detected. The aim of the reported method is to detect, with a SYBR Green technology, a broad range of noroviruses with a control for inhibition. Results A SYBR Green real-time RT-PCR assay was developed making use of a foreign internal RNA control added in the same tube. This assay is able to detect human and bovine noroviruses belonging to genogroups I, II and III and to distinguish between norovirus and internal control amplicons using melting curve analysis. A 10-fold dilution of samples appears to be the method of choice to remove inhibition. This assay was validated with human and bovine stool samples previously tested for norovirus by conventional RT-PCR. Conclusion This SYBR Green real-time RT-PCR assay allows the detection of the most important human and bovine noroviruses in the same assay, and avoids false negative results making use of an internal control. Melting curves allow the discrimination between the internal control and norovirus amplicons. It gives preliminary information about the species of origin. The sensitivity of the developed assay is higher than conventional RT-PCR and a 10-fold dilution of samples showed a better efficiency and reproducibility to remove RT-PCR inhibition than addition of bovine serum albumin.

  10. Detection of human papillomavirus in pterygium and conjunctival papilloma by hybrid capture II and PCR assays.

    Science.gov (United States)

    Takamura, Y; Kubo, E; Tsuzuki, S; Akagi, Y

    2008-11-01

    To elucidate the putative role of human papillomavirus (HPV) infection in pterygium and conjunctival papilloma. Hybrid capture II (HC-II) and polymerase chain reaction (PCR) assays were performed to detect HPV in pterygium (42 samples obtained from 40 patients) and conjunctival papilloma (8 samples from 6 patients). The amount of HPV DNA was evaluated by measurement of relative light units (RLUs) on a luminometer. All papilloma samples were positive for HPV DNA by PCR and HC-II. The RLU values for specimens of recurrent and re-recurrent papilloma were markedly higher than those for specimens of primary lesions. HPV was detected by PCR in 2 of 42 (4.8%) beta-globin-positive pterygium specimens, whereas HC-II showed that HPV was negative in all pterygium samples. Our results support the hypothesis that HPV DNA is associated with the pathogenesis of conjunctival papilloma, but not pterygium. RLU measurement by HC-II may serve as a marker for evaluating the activity of HPV in conjunctival tumours.

  11. Validation of a multiplex PCR assay for the forensic identification of Indian crocodiles.

    Science.gov (United States)

    Meganathan, Poorlin Ramakodi; Dubey, Bhawna; Jogayya, Kothakota Naga; Haque, Ikramul

    2011-09-01

    A dependable and efficient wildlife species identification system is essential for swift dispensation of the justice linking wildlife crimes. Development of molecular techniques is befitting the need of the time. The forensic laboratories often receive highly ill-treated samples for identification purposes, and thus, validation of any novel methodology is necessary for forensic usage. We validate a novel multiplex polymerase chain reaction assay, developed at this laboratory for the forensic identification of three Indian crocodiles, Crocodylus palustris, Crocodylus porosus, and Gavialis gangeticus, following the guidelines of Scientific Working Group on DNA Analysis Methods. The multiplex PCR was tested for its specificity, reproducibility, sensitivity, and stability. This study also includes the samples treated with various chemical substances and exposed to various environmental regimes. The result of this validation study promises this technique to be an efficient identification tool for Indian crocodiles and therefore is recommended for forensic purposes.

  12. Improved PCR assay for the species-specific identification and quantitation of Legionella pneumophila in water.

    Science.gov (United States)

    Cho, Min Seok; Ahn, Tae-Young; Joh, Kiseong; Lee, Eui Seok; Park, Dong Suk

    2015-11-01

    Legionellosis outbreak is a major global health care problem. However, current Legionella risk assessments may be compromised by uncertainties in Legionella detection methods, infectious dose, and strain infectivity. These limitations may place public health at significant risk, leading to significant monetary losses in health care. However, there are still unmet needs for its rapid identification and monitoring of legionellae in water systems. Therefore, in the present study, a primer set was designed based on a LysR-type transcriptional regulator (LTTR) family protein gene of Legionella pneumophila subsp. pneumophila str. Philadelphia 1 because it was found that this gene is structurally diverse among species through BLAST searches. The specificity of the primer set was evaluated using genomic DNA from 6 strains of L. pneumophila, 5 type strains of other related Legionella species, and other 29 reference pathogenic bacteria. The primer set used in the PCR assay amplified a 264-bp product for only targeted six strains of L. pneumophila. The assay was also able to detect at least 1.39 × 10(3) copies/μl of cloned amplified target DNA using purified DNA or 7.4 × 10(0) colony-forming unit per reaction when using calibrated cell suspension. In addition, the sensitivity and specificity of this assay were confirmed by successful detection of Legionella pneumophila in environmental water samples.

  13. Development and inter-laboratory assessment of droplet digital PCR assays for multiplex quantification of 15 genetically modified soybean lines.

    Science.gov (United States)

    Košir, Alexandra Bogožalec; Spilsberg, Bjørn; Holst-Jensen, Arne; Žel, Jana; Dobnik, David

    2017-08-17

    Quantification of genetically modified organisms (GMOs) in food and feed products is often required for their labelling or for tolerance thresholds. Standard-curve-based simplex quantitative polymerase chain reaction (qPCR) is the prevailing technology, which is often combined with screening analysis. With the rapidly growing number of GMOs on the world market, qPCR analysis becomes laborious and expensive. Innovative cost-effective approaches are therefore urgently needed. Here, we report the development and inter-laboratory assessment of multiplex assays to quantify GMO soybean using droplet digital PCR (ddPCR). The assays were developed to facilitate testing of foods and feed for compliance with current GMO regulations in the European Union (EU). Within the EU, the threshold for labelling is 0.9% for authorised GMOs per ingredient. Furthermore, the EU has set a technical zero tolerance limit of 0.1% for certain unauthorised GMOs. The novel multiplex ddPCR assays developed target 11 GMO soybean lines that are currently authorised, and four that are tolerated, pending authorisation in the EU. Potential significant improvements in cost efficiency are demonstrated. Performance was assessed for the critical parameters, including limits of detection and quantification, and trueness, repeatability, and robustness. Inter-laboratory performance was also determined on a number of proficiency programme and real-life samples.

  14. 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 IS1311, 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.

  15. Comparison of three multiplex PCR assays for the detection of respiratory viral infections: evaluation of xTAG respiratory virus panel fast assay, RespiFinder 19 assay and RespiFinder SMART 22 assay

    Directory of Open Access Journals (Sweden)

    Dabisch-Ruthe Mareike

    2012-07-01

    Full Text Available Abstract Background A broad spectrum of pathogens is causative for respiratory tract infections, but symptoms are mostly similar. Therefore, the identification of the causative viruses and bacteria is only feasible using multiplex PCR or several monoplex PCR tests in parallel. Methods The analytical sensitivity of three multiplex PCR assays, RespiFinder-19, RespiFinder-SMART-22 and xTAG-Respiratory-Virus-Panel-Fast-Assay (RVP, were compared to monoplex real-time PCR with quantified standardized control material. All assays include the most common respiratory pathogens. Results To compare the analytical sensitivity of the multiplex assays, samples were inoculated with 13 different quantified viruses in the range of 101 to 105 copies/ml. Concordant results were received for rhinovirus, whereas the RVP detected influenzavirus, RSV and hMPV more frequently in low concentrations. The RespiFinder-19 and the RespiFinder-SMART-22 showed a higher analytical sensitivity for adenoviruses and coronaviruses, whereas the RVP was incapable to detect adenovirus and coronavirus in concentrations of 104 copies/ml. The RespiFinder-19 and RespiFinder-SMART-22A did not detect influenzaviruses (104 copies/ml and RSV (103 copies/ml. The detection of all 13 viruses in one sample was only achieved using monoplex PCR. To analyze possible competitive amplification reactions between the different viruses, samples were further inoculated with only 4 different viruses in one sample. Compared to the detection of 13 viruses in parallel, only a few differences were found. The incidence of respiratory viruses was compared in tracheal secretion (TS samples (n = 100 of mechanically ventilated patients in winter (n = 50 and summer (n = 50. In winter, respiratory viruses were detected in 32 TS samples (64% by RespiFinder-19, whereas the detection rate with RVP was only 22%. The most frequent viruses were adenovirus (32% and PIV-2 (20%. Multiple infections were detected

  16. Miniprimer PCR assay targeting multiple genes: a new rapid and reliable tool for genotyping Pantoea stewartii subsp. stewartii.

    Science.gov (United States)

    Xu, R; Chen, Q; Robleh Djama, Z; Tambong, J T

    2010-02-01

    Development of a 'miniprimer' PCR assay for genotyping Pantoea stewartii subsp. stewartii, the causal agent of the Stewart's bacterial wilt on maize. Four 10-nucleotide (10-nt) 'miniprimer' sets were designed and evaluated in the presence of Titanium Taq DNA polymerase. Under optimal reaction conditions, the miniprimer pair Uni-BacF-10/Uni-BacR-10 reproducibly generated identical banding patterns among 10 strains of P. stewartii subsp. stewartii, different patterns from strains of P. stewartii subsp. indologenes, other Panteoa species, Clavibacter michiganensis, Pectobacterium spp., Pseudomonas spp. and other bacterial species. The amplicons of Pantoea stewartii subsp. stewartii were cloned and sequenced to identify genes or DNA fragments that are targeted by the miniprimer PCR assay. Of the 14 'clone types' identified, sequences of a 1.23-kb fragment had a 99.8% similarity to part of the Pantoea stewartii zeaxanthin diglucoside biosynthetic operon (AY166713). Other dominant cloned fragments included a 411-bp amplicon that exhibited 99.8% similarity to the psaU gene (syn:ysaU; GQ249669), a type III protein-secretion system complex of P. stewartii subsp. stewartii strain DC283, and a 548-bp fragment showed 63% homology to the Asp/Glu racemase encoding gene in Erwinia tasmaniensis strain ET1/99. The miniprimer PCR assay reported here is highly discriminatory and reproducible in genotyping Pantoea stewartii subsp. stewartii. This miniprimer PCR assay could be a new reliable and rapid tool for fingerprinting the Stewart's wilt pathogen of maize.

  17. 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.

  18. Performance Assessment of Human and Cattle Associated Quantitative Real-time PCR Assays - slides

    Science.gov (United States)

    The presentation overview is (1) Single laboratory performance assessment of human- and cattle associated PCR assays and (2) A Field Study: Evaluation of two human fecal waste management practices in Ohio watershed.

  19. A quadruplex PCR (qxPCR) assay for adulteration in dairy products.

    Science.gov (United States)

    Agrimonti, Caterina; Pirondini, Andrea; Marmiroli, Marta; Marmiroli, Nelson

    2015-11-15

    This study describes the development of a quadruplex quantitative Real Time PCR (qxPCR) based on SYBR®GreenER chemistry, for rapid identification of DNA of cow, goat, sheep and buffalo in dairy products, and for quantification of cow DNA in these products. The platform was applied to: (i) mixes of milks at fixed percentages; (ii) cheeses prepared with the same mixes; (iii) commercial dairy products. The methodology enabled the detection of DNA from cow in mixes of milk and cheeses with a limit of detection (LOD) of 0.1%. When applied to commercial dairy products the qxPCR gave results comparable with each single-plex Real Time PCR. A good correlation (R(2)>0.9) between peaks' area of derivative of melting curves of amplicons and percentages of cow milk in milk mixes and cheeses, allows for an estimation of cow DNA in a dynamic range varying from 0.1-5% to 1-25%.

  20. The sensitivity and specificity of a reverse transcription-polymerase chain reaction assay for the avian pneumovirus (Colorado strain).

    Science.gov (United States)

    Pedersen, J C; Reynolds, D L; Ali, A

    2000-01-01

    A reverse transcription-polymerase chain reaction (RT-PCR) assay for the detection of avian pneumovirus (APV), Colorado strain (US/CO), was evaluated for sensitivity and specificity. The single-tube RT-PCR assay utilized primers developed from the matrix (M) gene sequence of the US/CO APV. The RT-PCR amplified the US/CO APV but did not amplify other pneumoviruses, including the avian pneumoviruses subgroups A and B. The RT-PCR was capable of detecting between 10(0.25) mean tissue culture infective dose (TCID50) and 10(-0.44) TCID50 of the US/CO APV. These results have demonstrated that the single-tube RT-PCR assay is a specific and sensitive assay for the detection of US/CO APV.

  1. Real-Time PCR Assay To Detect Smallpox Virus

    Science.gov (United States)

    Sofi Ibrahim, M.; Kulesh, David A.; Saleh, Sharron S.; Damon, Inger K.; Esposito, Joseph J.; Schmaljohn, Alan L.; Jahrling, Peter B.

    2003-01-01

    We developed a highly sensitive and specific assay for the rapid detection of smallpox virus DNA on both the Smart Cycler and LightCycler platforms. The assay is based on TaqMan chemistry with the orthopoxvirus hemagglutinin gene used as the target sequence. With genomic DNA purified from variola virus Bangladesh 1975, the limit of detection was estimated to be approximately 25 copies on both machines. The assay was evaluated in a blinded study with 322 coded samples that included genomic DNA from 48 different isolates of variola virus; 25 different strains and isolates of camelpox, cowpox, ectromelia, gerbilpox, herpes, monkeypox, myxoma, rabbitpox, raccoonpox, skunkpox, vaccinia, and varicella-zoster viruses; and two rickettsial species at concentrations mostly ranging from 100 fg/μl to 1 ng/μl. Contained within those 322 samples were variola virus DNA, obtained from purified viral preparations, at concentrations of 1 fg/μl to 1 ng/μl. On the Smart Cycler platform, 2 samples with false-positive results were detected among the 116 samples not containing variola virus tested; i.e., the overall specificity of the assay was 98.3%. On the LightCycler platform, five samples with false-positive results were detected (overall specificity, 95.7%). Of the 206 samples that contained variola virus DNA ranging in concentrations from 100 fg/μl to 1 ng/μl, 8 samples were considered negative on the Smart Cycler platform and 1 sample was considered negative on the LightCycler platform. Thus, the clinical sensitivities were 96.1% for the Smart Cycler instrument and 99.5% for the LightCycler instrument. The vast majority of these samples were derived from virus-infected cell cultures and variola virus-infected tissues; thus, the DNA material contained both viral DNA and cellular DNA. Of the 43 samples that contained purified variola virus DNA ranging in concentration from 1 fg/μl to 1 ng/μl, the assay correctly detected the virus in all 43 samples on both the Smart Cycler

  2. A New Lab Developed Real Time PCR Assay for Direct Detection of C. Difficle from Stool Sample without DNA Extraction.

    Science.gov (United States)

    Li, Brandon

    2016-09-01

    Clostridium difficile is a major cause of nosocomial antibiotic-associated infectious diarrhea and pseudomembranous colitis. Detection of C. difficile by anaerobic bacterial culture and/or cytotoxicity assays has been largely replaced by rapid enzyme immunoassays (EIA). However, due to the lack of sensitivity of stool EIA, we developed a multiplex real-time PCR assay targeting the C. difficile toxin genes tcdB. stool samples from hospitalized pediatric patients suspected of having C. difficile-associated disease were prospectively collected. Three testing modalities were evaluated, including enriched culture, cepheid Xpert and real-time Pcr (tcdB) on stool samples performed with tcdB gene-specific primers and hydrolysis probes. A total of 150 de-identified clinical specimen were analyzed. The sensitivities of stool real-time Pcr were 95% against cepheid Xpert C. difficile and 93% against enriched culture respectively, with a specificity of 97% and 94%. The lower limit of detection of the stool real-time PCR was 0.5 cFU/ml of per reaction for tcdB. Direct detection of C. difficile toxin genes in stool samples by real-time Pcr showed performance comparable to enriched culture. Real-time PCR of DNA from stool samples is a rapid and cost-effective diagnostic modality for patients that should facilitate appropriate patient management.

  3. A New Lab Developed Real Time PCR Assay for Direct Detection of C. Difficle from Stool Sample without DNA Extraction

    Science.gov (United States)

    Li, Brandon

    2016-01-01

    Clostridium difficile is a major cause of nosocomial antibiotic-associated infectious diarrhea and pseudomembranous colitis. Detection of C. difficile by anaerobic bacterial culture and/or cytotoxicity assays has been largely replaced by rapid enzyme immunoassays (EIA). However, due to the lack of sensitivity of stool EIA, we developed a multiplex real-time PCR assay targeting the C. difficile toxin genes tcdB. stool samples from hospitalized pediatric patients suspected of having C. difficile-associated disease were prospectively collected. Three testing modalities were evaluated, including enriched culture, cepheid Xpert and real-time Pcr (tcdB) on stool samples performed with tcdB gene-specific primers and hydrolysis probes. A total of 150 de-identified clinical specimen were analyzed. The sensitivities of stool real-time Pcr were 95% against cepheid Xpert C. difficile and 93% against enriched culture respectively, with a specificity of 97% and 94%. The lower limit of detection of the stool real-time PCR was 0.5 cFU/ml of per reaction for tcdB. Direct detection of C. difficile toxin genes in stool samples by real-time Pcr showed performance comparable to enriched culture. Real-time PCR of DNA from stool samples is a rapid and cost-effective diagnostic modality for patients that should facilitate appropriate patient management. PMID:27829823

  4. A multiplex reverse transcription-polymerase chain reaction assay for Newcastle disease virus and avian pneumovirus (Colorado strain).

    Science.gov (United States)

    Ali, A; Reynolds, D L

    2000-01-01

    Newcastle disease virus (NDV) and avian pneumovirus (APV) cause Newcastle disease and rhinotracheitis respectively, in turkeys. Both of these viruses infect the respiratory system. A one-tube, multiplex, reverse transcription-polymerase chain reaction (RT-PCR) assay for the detection of both NDV and Colorado strain of APV (APV-Col) was developed and evaluated. The primers, specific for each virus, were designed from the matrix protein gene of APV-Col and the fusion protein gene of NDV to amplify products of 631 and 309 nucleotides, respectively. The multiplex RT-PCR assay, for detecting both viruses simultaneously, was compared with the single-virus RT-PCR assays for its sensitivity and specificity. The specific primers amplified products of predicted size from each virus in the multiplex as well as the single-virus RT-PCR assays. The multiplex RT-PCR assay was determined to be equivalent to the single-virus RT-PCR assays for detecting both NDV and APV-Col. This multiplex RT-PCR assay proved to be a sensitive method for the simultaneous and rapid detection of NDV and APV-Col. This assay has the potential for clinical diagnostic applications.

  5. Validation of real time PCR assays for use in routine diagnostics of pig diarrhoea

    DEFF Research Database (Denmark)

    Ståhl, Marie; Hjulsager, Charlotte Kristiane; Breum, Solvej Østergaard

    At the National Veterinary Institute in Denmark we want to optimize routine diagnostic analyses by screening samples simultaneously for several agents by real time PCR. Here we present the validation of real time PCR assays for E. coli F4. E coli F18 and Lawsonia intracellularis2 in pig feces...

  6. A multiplex real-time PCR assay for routine diagnosis of bacterial vaginosis

    NARCIS (Netherlands)

    Kusters, J. G.; Reuland, E. A.; Bouter, S.; Koenig, P.; Dorigo-Zetsma, J. W.

    2015-01-01

    A semi-quantitative multiplex PCR assay for the diagnosis of bacterial vaginosis (BV) was evaluated in a prospective study in a population of Dutch women with complaints of abnormal vaginal discharge. The PCR targets Gardnerella vaginalis, Atopobium vaginae, Megasphaera phylotype 1, Lactobacillus

  7. A multiplex real-time PCR assay for routine diagnosis of bacterial vaginosis

    NARCIS (Netherlands)

    Kusters, J. G.; Reuland, E. A.; Bouter, S.; Koenig, P.; Dorigo-Zetsma, J. W.

    2015-01-01

    A semi-quantitative multiplex PCR assay for the diagnosis of bacterial vaginosis (BV) was evaluated in a prospective study in a population of Dutch women with complaints of abnormal vaginal discharge. The PCR targets Gardnerella vaginalis, Atopobium vaginae, Megasphaera phylotype 1, Lactobacillus cr

  8. Evaluation of a PCR-restriction fragment length polymorphism (PCR-RFLP) assay for molecular epidemiological study of Shiga toxin-producing Escherichia coli.

    Science.gov (United States)

    Sugimoto, Norihiko; Shima, Kensuke; Hinenoya, Atsushi; Asakura, Masahiro; Matsuhisa, Akio; Watanabe, Haruo; Yamasaki, Shinji

    2011-07-01

    In this study, we have evaluated our recently developed polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay for the molecular subtyping of Shiga toxin-producing Escherichia coli (STEC). A total of 200 STEC strains including O157 (n=100), O26 (n=50), O111 (n=10), and non-O26/O111/O157 (n=40) serogroups isolated during 2005-2006 in Japan, which were identified to be clonally different by pulsed-field gel electrophoresis (PFGE) were further analyzed by the PCR-RFLP assay in comparison to PFGE. Ninety-five of O157, 48 of O26, five of O111 and 19 of non-O26/O111/O157 STEC strains yielded one to three amplicons ranging from 6.0 to 15.5 kb in size by the specific primer set targeting region V which is located in the upstream of stx genes. These strains were classified into 41 (O157), 8 (O26), 4 (O111) and 17 (non-O26/O111/O157) groups based on the RFLP patterns obtained by subsequent restriction digestion, respectively. Although the discriminatory power of PCR-RFLP assay was somewhat less than that of PFGE, it is more convenient for molecular subtyping of STEC strains especially for O157, the most important serogroup implicated in human diseases, as well as to identify the outbreak-associated isolates because of its simplicity, rapidity, ease and good reproducibility.

  9. Detection and quantification by PCR assay of the biocontrol agent Pantoea agglomerans CPA-2 on apples.

    Science.gov (United States)

    Soto-Muñoz, Lourdes; Teixidó, Neus; Usall, Josep; Viñas, Inmaculada; Torres, Rosario

    2014-04-03

    The registration of biological control agents requires the development of monitoring systems to detect and quantify the agent in the environment. Pantoea agglomerans CPA-2 is an effective biocontrol agent for postharvest diseases of citrus and pome fruits. The monitoring of CPA-2 in postharvest semi-commercial trials was evaluated by Rodac impression plates and the colonies isolated were confirmed by conventional PCR using the SCAR primers PAGA1 and PAGB1. Samples were taken from different surfaces that had contact with CPA-2, the surrounding environment and working clothes worn by handlers. Moreover, population dynamics of the strain CPA-2 were determined on apple surfaces using both the classical plating technique and real-time quantitative PCR (qPCR). A qPCR assay using a 3'-minor groove-binding (MGB) probe was developed for the specific detection and quantification of P. agglomerans strain CPA-2. Based on the nucleotide sequence of a SCAR fragment of CPA-2, one primer set and TaqMan MGB probe were designed. The primers SP2-F/SP2-R and the TaqMan MGB probe showed a specific detection of strain CPA-2 on apple surfaces, which was verified tested against purified DNA from 17 strains of P. agglomerans, 4 related Pantoea species, and 21 bacterial strains from other genera isolated from whole and also freshly-cut fruit and vegetables. The detection level was approximately 10(3) cells per reaction, and the standard curve was linear within a range of 5log units. Results from semi-commercial trials showed that CPA-2 had a low impact. The maximum persistence of P. agglomerans CPA-2 was not longer than 5days in plastic boxes stored at 0°C. Significant differences in CPA-2 population level dynamics were observed in results obtained by qPCR and dilution plating. These differences may indicate the presence of non-degraded DNA from non-viable cells. In conclusion, qPCR is a novel potential tool to quickly and specifically monitor recent surface colonisation by CPA-2

  10. A diagnostic polymerase chain reaction assay for Zika virus.

    Science.gov (United States)

    Balm, Michelle N D; Lee, Chun Kiat; Lee, Hong Kai; Chiu, Lily; Koay, Evelyn S C; Tang, Julian W

    2012-09-01

    Zika virus (ZIKV) is a mosquito-borne flavivirus. Infection results in a dengue-like illness with fever, headache, malaise, and a maculopapular rash. Nearly all cases are mild and self-limiting but in 2007, a large outbreak of ZIKV was reported from the island of Yap (in Micronesia, northwest of Indonesia). Singapore is already endemic for dengue, and its impact on public health and economic burden is significant. Other dengue-like infections (e.g., Chikungunya virus) are present. Yet only 10% of reported dengue cases have laboratory confirmation. The identification and control of other dengue-like, mosquito-transmitted infections is thus important for the health of Singapore's population, as well as its economy. Given that ZIKV shares the same Aedes mosquito vector with both dengue and Chikungunya, it is possible that this virus is present in Singapore and causing some of the mild dengue-like illness. A specific and sensitive one-step, reverse transcription polymerase chain reaction (RT-PCR) with an internal control (IC) was designed and tested on 88 archived samples of dengue-negative, Chikungunya-negative sera from patients presenting to our hospital with a dengue-like illness, to determine the presence of ZIKV in Singapore. The assay was specific for detection of ZIKV and displayed a lower limit of detection (LoD) of 140 copies viral RNA/reaction when tested on synthetic RNA standards prepared using pooled negative patient plasma. Of the 88 samples tested, none were positive for ZIKV RNA, however, the vast majority of these were from patients admitted to hospital and further study may be warranted in community-based environments.

  11. Detection of Alternaria fungal contamination in cereal grains by a polymerase chain reaction-based assay.

    Science.gov (United States)

    Zur, Gideon; Shimoni, Eyal; Hallerman, Eric; Kashi, Yechezkel

    2002-09-01

    Alternaria sp. are important fungal contaminants of grain products; they secrete four structural classes of compounds that are toxic or carcinogenic to plants and animals and cause considerable economic losses to growers and the food-processing industry. Alternaria toxins have been detected by high-performance liquid chromatography (HPLC), enzyme-linked immunosorbent assay, and other techniques. Here, we report the development of a polymerase chain reaction (PCR)-based method for the detection of Alternaria DNA. PCR primers were designed to anneal to the ITS1 and ITS2 regions of the 5.8S rDNA gene of Alternaria alternata or Alternaria solani but not to other microbial or plant DNA. We compared the sensitivity of PCR in detecting Alternaria DNA, that of the HPLC method in detecting Alternaria alternariol and alternariol methyl ether toxins, and that of the morphological examination of mycelia and conidia in experimentally infested corn samples. The sensitivity of toxin detection for HPLC was above the level of contamination in a set of commercially obtained grain samples, resulting in negative scores for all samples, while the PCR-based method and mold growth plating followed by morphological identification of Alternaria gave parallel, positive results for 8 of 10 samples. The PCR assay required just 8 h, enabling the rapid and simultaneous testing of many samples at a low cost. PCR-based evidence for the presence of Alternaria DNA followed by positive assay results for Alternaria toxins would support the rejection of a shipment of grain.

  12. Sensitive detection of novel Indian isolate of BTV 21 using ns1 gene based real-time PCR assay

    Directory of Open Access Journals (Sweden)

    Gaya Prasad

    2013-06-01

    Full Text Available Aim: The study was conducted to develop ns1 gene based sensitive real-time RT-PCR assay for diagnosis of India isolates of bluetongue virus (BTV. Materials and Methods: The BTV serotype 21 isolate (KMNO7 was isolated from Andhra Pradesh and propagated in BHK-21 cell line in our laboratory. The Nucleic acid (dsRNA of virus was extracted using Trizol method and cDNA was prepared using a standard protocol. The cDNA was allowed to ns1 gene based group specific PCR to confirm the isolate as BTV. The viral RNA was diluted 10 folds and the detection limit of ns1 gene based RT-PCR was determined. Finally the tenfold diluted viral RNA was subjected to real-time RT-PCR using ns1 gene primer and Taq man probe to standardized the reaction and determine the detection limit. Results: The ns1 gene based group specific PCR showed a single 366bp amplicon in agarose gel electrophoresis confirmed the sample as BTV. The ns1 gene RT-PCR using tenfold diluted viral RNA showed the detection limit of 70.0 fg in 1%agarose gel electrophoresis. The ns1 gene based real time RT-PCR was successfully standardized and the detection limit was found to be 7.0 fg. Conclusion: The ns1 gene based real-time RT-PCR was successfully standardized and it was found to be 10 times more sensitive than conventional RT-PCR. Key words: bluetongue, BTV21, RT-PCR, Real time RT-PCR, ns1 gene [Vet World 2013; 6(8.000: 554-557

  13. Detection of pathogenic Leptospira spp. By polymerase chain reaction reaction (PCR targeting ligB gene

    Directory of Open Access Journals (Sweden)

    Fariba Fotohi

    2014-10-01

    Full Text Available Background: Leptospirosis is an emerging infectious disease and is considered to be the most widespread zoonotic disease in the world. LigB is an immunogenic outer membrane protein. The leptospiral ligB gene expressed only in pathogenic Leptospira spp. The aim of this study was molecular diagnosis of pathogen Leptospires by PCR based on ligB gene. Materials and Methods: Five pathogenic Leptospires: L. canicola, L. grippotyphosa, L. pomona, L. icterohaemorrhagiae, L. serjoe hardjo and saprophytic L. biflexa were used in this study. The bacteria were inoculated into the selective culture medium and extraction of the genomic DNA was performed by standard Phenol-Chlorophorm method. The specific primers for proliferation of ligB gene were designed. The specificity and sensitivity of PCR method was evaluated. Results: PCR product was 1041bp which indicated proliferation of ligB gene which was supported using electrophoresis. The PCR based on ligB gene detected all pathogenic reference serovars of Leptospira spp. tested. No PCR products were amplified from the non-pathogenic L. biflexa. Conclusion: Considering the spread of Leptosperosis in moderate and hot areas which have high rate of fall, a proper molecular diagnostic test with high specificity and sensitivity such as PCR is essential. PCR assay with high specificity and sensitivity may prove to be a rapid method for diagnosing acute leptospirosis. The results suggested that the PCR based on ligB gene can be used for detection of pathogenic leptospires.

  14. Comparison of PCR,DIA and Pathogenicity Assay for Detection of Xanthomonas axonopodis pv.citri,the Causal Agent of Citrus Bacterial Canker Disease

    Institute of Scientific and Technical Information of China (English)

    WANG Zhong-kang; SUN Xian-yun; YIN You-ping; ZHOU Chang-yong; XIA Yu-xian

    2004-01-01

    Polymerase chain reaction (PCR) approach based on newly designed primers, JYF5/JYR5, was applied for specific detection of Xanthomonas axonopodis pv.citri(Xac). The efficiency and reliability of PCR method were compared with dot immunobinding assay (DIA) and classical pathogenicity test techniques for detecting suspensions of pure cells of Xac and soaking sap of citrus tissues. Detection sensitivity of PCR was about 4.5 cells or 1.56 pg target DNA per reaction which was higher than that of DIA (ca. 450 cells per dot).These three techniques (PCR assay, DIA and Pathogenecity test) could always detect Xac from symptomatic citrus samples. Different performances were obtained from citrus materials without symptoms, and the positive detection frequency was PCR, DIA and pathogenicity test.

  15. Evaluation of 11 PCR assays for species-level identification of Campylobacter jejuni and Campylobacter coli

    DEFF Research Database (Denmark)

    On, Stephen L.W.; Jordan, Penelope J.

    2003-01-01

    We examined the sensitivity and specificity of 11 PCR assays described for the species identification of Campylobacter jejuni and Campylobacter coli by using 111 type, reference, and field strains of C. jejuni, C. coli, and Campylobacter lari. For six assays, an additional 21 type strains...

  16. Rapid differential diagnosis between extrapulmonary tuberculosis and focal complications of brucellosis using a multiplex real-time PCR assay.

    Directory of Open Access Journals (Sweden)

    María Isabel Queipo-Ortuño

    Full Text Available BACKGROUND: Arduous to differ clinically, extrapulmonary tuberculosis and focal complications of brucellosis remain important causes of morbidity and mortality in many countries. We developed and applied a multiplex real-time PCR assay (M RT-PCR for the simultaneous detection of Mycobacterium tuberculosis complex and Brucella spp. METHODOLOGY: Conventional microbiological techniques and M RT-PCR for M. tuberculosis complex and Brucella spp were performed on 45 clinical specimens from patients with focal complications of brucellosis or extrapulmonary tuberculosis and 26 control samples. Fragments of 207 bp and 164 bp from the conserved region of the genes coding for an immunogenic membrane protein of 31 kDa of B. abortus (BCSP31 and the intergenic region SenX3-RegX3 were used for the identification of Brucella and M. tuberculosis complex, respectively. CONCLUSIONS: The detection limit of the M RT-PCR was 2 genomes per reaction for both pathogens and the intra- and inter-assay coefficients of variation were 0.44% and 0.93% for Brucella and 0.58% and 1.12% for Mycobacterium. M RT-PCR correctly identified 42 of the 45 samples from patients with tuberculosis or brucellosis and was negative in all the controls. Thus, the overall sensitivity, specificity, PPV and NPV values of the M RT PCR assay were 93.3%, 100%, 100% and 89.7%, respectively, with an accuracy of 95.8% (95% CI, 91.1%-100%. Since M RT-PCR is highly reproducible and more rapid and sensitive than conventional microbiological tests, this technique could be a promising and practical approach for the differential diagnosis between extrapulmonary tuberculosis and focal complications of brucellosis.

  17. Establishment of a 10-Plex Quantitative Fluorescent-PCR Assay for rapid diagnosis of sex chromosome aneuploidies.

    Directory of Open Access Journals (Sweden)

    Xingmei Xie

    Full Text Available Sex chromosome aneuploidies occur commonly in the general population, with an incidence of 1 in 400 newborns. However, no tests specifically targeting sex chromosomes have been carried out in prenatal diagnosis or newborn screening, resulting in late recognition of these diseases. In this study, a rapid diagnostic method for sex chromosome aneuploidies was established using Quantitative Fluorescent-PCR (QF-PCR. Ten markers were included in one multiplex QF-PCR assay, including two sex determination genes (AMXY and SRY, five X-linked short tandem repeats (STRs; DXS1053, DXS981, DXS6809, DXS1187, and DXS8377, one X/Y-common STR (X22, and two autosomal STRs (D13S305 and D21S11. Retrospective tests of 70 cases with known cytogenetic results indicated that the 10-plex QF-PCR assay could well determine sex chromosome copy numbers by both allelic peak numbers and a sex chromosome dosage calculation with the autosomal STRs as internal controls. Prospective comparison with cytogenetic karyotyping on 534 cases confirmed that the 10-plex QF-PCR assay could be well employed for sex chromosome aneuploidy diagnosis in at least the Chinese Han population. This is the first QF-PCR test for the diagnosis of sex chromosome aneuploidies in the Chinese population. This test is superior to previous designs by including up to 8 sex-linked markers covering different parts of sex chromosomes as well as employing internal controls for copy number dosage calculation in a single PCR reaction. Due to simple technique and data analysis, as well as easy implementation within routine clinical services, this method is of great clinical application value and could be widely applied.

  18. Establishment of a 10-Plex Quantitative Fluorescent-PCR Assay for rapid diagnosis of sex chromosome aneuploidies.

    Science.gov (United States)

    Xie, Xingmei; Liang, Qiaoyi

    2014-01-01

    Sex chromosome aneuploidies occur commonly in the general population, with an incidence of 1 in 400 newborns. However, no tests specifically targeting sex chromosomes have been carried out in prenatal diagnosis or newborn screening, resulting in late recognition of these diseases. In this study, a rapid diagnostic method for sex chromosome aneuploidies was established using Quantitative Fluorescent-PCR (QF-PCR). Ten markers were included in one multiplex QF-PCR assay, including two sex determination genes (AMXY and SRY), five X-linked short tandem repeats (STRs; DXS1053, DXS981, DXS6809, DXS1187, and DXS8377), one X/Y-common STR (X22), and two autosomal STRs (D13S305 and D21S11). Retrospective tests of 70 cases with known cytogenetic results indicated that the 10-plex QF-PCR assay could well determine sex chromosome copy numbers by both allelic peak numbers and a sex chromosome dosage calculation with the autosomal STRs as internal controls. Prospective comparison with cytogenetic karyotyping on 534 cases confirmed that the 10-plex QF-PCR assay could be well employed for sex chromosome aneuploidy diagnosis in at least the Chinese Han population. This is the first QF-PCR test for the diagnosis of sex chromosome aneuploidies in the Chinese population. This test is superior to previous designs by including up to 8 sex-linked markers covering different parts of sex chromosomes as well as employing internal controls for copy number dosage calculation in a single PCR reaction. Due to simple technique and data analysis, as well as easy implementation within routine clinical services, this method is of great clinical application value and could be widely applied.

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

    Science.gov (United States)

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

    2014-02-01

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

  20. PCR-Reverse Blot Hybridization Assay for Screening and Identification of Pathogens in Sepsis

    OpenAIRE

    Choi, Yeonim; Wang, Hye-young; Lee, Gyusang; Park, Soon-Deok; Jeon, Bo-Young; Uh, Young; Kim, Jong Bae; Lee, Hyeyoung

    2013-01-01

    Rapid and accurate identification of the pathogens involved in bloodstream infections is crucial for the prompt initiation of appropriate therapy, as this can decrease morbidity and mortality rates. A PCR-reverse blot hybridization assay for sepsis, the reverse blot hybridization assay (REBA) Sepsis-ID test, was developed; it uses pan-probes to distinguish Gram-positive and -negative bacteria and fungi. In addition, the assay was designed to identify bacteria and fungi using six genus-specifi...

  1. Quantitative assay of photoinduced DNA strand breaks by real-time PCR.

    Science.gov (United States)

    Wiczk, Justyna; Westphal, Kinga; Rak, Janusz

    2016-09-05

    Real-time PCR (qPCR) - a modern methodology primarily used for studying gene expression has been employed for the quantitative assay of an important class of DNA damage - single strand breaks. These DNA lesions which may lead to highly cytotoxic double strand breaks were quantified in a model system where double stranded DNA was sensitized to UV photons by labeling with 5-bromo-2'-deoxyuridine. The amount of breaks formed due to irradiation with several doses of 320nm photons was assayed by two independent methods: LC-MS and qPCR. A very good agreement between the relative damage measured by the two completely different analytical tools proves the applicability of qPCR for the quantitative analysis of SSBs. Our results suggest that the popularity of the hitherto underestimated though accurate and site-specific technique of real-time PCR may increase in future DNA damage studies.

  2. How to evaluate PCR assays for the detection of low-level DNA

    DEFF Research Database (Denmark)

    Banch-Clausen, Frederik; Urhammer, Emil; Rieneck, Klaus

    2015-01-01

    distribution describing parameters for singleplex real-time PCR-based detection of low-level DNA. The model was tested against experimental data of diluted cell-free foetal DNA. Also, the model was compared with a simplified formula to enable easy predictions. The model predicted outcomes that were......High sensitivity of PCR-based detection of very low copy number DNA targets is crucial. Much focus has been on design of PCR primers and optimization of the amplification conditions. Very important are also the criteria used for determining the outcome of a PCR assay, e.g. how many replicates...... are needed and how many of these should be positive or what amount of template should be used? We developed a mathematical model to obtain a simple tool for quick PCR assay evaluation before laboratory optimization and validation procedures. The model was based on the Poisson distribution and the Binomial...

  3. A novel comprehensive set of fungal Real time PCR assays (fuPCR) for the detection of fungi in immunocompromised haematological patients-A pilot study.

    Science.gov (United States)

    Rahn, Sebastian; Schuck, Anna; Kondakci, Mustafa; Haas, Rainer; Neuhausen, Nicole; Pfeffer, Klaus; Henrich, Birgit

    2016-12-01

    Fungal infections are recognized in an increasing number of patients with immunological deficits and are associated with high rates of mortality (Brown et al., 2012a). In this pilot-study, a rapid Real time PCR (fuPCR) was designed for the detection and differentiation of fungal pathogens in clinical specimens of haematological patients. The fuPCR, targeting the internal transcribed spacer region 2 (ITS2) of rDNA region, is comprised of seven multiplex reactions, which were shown to be specific and sensitive for a comprehensive spectrum of clinically relevant fungal species. This was validated by testing respective fungal DNAs in each fuPCR reaction and 28 respiratory samples of fungal pneumonia-proven patients. Clinical sample sets of throat swab, EDTA-blood and blood sera from 50 patients with severe haematological malignancies, including haematopoietic stem cell transfer (HSCT), and samples from 30 healthy individuals were then analysed. In a first step, 198 samples of immunosuppressed patients were solely examined by fuPCR; and 50.8% (33/65) respiratory swabs, 4.8% (3/63) EDTA blood samples and 1.4% (1/70) blood serum samples were tested positive. In a second step, 56 respiratory samples of immunosuppressed patients and 30 of healthy individuals were simultaneously analysed by fuPCR and standard cultivation techniques. By both methods 30.4% (17/56) swabs of the immunocompromised patients were tested positive, 37.5% (21/56) were tested negative and 32.1% (18/56) were tested fuPCR positive and culture negative. In analysing the blood samples of the immunocompromised patients 5.4% (3/56) EDTA blood samples and 16.1% (9/56) sera samples were tested fuPCR-positive, whereas all samples of 30 healthy individuals with no signs of immunological deficits were tested negative by fuPCR. 38.9% (14/36) of the fungi detected in respiratory samples of the immunosuppressed patients, belonged to Candida spp., 47.2% (17/36) to Saccharomyces spp., 5.6% (2/36) to Cladosporium spp

  4. Monitoring infection: from blood culture to polymerase chain reaction (PCR).

    Science.gov (United States)

    Book, Malte; Lehmann, Lutz Eric; Zhang, XiangHong; Stüber, Frank

    2013-06-01

    In patients with sepsis, diagnosis of blood stream infection (BSI) is a key concern to the therapist. Direct verification of pathogens in the blood stream executed by blood cultures (BC) still is regarded as the gold standard up to date. The quickest possible initiation of an appropriate antimicrobial therapy is a cornerstone of an effective therapy. Moreover, in this view BC can also serve to identify antimicrobial agents to target the pathogen. However, when employing BC the time needed until microbiological results are available ranges from 24 up to 72 h. Moreover, infections caused by multiple pathogens often remain undetected and concurrent antibiotic therapy may lower the overall sensitivity. Alternative pathogen characterization can be performed by polymerase chain reaction (PCR) based amplification methods. Results using PCR can be obtained within 6-8 h. Therefore, the time delay until an appropriate therapy can be reduced enormously. Moreover, these methods have the potential to enhance the sensitivity in the diagnosis of blood stream infections. Therefore, PCR based methods might be a valuable adjunct to present procedures of diagnosing bacteraemia.

  5. Viability-qPCR for detecting Legionella: Comparison of two assays based on different amplicon lengths.

    Science.gov (United States)

    Ditommaso, Savina; Giacomuzzi, Monica; Ricciardi, Elisa; Zotti, Carla M

    2015-08-01

    Two different real-time quantitative PCR (PMA-qPCR) assays were applied for quantification of Legionella spp. by targeting a long amplicon (approx 400 bp) of 16S rRNA gene and a short amplicon (approx. 100 bp) of 5S rRNA gene. Purified DNA extracts from pure cultures of Legionella spp. and from environmental water samples were quantified. Application of the two assays to quantify Legionella in artificially contaminated water achieved that both assays were able to detect Legionella over a linear range of 10 to 10(5) cells ml(-1). A statistical analysis of the standard curves showed that both assays were linear with a good correlation coefficient (R(2) = 0.99) between the Ct and the copy number. Amplification with the reference assay was the most effective for detecting low copy numbers (1 bacterium per PCR mixture). Using selective quantification of viable Legionella by the PMA-qPCR method we obtained a greater inhibition of the amplification of the 400-bp 16S gene fragment (Δlog(10) = 3.74 ± 0.39 log(10) GU ml(-1)). A complete inhibition of the PCR signal was obtained when heat-killed cells in a concentration below 1 × 10(5) cells ml(-1) were pretreated with PMA. Analysing short amplicon sizes led to only 2.08 log reductions in the Legionella dead-cell signal. When we tested environmental water samples, the two qPCR assays were in good agreement according to the kappa index (0.741). Applying qPCR combined with PMA treatment, we also obtained a good agreement (kappa index 0.615). The comparison of quantitative results shows that both assays yielded the same quantification sensitivity (mean log = 4.59 vs mean log = 4.31).

  6. Development and validation of PCR-based assays for diagnosis of American cutaneous leishmaniasis and identification of the parasite species.

    Science.gov (United States)

    Graça, Grazielle Cardoso da; Volpini, Angela Cristina; Romero, Gustavo Adolfo Sierra; Oliveira Neto, Manoel Paes de; Hueb, Marcia; Porrozzi, Renato; Boité, Mariana Côrtes; Cupolillo, Elisa

    2012-08-01

    In this study, PCR assays targeting different Leishmania heat-shock protein 70 gene (hsp70) regions, producing fragments ranging in size from 230-390 bp were developed and evaluated to determine their potential as a tool for the specific molecular diagnosis of cutaneous leishmaniasis (CL). A total of 70 Leishmania strains were analysed, including seven reference strains (RS) and 63 previously typed strains. Analysis of the RS indicated a specific region of 234 bp in the hsp70 gene as a valid target that was highly sensitive for detection of Leishmania species DNA with capacity of distinguishing all analyzed species, after polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). This PCR assay was compared with other PCR targets used for the molecular diagnosis of leishmaniasis: hsp70 (1400-bp region), internal transcribed spacer (ITS)1 and glucose-6-phosphate dehydrogenase (G6pd). A good agreement among the methods was observed concerning the Leishmania species identification. Moreover, to evaluate the potential for molecular diagnosis, we compared the PCR targets hsp70-234 bp, ITS1, G6pd and mkDNA using a panel of 99 DNA samples from tissue fragments collected from patients with confirmed CL. Both PCR-hsp70-234 bp and PCR-ITS1 detected Leishmania DNA in more than 70% of the samples. However, using hsp70-234 bp PCR-RFLP, identification of all of the Leishmania species associated with CL in Brazil can be achieved employing a simpler and cheaper electrophoresis protocol.

  7. Development and validation of PCR-based assays for diagnosis of American cutaneous leishmaniasis and identificatio nof the parasite species

    Directory of Open Access Journals (Sweden)

    Grazielle Cardoso da Graça

    2012-08-01

    Full Text Available In this study, PCR assays targeting different Leishmania heat-shock protein 70 gene (hsp70 regions, producing fragments ranging in size from 230-390 bp were developed and evaluated to determine their potential as a tool for the specific molecular diagnosis of cutaneous leishmaniasis (CL. A total of 70 Leishmania strains were analysed, including seven reference strains (RS and 63 previously typed strains. Analysis of the RS indicated a specific region of 234 bp in the hsp70 gene as a valid target that was highly sensitive for detection of Leishmania species DNA with capacity of distinguishing all analyzed species, after polymerase chain reaction-restriction fragment length polymorfism (PCR-RFLP. This PCR assay was compared with other PCR targets used for the molecular diagnosis of leishmaniasis: hsp70 (1400-bp region, internal transcribed spacer (ITS1 and glucose-6-phosphate dehydrogenase (G6pd. A good agreement among the methods was observed concerning the Leishmania species identification. Moreover, to evaluate the potential for molecular diagnosis, we compared the PCR targets hsp70-234 bp, ITS1, G6pd and mkDNA using a panel of 99 DNA samples from tissue fragments collected from patients with confirmed CL. Both PCR-hsp70-234 bp and PCR-ITS1 detected Leishmania DNA in more than 70% of the samples. However, using hsp70-234 bp PCR-RFLP, identification of all of the Leishmania species associated with CL in Brazil can be achieved employing a simpler and cheaper electrophoresis protocol.

  8. Differentiating Botulinum Neurotoxin-Producing Clostridia with a Simple, Multiplex PCR Assay.

    Science.gov (United States)

    Williamson, Charles H D; Vazquez, Adam J; Hill, Karen; Smith, Theresa J; Nottingham, Roxanne; Stone, Nathan E; Sobek, Colin J; Cocking, Jill H; Fernández, Rafael A; Caballero, Patricia A; Leiser, Owen P; Keim, Paul; Sahl, Jason W

    2017-09-15

    Diverse members of the genus Clostridium produce botulinum neurotoxins (BoNTs), which cause a flaccid paralysis known as botulism. While multiple species of clostridia produce BoNTs, the majority of human botulism cases have been attributed to Clostridium botulinum groups I and II. Recent comparative genomic studies have demonstrated the genomic diversity within these BoNT-producing species. This report introduces a multiplex PCR assay for differentiating members of C. botulinum group I, C. sporogenes, and two major subgroups within C. botulinum group II. Coding region sequences unique to each of the four species/subgroups were identified by in silico analyses of thousands of genome assemblies, and PCR primers were designed to amplify each marker. The resulting multiplex PCR assay correctly assigned 41 tested isolates to the appropriate species or subgroup. A separate PCR assay to determine the presence of the ntnh gene (a gene associated with the botulinum neurotoxin gene cluster) was developed and validated. The ntnh gene PCR assay provides information about the presence or absence of the botulinum neurotoxin gene cluster and the type of gene cluster present (ha positive [ha(+)] or orfX(+)). The increased availability of whole-genome sequence data and comparative genomic tools enabled the design of these assays, which provide valuable information for characterizing BoNT-producing clostridia. The PCR assays are rapid, inexpensive tests that can be applied to a variety of sample types to assign isolates to species/subgroups and to detect clostridia with botulinum neurotoxin gene (bont) clusters.IMPORTANCE Diverse clostridia produce the botulinum neurotoxin, one of the most potent known neurotoxins. In this study, a multiplex PCR assay was developed to differentiate clostridia that are most commonly isolated in connection with human botulism cases: C. botulinum group I, C. sporogenes, and two major subgroups within C. botulinum group II. Since BoNT-producing and

  9. RT-PCR assay for detection of Lassa virus and related Old World arenaviruses targeting the L gene.

    Science.gov (United States)

    Vieth, Simon; Drosten, Christian; Lenz, Oliver; Vincent, Martin; Omilabu, Sunday; Hass, Meike; Becker-Ziaja, Beate; ter Meulen, Jan; Nichol, Stuart T; Schmitz, Herbert; Günther, Stephan

    2007-12-01

    This study describes an RT-PCR assay targeting the L RNA segment of arenaviruses. Conserved regions were identified in the polymerase domain of the L gene on the basis of published sequences for Lassa virus, lymphocytic choriomeningitis virus (LCMV), Pichinde virus and Tacaribe virus, as well as 15 novel sequences for Lassa virus, LCMV, Ippy virus, Mobala virus and Mopeia virus determined in this study. Using these regions as target sites, a PCR assay for detection of all known Old World arenaviruses was developed and optimized. The concentration that yields 95% positive results in a set of replicate tests (95% detection limit) was determined to be 4290 copies of Lassa virus L RNA per ml of serum, corresponding to 30 copies per reaction. The ability of the assay to detect various Old World arenaviruses was demonstrated with in vitro transcribed RNA, material from infected cell cultures and samples from patients with Lassa fever and monkeys with LCMV-associated callitrichid hepatitis. The L gene PCR assay may be applicable: (i) as a complementary diagnostic test for Lassa virus and LCMV; (ii) to identify unknown Old World arenaviruses suspected as aetiological agents of disease; and (iii) for screening of potential reservoir hosts for unknown Old World arenaviruses.

  10. A real time polymerase chain reaction assay for quantification of Edwardsiella ictaluri in catfish pond water and genetic homogeneity of diagnostic case isolates from Mississippi

    Science.gov (United States)

    A quantitative polymerase chain reaction (qPCR) assay was developed for the detection and quantification of Edwardsiella ictaluri in channel catfish Ictalurus punctatus pond water using modifications to a published E. ictaluri–specific qPCR assay and previously established protocols for the molecula...

  11. A Color-Reaction-Based Biochip Detection Assay for RIF and INH Resistance of Clinical Mycobacterial Specimens.

    Science.gov (United States)

    Xue, Wenfei; Peng, Jingfu; Yu, Xiaoli; Zhang, Shulin; Zhou, Boping; Jiang, Danqing; Chen, Jianbo; Ding, Bingbing; Zhu, Bin; Li, Yao

    2016-01-01

    The widespread occurrence of drug-resistant Mycobacterium tuberculosis places importance on the detection of TB (tuberculosis) drug susceptibility. Conventional drug susceptibility testing (DST) is a lengthy process. We developed a rapid enzymatic color-reaction-based biochip assay. The process included asymmetric multiplex PCR/templex PCR, biochip hybridization, and an enzymatic color reaction, with specific software for data operating. Templex PCR (tem- PCR) was applied to avoid interference between different primers in conventional multiplex- PCR. We applied this assay to 276 clinical specimens (including 27 sputum, 4 alveolar lavage fluid, 2 pleural effusion, and 243 culture isolate specimens; 40 of the 276 were non-tuberculosis mycobacteria specimens and 236 were M. tuberculosis specimens). The testing process took 4.5 h. A sensitivity of 50 copies per PCR was achieved, while the sensitivity was 500 copies per PCR when tem-PCR was used. Allele sequences could be detected in mixed samples at a proportion of 10%. Detection results showed a concordance rate of 97.46% (230/236) in rifampicin resistance detection (sensitivity 95.40%, specificity 98.66%) and 96.19% (227/236) in isoniazid (sensitivity 93.59%, specificity 97.47%) detection with those of DST assay. Concordance rates of testing results for sputum, alveolar lavage fluid, and pleural effusion specimens were 100%. The assay provides a potential choice for TB diagnosis and treatment.

  12. Simultaneous detection of bovine and porcine DNA in pharmaceutical gelatin capsules by duplex PCR assay for Halal authentication.

    Science.gov (United States)

    Nikzad, Jafar; Shahhosseini, Soraya; Tabarzad, Maryam; Nafissi-Varcheh, Nastaran; Torshabi, Maryam

    2017-02-14

    In the pharmaceutical industry, hard- and soft-shelled capsules are typically made from gelatin, commonly derived from bovine and porcine sources. To ensure that pharmaceutical products comply with halal regulations in Muslim countries (no porcine products allowed), development of a valid, reliable, quick, and most importantly, cost-effective tests are of utmost importance. We developed a species-specific duplex polymerase chain reaction (PCR) assay targeting 149 bp porcine and 271 bp bovine mitochondrial DNA (mtDNA) to simultaneously detect both porcine and bovine DNA (in one reaction at the same time) in gelatin. Some additional simplex PCR tests (targeting 126 bp bovine and 212 bp porcine mtDNA) and real-time PCR using a commercially available kit (for identification of porcine DNA) were used to verify the selectivity and sensitivity of our duplex PCR. After optimization of DNA extraction and PCR methods, hard/soft pharmaceutical gelatin capsules (containing drug) were tested for the presence of porcine and/or bovine DNA. Duplex PCR detected the presence of as little as 0.1% porcine DNA, which was more accurate than the commercially available kit. Of all gelatin capsules tested (n = 24), 50% contained porcine DNA (pure porcine gelatin alone or in combination with bovine gelatin). Duplex PCR presents an easy-to-follow, quick, low-cost and reliable method to simultaneously detect porcine and bovine DNAs (>100 bp) in minute amounts in highly processed gelatin-containing pharmaceutical products (with a 0.1% sensitivity for porcine DNA) which may be used for halal authentication. Simultaneous detection of porcine and bovine DNA in gelatin capsules by duplex PCR.

  13. Effective characterization of Salmonella Enteritidis by most probable number (MPN) followed by multiplex polymerase chain reaction (PCR) methods.

    Science.gov (United States)

    Zappelini, Lincohn; Martone-Rocha, Solange; Dropa, Milena; Matté, Maria Helena; Tiba, Monique Ribeiro; Breternitz, Bruna Suellen; Razzolini, Maria Tereza Pepe

    2017-02-01

    Nontyphoidal Salmonella (NTS) is a relevant pathogen involved in gastroenteritis outbreaks worldwide. In this study, we determined the capacity to combine the most probable number (MPN) and multiplex polymerase chain reaction (PCR) methods to characterize the most important Salmonella serotypes in raw sewage. A total of 499 isolates were recovered from 27 raw sewage samples and screened using two previously described multiplex PCR methods. From those, 123 isolates were selected based on PCR banding pattern-identical or similar to Salmonella Enteritidis and Salmonella Typhimurium-and submitted to conventional serotyping. Results showed that both PCR assays correctly serotyped Salmonella Enteritidis, however, they presented ambiguous results for Salmonella Typhimurium identification. These data highlight that MPN and multiplex PCR can be useful methods to describe microbial quality in raw sewage and suggest two new PCR patterns for Salmonella Enteritidis identification.

  14. Development and evaluation of TaqMan real-time PCR assay for detection of beak and feather disease virus.

    Science.gov (United States)

    Černíková, Lenka; Vitásková, Eliška; Nagy, Alexander

    2017-03-02

    Psittacine beak and feather disease (PBFD) is one of the most significant viral diseases in psittacine birds. The aim of the presented study was to develop a highly specific and sensitive TaqMan real-time PCR assay for universal detection of beak and feather disease virus (BFDV). Primers and a hydrolysis probe were selected on the highly conserved regions belonging to the ORF1 of the BFDV genome which were identified by aligning 814 genomic sequences downloaded from the GenBank database. The evaluation of the reaction parameters suggested a reaction efficiency of 97.1%, with consistent detection of 10(1) virus copies/μl of nucleic acid extract. The low values of standard deviation and coefficient of variation indicate a high degree of reproducibility and repeatability. The diagnostic applicability of the assay was proven on 36 BFDV positive and 107 negative specimens of psittacine origin representing 28 species. The assay showed a 100% ability to detect distinct genetic variants of the virus. Our data suggest that the presented TaqMan real-time PCR represents a specific, sensitive and reliable assay facilitating the molecular detection of BFDV.

  15. Real-time PCR assays for hepatitis B virus DNA quantification may require two different targets.

    Science.gov (United States)

    Liu, Chao; Chang, Le; Jia, Tingting; Guo, Fei; Zhang, Lu; Ji, Huimin; Zhao, Junpeng; Wang, Lunan

    2017-05-12

    Quantification Hepatitis B virus (HBV) DNA plays a critical role in the management of chronic HBV infections. However, HBV is a DNA virus with high levels of genetic variation, and drug-resistant mutations have emerged with the use of antiviral drugs. If a mutation caused a sequence mismatched in the primer or probe of a commercial DNA quantification kit, this would lead to an underestimation of the viral load of the sample. The aim of this study was to determine whether commercial kits, which use only one pair of primers and a single probe, accurately quantify the HBV DNA levels and to develop an improved duplex real-time PCR assay. We developed a new duplex real-time PCR assay that used two pairs of primers and two probes based on the conserved S and C regions of the HBV genome. We performed HBV DNA quantitative detection of HBV samples and compared the results of our duplex real-time PCR assays with the COBAS TaqMan HBV Test version 2 and Daan real-time PCR assays. The target region of the discordant sample was amplified, sequenced, and validated using plasmid. The results of the duplex real-time PCR were in good accordance with the commercial COBAS TaqMan HBV Test version 2 and Daan real-time PCR assays. We showed that two samples from Chinese HBV infections underestimated viral loads when quantified by the Roche kit because of a mismatch between the viral sequence and the reverse primer of the Roche kit. The HBV DNA levels of six samples were undervalued by duplex real-time PCR assays of the C region because of mutations in the primer of C region. We developed a new duplex real-time PCR assay, and the results of this assay were similar to the results of commercial kits. The HBV DNA level could be undervalued when using the COBAS TaqMan HBV Test version 2 for Chinese HBV infections owing to a mismatch with the primer/probe. A duplex real-time PCR assay based on the S and C regions could solve this problem to some extent.

  16. A duplex real-time RT-PCR assay for profiling inhibitors of four dengue serotypes.

    Science.gov (United States)

    Gong, Edwin Yunhao; Smets, Alexandra; Verheyen, Nick; Clynhens, Marleen; Gustin, Emmanuel; Lory, Pedro; Kraus, Guenter

    2013-01-01

    We have developed a duplex real-time RT-PCR assay for profiling antiviral inhibitors of four dengue virus (DENV) serotypes. In this assay, the primers and the probe for amplifying DENV were designed in the conserved regions of the genome after aligned more than 300 nucleotide sequences of four dengue serotypes deposited in the GeneBank. To discriminate the antiviral activity from the cytotoxicity of compounds, a housekeeping gene of the Vero cells, β-actin, was used to design the primers and the probe for the second set of PCR as an internal control, which is used to normalize the RNA levels of dengue-specific PCR due to the cellular toxicity of test compounds. For compound profiling, the duplex PCR is performed using LightCycler(®) in a single tube to simultaneously amplify both the dengue target gene and the Vero cell housekeeping gene from the compound-treated Vero cell lysates. This assay was validated against a panel of reference compounds. The results show that the universal primers and probe in this duplex RT-PCR assay can efficiently amplify all four dengue serotypes and that the PCR efficiency for both the dengue target gene and the Vero cells β-actin gene is 100%.

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

    Science.gov (United States)

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

    2015-06-21

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

  18. [Detection of human enteroviruses with real-time PCR assay using TaqMan fluorescent probe].

    Science.gov (United States)

    Leś, Katarzyna; Przybylski, Maciej; Dzieciatkowski, Tomasz; Młynarczyk, Grazyna

    2010-01-01

    Infections with human enteroviruses are common worldwide and cause a wide range of signs and symptoms. Nowadays in current diagnostics procedures older virological methods, such virus isolation in a cell cultures and seroneutralisation assay, are replaced with molecular biology tests. The aim of the study was development of real-time PCR assay for detection of human adenoviruses. DNA isolated from MK2 cell line infected with nineteen different enterovirus strains was used for development of a qualitative real-time PCR assay using primers targeting a conserved region of the 5'UTR region and a specific TaqMan probe. The analytical sensitivity of real-time PCR assay was tested using serial dilutions of Coxackie A9 cDNA in range between 10 degrees and 10(-8). For comparison typical end-point detected RT-PCR for enterovirus detection with the same cDNA dilutions was made. The sensitivity of novel method was about ten thousand-fold higher than older one. The conclusion is that real-time PCR is very advisable in diagnostics of diseases caused with enteroviruses. The high level of sensitivity, specificity, accuracy, and rapidity provided by this assay are favorable for the use in the detection of enteroviral RNA in clinical specimens, especially from neuroinfections.

  19. Development and preliminary evaluation of a real-time PCR assay for Halioticida noduliformans in abalone tissues.

    Science.gov (United States)

    Greeff, Mariska R; Christison, Kevin W; Macey, Brett M

    2012-06-13

    Abalone Haliotis midae exhibiting typical clinical signs of tubercle mycosis were discovered in South African culture facilities in 2006, posing a significant threat to the industry. The fungus responsible for the outbreak was identified as a Peronosporomycete, Halioticida noduliformans. Currently, histopathology and gross observation are used to diagnose this disease, but these 2 methods are neither rapid nor sensitive enough to provide accurate and reliable diagnosis. Real-time quantitative PCR (qPCR) is a rapid and reliable method for the detection and quantification of a variety of pathogens, so therefore we aimed to develop a qPCR assay for species-specific detection and quantification of H. noduliformans. Effective extraction of H. noduliformans genomic DNA from laboratory grown cultures, as well as from spiked abalone tissues, was accomplished by grinding samples using a pellet pestle followed by heat lysis in the presence of Chelax-100 beads. A set of oligonucleotide primers was designed to specifically amplify H. noduliformans DNA in the large subunit (LSU) rRNA gene, and tested for cross-reactivity to DNA extracted from related and non-related fungi isolated from seaweeds, crustaceans and healthy abalone; no cross-amplification was detected. When performing PCR assays in an abalone tissue matrix, an environment designed to be a non-sterile simulation of environmental conditions, no amplification occurred in the negative controls. The qPCR assay sensitivity was determined to be approximately 0.28 pg of fungal DNA (~2.3 spores) in a 25 µl reaction volume. Our qPCR technique will be useful for monitoring and quantifying H. noduliformans for the surveillance and management of abalone tubercle mycosis in South Africa.

  20. Investigation of polymerase chain reaction assays to improve detection of bacterial involvement in bovine respiratory disease.

    Science.gov (United States)

    Bell, Colin J; Blackburn, Paul; Elliott, Mark; Patterson, Tony I A P; Ellison, Sean; Lahuerta-Marin, Angela; Ball, Hywel J

    2014-09-01

    Bovine respiratory disease (BRD) causes severe economic losses to the cattle farming industry worldwide. The major bacterial organisms contributing to the BRD complex are Mannheimia haemolytica, Histophilus somni, Mycoplasma bovis, Pasteurella multocida, and Trueperella pyogenes. The postmortem detection of these organisms in pneumonic lung tissue is generally conducted using standard culture-based techniques where the presence of therapeutic antibiotics in the tissue can inhibit bacterial isolation. In the current study, conventional and real-time polymerase chain reaction (PCR) assays were used to assess the prevalence of these 5 organisms in grossly pneumonic lung samples from 150 animals submitted for postmortem examination, and the results were compared with those obtained using culture techniques. Mannheimia haemolytica was detected in 51 cases (34%) by PCR and in 33 cases (22%) by culture, H. somni was detected in 35 cases (23.3%) by PCR and in 6 cases (4%) by culture, Myc. bovis was detected in 53 cases (35.3%) by PCR and in 29 cases (19.3%) by culture, P. multocida was detected in 50 cases (33.3%) by PCR and in 31 cases (20.7%) by culture, and T. pyogenes was detected in 42 cases (28%) by PCR and in 31 cases (20.7%) by culture, with all differences being statistically significant. The PCR assays indicated positive results for 111 cases (74%) whereas 82 cases (54.6%) were culture positive. The PCR assays have demonstrated a significantly higher rate of detection of all 5 organisms in cases of pneumonia in cattle in Northern Ireland than was detected by current standard procedures.

  1. Real-time reverse transcription-polymerase chain reaction assays for identification of wild poliovirus 1 & 3

    OpenAIRE

    Sharma, Deepa K.; Nalavade, Uma P.; Deshpande, Jagadish M.

    2015-01-01

    Background & objectives: The poliovirus serotype identification and intratypic differentiation by real-time reverse transcription-polymerase chain reaction (rRT-PCR) assay is suitable for serotype mixtures but not for intratypic mixtures of wild and vaccine poliovirus strains. This study was undertaken to develop wild poliovirus 1 and 3 (WPV1 and WPV3) specific rRT-PCR assays for use. Methods: Specific primers and probes for rRT-PCR were designed based on VP1 sequences of WPV1 and WPV3 isolat...

  2. Comparative analysis of cultural isolation and PCR based assay for detection of Campylobacter jejuni in food and faecal samples

    Directory of Open Access Journals (Sweden)

    Harkanwaldeep Singh

    2011-03-01

    Full Text Available In the present study, the efficacy of polymerase chain reaction (PCR based on mapA gene of C. jejuni was tested for detection of Campylobacter jejuni in naturally infected as well as spiked faecal and food samples of human and animal origin. Simultaneously, all the samples were subjected to the cultural isolation of organism and biochemical characterization. The positive samples resulted in the amplification of a DNA fragment of size ~589 bp in PCR assay whereas the absence of such amplicon in DNA extracted from E. coli, Listeria, Salmonella and Staphylococcus confirmed the specificity of the primers. Of randomly collected 143 faecal samples comprising human diarrheic stools (43, cattle diarrheic faeces (48 and poultry faecal swabs (52 only 4, 3 and 8, respectively, could be detected by isolation whereas 6, 3 and 10, respectively, were found positive by PCR. However, among food samples viz. beef (30, milk (35, cheese (30, only one beef sample was detected both by culture as well as PCR. Additionally, PCR was found to be more sensitive for C. jejuni detection in spiked faecal and food samples (96.1% each as relative to culture isolation which could detect the organism in 86.7% and 80% samples, respectively. The results depicted the superior efficacy of PCR for rapid screening of samples owing to its high sensitivity, specificity and automation potential.

  3. Rapid and Quantitative Detection of Leifsonia xyli subsp. xyli in Sugarcane Stalk Juice Using a Real-Time Fluorescent (TaqMan) PCR Assay

    Science.gov (United States)

    Fu, Hua-Ying; Sun, Sheng-Ren; Wang, Jin-Da; Ahmad, Kashif; Wang, Heng-Bo; Chen, Ru-Kai

    2016-01-01

    Ratoon stunting disease (RSD) of sugarcane, one of the most important diseases seriously affecting the productivity of sugarcane crops, was caused by the bacterial agent Leifsonia xyli subsp. xyli (Lxx). A TaqMan probe-based real-time quantitative polymerase chain reaction (qPCR) assay was established in this study for the quantification of Lxx detection in sugarcane stalk juice. A pair of PCR primers (Pat1-QF/Pat1-QR) and a fluorogenic probe (Pat1-QP) targeting the Part1 gene of Lxx were used for the qPCR assay. The assay had a detection limit of 100 copies of plasmid DNA and 100 fg of Lxx genomic DNA, which was 100-fold more sensitive than the conventional PCR. Fifty (28.7%) of 174 stalk juice samples from two field trials were tested to be positive by qPCR assay, whereas, by conventional PCR, only 12.1% (21/174) were tested to be positive with a published primer pair CxxITSf#5/CxxITSr#5 and 15.5% (27/174) were tested to be positive with a newly designed primer pair Pat1-F2/Pat1-R2. The new qPCR assay can be used as an alternative to current diagnostic methods for Lxx, especially when dealing with certificating a large number of healthy cane seedlings and determining disease incidence accurately in commercial fields.

  4. Rapid and Quantitative Detection of Leifsonia xyli subsp. xyli in Sugarcane Stalk Juice Using a Real-Time Fluorescent (TaqMan PCR Assay

    Directory of Open Access Journals (Sweden)

    Hua-Ying Fu

    2016-01-01

    Full Text Available Ratoon stunting disease (RSD of sugarcane, one of the most important diseases seriously affecting the productivity of sugarcane crops, was caused by the bacterial agent Leifsonia xyli subsp. xyli (Lxx. A TaqMan probe-based real-time quantitative polymerase chain reaction (qPCR assay was established in this study for the quantification of Lxx detection in sugarcane stalk juice. A pair of PCR primers (Pat1-QF/Pat1-QR and a fluorogenic probe (Pat1-QP targeting the Part1 gene of Lxx were used for the qPCR assay. The assay had a detection limit of 100 copies of plasmid DNA and 100 fg of Lxx genomic DNA, which was 100-fold more sensitive than the conventional PCR. Fifty (28.7% of 174 stalk juice samples from two field trials were tested to be positive by qPCR assay, whereas, by conventional PCR, only 12.1% (21/174 were tested to be positive with a published primer pair CxxITSf#5/CxxITSr#5 and 15.5% (27/174 were tested to be positive with a newly designed primer pair Pat1-F2/Pat1-R2. The new qPCR assay can be used as an alternative to current diagnostic methods for Lxx, especially when dealing with certificating a large number of healthy cane seedlings and determining disease incidence accurately in commercial fields.

  5. 沙门氏致病菌标准阳性模板的构建及实时荧光定量聚合酶链式反应检测%Construction of Standard Positive Template and Development of a Real-Time Fluorescence Quantitative Polymerase Chain Reaction (FQ-PCR) Assay for PathogenicSalmonella spp.

    Institute of Scientific and Technical Information of China (English)

    陈晨; 邵彪; 陈刚; 黄伟东

    2014-01-01

    Objective: To construct recombinant plasmids for use as standard positive template and establish a real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) assay for determining pathogenicSalmonellaspp. in foods. Methods: Primers and Taqman probe were designed and synthesized with the specific fragment of InvA gene as the target sequence. Recombinant plasmids were constructed by inserting the target gene into PGM-T carriers. Real-time fluorescent quantitative PCR method was established and its sensitivity, specificity, repeatability and accuracy were investigated. Results: The recombinant plasmids constructed using the specific sequence of pathogenicSalmonella spp. could be used as a standard positive template for fluorescent quantitative PCR. The standard curve wasY=-3.151 lgX+42.86 (R2=0.999), and the sensitivity of the method was 80 copies per reaction. It was specific to detect Salmonellaspp. with good repeatability (the inter-batch and intra-batch coefficients of variation were both less than 5%). Conclusion: The FQ-PCR method allows qualitative and quantitative detection of pathogenicSalmonellaspp.%目的:构建重组质粒作为标准阳性模板,建立食品中沙门氏致病菌实时荧光定量聚合酶链式反应检测方法。方法:以致病性沙门氏菌invA基因上特异性片段为目标,设计并合成引物和TaqMan探针,将目标片段连接到PGM-T载体上构建重组质粒,建立实时荧光定量检测体系,并考察方法的灵敏性、特异性、重复性和准确性。结果:构建出致病性沙门氏菌特异性基因片段的重组质粒,能够作为实时荧光定量聚合酶链式反应检测方法的标准阳性模板,标准曲线方程为Y=-3.151 lgX+42.86(R2=0.999),灵敏度80拷贝反应体系,能特异区分沙门氏菌与类型的细菌,同时,批内和批间的变异系数均小于5%,具有良好的重复性。结论:本方法能够实现对食品

  6. Tetraplex PCR assay involving double gene-sites discriminates beef and buffalo in Malaysian meat curry and burger products.

    Science.gov (United States)

    Hossain, M A Motalib; Ali, Md Eaqub; Hamid, Sharifah Bee Abd; Hossain, S M Azad; Asing; Nizar, Nina Naquiah Ahmad; Uddin, Mohammad Nasir; Ali, Lokman; Asaduzzaman, Md; Akanda, Md Jahurul Haque

    2017-06-01

    Replacement of beef by buffalo and vice versa is frequent in global markets, but their authentication is challenging in processed foods due to the fragmentation of most biomarkers including DNA. The shortening of target sequences through use of two target sites might ameliorate assay reliability because it is highly unlikely that both targets will be lost during food processing. For the first time, we report a tetraplex polymerase chain reaction (PCR) assay targeting two different DNA regions in beef (106 and 120-bp) and buffalo (90 and 138-bp) mitochondrial genes to discriminate beef and buffalo in processed foods. All targets were stable under boiling, autoclaving and microwave cooking conditions. A survey in Malaysian markets revealed 71% beef curries contained buffalo but there was no buffalo in beef burgers. The assay detected down to 0.01ng DNA and 1% meat in admixed and burger products.

  7. Development of a real-time PCR assay for the rapid detection of Acinetobacter baumannii from whole blood samples.

    Science.gov (United States)

    De Gregorio, Eliana; Roscetto, Emanuela; Iula, Vita Dora; Martinucci, Marianna; Zarrilli, Raffaele; Di Nocera, Pier Paolo; Catania, Maria Rosaria

    2015-04-01

    Acinetobacter baumannii is a multidrug-resistant pathogen associated with severe infections in hospitalized patients, including pneumonia, urinary and bloodstream infections. Rapid detection of A. baumannii infection is crucial for timely treatment of septicemic patients. The aim of the present study was to develop a specific marker for a quantitative polymerase chain reaction (PCR) assay for the detection of A. baumannii. The target gene chosen is the biofilm-associated protein (bap) gene, encoding a cell surface protein involved in biofilm formation. The assay is specific for A. baumannii, allowing its discrimination from different species of Acinetobacter and other clinically relevant bacterial pathogens. The assay is able to detect one genomic copy of A. baumannii, corresponding to 4 fg of purified DNA, and 20 colony-forming units/ml using DNA extracted from spiked whole blood samples.

  8. Identifying of meat species using polymerase chain reaction (PCR)

    Science.gov (United States)

    Foong, Chow Ming; Sani, Norrakiah Abdullah

    2013-11-01

    Meat has been widely consumed as an important protein source in daily life of human. Furthermore, with busy and intense urban lifestyle, processed food is now one of the main protein sources of one's diet. Consumers rely on the food labeling to decide if the meat product purchased is safe and reliable. Therefore, it is important to ensure the food labeling is done in a correct manner to avoid consumer fraud. More consumers are now concern about the food quality and safety as compared to before. This study described the meat species identification and detection method using Polymerase Chain Reaction (PCR) in 8 types of meats (cattle, buffalo, goat, sheep, chicken, duck, pork and horse). The objective of this study is to decide on the specificity of oligonucleotide sequences obtained from previous study. There were 5 proposed oligonucleotide primer in this study. The main important finding in this work is the specificity of oligonucleotide primers to raw meats. It if found that the oligonucleotide primers proposed were not specific to the local raw meat species. Therefore, further study is needed to obtain a species-specific oligonucletide primers for PCR, in order to be applied in food product testing.

  9. Identifying of meat species using polymerase chain reaction (PCR)

    Energy Technology Data Exchange (ETDEWEB)

    Foong, Chow Ming; Sani, Norrakiah Abdullah [School of Chemical Sciences and Food Technology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600, Bangi, Selangor (Malaysia)

    2013-11-27

    Meat has been widely consumed as an important protein source in daily life of human. Furthermore, with busy and intense urban lifestyle, processed food is now one of the main protein sources of one’s diet. Consumers rely on the food labeling to decide if the meat product purchased is safe and reliable. Therefore, it is important to ensure the food labeling is done in a correct manner to avoid consumer fraud. More consumers are now concern about the food quality and safety as compared to before. This study described the meat species identification and detection method using Polymerase Chain Reaction (PCR) in 8 types of meats (cattle, buffalo, goat, sheep, chicken, duck, pork and horse). The objective of this study is to decide on the specificity of oligonucleotide sequences obtained from previous study. There were 5 proposed oligonucleotide primer in this study. The main important finding in this work is the specificity of oligonucleotide primers to raw meats. It if found that the oligonucleotide primers proposed were not specific to the local raw meat species. Therefore, further study is needed to obtain a species-specific oligonucletide primers for PCR, in order to be applied in food product testing.

  10. In silico and in vitro evaluation of PCR-based assays for the detection of Bacillus anthracis chromosomal signature sequences.

    Science.gov (United States)

    Ågren, Joakim; Hamidjaja, Raditijo A; Hansen, Trine; Ruuls, Robin; Thierry, Simon; Vigre, Håkan; Janse, Ingmar; Sundström, Anders; Segerman, Bo; Koene, Miriam; Löfström, Charlotta; Van Rotterdam, Bart; Derzelle, Sylviane

    2013-11-15

    Bacillus anthracis, the causative agent of anthrax, is a zoonotic pathogen that is relatively common throughout the world and may cause life threatening diseases in animals and humans. There are many PCR-based assays in use for the detection of B. anthracis. While most of the developed assays rely on unique markers present on virulence plasmids pXO1 and pXO2, relatively few assays incorporate chromosomal DNA markers due to the close relatedness of B. anthracis to the B. cereus group strains. For the detection of chromosomal DNA, different genes have been used, such as BA813, rpoB, gyrA, plcR, S-layer, and prophage-lambda. Following a review of the literature, an in silico analysis of all signature sequences reported for identification of B. anthracis was conducted. Published primer and probe sequences were compared for specificity against 134 available Bacillus spp. genomes. Although many of the chromosomal targets evaluated are claimed to be specific to B. anthracis, cross-reactions with closely related B. cereus and B. thuringiensis strains were often observed. Of the 35 investigated PCR assays, only 4 were 100% specific for the B. anthracis chromosome. An interlaboratory ring trial among five European laboratories was then performed to evaluate six assays, including the WHO recommended procedures, using a collection of 90 Bacillus strains. Three assays performed adequately, yielding no false positive or negative results. All three assays target chromosomal markers located within the lambdaBa03 prophage region (PL3, BA5345, and BA5357). Detection limit was further assessed for one of these highly specific assays.

  11. A PCR-high-resolution melt assay for rapid differentiation of nontypeable Haemophilus influenzae and Haemophilus haemolyticus.

    Science.gov (United States)

    Pickering, Janessa; Binks, Michael J; Beissbarth, Jemima; Hare, Kim M; Kirkham, Lea-Ann S; Smith-Vaughan, Heidi

    2014-02-01

    We have developed a PCR-high-resolution melt (PCR-HRM) assay to discriminate nontypeable Haemophilus influenzae (NTHi) colonies from Haemophilus haemolyticus. This method is rapid and robust, with 96% sensitivity and 92% specificity compared to the hpd#3 assay. PCR-HRM is ideal for high-throughput screening for NTHi surveillance and clinical trials.

  12. Diagnosis of ocular toxoplasmosis by two polymerase chain reaction (PCR) examinations: qualitative multiplex and quantitative real-time.

    Science.gov (United States)

    Sugita, Sunao; Ogawa, Manabu; Inoue, Shizu; Shimizu, Norio; Mochizuki, Manabu

    2011-09-01

    To establish a two-step polymerase chain reaction (PCR) diagnostic system for ocular toxoplasmosis. A total of 13 ocular fluid samples (11 aqueous humor and 2 vitreous fluid) were collected from 13 patients with clinically suspected ocular toxoplasmosis. Ten ocular samples from other uveitis patients and 20 samples from subjects without ocular inflammation were used as controls. Two polymerase chain reaction (PCR) methods, i.e., qualitative multiplex PCR and quantitative real-time PCR, were used to measure the toxoplasma genome (T. gondii B1 gene). Qualitative multiplex PCR detected T. gondii B1 gene in the ocular fluids of 11 out of 13 patients with clinically suspected ocular toxoplasmosis. In real-time PCR, we detected high copy numbers of T. gondii DNA (5.1 × 10(2)-2.1 × 10(6) copies/mL) in a total of 10 patients (10/13, 77%). Only ocular toxoplasmosis scar lesions were observed in the three real-time PCR-negative patients. PCR assay results for the samples from the two control groups were all negative. The two-step PCR examination to detect toxoplasma DNA is a useful tool for diagnosing ocular toxoplasmosis.

  13. PCR Assay Using Cerebrospinal Fluid for Diagnosis of Cerebral Toxoplasmosis in Brazilian AIDS patients

    Science.gov (United States)

    Vidal, José E.; Colombo, Fabio Antonio; Penalva de Oliveira, Augusto C.; Focaccia, Roberto; Pereira-Chioccola, Vera Lucia

    2004-01-01

    Highly active antiretroviral therapy has decreased the incidence of opportunistic infections in the central nervous system in AIDS patients. However, neurological abnormalities still remain important causes of mortality and morbidity in developing countries. In Brazil, cerebral toxoplasmosis is the most common cerebral mass lesion in AIDS patients. For these reasons, early, inexpensive, and sensitive diagnostic tests must be evaluated. The aim of this study was to evaluate PCR, using cerebrospinal fluid (CSF) samples to detect Toxoplasma gondii DNA, and to determine if the association of PCR with immunological assays can contribute to a timely diagnosis. We studied two sample groups. First, we analyzed stored CSF samples from 29 newborns and from 39 adults with AIDS without a definitive diagnosis of toxoplasmosis. The goal of this step was to standardize the methodology with a simple and economical procedure to recover the T. gondii DNA. Next, we prospectively evaluated CSF samples from 12 AIDS patients with a first episode of cerebral toxoplasmosis and 18 AIDS patients with other neurological opportunistic diseases and without previous cerebral toxoplasmosis. In all PCR samples, an indirect immunofluorescent assay and an enzyme-linked immunosorbent assay were performed. Samples from all patients with cerebral toxoplasmosis presented positive PCR results (sensitivity, 100%), and a sample from one of the 18 AIDS patients with other neurological diseases also presented positive PCR results (specificity, 94.4%). These findings suggest the clinical utility of PCR in the diagnosis of cerebral toxoplasmosis in developing countries. PMID:15472338

  14. How to evaluate PCR assays for the detection of low-level DNA

    DEFF Research Database (Denmark)

    Banch-Clausen, Frederik; Urhammer, Emil; Rieneck, Klaus

    2015-01-01

    are needed and how many of these should be positive or what amount of template should be used? We developed a mathematical model to obtain a simple tool for quick PCR assay evaluation before laboratory optimization and validation procedures. The model was based on the Poisson distribution and the Binomial...... not significantly different from experimental data generated by testing of cell-free foetal DNA. Also, the simplified formula was applicable for fast and accurate assay evaluation. In conclusion, the model can be applied for evaluation of sensitivity of real-time PCR-based detection of low-level DNA, and may also...

  15. Quantitative Analysis of Epstein-Barr Virus Load by Using a Real-Time PCR Assay

    OpenAIRE

    1999-01-01

    To measure the virus load in patients with symptomatic Epstein-Barr virus (EBV) infections, we used a real-time PCR assay to quantify the amount of EBV DNA in blood. The real-time PCR assay could detect from 2 to over 107 copies of EBV DNA with a wide linear range. We estimated the virus load in peripheral blood mononuclear cells (PBMNC) from patients with symptomatic EBV infections. The mean EBV-DNA copy number in the PBMNC was 103.7 copies/μg of DNA in patients with EBV-related lymphoprolif...

  16. Development of a TaqMan real-time PCR assay for quantification of airborne conidia of Botrytis squamosa and management of botrytis leaf blight of onion.

    Science.gov (United States)

    Carisse, O; Tremblay, D M; Lévesque, C A; Gindro, K; Ward, P; Houde, A

    2009-11-01

    The use of a DNA-based method for quantifying airborne inoculum of Botrytis squamosa, a damaging pathogen of onion, was investigated. A method for purifying DNA from conidia collected using rotating-arm samplers and quantifying it using a TaqMan real-time quantitative polymerase chain reaction (qPCR) assay is described. The sensitivity of the qPCR assay was high, with a detection limit of 2 conidia/rod. A linear relationship between numbers of conidia counted with a compound microscope and those determined with the qPCR assay was obtained. Receiver operating characteristic curve analysis was used to evaluate the reliability of the two methods of conidia quantification (microscope examination and qPCR assay) to predict the risk of disease being below or above a damage threshold (D(th)). In total, 142 field samples from commercial onion fields were analyzed. At damage thresholds of 5 or 10 lesions/leaf, conidia quantification with the qPCR assay was more reliable at predicting disease risk than conidia quantification based on microscope counts. The proportion of decisions where the disease was present and predicted was higher for the qPCR assay than for the microscope counts, with values of 0.95 and 0.89 compared with 0.79 and 0.81 for D(th) of 5 and 10 lesions/leaf, respectively. The proportion of decisions where the disease was present but not predicted was lower for the qPCR assay than for microscope counts, with values of 0.05 and 0.11 compared with 0.20 and 0.19 for D(th) of 5 and 10 lesions/leaf, respectively. The results demonstrated that this new qPCR assay was reliable for quantifying B. squamosa airborne inoculum in commercial onion fields and that molecular conidia quantification could be used as a component of a risk management system for Botrytis leaf blight.

  17. Mycoplasma bovis real-time polymerase chain reaction assay validation and diagnostic performance.

    Science.gov (United States)

    Clothier, Kristin A; Jordan, Dianna M; Thompson, Curtis J; Kinyon, Joann M; Frana, Timothy S; Strait, Erin L

    2010-11-01

    Mycoplasma bovis is an important bacterial pathogen in cattle, producing a variety of clinical diseases. The organism, which requires specialized culture conditions and extended incubation times to isolate and identify, is frequently associated with concurrent infection with other pathogens which can potentially be more easily identified. Real-time polymerase chain reaction (real-time PCR) is a valuable diagnostic technique that can rapidly identify infectious agents in clinical specimens. A real-time PCR assay was designed based on the uvrC gene to identify M. bovis in diagnostic samples. Using culture as the gold standard test, the assay performed well in a variety of diagnostic matrices. Initial validation testing was conducted on 122 milk samples (sensitivity: 88.9% [95% confidence interval (CI): 68.4-100%], specificity: 100%); 154 lung tissues (sensitivity: 89.0% [95% CI: 83.1-94.9%], specificity: 97.8% [95% CI: 93.5-100%]); 70 joint tissue/fluid specimens (sensitivity: 92.3% [95% CI: 82.1-100%], specificity: 95.5% [95% CI: 89.3-100%]); and 26 nasal swabs (sensitivity: 75.0% [95% CI: 45.0-100%], specificity: 83.3% [95% CI: 66.1-100%]). Low numbers of other sample matrices showed good agreement between results of culture and PCR. A review of clinical cases from 2009 revealed that, in general, PCR was used much more frequently than culture and provided useful diagnostic information in conjunction with clinical signs, signalment, and gross and histopathologic lesions. Diagnostic performance of the real-time PCR assay developed as a testing method indicates that it is a rapid, accurate assay that is adaptable to a variety of PCR platforms and can provide reliable results on an array of clinical samples.

  18. Quantitative real-time PCR (qPCR) assay for human-dog-cat species identification and nuclear DNA quantification.

    Science.gov (United States)

    Kanthaswamy, S; Premasuthan, A; Ng, J; Satkoski, J; Goyal, V

    2012-03-01

    In the United States, human forensic evidence collected from crime scenes is usually comingled with biomaterial of canine and feline origins. Knowledge of the concentration of nuclear DNA extracted from a crime scene biological sample and the species from which the sample originated is essential for DNA profiling. The ability to accurately detect and quantify target DNA in mixed-species samples is crucial when target DNA may be overwhelmed by non-target DNA. We have designed and evaluated a species-specific (human, dog and cat) nuclear DNA identification assay based on the TaqMan(®) quantitative real-time PCR (qPCR) technology that can simultaneously detect and measure minute quantities of DNA specific to either humans, dogs and/or cats. The fluorogenic triplex assay employs primers and hydrolysis probes that target the human TH01 locus as well as the dog and cat Melanocortin 1 Receptor (MC1R) sequences in a species-specific manner. We also demonstrate that the assay is a highly sensitive, reliable and robust method for identifying and quantifying mixed-species templates of human-dog-cat origin with as little as 0.4 pg of human and cat nuclear DNA, respectively, and 4.0 pg of dog nuclear DNA.

  19. Diagnosis of herpes simplex virus-1 keratitis using Giemsa stain, immunofluorescence assay, and polymerase chain reaction assay on corneal scrapings

    Science.gov (United States)

    Farhatullah, S; Kaza, S; Athmanathan, S; Garg, P; Reddy, S B; Sharma, S

    2004-01-01

    Aims: To evaluate three tests used routinely for the diagnosis of herpes simplex virus (HSV) keratitis. Methods: Corneal scrapings from 28 patients with clinically typical dendritic corneal ulcer suggestive of HSV keratitis, and 30 patients with clinically non-viral corneal ulcers, were tested by (i) Giemsa stain for multinucleated giant cells, (ii) immunofluorescence assay (IFA) for HSV-1 antigen, and (iii) polymerase chain reaction (PCR) for HSV-1 DNA, by investigators masked to clinical diagnosis. The control subjects were also investigated by smears and cultures for bacteria, fungus, and Acanthamoeba. Results: The specificity and positive predictive values of all three tests for the diagnosis of HSV keratitis were between 95–100%. The sensitivity of IFA and PCR was 78.6% and 81.2%, respectively, and the difference was not significant; however, their sensitivity and negative predictive value were significantly higher than Giemsa stain. Conclusions: While a combination of IFA and PCR constitute the choice of tests in clinically suspected cases of HSV keratitis, multinucleated giant cells in Giemsa stain can pre-empt testing by IFA and PCR in otherwise atypical cases of HSV keratitis. PMID:14693792

  20. A fluorescence-based quantitative real-time PCR assay for accurate Pocillopora damicornis species identification

    Science.gov (United States)

    Thomas, Luke; Stat, Michael; Evans, Richard D.; Kennington, W. Jason

    2016-09-01

    Pocillopora damicornis is one of the most extensively studied coral species globally, but high levels of phenotypic plasticity within the genus make species identification based on morphology alone unreliable. As a result, there is a compelling need to develop cheap and time-effective molecular techniques capable of accurately distinguishing P. damicornis from other congeneric species. Here, we develop a fluorescence-based quantitative real-time PCR (qPCR) assay to genotype a single nucleotide polymorphism that accurately distinguishes P. damicornis from other morphologically similar Pocillopora species. We trial the assay across colonies representing multiple Pocillopora species and then apply the assay to screen samples of Pocillopora spp. collected at regional scales along the coastline of Western Australia. This assay offers a cheap and time-effective alternative to Sanger sequencing and has broad applications including studies on gene flow, dispersal, recruitment and physiological thresholds of P. damicornis.

  1. Evaluation of Various Campylobacter-Specific Quantitative PCR (qPCR) Assays for Detection and Enumeration of Campylobacteraceae in Irrigation Water and Wastewater via a Miniaturized Most-Probable-Number-qPCR Assay.

    Science.gov (United States)

    Banting, Graham S; Braithwaite, Shannon; Scott, Candis; Kim, Jinyong; Jeon, Byeonghwa; Ashbolt, Nicholas; Ruecker, Norma; Tymensen, Lisa; Charest, Jollin; Pintar, Katarina; Checkley, Sylvia; Neumann, Norman F

    2016-08-01

    Campylobacter spp. are the leading cause of bacterial gastroenteritis worldwide, and water is increasingly seen as a risk factor in transmission. Here we describe a most-probable-number (MPN)-quantitative PCR (qPCR) assay in which water samples are centrifuged and aliquoted into microtiter plates and the bacteria are enumerated by qPCR. We observed that commonly used Campylobacter molecular assays produced vastly different detection rates. In irrigation water samples, detection rates varied depending upon the PCR assay and culture method used, as follows: 0% by the de Boer Lv1-16S qPCR assay, 2.5% by the Van Dyke 16S and Jensen glyA qPCR assays, and 75% by the Linton 16S endpoint PCR when cultured at 37°C. Primer/probe specificity was the major confounder, with Arcobacter spp. routinely yielding false-positive results. The primers and PCR conditions described by Van Dyke et al. (M. I. Van Dyke, V. K. Morton, N. L. McLellan, and P. M. Huck, J Appl Microbiol 109:1053-1066, 2010, http://dx.doi.org/10.1111/j.1365-2672.2010.04730.x) proved to be the most sensitive and specific for Campylobacter detection in water. Campylobacter occurrence in irrigation water was found to be very low (Campylobacter-specific qPCR was used, with the most commonly detected species being C. jejuni, C. coli, and C. lari Campylobacters in raw sewage were present at ∼10(2)/100 ml, with incubation at 42°C required for reducing microbial growth competition from arcobacters. Overall, when Campylobacter prevalence and/or concentration in water is reported using molecular methods, considerable validation is recommended when adapting methods largely developed for clinical applications. Furthermore, combining MPN methods with molecular biology-based detection algorithms allows for the detection and quantification of Campylobacter spp. in environmental samples and is potentially suited to quantitative microbial risk assessment for improved public health disease prevention related to food and water

  2. Comparison of histopathology and PCR based assay for detection of experimentally induced toxoplasmosis in murine model

    Institute of Scientific and Technical Information of China (English)

    Vikrant Sudan; A K Tewari; R Singh; Harkirat Singh

    2015-01-01

    Objective:To compare histopathology and PCR based detection in diagnosis of experimentally induced toxoplasmosis of RH human strain of the parasite in murine models. Methods:A comparison of histopathology and PCR based detection was done to diagnose experimentally induced toxoplasmosis in ten inbred swiss albino mice after intraperitoneal inoculation of 100 tachyzoites of laboratory mantained human RH strain of the parasite. Tissue samples from lung, liver, spleen, brain, heart and kidney were taken and processed for histopathological examination while all the samples also were subjected to PCR, using primers directed to the multicopy of SAG 3 gene, in dublicates. Results: Histopathology revealed presence of tachyzoites only in liver while along with lung, liver, spleen and brain tissue yielded desired positive PCR amplicons. Conclusions:The SAG 3 based PCR is able to diagnose toxoplasmosis in those tissues which are declared negative by histopathological assay.

  3. Validity of a PCR assay in CSF for the diagnosis of neurocysticercosis

    Science.gov (United States)

    Campoverde, Alfredo; Romo, Matthew L.; García, Lorena; Piedra, Luis M.; Pacurucu, Mónica; López, Nelson; Aguilar, Jenner; López, Sebastian; Vintimilla, Luis C.; Toral, Ana M.; Peña-Tapia, Pablo

    2017-01-01

    Objective: To prospectively evaluate the validity of a PCR assay in CSF for the diagnosis of neurocysticercosis (NC). Methods: We conducted a multicenter, prospective case-control study, recruiting participants from 5 hospitals in Cuenca, Ecuador, from January 2015 to February 2016. Cases fulfilled validated diagnostic criteria for NC. For each case, a neurosurgical patient who did not fulfill the diagnostic criteria for NC was selected as a control. CT and MRI, as well as a CSF sample, were collected from both cases and controls. The diagnostic criteria to identify cases were used as a reference standard. Results: Overall, 36 case and 36 control participants were enrolled. PCR had a sensitivity of 72.2% (95% confidence interval [CI] 54.8%–85.8%) and a specificity of 100.0% (95% CI 90.3%–100.0%). For parenchymal NC, PCR had a sensitivity of 42.9% (95% CI 17.7%–71.1%), and for extraparenchymal NC, PCR had a sensitivity of 90.9% (95% CI 70.8%–98.9%). Conclusions: This study demonstrated the usefulness of this PCR assay in CSF for the diagnosis of NC. PCR may be particularly helpful for diagnosing extraparenchymal NC when neuroimaging techniques have failed. Classification of evidence: This study provides Class III evidence that CSF PCR can accurately identify patients with extraparenchymal NC. PMID:28105460

  4. An Improved PCR-RFLP Assay for Detection and Genotyping of Asymptomatic Giardia lamblia Infection in a Resource-Poor Setting.

    Science.gov (United States)

    Hawash, Yoursry; Ghonaim, M M; Al-Shehri, S S

    2016-02-01

    Laboratory workers, in resource-poor countries, still consider PCR detection of Giardia lamblia more costly and more time-consuming than the classical parasitological techniques. Based on 2 published primers, an in-house one-round touchdown PCR-RFLP assay was developed. The assay was validated with an internal amplification control included in reactions. Performance of the assay was assessed with DNA samples of various purities, 91 control fecal samples with various parasite load, and 472 samples of unknown results. Two cysts per reaction were enough for PCR detection by the assay with exhibited specificity (Sp) and sensitivity (Se) of 100% and 93%, respectively. Taking a published small subunit rRNA reference PCR test results (6%; 29/472) as a nominated gold standard, G. lamblia was identified in 5.9% (28/472), 5.2%, (25/472), and 3.6% (17/472) by PCR assay, RIDA(®) Quick Giardia antigen detection test (R-Biopharm, Darmstadt, Germany), and iodine-stained smear microscopy, respectively. The percent agreements (kappa values) of 99.7% (0.745), 98.9% (0.900), and 97.7% (0.981) were exhibited between the assay results and that of the reference PCR, immunoassay, and microscopy, respectively. Restriction digestion of the 28 Giardia-positive samples revealed genotype A pattern in 12 and genotype B profile in 16 samples. The PCR assay with the described format and exhibited performance has a great potential to be adopted in basic clinical laboratories as a detection tool for G. lamblia especially in asymptomatic infections. This potential is increased more in particular situations where identification of the parasite genotype represents a major requirement as in epidemiological studies and infection outbreaks.

  5. Development and validation of a multiplex reverse transcription PCR assay for simultaneous detection of three papaya viruses.

    Science.gov (United States)

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

    2014-10-21

    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.

  6. Detection of Cryptococcus neoformans DNA in Tissue Samples by Nested and Real-Time PCR Assays

    Science.gov (United States)

    Bialek, Ralf; Weiss, Michael; Bekure-Nemariam, Kubrom; Najvar, Laura K.; Alberdi, Maria B.; Graybill, John R.; Reischl, Udo

    2002-01-01

    Two PCR protocols targeting the 18S rRNA gene of Cryptococcus neoformans were established, compared, and evaluated in murine cryptococcal meningitis. One protocol was designed as a nested PCR to be performed in conventional block thermal cyclers. The other protocol was designed as a quantitative single-round PCR adapted to LightCycler technology. One hundred brain homogenates and dilutions originating from 20 ICR mice treated with different azoles were examined. A fungal burden of 3 × 101 to 2.9 × 104 CFU per mg of brain tissue was determined by quantitative culture. Specific PCR products were amplified by the conventional and the LightCycler methods in 86 and 87 samples, respectively, with products identified by DNA sequencing and real-time fluorescence detection. An analytical sensitivity of 1 CFU of C. neoformans per mg of brain tissue and less than 10 CFU per volume used for extraction was observed for both PCR protocols, while homogenates of 70 organs from mice infected with other fungi were PCR negative. Specificity testing was performed with genomic DNA from 31 hymenomycetous fungal species and from the ustilaginomycetous yeast Malassezia furfur, which are phylogenetically related to C. neoformans. Twenty-four strains, including species of human skin flora like M. furfur and Trichosporon spp., were PCR negative. Amplification was observed with Cryptococcus amylolentus, Filobasidiella depauperata, Cryptococcus laurentii, and five species unrelated to clinical specimens. LightCycler PCR products from F. depauperata and Trichosporon faecale could be clearly discriminated by melting curve analysis. The sensitive and specific nested PCR assay as well as the rapid and quantitative LightCycler PCR assay might be useful for the diagnosis and monitoring of human cryptococcal infections. PMID:11874894

  7. Use of a Combined Duplex PCR/Dot Blot Assay for more sensitive genetic characterization

    Directory of Open Access Journals (Sweden)

    Erin Curry

    2008-01-01

    Full Text Available A reliable and sensitive method of genetic analysis is necessary to detect multiple specific nucleic acid sequences from samples containing limited template. The most widely utilized method of specific gene detection, polymerase chain reaction (PCR, imparts inconsistent results when assessing samples with restricted template, especially in a multiplex reaction when copies of target genes are unequal. This study aimed to compare two methods of PCR product analysis, fluorescent detection following agarose gel electrophoresis or dot blot hybridization with chemiluminescent evaluation, in the detection of a single copy gene (SRY and a multicopy gene (β-actin. Bovine embryo sex determination was employed to exploit the limited DNA template available and the target genes of unequal copies. Primers were used either independently or together in a duplex reaction with purified bovine genomic DNA or DNA isolated from embryos. When used independently, SRY and β-actin products were detected on a gel at the equivalent of 4-cell or 1-cell of DNA, respectively; however, the duplex reaction produced visible SRY bands at the 256 cell DNA equivalent and β-actin products at the 64 cell DNA equivalent. Upon blotting and hybridization of the duplex PCR reaction, product was visible at the 1–4 cell DNA equivalent. Duplex PCR was also conducted on 186 bovine embryos and product was subjected to gel electrophoresis or dot-blot hybridization in duplicate. Using PCR alone, sex determination was not possible for 22.6% of the samples. Using PCR combined with dot blot hybridization, 100.0% of the samples exhibited either both the male specific and β-actin products or the β-actin signal alone, indicating that the reaction worked in all samples. This study demonstrated that PCR amplification followed by dot blot hybridization provided more conclusive results in the evaluation of samples with low DNA concentrations and target genes of unequal copies.

  8. Identification and Differentiation of Verticillium Species and V. longisporum Lineages by Simplex and Multiplex PCR Assays

    Science.gov (United States)

    Inderbitzin, Patrik; Davis, R. Michael; Bostock, Richard M.; Subbarao, Krishna V.

    2013-01-01

    Accurate species identification is essential for effective plant disease management, but is challenging in fungi including Verticillium sensu stricto (Ascomycota, Sordariomycetes, Plectosphaerellaceae), a small genus of ten species that includes important plant pathogens. Here we present fifteen PCR assays for the identification of all recognized Verticillium species and the three lineages of the diploid hybrid V. longisporum. The assays were based on DNA sequence data from the ribosomal internal transcribed spacer region, and coding and non-coding regions of actin, elongation factor 1-alpha, glyceraldehyde-3-phosphate dehydrogenase and tryptophan synthase genes. The eleven single target (simplex) PCR assays resulted in amplicons of diagnostic size for V. alfalfae, V. albo-atrum, V. dahliae including V. longisporum lineage A1/D3, V. isaacii, V. klebahnii, V. nonalfalfae, V. nubilum, V. tricorpus, V. zaregamsianum, and Species A1 and Species D1, the two undescribed ancestors of V. longisporum. The four multiple target (multiplex) PCR assays simultaneously differentiated the species or lineages within the following four groups: Verticillium albo-atrum, V. alfalfae and V. nonalfalfae; Verticillium dahliae and V. longisporum lineages A1/D1, A1/D2 and A1/D3; Verticillium dahliae including V. longisporum lineage A1/D3, V. isaacii, V. klebahnii and V. tricorpus; Verticillium isaacii, V. klebahnii and V. tricorpus. Since V. dahliae is a parent of two of the three lineages of the diploid hybrid V. longisporum, no simplex PCR assay is able to differentiate V. dahliae from all V. longisporum lineages. PCR assays were tested with fungal DNA extracts from pure cultures, and were not evaluated for detection and quantification of Verticillium species from plant or soil samples. The DNA sequence alignments are provided and can be used for the design of additional primers. PMID:23823707

  9. Promising Nucleic Acid Lateral Flow Assay Plus PCR for Shiga Toxin-Producing Escherichia coli.

    Science.gov (United States)

    Terao, Yoshitaka; Takeshita, Kana; Nishiyama, Yasutaka; Morishita, Naoki; Matsumoto, Takashi; Morimatsu, Fumiki

    2015-08-01

    Shiga toxin (Stx)-producing Escherichia coli (STEC) is a frequent cause of foodborne infections, and methods for rapid and reliable detection of STEC are needed. A nucleic acid lateral flow assay (NALFA) plus PCR was evaluated for detecting STEC after enrichment. When cell suspensions of 45 STEC strains, 14 non-STEC strains, and 13 non-E. coli strains were tested with the NALFA plus PCR, all of the STEC strains yielded positive results, and all of the non-STEC and non-E. coli strains yielded negative results. The lower detection limit for the STEC strains ranged from 0.1 to 1 pg of genomic DNA (about 20 to 200 CFU) per test, and the NALFA plus PCR was able to detect Stx1- and Stx2-producing E. coli strains with similar sensitivities. The ability of the NALFA plus PCR to detect STEC in enrichment cultures of radish sprouts, tomato, raw ground beef, and beef liver inoculated with 10-fold serially diluted STEC cultures was comparable to that of a real-time PCR assay (at a level of 100 to 100,000 CFU/ml in enrichment culture). The bacterial inoculation test in raw ground beef revealed that the lower detection limit of the NALFA plus PCR was also comparable to that obtained with a real-time PCR assay that followed the U.S. Department of Agriculture guidelines. Although further evaluation is required, these results suggest that the NALFA plus PCR is a specific and sensitive method for detecting STEC in a food manufacturing plant.

  10. Cytomegalovirus DNA quantification using an automated platform for nucleic acid extraction and real-time PCR assay setup.

    Science.gov (United States)

    Forman, Michael; Wilson, Andy; Valsamakis, Alexandra

    2011-07-01

    Analytical performance characteristics of the QIAsymphony RGQ system with artus cytomegalovirus (CMV) reagents were determined. Measurable range spanned 2.0 to ≥ 7.0 log(10) copies/ml. The detection limit was 23 copies/ml. Intrarun and interrun coefficients of variation were ≤ 2.1% at 3.0 and 5.0 log(10) copies/ml. In clinical specimens, RGQ values were ~0.2 log(10) copies/ml higher than those in an assay using a BioRobot M48 extraction/manual reaction setup/7500 Real-Time PCR instrument. No cross-contamination was observed.

  11. Evaluation of Various Campylobacter-Specific Quantitative PCR (qPCR) Assays for Detection and Enumeration of Campylobacteraceae in Irrigation Water and Wastewater via a Miniaturized Most-Probable-Number–qPCR Assay

    Science.gov (United States)

    Banting, Graham S.; Braithwaite, Shannon; Scott, Candis; Kim, Jinyong; Jeon, Byeonghwa; Ashbolt, Nicholas; Ruecker, Norma; Tymensen, Lisa; Charest, Jollin; Pintar, Katarina; Checkley, Sylvia

    2016-01-01

    ABSTRACT Campylobacter spp. are the leading cause of bacterial gastroenteritis worldwide, and water is increasingly seen as a risk factor in transmission. Here we describe a most-probable-number (MPN)–quantitative PCR (qPCR) assay in which water samples are centrifuged and aliquoted into microtiter plates and the bacteria are enumerated by qPCR. We observed that commonly used Campylobacter molecular assays produced vastly different detection rates. In irrigation water samples, detection rates varied depending upon the PCR assay and culture method used, as follows: 0% by the de Boer Lv1-16S qPCR assay, 2.5% by the Van Dyke 16S and Jensen glyA qPCR assays, and 75% by the Linton 16S endpoint PCR when cultured at 37°C. Primer/probe specificity was the major confounder, with Arcobacter spp. routinely yielding false-positive results. The primers and PCR conditions described by Van Dyke et al. (M. I. Van Dyke, V. K. Morton, N. L. McLellan, and P. M. Huck, J Appl Microbiol 109:1053–1066, 2010, http://dx.doi.org/10.1111/j.1365-2672.2010.04730.x) proved to be the most sensitive and specific for Campylobacter detection in water. Campylobacter occurrence in irrigation water was found to be very low (arcobacters. Overall, when Campylobacter prevalence and/or concentration in water is reported using molecular methods, considerable validation is recommended when adapting methods largely developed for clinical applications. Furthermore, combining MPN methods with molecular biology-based detection algorithms allows for the detection and quantification of Campylobacter spp. in environmental samples and is potentially suited to quantitative microbial risk assessment for improved public health disease prevention related to food and water exposures. IMPORTANCE The results of this study demonstrate the importance of assay validation upon data interpretation of environmental monitoring for Campylobacter when using molecular biology-based assays. Previous studies describing

  12. A TaqMan-based real-time PCR assay for porcine parvovirus 4 detection and quantification in reproductive tissues of sows

    Science.gov (United States)

    Porcine parvovirus 4 (PPV4) is a DNA virus, and a member of the Parvoviridae family within the Bocavirus genera. It was recently detected in swine, but its epidemiology and pathology remain unclear. A TaqMan-based real-time polymerase chain reaction (qPCR) assay targeting a conserved region of the O...

  13. Real-time PCR assays for detection and quactification of Edwardsiella tarda, Edwardsiella piscicida, Edwardsiella piscicida-like sp. in catfish tissues and pond water

    Science.gov (United States)

    Researchers have proposed the adoption of 3 distinct genetic taxa among bacteria previously classified as Edwardsiella tarda; namely E. tarda, E. piscicida, and a taxon presently termed E. piscicida–like. Individual real-time polymerase chain reaction (qPCR) assays were developed, based on published...

  14. Real-time PCR assays for detection and quactification of Edwardsiella tarda, Edwardsiella piscicida, Edwardsiella piscicida-like sp. in catfish tissues and pond water

    Science.gov (United States)

    Researchers have proposed the adoption of 3 distinct genetic taxa among bacteria previously classified as Edwardsiella tarda; namely E. tarda, E. piscicida, and a taxon presently termed E. piscicida–like. Individual real-time polymerase chain reaction (qPCR) assays were developed, based on published...

  15. 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

  16. Determining lower limits of detection of digital PCR assays for cancer-related gene mutations

    Directory of Open Access Journals (Sweden)

    Coren A. Milbury

    2014-09-01

    Full Text Available Digital PCR offers very high sensitivity compared to many other technologies for processing molecular detection assays. Herein, a process is outlined for determining the lower limit of detection (LoD of two droplet-based digital PCR assays for point mutations of the epidermal growth factor receptor (EGFR gene. Hydrolysis probe mutation-detection assays for EGFR p.L858R and p.T790M mutations were characterized in detail. Furthermore, sixteen additional cancer-related mutation assays were explored by the same approach. For the EGFR L8585R assay, the assay sensitivity is extremely good, and thus, the LoD is limited by the amount of amplifiable DNA that is analyzed. With 95% confidence limits, the LoD is one mutant in 180,000 wild-type molecules for the evaluation of 3.3 μg of genomic DNA, and detection of one mutant molecule in over 4 million wild-type molecules was achieved when 70 million copies of DNA were processed. The measured false-positive rate for the EGFR L8585R assay is one in 14 million, which indicates the theoretical LoD if an unlimited amount of DNA is evaluated. For the EFGR T790M assay, the LoD is one mutant in 13,000 for analysis of a 3.3 μg sample of genomic DNA, and the dPCR assay limit sensitivity approaches one mutant in 22,000 wild-type molecules.

  17. Nested polymerase chain reaction (PCR) targeting 16S rDNA for bacterial identification in empyema.

    Science.gov (United States)

    Prasad, Rajniti; Kumari, Chhaya; Das, B K; Nath, Gopal

    2014-05-01

    Empyema in children causes significant morbidity and mortality. However, identification of organisms is a major concern. To detect bacterial pathogens in pus specimens of children with empyema by 16S rDNA nested polymerase chain reaction (PCR) and correlate it with culture and sensitivity. Sixty-six children admitted to the paediatric ward with a diagnosis of empyema were enrolled prospectively. Aspirated pus was subjected to cytochemical examination, culture and sensitivity, and nested PCR targeting 16S rDNA using a universal eubacterial primer. Mean (SD) age was 5·8 (1·8) years (range 1-13). Analysis of aspirated pus demonstrated total leucocyte count >1000×10(6)/L, elevated protein (≧20 g/L) and decreased glucose (≤2·2 mmol/L) in 80·3%, 98·5% and 100%, respectively. Gram-positive cocci were detected in 29 (43·9%) and Gram-negative bacilli in two patients. Nested PCR for the presence of bacterial pathogens was positive in 50·0%, compared with 36·3% for culture. 16S rDNA PCR improves rates of detection of bacteria in pleural fluid, and can detect bacterial species in a single assay as well as identifying unusual and unexpected causal agents.

  18. A clinical comparative study of polymerase chain reaction assay for diagnosis of pneumocystis pneumonia in non-AIDS patients

    Institute of Scientific and Technical Information of China (English)

    MU Xiang-dong; WANG Guang-fa; SU Li

    2011-01-01

    Background Pneurnocystis jirovecii pneumonia (PCP) is one of the most common and fatal infections in non-AIDS immunocompromised patients,which is difficult to diagnose by traditional morphologic methods.This study evaluated polymerase chain reaction (PCR) assays of Pneumocystis jirovecii mitochondrial large subunits ribosomal RNA in sputum and bronchioalveolar lavage fluid (BALF) for diagnosing PCP.Methods Sputum and BALF specimens from two groups were collected:one group (PCP group) included 20 patients definitely diagnosed of PCP by Gomori methenamine silver (GMS) stains of BALF;the other group (non-PCP group) included 40 patients.Each specimen was examined by GMS stains and PCR assays.Results GMS stains of BALF in PCP group were 100% positive (20/20),GMS stains of sputum in PCP group were 35% positive (7/20);GMS stains of BALF in non-PCP group were 100% negative (40/40),GMS stains of sputum in non-PCP group were 100% negative (40/40).PCR assays of BALF in PCP group were 100% positive (20/20),PCR assays of sputum in PCP group were 100% positive (20/20);PCR assays of BALF in non-PCP group were 100% negative (40/40),PCR assays of sputum in non-PCP group were 100% negative (40/40).Sensitivity and specificity of PCR assays of sputum and BALF were both 100%;positive and negative predictive values were also both 100%.Conclusion The diagnostic value of PCR assays of Pneumocystisjirovecii mitochondrial large subunits ribosomal RNA on sputum and BALF for pneumocystis pneumonia are both high and equivalent.

  19. Novel PCR Assays Complement Laser Biosensor-Based Method and Facilitate Listeria Species Detection from Food.

    Science.gov (United States)

    Kim, Kwang-Pyo; Singh, Atul K; Bai, Xingjian; Leprun, Lena; Bhunia, Arun K

    2015-09-08

    The goal of this study was to develop the Listeria species-specific PCR assays based on a house-keeping gene (lmo1634) encoding alcohol acetaldehyde dehydrogenase (Aad), previously designated as Listeria adhesion protein (LAP), and compare results with a label-free light scattering sensor, BARDOT (bacterial rapid detection using optical scattering technology). PCR primer sets targeting the lap genes from the species of Listeria sensu stricto were designed and tested with 47 Listeria and 8 non-Listeria strains. The resulting PCR primer sets detected either all species of Listeria sensu stricto or individual L. innocua, L. ivanovii and L. seeligeri, L. welshimeri, and L. marthii without producing any amplified products from other bacteria tested. The PCR assays with Listeria sensu stricto-specific primers also successfully detected all species of Listeria sensu stricto and/or Listeria innocua from mixed culture-inoculated food samples, and each bacterium in food was verified by using the light scattering sensor that generated unique scatter signature for each species of Listeria tested. The PCR assays based on the house-keeping gene aad (lap) can be used for detection of either all species of Listeria sensu stricto or certain individual Listeria species in a mixture from food with a detection limit of about 10⁴ CFU/mL.

  20. A Multiplexed, Probe-Based Quantitative PCR Assay for DNA of Phytophthora sojae

    Science.gov (United States)

    Phytophthora sojae (Kaufm. & Gerd.) causes seed rot, pre- and post-emergence damping off, and sometimes foliar blight in soybean (Glycine max). Crop loss may approach 100% with susceptible cultivars. We report here the development of a unique quantitative PCR assay specific to DNA of P. sojae, and a...

  1. Detection of pork adulteration by highly-specific PCR assay of mitochondrial D-loop.

    Science.gov (United States)

    Karabasanavar, Nagappa S; Singh, S P; Kumar, Deepak; Shebannavar, Sunil N

    2014-02-15

    We describe a highly specific PCR assay for the authentic identification of pork. Accurate detection of tissues derived from pig (Sus scrofa) was accomplished by using newly designed primers targeting porcine mitochondrial displacement (D-loop) region that yielded an unique amplicon of 712 base pairs (bp). Possibility of cross-amplification was precluded by testing as many as 24 animal species (mammals, birds, rodent and fish). Suitability of PCR assay was confirmed in raw (n = 20), cooked (60, 80 and 100 °C), autoclaved (121 °C) and micro-oven processed pork. Sensitivity of detection of pork in other species meat using unique pig-specific PCR was established to be at 0.1%; limit of detection (LOD) of pig DNA was 10 pg (pico grams). The technique can be used for the authentication of raw, processed and adulterated pork and products under the circumstances of food adulteration related disputes or forensic detection of origin of pig species.

  2. Evaluation of a novel PCR-based diagnostic assay for detection of Mycobacterium tuberculosis in sputum samples.

    Science.gov (United States)

    Maher, M; Glennon, M; Martinazzo, G; Turchetti, E; Marcolini, S; Smith, T; Dawson, M T

    1996-01-01

    We report on a PCR-based assay we have developed for the detection of Mycobacterium tuberculosis in sputum samples. One hundred sputum specimens, which included 34 culture-positive and 66 culture-negative specimens, were evaluated with this system. Of the 34 culture-positive specimens, 31 were PCR positive, and 60 of the culture-negative specimens were PCR negative. An internal standard has been included in the assay system to monitor PCR inhibition and to confirm the reliability of the PCR assay. PMID:8862607

  3. Duplex real-time PCR assay for rapid detection of ampicillin-resistant Enterococcus faecium.

    Science.gov (United States)

    Mohn, Stein Christian; Ulvik, Arve; Jureen, Roland; Willems, Rob J L; Top, Janetta; Leavis, Helen; Harthug, Stig; Langeland, Nina

    2004-02-01

    Rapid and accurate identification of carriers of resistant microorganisms is an important aspect of efficient infection control in hospitals. Traditional identification methods of antibiotic-resistant bacteria usually take at least 3 to 4 days after sampling. A duplex real-time PCR assay was developed for rapid detection of ampicillin-resistant Enterococcus faecium (ARE). Primers and probes that are used in this assay specifically detected the D-Ala-D-Ala ligase gene of E. faecium and the modified penicillin-binding protein 5 gene (pbp5) carrying the Glu-to-Val substitution at position 629 (Val-629) in a set of 129 tested E. faecium strains with known pbp5 sequence. Presence of the Val-629 in the strain set from 11 different countries was highly correlated with ampicillin resistance. In a screening of hospitalized patients, the real-time PCR assay yielded a sensitivity and a specificity for the detection of ARE colonization of 95% and 100%, respectively. The results were obtained 4 h after samples were harvested from overnight broth of rectal swab samples, identifying both species and the resistance marker mutation in pbp5. This novel assay reliably identifies ARE 2 to 3 days more quickly than traditional culture methods, thereby increasing laboratory throughput, making it useful for rectal screening of ARE. The assay demonstrates the advantages of real-time PCR for detection of nosocomial pathogens.

  4. Strategies to develop strain-specific PCR based assays for probiotics.

    Science.gov (United States)

    Treven, P

    2015-01-01

    Since health benefits conferred by probiotics are strain-specific, identification to the strain level is mandatory to allow the monitoring of the presence and the abundance of specific probiotic in a product or in a gastrointestinal tract. Compared to standard plate counts, the reduced duration of the assays and higher specificity makes PCR-based methods (standard PCR and quantitative PCR) very appropriate for detection or quantification of probiotics. Development of strain-specific assay consists of 4 main stages: (1) strain-specific marker identification; (2) construction of potential strain-specific primers; (3) validation on DNA from pure cultures of target and related strains; and (4) validation on spiked samples. The most important and also the most challenging step is the identification of strain-specific sequences, which can be subsequently targeted by specific primers or probes. Such regions can be identified on sequences derived from 16S-23S internally transcribed spacers, randomly amplified polymorphic DNA, representational difference analysis and suppression subtractive hybridisation. Already known phenotypic or genotypic characteristics of the target strain can also be used to develop the strain-specific assay. However, the initial stage of strain-specific assay development can be replaced by comparative genomics analysis of target genome with related genomes in public databases. Advances in whole genome sequencing (WGS) have resulted in a cost reduction for bacterial genome sequencing and consequently have made this approach available to most laboratories. In the present paper I reviewed the available literature on PCR and qPCR assays developed for detection of a specific probiotic strain and discussed future WGS and comparative genomics-based approaches.

  5. Multiplex polymerase chain reaction (PCR) on a SU-8 chip

    DEFF Research Database (Denmark)

    Christensen, Troels Balmer; Bang, Dang Duong; Wolff, Anders

    2008-01-01

    We present the detection of Campylobacter at species level using multiplex PCR in a micro fabricated PCR chip. The chip is based on the polymer SU-8 that allows integration with different microfluidic components, e.g., sample pre-treatment before PCR, and DNA detection simultaneously with or afte...

  6. Usefulness of PCR-based assays to assess drug efficacy in Chagas disease chemotherapy: value and limitations.

    Science.gov (United States)

    Britto, Constança Carvalho

    2009-07-01

    One major goal of research on Chagas disease is the development of effective chemotherapy to eliminate the infection from individuals who have not yet developed cardiac and/or digestive disease manifestations. Cure evaluation is the more complex aspect of its treatment, often leading to diverse and controversial results. The absence of reliable methods or a diagnostic gold standard to assess etiologic treatment efficacy still constitutes a major challenge. In an effort to develop more sensitive tools, polymerase chain reaction (PCR)-based assays were introduced to detect low amounts of Trypanosoma cruzi DNA in blood samples from chagasic patients, thus improving the diagnosis and follow-up evaluation after chemotherapy. In this article, I review the main problems concerning drug efficacy and criteria used for cure estimation in treated chagasic patients, and the work conducted by different groups on developing PCR methodologies to monitor treatment outcome of congenital infections as well as recent and late chronic T. cruzi infections.

  7. Real-time PCR assay for detection and enumeration of Dekkera bruxellensis in wine.

    Science.gov (United States)

    Phister, Trevor G; Mills, David A

    2003-12-01

    Traditional methods to detect the spoilage yeast Dekkera bruxellensis from wine involve lengthy enrichments. To overcome this difficulty, we developed a quantitative real-time PCR method to directly detect and enumerate D. bruxellensis in wine. Specific PCR primers to D. bruxellensis were designed to the 26S rRNA gene, and nontarget yeast and bacteria common to the winery environment were not amplified. The assay was linear over a range of cell concentrations (6 log units) and could detect as little as 1 cell per ml in wine. The addition of large amounts of nontarget yeasts did not impact the efficiency of the assay. This method will be helpful to identify possible routes of D. bruxellensis infection in winery environments. Moreover, the time involved in performing the assay (3 h) should enable winemakers to more quickly make wine processing decisions in order to reduce the threat of spoilage by D. bruxellensis.

  8. Diagnostic Accuracy of Real-Time PCR Assays Targeting 16S rRNA and lipl32 Genes for Human Leptospirosis in Thailand: A Case-Control Study

    Science.gov (United States)

    Thaipadunpanit, Janjira; Chierakul, Wirongrong; Wuthiekanun, Vanaporn; Limmathurotsakul, Direk; Amornchai, Premjit; Boonslip, Siriphan; Smythe, Lee D.; Limpaiboon, Roongrueng; Hoffmaster, Alex R.; Day, Nicholas P. J.; Peacock, Sharon J.

    2011-01-01

    Background Rapid PCR-based tests for the diagnosis of leptospirosis can provide information that contributes towards early patient management, but these have not been adopted in Thailand. Here, we compare the diagnostic sensitivity and specificity of two real-time PCR assays targeting rrs or lipL32 for the diagnosis of leptospirosis in northeast Thailand. Methods/Principal Findings A case-control study of 266 patients (133 cases of leptospirosis and 133 controls) was constructed to evaluate the diagnostic sensitivity and specificity (DSe & DSp) of both PCR assays. The median duration of illness prior to admission of cases was 4 days (IQR 2–5 days; range 1–12 days). DSe and DSp were determined using positive culture and/or microscopic agglutination test (MAT) as the gold standard. The DSe was higher for the rrs assay than the lipL32 assay (56%, (95% CI 47–64%) versus 43%, (95% CI 34–52%), p<0.001). No cases were positive for the lipL32 assay alone. There was borderline evidence to suggest that the DSp of the rrs assay was lower than the lipL32 assay (90% (95% CI 83–94%) versus 93%, (95%CI 88–97%), p = 0.06). Nine controls gave positive reactions for both assays and 5 controls gave a positive reaction for the rrs assay alone. The DSe of the rrs and lipL32 assays were high in the subgroup of 39 patients who were culture positive for Leptospira spp. (95% and 87%, respectively, p = 0.25). Conclusions/Significance Early detection of Leptospira using PCR is possible for more than half of patients presenting with leptospirosis and could contribute to individual patient care. PMID:21283633

  9. A facile and specific assay for quantifying microRNA by an optimized RT-qPCR approach.

    Directory of Open Access Journals (Sweden)

    Qian Mei

    Full Text Available BACKGROUND: The spatiotemporal expression patterns of microRNAs (miRNAs are important to the verification of their predicted function. RT-qPCR is the accepted technique for the quantification of miRNA expression; however, stem-loop RT-PCR and poly(T-adapter assay, the two most frequently used methods, are not very convenient in practice and have poor specificity, respectively. RESULTS: We have developed an optimal approach that integrates these two methods and allows specific and rapid detection of tiny amounts of sample RNA and reduces costs relative to other techniques. miRNAs of the same sample are polyuridylated and reverse transcribed into cDNAs using a universal poly(A-stem-loop RT primer and then used as templates for SYBR® Green real-time PCR. The technique has a dynamic range of eight orders of magnitude with a sensitivity of up to 0.2 fM miRNA or as little as 10 pg of total RNA. Virtually no cross-reaction is observed among the closely-related miRNA family members and with miRNAs that have only a single nucleotide difference in this highly specific assay. The spatial constraint of the stem-loop structure of the modified RT primer allowed detection of miRNAs directly from cell lysates without laborious total RNA isolation, and the poly(U tail made it possible to use multiplex RT reactions of mRNA and miRNAs in the same run. CONCLUSIONS: The cost-effective RT-qPCR of miRNAs with poly(A-stem-loop RT primer is simple to perform and highly specific, which is especially important for samples that are precious and/or difficult to obtain.

  10. 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.

  11. Diagnosis of Genus Helicobacter through a hemi-nested PCR assay o

    Directory of Open Access Journals (Sweden)

    Heping Qin

    2016-05-01

    Full Text Available The present study aimed to establish a genus-specific PCR-based assay to detect helicobacters using 16S rRNA gene as the target template. We designed the hemi-nested primers based on sequences of 16S rRNA gene of 34 types of Helicobacter species. The inclusivity, sensitivity, and specificity of the PCR assay using these primers were examined in three different models, comprising feces simulated samples, BLAB/c mice infection model and clinic patients samples. The detection sensitivity of Helicobacter pylori, Helicobacter hepaticus and Helicobacter bilis strains from feces simulated samples was all 102 CFU/ml. We successfully detected H. hepaticus and H. bilis in the liver, cecum and feces of experimentally infected mice. H. pylori was successfully detected in the feces samples from 3 patients infected with H. pylori while not in the feces samples from 3 healthy human. However, the C97/C05–C97/C98 PCR assay detected H. pylori in the 2 positive samples. Due to the PCR assay’s excellent inclusivity, high sensitivity and specificity it may be used to detect the presence of Helicobacters.

  12. Novel assay of competitively differentiated polymerase chain reaction for screening point mutation of hepatitis B virus

    Institute of Scientific and Technical Information of China (English)

    Xiao-Mou Peng; Xue-Juan Chen; Jian-Guo Li; Lin Gu; Yang-Su Huang; Zhi-Liang Gao

    2003-01-01

    AIM: Point mutation, one of the commonest gene mutations,is the most important molecular pathogenesis of cancer and chronic infection. The commonest methods for detection of point mutation are based on polymerase chain reaction (PCR). These techniques, however, cannot be used in large scale screening since they are neither accurate nor simple.For this reason, this study established a novel method of competitively differentiated PCR (CD-PCR) for screening point mutation in clinical practice.METHODS: Two competitively differentiated primers for mutant-type and wild-type templates respectively with an identically complemented region in 3′ end except for last 2base pairs and a different non-complemented region in 5′end were designed. Thus, competitive amplification might be carried out at a lower annealing temperature at first, and then differentiated amplification at a higher annealing temperature when primers could not combine with initial templates. The amplification was performed in one-tube.The products of CD-PCR were detected using microplate hybridization assay. CD-PCR was evaluated by detecting G1896A variant of hepatitis B virus (HBV) in form of recombinant plasmids and in sera from patients with hepatitis B, and compared with allele-specific PCR (AS-PCR) and competitive AS-PCR.RESULTS: CD-PCR was successfully established. It could clearly distinguish wild-type and mutant-type plasmid DNA of G1896A variant when the amount of plasmid DNA was between 102-108copies/reaction, while for AS-PCR and competitive AS-PCR, the DNA amount was between 102-104copies/reaction. CD-PCR could detect one copy of G1896A variant among 10-100 copies of wild-type plasmid DNA. The specificity of CD-PCR was higher than those of AS-PCR and competitive AS-PCR in the detection of HBV G1896A variant in sera from patients with hepatitis B. CD-PCR was independent of the amount of HBV DNA in serum. HBV G1896A variant was more often found in HBeAg (-) patients with a lower level of

  13. Acanthamoeba can be differentiated by the polymerase chain reaction and simple plating assays.

    Science.gov (United States)

    Khan, N A; Jarroll, E L; Paget, T A

    2001-09-01

    Acanthamoeba are opportunistic pathogens with invasive and noninvasive species. For clinical purposes it is important to differentiate potentially pathogenic from nonpathogenic isolates. For the rapid and sensitive identification of Acanthamoeba at the genus level, we used a polymerase chain reaction (PCR)-based method which detected as few as five cells. Further, we tested nine isolates of Acanthamoeba for their ability to produce cytopathic effects (CPE) on corneal epithelial cells. On the basis of the results, Acanthamoeba were divided into pathogenic or nonpathogenic groups. However, because CPE assays are not available to every diagnostic laboratory, we developed a simple plating assay based on osmotolerance which correlated well with the CPE assays. Pathogenic Acanthamoeba showed growth on higher osmolarity (agar plates containing one molar mannitol), while growth of nonpathogens was inhibited on these plates. In conclusion, we have developed methods for the rapid identification and differentiation of Acanthamoeba.

  14. Evaluation of a new multiplex polymerase chain reaction assay STDFinder for the simultaneous detection of 7 sexually transmitted disease pathogens.

    Science.gov (United States)

    Muvunyi, Claude Mambo; Dhont, Nathalie; Verhelst, Rita; Crucitti, Tania; Reijans, Martin; Mulders, Brit; Simons, Guus; Temmerman, Marleen; Claeys, Geert; Padalko, Elizaveta

    2011-09-01

    We evaluated a new multiplex polymerase chain reaction (mPCR), "STDFinder assay", a novel multiplex ligation-dependent probe amplification (MLPA) assay for the simultaneous detection of 7 clinically relevant pathogens of STDs, i.e., Neisseria gonorrhoeae, Chlamydia trachomatis, Trichomonas vaginalis, Mycoplasma genitalium, Treponema pallidum, and herpes simplex virus type 1 and 2 (HSV-1 and HSV-2). An internal amplification control was included in the mPCR reaction. The limits of detection for the STDFinder assay varied among the 7 target organisms from 1 to 20 copies per MLPA assay. There were no cross-reactions among any of the probes. Two hundred and forty-two vaginal swabs and an additional 80 specimens with known results for N. gonorrhoeae and C. trachomatis, obtained from infertile women seen at an infertility research clinic at the Kigali Teaching Hospital in Rwanda, were tested by STDFinder assay and the results were confirmed by single real-time PCR using different species-specific targets. Compared to the reference standard, the STDFinder assay showed specificities and sensitivities of 100% and 100%, respectively, for N. gonorrhoeae, C. trachomatis, and M. genitalium; 90.2% and 100%, respectively, for Trichomonas vaginalis; and 96.1% and 100%, respectively, for HSV-2. No specimen was found to be positive for HSV-1 by either the STDFinder assay or the comparator method. Similarly, the sensitivity for Treponema pallidum could not be calculated due to the absence of any Treponema pallidum-positive samples. In conclusion, the STDFinder assays have comparable clinical sensitivity to the conventional mono and duplex real-time PCR assay and are suitable for the routine detection of a broad spectrum of these STDs at relatively low cost due to multiplexing.

  15. Diagnosis of enzootic pneumonia in Danish cattle: reverse transcription-polymerase chain reaction assay for detection of bovine respiratory syncytial virus in naturally and experimentally infected cattle

    DEFF Research Database (Denmark)

    Larsen, Lars Erik; Tjørnehøj, Kirsten; Viuff, B.;

    1999-01-01

    A reverse transcription-polymerase chain reaction (RT-PCR) assay was developed for detection of bovine respiratory syncytial virus (BRSV) in lung tissue of naturally and experimentally infected cattle. Primers were selected from the gene coding the F fusion protein, which is relatively conserved...... among BRSV isolates. The RT-PCR assay was highly specific, it yielded positive reactions only when performed on BRSV-infected cell cultures or tissues. The detection limit of the RT-PCR assay was assessed as 5 TCID50. BRSV was detected in tissues of the respiratory tract and in the tracheobroncheal....... (7%), and Pasteurella haemolytica (7%) were the most common bacterial agents found in the lungs. BRSV was identified using a conventional antigen enzyme-linked immunosorbent assay (ELISA) in 23 (17%) animals. The established BRSV-specific RT-PCR assay yielded positive results for the same 23 animals...

  16. Diagnosis of enzootic pneumonia in Danish cattle: reverse transcription-polymerase chain reaction assay for detection of bovine respiratory syncytial virus in naturally and experimentally infected cattle

    DEFF Research Database (Denmark)

    Larsen, Lars Erik; Tjørnehøj, Kirsten; Viuff, B.

    1999-01-01

    A reverse transcription-polymerase chain reaction (RT-PCR) assay was developed for detection of bovine respiratory syncytial virus (BRSV) in lung tissue of naturally and experimentally infected cattle. Primers were selected from the gene coding the F fusion protein, which is relatively conserved...... among BRSV isolates. The RT-PCR assay was highly specific, it yielded positive reactions only when performed on BRSV-infected cell cultures or tissues. The detection limit of the RT-PCR assay was assessed as 5 TCID50. BRSV was detected in tissues of the respiratory tract and in the tracheobroncheal....... (7%), and Pasteurella haemolytica (7%) were the most common bacterial agents found in the lungs. BRSV was identified using a conventional antigen enzyme-linked immunosorbent assay (ELISA) in 23 (17%) animals. The established BRSV-specific RT-PCR assay yielded positive results for the same 23 animals...

  17. A multiplex, internally controlled real-time PCR assay for detection of toxigenic Clostridium difficile and identification of hypervirulent strain 027/ST-1

    DEFF Research Database (Denmark)

    Hoegh, A M; Nielsen, J B; Lester, A

    2012-01-01

    The purpose of this study was to validate a multiplex real-time PCR assay capable of detecting toxigenic Clostridium difficile and simultaneously identifying C. difficile ribotype 027/ST-1 by targeting the toxin genes tcdA, tcdB and cdtA in one reaction and in a separate reaction identifying the Δ...... to confirm the correct identification of the Δ117 deletion in tcdC and C. difficile ribotype 027/ST-1, respectively. The PCR assay displayed a sensitivity, specificity, PPV and NPV of 99.0%, 97.4%, 87.4% and 99.8%, respectively, compared to toxigenic culture on 665 samples evaluable both by PCR and culture....... Sequencing of tcdC, ribotyping and MLST of cultured isolates validated the genotyping assay and confirmed the ability of the assay to correctly identify C. difficile ribotype 027/ST-1 in our current epidemiological setting. We describe the use of a combination of two separate PCR assays for sensitive...

  18. Assessment of Legionella pneumophila in recreational spring water with quantitative PCR (Taqman) assay.

    Science.gov (United States)

    Shen, Shu-Min; Chou, Ming-Yuan; Hsu, Bing-Mu; Ji, Wen-Tsai; Hsu, Tsui-Kang; Tsai, Hsiu-Feng; Huang, Yu-Li; Chiu, Yi-Chou; Kao, Erl-Shyh; Kao, Po-Min; Fan, Cheng-Wei

    2015-07-01

    Legionella spp. are common in various natural and man-made aquatic environments. Recreational hot spring is frequently reported as an infection hotspot because of various factors such as temperature and humidity. Although polymerase chain reaction (PCR) had been used for detecting Legionella, several inhibitors such as humic substances, calcium, and melanin in the recreational spring water may interfere with the reaction thus resulting in risk underestimation. The purpose of this study was to compare the efficiencies of conventional and Taqman quantitative PCR (qPCR) on detecting Legionella pneumophila in spring facilities and in receiving water. In the results, Taqman PCR had much better efficiency on specifying the pathogen in both river and spring samples. L. pneumophila was detected in all of the 27 river water samples and 45 of the 48 hot spring water samples. The estimated L. pneumophela concentrations ranged between 1.0 × 10(2) and 3.3 × 10(5) cells/l in river water and 72.1-5.7 × 10(6) cells/l in hot spring water. Total coliforms and turbidity were significantly correlated with concentrations of L. pneumophila in positive water samples. Significant difference was also found in water temperature between the presence/absence of L. pneumophila. Our results suggest that conventional PCR may be not enough for detecting L. pneumophila particularly in the aquatic environments full of reaction inhibitors.

  19. FungiQuant: A broad-coverage fungal quantitative real-time PCR assay

    Directory of Open Access Journals (Sweden)

    Liu Cindy M

    2012-11-01

    Full Text Available Abstract Background Fungal load quantification is a critical component of fungal community analyses. Limitation of current approaches for quantifying the fungal component in the human microbiome suggests the need for new broad-coverage techniques. Methods We analyzed 2,085 18S rRNA gene sequences from the SILVA database for assay design. We generated and quantified plasmid standards using a qPCR-based approach. We evaluated assay coverage against 4,968 sequences and performed assay validation following the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE guidelines. Results We designed FungiQuant, a TaqMan® qPCR assay targeting a 351 bp region in the fungal 18S rRNA gene. Our in silico analysis showed that FungiQuant is a perfect sequence match to 90.0% of the 2,617 fungal species analyzed. We showed that FungiQuant’s is 100% sensitive and its amplification efficiencies ranged from 76.3% to 114.5%, with r2-values of >0.99 against the 69 fungal species tested. Additionally, FungiQuant inter- and intra-run coefficients of variance ranged from Conclusions FungiQuant has comprehensive coverage against diverse fungi and is a robust quantification and detection tool for delineating between true fungal detection and non-target human DNA.

  20. Comparison and evaluation of conventional RT-PCR, SYBR green I and TaqMan real-time RT-PCR assays for the detection of porcine epidemic diarrhea virus.

    Science.gov (United States)

    Zhou, Xinrong; Zhang, Tiansheng; Song, Deping; Huang, Tao; Peng, Qi; Chen, Yanjun; Li, Anqi; Zhang, Fanfan; Wu, Qiong; Ye, Yu; Tang, Yuxin

    2017-06-01

    Porcine epidemic diarrhea (PED) caused by porcine epidemic diarrhea virus (PEDV) is a highly contagious intestinal disease, resulting in substantial economic losses to the swine industry worldwide. In this study, three assays, namely a conventional reverse transcription-polymerase chain reaction (RT-PCR), a SYBR Green I real-time RT-PCR and a TaqMan real-time RT-PCR targeting the highly conserved M gene of PEDV, were developed and evaluated. Then, the analytical specificity, sensitivity and reproducibility of these assays were determined and compared. The TaqMan real-time RT-PCR was 100-fold and 10,000-fold more sensitive than that of the SYBR Green I real-time RT-PCR and the conventional RT-PCR, respectively. The analytical sensitivity of TaqMan real-time RT-PCR was 10 copies/μl of target gene and no cross amplification with other viruses tested was observed. With the features of high specificity, sensitivity, and reproducibility, the TaqMan real-time RT-PCR established in this study could be a useful tool for clinical diagnosis, epidemiological surveys and outbreak investigations of PED. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. Development of a novel real-time qPCR assay for the dual detection of canine and phocine distemper virus

    DEFF Research Database (Denmark)

    Nielsen, Linette Buxbom; Hjulsager, Charlotte Kristiane; Larsen, Helene

    conventional PCR assays with real-time PCR assays to obtain a uniform assay palette. The present work describes the development of a novel real-time RT-qPCR assay for the dual detection of canine and phocine distemper virus. The assay is relevant for the future detection of outbreaks of canine distemper virus...

  2. Development of a Multiplex Real-Time PCR Assay for the Detection of Treponema pallidum, HCV, HIV-1, and HBV.

    Science.gov (United States)

    Zhou, Li; Gong, Rui; Lu, Xuan; Zhang, Yi; Tang, Jingfeng

    2015-01-01

    Treponema pallidum, hepatitis C virus (HCV), human immunodeficiency virus (HIV)-1, and hepatitis B virus (HBV) are major causes of sexually transmitted diseases passed through blood contact. The development of a sensitive and efficient method for detection is critical for early diagnosis and for large-scale screening of blood specimens in China. This study aims to establish an assay to detect these pathogens in clinical serum specimens. We established a TaqMan-locked nucleic acid (LNA) real-time polymerase chain reaction (PCR) assay for rapid, sensitive, specific, quantitative, and simultaneous detection and identification. The copy numbers of standards of these 4 pathogens were quantified. Standard curves were generated by determining the mean cycle threshold values versus 10-fold serial dilutions of standards over a range of 10(6) to 10(1) copies/μL, with the lowest detection limit of the assay being 10(1) copies/μL. The assay was applied to 328 clinical specimens and compared with enzyme-linked immunosorbent assay (ELISA) and commercial nucleic acid testing (NAT) methods. The assay identified 39 T. pallidum-, 96 HCV-, 13 HIV-1-, 123 HBV-, 5 HBV/HCV-, 1 T. pallidum/HBV-, 1 HIV-1/HCV-, and 1 HIV-1/T. pallidum-positive specimens. The high sensitivity of the assay confers strong potential for its use as a highly reliable, cost-effective, and useful molecular diagnostic tool for large-scale screening of clinical specimens. This assay will assist in the study of the pathogenesis and epidemiology of sexually transmitted blood diseases.

  3. Evaluation of a PCR assay for identification and differentiation of Campylobacter fetus subspecies

    DEFF Research Database (Denmark)

    Hum, S.; Quinn, K.; Brunner, J.

    1997-01-01

    Objective To evaluate a polymerase chain reaction assay for identification of Campylobacter fetus and differentiation of the defined subspecies. Design Characterisation of bacterial strains by traditional phenotyping, polymerase chain reaction, a probabilistic identification scheme and macrorestr......Objective To evaluate a polymerase chain reaction assay for identification of Campylobacter fetus and differentiation of the defined subspecies. Design Characterisation of bacterial strains by traditional phenotyping, polymerase chain reaction, a probabilistic identification scheme...... and macrorestriction profiling using pulsed field gel electrophoresis. Procedure The results of identification of 99 bacterial strains as determined by conventional phenotyping or by polymerase chain reaction were compared. Two of these were type strains of C fetus subsp fetus and C fetus subsp venerealis......; the remaining strains were field isolates putatively identified as C fetus. In cases where the subspecies identity was disputed, isolates were identified by means of a probabilistic identification scheme and by macrorestriction profiling. Results The agreement between strain identities initially suggested...

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

    Science.gov (United States)

    Prithiviraj, Jothikumar; Hill, Vincent; Jothikumar, Narayanan

    2012-04-20

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

  5. Novel and sensitive qPCR assays for the detection and identification of aspergillosis causing species.

    Science.gov (United States)

    Paholcsek, Melinda; Leiter, Eva; Markovics, Arnold; Biró, Sándor

    2014-09-01

    Despite concerted efforts, diagnosis of aspergillosis is still a great challenge to clinical microbiology laboratories. Along with the requirement for high sensitivity and specificity, species-specific identification is important. We developed rapid, sensitive and species-specific qPCR assays using the TaqMan technology for the detection and identification of Aspergillus fumigatus and Aspergillus terreus. The assays were designed to target orthologs of the Streptomyces factor C gene that are only found in a few species of filamentous fungi. Fungi acquired this gene through horizontal gene transfer and divergence of the gene allows identification of species. The assays have potential as a molecular diagnosis tool for the early detection of fungal infection caused by Aspergillus fumigatus and Aspergillus terreus, which merits future diagnostic studies. The assays were sensitive enough to detect a few genomic equivalents in blood samples.

  6. Development of a real-time PCR assay for the direct detection of Candida species causing Vulvovaginal candidiasis.

    Science.gov (United States)

    Tardif, Keith D; Schlaberg, Robert

    2017-01-25

    Identification of Candida species by traditional methods can be time-consuming and have limited analytical sensitivity. We developed a multiplex real-time PCR assay for detection and differentiation of Candida species causing vulvovaginal candidiasis (VVC). Overall, this PCR assay is a powerful diagnostic tool offering superior accuracy, sensitivity, and specificity.

  7. Comparison of cell-based and PCR-based assays as methods for measuring infectivity of Tulane virus.

    Science.gov (United States)

    Shan, Lei; Yang, David; Wang, Dapeng; Tian, Peng

    2016-05-01

    In this study, we used Tulane virus (TV) as a surrogate for HuNoV to evaluate for correlation between two cell-based assays and three PCR-based assays. Specifically, the cell-based plaque and TCID50 assays measure for infectious virus particles, while the PCR-based RNase exposure, porcine gastric mucin in-situ-capture qRT-PCR (PGM-ISC-qRT-PCR), and antibody in-situ-capture qRT-PCR (Ab-ISC-qRT-PCR) assays measure for an amplicon within encapsidated viral genome. Ten batches of viral stocks ranging from 3.41 × 10(5) to 6.67 × 10(6) plaque forming units (PFUs) were used for side by side comparison with PFU as a reference. The results indicate that one PFU was equivalent to 6.69 ± 2.34 TCID50 units, 9.75 ± 10.87 RNase-untreated genomic copies (GCs), 2.87 ± 3.05 RNase-treated GCs, 0.07 ± 0.07 PGM-ISC-qRT-PCR GCs, and 0.52 ± 0.39 Ab-ISC-qRT-PCR GCs. We observed that while the cell-based assays were consistent with each other, the TCID50 assay was more sensitive than the plaque assay. In contrast, the PCR-based assays were not always consistent with the cell-based assays. The very high variations in GCs as measured by both ISC-RT-qPCR assays made them difficult to correlate against the relatively small variations (<20-fold) in the PFUs or TCID50 units as measured by the cell-based assays.

  8. Rapid identification of Acinetobacter baumannii, Acinetobacter nosocomialis and Acinetobacter pittii with a multiplex PCR assay.

    Science.gov (United States)

    Chen, Te-Li; Lee, Yi-Tzu; Kuo, Shu-Chen; Yang, Su-Pen; Fung, Chang-Phone; Lee, Shou-Dong

    2014-09-01

    Acinetobacter baumannii, Acinetobacter nosocomialis and Acinetobacter pittii are clinically relevant members of the Acinetobacter calcoaceticus-A. baumannii (Acb) complex and important nosocomial pathogens. These three species are genetically closely related and phenotypically similar; however, they differ in their epidemiology, antibiotic resistance and pathogenicity. In this study, we investigated the use of a multiplex PCR-based assay designed to detect internal fragments of the 16S-23S rRNA intergenic region and the gyrB and recA genes. The assay was capable of differentiating A. baumannii, A. nosocomialis and A. pittii in a reliable manner. In 23 different reference strains and 89 clinical isolates of Acinetobacter species, the assay accurately identified clinically relevant Acb complex species except those 'between 1 and 3' or 'close to 13TU'. None of the non-Acb complex species was misidentified. In an analysis of 1034 positive blood cultures, the assay had a sensitivity of 92.4 % and specificity of 98.2 % for Acb complex identification. Our results show that a single multiplex PCR assay can reliably differentiate clinically relevant Acb complex species. Thus, this method may be used to better understand the clinical differences between infections caused by these species.

  9. Development and comparison of a real-time PCR assay for detection of Dichelobacter nodosus with culturing and conventional PCR: harmonisation between three laboratories

    DEFF Research Database (Denmark)

    Frosth, Sara; Slettemeås, Jannice S.; Jørgensen, Hannah J.

    2012-01-01

    BACKGROUND: Ovine footrot is a contagious disease with worldwide occurrence in sheep. The main causative agent is the fastidious bacterium Dichelobacter nodosus. In Scandinavia, footrot was first diagnosed in Sweden in 2004 and later also in Norway and Denmark. Clinical examination of sheep feet...... a TaqMan-based real-time PCR assay for detection of D. nodosus and to compare its performance with culturing and conventional PCR. METHODS: A D. nodosus-specific TaqMan based real-time PCR assay targeting the 16S rRNA gene was designed. The inclusivity and exclusivity (specificity) of the assay...... was tested using 55 bacterial and two fungal strains. To evaluate the sensitivity and harmonisation of results between different laboratories, aliquots of a single DNA preparation were analysed at three Scandinavian laboratories. The developed real-time PCR assay was compared to culturing by analysing 126...

  10. A PCR assay for gender assignment in dugong (Dugong dugon) and West Indian manatee (Trichechus manatus).

    Science.gov (United States)

    McHale, M; Broderick, D; Ovenden, J R; Lanyon, J M

    2008-05-01

    Gender assignment for some aquatic mammals in the field is difficult. Molecular sexing from tissue biopsies is possible as males are heterogametic. Here we describe a multiplex PCR assay that amplifies the male specific SRY gene and differentiates ZFX and ZFY gametologues in two sirenian species, dugong (Dugong dugon) and West Indian manatee (Trichechus manatus). The assay was validated with animals of known gender and proved accurate and robust to experimental failure. © 2007 The State of Queensland Through its Department of Primary Industries and Fisheries.

  11. A real-time PCR assay for the detection of atypical strains of Chlamydiaceae from pigeons.

    Directory of Open Access Journals (Sweden)

    Aleksandar Zocevic

    Full Text Available Recent evidence of the occurrence of atypical Chlamydiaceae strains in pigeons, different from the established Chlamydiaceae, requires the development of a specific and rapid detection tool to investigate their prevalence and significance. Here is described a new real-time PCR assay that allows specific detection of atypical Chlamydiaceae from pigeons. The assay has been used to assess the dissemination of these strains in field samples collected from Parisian pigeon populations in 2009. The results suggest a limited dissemination compared to the usually higher prevalence of Chlamydia psittaci that is the main species associated with avian chlamydiosis.

  12. Comparison of real-time SYBR green dengue assay with real-time taqman RT-PCR dengue assay and the conventional nested PCR for diagnosis of primary and secondary dengue infection

    OpenAIRE

    Damodar Paudel; Richard Jarman; Kriengsak Limkittikul; Chonticha Klungthong; Supat Chamnanchanunt; Ananda Nisalak; Robert Gibbons; Watcharee Chokejindachai

    2011-01-01

    Background : Dengue fever and dengue hemorrhagic fever are caused by dengue virus. Dengue infection remains a burning problem of many countries. To diagnose acute dengue in the early phase we improve the low cost, rapid SYBR green real time assay and compared the sensitivity and specificity with real time Taqman® assay and conventional nested PCR assay. Aims: To develop low cost, rapid and reliable real time SYBR green diagnostic dengue assay and compare with Taqman real-time assay and conven...

  13. HLA-DQA1 typing in Danes by two polymerase chain reaction (PCR) based methods

    DEFF Research Database (Denmark)

    Cowland, J B; Madsen, H O; Morling, N

    1995-01-01

    A total of 280 persons were HLA-DQA1 typed by two different polymerase chain reaction (PCR) based methods; (i) a reverse dot-blot (RDB) method, which can differentiate between six alleles, and (ii) a combined PCR-restriction fragment length polymorphism (PCR-RFLP) and allele-specific amplification...

  14. Testing the feasibility of DNA typing for human identification by PCR and an oligonucleotide ligation assay

    Energy Technology Data Exchange (ETDEWEB)

    Delahunty, C.; Ankener, W.; Deng, Qiang [Univ. of Washington, Seattle, WA (United States)] [and others

    1996-06-01

    The use of DNA typing in human genome analysis is increasing and finding widespread application in the area of forensic and paternity testing. In this report, we explore the feasibility of typing single nucleotide polymorphisms (SNPs) by using a semiautomated method for analyzing human DNA samples. In this approach, PCR is used to amplify segments of human DNA containing a common SNP. Allelic nucleotides in the amplified product are then typed by a calorimetric implementation of the oligonucleotide ligation assay (OLA). The results of the combined assay, PCR/OLA, are read directly by a spectrophotometer; the absorbances are compiled and the genotypes are automatically determined. A panel of 20 markers has been developed for DNA typing and has been tested using a sample panel from the CEPH pedigrees (CEPH parents). The results of this typing, as well as the potential to apply this method to larger populations, are discussed. 62 refs., 2 figs., 4 tabs.

  15. Multiplex quantification of four DNA targets in one reaction with Bio-Rad droplet digital PCR system for GMO detection

    Science.gov (United States)

    Dobnik, David; Štebih, Dejan; Blejec, Andrej; Morisset, Dany; Žel, Jana

    2016-10-01

    The advantages of the digital PCR technology are already well documented until now. One way to achieve better cost efficiency of the technique is to use it in a multiplexing strategy. Droplet digital PCR platforms, which include two fluorescence filters, support at least duplex reactions and with some developments and optimization higher multiplexing is possible. The present study not only shows a development of multiplex assays in droplet digital PCR, but also presents a first thorough evaluation of several parameters in such multiplex digital PCR. Two 4-plex assays were developed for quantification of 8 different DNA targets (7 genetically modified maize events and maize endogene). Per assay, two of the targets were labelled with one fluorophore and two with another. As current analysis software does not support analysis of more than duplex, a new R- and Shiny-based web application analysis tool (http://bit.ly/ddPCRmulti) was developed that automates the analysis of 4-plex results. In conclusion, the two developed multiplex assays are suitable for quantification of GMO maize events and the same approach can be used in any other field with a need for accurate and reliable quantification of multiple DNA targets.

  16. A LightCycler real-time PCR hybridization probe assay for detecting food-borne thermophilic Campylobacter

    DEFF Research Database (Denmark)

    Perelle, S.; Josefsen, Mathilde Hartmann; Hoorfar, Jeffrey

    2004-01-01

    Cycler real-time PCR assay (LC-PCR), which used fluorescent hybridization probes was developed. The test incorporated an internal amplification control co-amplified with the 16S rRNA gene of Campylobacter to monitor potential PCR inhibitors and ensure successful amplifications. The specificity study involving...

  17. Validation of a sensitive PCR assay for the detection of Chelonid fibropapilloma-associated herpesvirus in latent turtle infections.

    Science.gov (United States)

    Alfaro-Núñez, Alonzo; Gilbert, M Thomas P

    2014-09-01

    The Chelonid fibropapilloma-associated herpesvirus (CFPHV) is hypothesized to be the causative agent of fibropapillomatosis, a neoplastic disease in sea turtles, given its consistent detection by PCR in fibropapilloma tumours. CFPHV has also been detected recently by PCR in tissue samples from clinically healthy (non exhibiting fibropapilloma tumours) turtles, thus representing presumably latent infections of the pathogen. Given that template copy numbers of viruses in latent infections can be very low, extremely sensitive PCR assays are needed to optimize detection efficiency. In this study, efficiency of several PCR assays designed for CFPHV detection is explored and compared to a method published previously. The results show that adoption of a triplet set of singleplex PCR assays outperforms other methods, with an approximately 3-fold increase in detection success in comparison to the standard assay. Thus, a new assay for the detection of CFPHV DNA markers is presented, and adoption of its methodology is recommended in future CFPHV screens among sea turtles.

  18. Precise Quantitation of MicroRNA in a Single Cell with Droplet Digital PCR Based on Ligation Reaction.

    Science.gov (United States)

    Tian, Hui; Sun, Yuanyuan; Liu, Chenghui; Duan, Xinrui; Tang, Wei; Li, Zhengping

    2016-12-06

    MicroRNA (miRNA) analysis in a single cell is extremely important because it allows deep understanding of the exact correlation between the miRNAs and cell functions. Herein, we wish to report a highly sensitive and precisely quantitative assay for miRNA detection based on ligation-based droplet digital polymerase chain reaction (ddPCR), which permits the quantitation of miRNA in a single cell. In this ligation-based ddPCR assay, two target-specific oligonucleotide probes can be simply designed to be complementary to the half-sequence of the target miRNA, respectively, which avoids the sophisticated design of reverse transcription and provides high specificity to discriminate a single-base difference among miRNAs with simple operations. After the miRNA-templated ligation, the ddPCR partitions individual ligated products into a water-in-oil droplet and digitally counts the fluorescence-positive and negative droplets after PCR amplification for quantification of the target molecules, which possesses the power of precise quantitation and robustness to variation in PCR efficiency. By integrating the advantages of the precise quantification of ddPCR and the simplicity of the ligation-based PCR, the proposed method can sensitively measure let-7a miRNA with a detection limit of 20 aM (12 copies per microliter), and even a single-base difference can be discriminated in let-7 family members. More importantly, due to its high selectivity and sensitivity, the proposed method can achieve precise quantitation of miRNAs in single-cell lysate. Therefore, the ligation-based ddPCR assay may serve as a useful tool to exactly reveal the miRNAs' actions in a single cell, which is of great importance for the study of miRNAs' biofunction as well as for the related biomedical studies.

  19. A diagnostic PCR assay for the detection of an Australian epidemic strain of Pseudomonas aeruginosa

    Directory of Open Access Journals (Sweden)

    Murphy Anna

    2010-07-01

    Full Text Available Abstract Background Chronic lung infection with the bacterium Pseudomonas aeruginosa is one of the hallmarks of cystic fibrosis (CF and is associated with worsening lung function, increased hospitalisation and reduced life expectancy. A virulent clonal strain of P. aeruginosa (Australian epidemic strain I; AES-I has been found to be widespread in CF patients in eastern Australia. Methods Suppression subtractive hybridization (SSH was employed to identify genetic sequences that are present in the AES-I strain but absent from the sequenced reference strain PAO1. We used PCR to evaluate the distribution of several of the AES-I loci amongst a collection of 188 P. aeruginosa isolates which was comprised of 35 AES-I isolates (as determined by PFGE, 78 non-AES-I CF isolates including other epidemic CF strains as well as 69 P. aeruginosa isolates from other clinical and environmental sources. Results We have identified a unique AES-I genetic locus that is present in all 35 AES-I isolates tested and not present in any of the other 153 P. aeruginosa strains examined. We have used this unique AES-I locus to develop a diagnostic PCR and a real-time PCR assay to detect the presence of P. aeruginosa and AES-I in patient sputum samples. Conclusions We have developed diagnostic PCR assays that are 100% sensitive and 100% specific for the P. aeruginosa strain AES-I. We have also shown that Whatman FTA® Elute cards may be used with PCR-based assays to rapidly detect the presence of P. aeruginosa strains in CF sputum.

  20. Establishment of realtime RT-PCR assay to detect polio virus in the Acute Flaccid Paralysis laboratory surveillance

    Directory of Open Access Journals (Sweden)

    Nike Susanti

    2016-07-01

    Polio Vaccine into Vaccine-Derived Poliovirus still continue. Since 1991, WHO has developedAcute Flaccid Paralysis (AFP laboratory based surveillance. In 2014, the polioviruses identification by real-timeReverse Transcriptase Polymerase Chain Reaction (rRT-PCR, has been introduced to National Polio Laboratory(NPL Center for Biomedical and Basic Technology of Health. The objective of the rRT-PCR application is to havefaster and better diagnostic methods to monitor the circulation and mutation of polio viruses.Methods: Isolate tested by rRT-PCR using a combination of primers and probe mentioned by WHO manual.The viral RNA is converted to cDNA using reverse transcriptase and amplified in a PCR reaction using Taqpolymerase. The PCR products are detected and identified by hybridization with specific probes. The combinationof primers and probes will result in the serotype identification and intratypic differentiation of poliovirus isolates.Results: In 2014 NPL Jakarta received 604 AFP cases through the surveillance system, five cases foundpositive for polio viruses by culture. All of the specimens were positive for polio vaccine viruses. Twocases were polio virus type P2 (40%, one cases polio virus type P1 (20%, 1 case polio virus type P3(20% and one case mix polio viruses type P1+P2 (20%.Conclusion: The real-time PCR assay was able to help the identification of polio viruses rapidly in Jakartalab. The test can be utilized for monitoring the population routinely immunized with OPV. (Health ScienceJournal of Indonesia 2016;7:27-31Keywords: ITD, Poliovirus, Identification, rRT-PCR

  1. Development and Preliminary Evaluation of a New Real-Time RT-PCR Assay For Detection of Peste des petits Ruminants Virus Genome.

    Science.gov (United States)

    Polci, A; Cosseddu, G M; Ancora, M; Pinoni, C; El Harrak, M; Sebhatu, T T; Ghebremeskel, E; Sghaier, S; Lelli, R; Monaco, F

    2015-06-01

    A duplex real-time reverse transcription-polymerase chain reaction (qRT-PCR) assay was developed for a simple and rapid diagnosis of Peste des petits ruminants (PPR). qRT-PCR primers and TaqMan probe were designed on a conserved region of nucleocapsid protein (Np) of PPR virus (PPRV) genome. An in vitro transcript of the target region was constructed and tested to determine analytical sensitivity. Commercial heterologous Armored RNA(®) was used as an internal positive control (IPC) for either RNA isolation or RT-PCR steps. The detection limit of the newly designed duplex real-time RT-PCR (qRT-PCR PPR_Np) was approximately 20 copies/μl with a 95% probability. No amplification signals were recorded when the qRT-PCR PPR_Np was applied to viruses closely related or clinically similar to PPRV- or to PPR-negative blood samples. A preliminary evaluation of the diagnostic performance was carried out by testing a group of 43 clinical specimens collected from distinct geographic areas of Africa and Middle East. qRT-PCR PPR_Np showed higher sensitivity than the conventional gel-based RT-PCR assays, which have been used as reference standards. Internal positive control made it possible to identify the occurrence of 5 false-negative results caused by the amplification failure, thus improving the accuracy of PPRV detection. © 2013 Blackwell Verlag GmbH.

  2. Evaluation of a commercial real-time polymerase chain reaction assay for detection of environmental contamination with Clostridium difficile.

    Science.gov (United States)

    Deshpande, A; Kundrapu, S; Sunkesula, V C K; Cadnum, J L; Fertelli, D; Donskey, C J

    2013-09-01

    Contaminated environmental surfaces are an important source for transmission of Clostridium difficile. However, there are no efficient and easy methods to assess contamination. The performance of a commercial real-time polymerase chain reaction (PCR) assay was evaluated for detection of environmental toxigenic C. difficile in comparison with anaerobic culture followed by toxin testing of isolates. For 66 sites sampled, PCR had a sensitivity of 17.39%, specificity 100%, positive predictive value 100% and negative predictive value 69.35%. Increasing the PCR cycle threshold (CT) value to 45 increased sensitivity to 52% without decreasing specificity. The commercial PCR assay is not sufficiently sensitive for environmental monitoring, but improved sensitivity might be possible through CT value modification.

  3. Development of PCR assays for detection of Trichomonas vaginalis in urine specimens.

    Science.gov (United States)

    Bandea, Claudiu I; Joseph, Kahaliah; Secor, Evan W; Jones, Laurie A; Igietseme, Joseph U; Sautter, Robert L; Hammerschlag, Margaret R; Fajman, Nancy N; Girardet, Rebecca G; Black, Carolyn M

    2013-04-01

    Trichomonas vaginalis infections are usually asymptomatic or can result in nonspecific clinical symptoms, which makes laboratory-based detection of this protozoan parasite essential for diagnosis and treatment. We report the development of a battery of highly sensitive and specific PCR assays for detection of T. vaginalis in urine, a noninvasive specimen, and development of a protocol for differentiating among Trichomonas species that commonly infect humans.

  4. Detection of Listeria monocytogenes in cheese with the magnetic immuno-polymerase chain reaction assay.

    Science.gov (United States)

    Fluit, A C; Torensma, R; Visser, M J; Aarsman, C J; Poppelier, M J; Keller, B H; Klapwijk, P; Verhoef, J

    1993-05-01

    A new detection system, the magnetic immuno-polymerase chain reaction (PCR) assay (MIPA) has been developed to detect Listeria monocytogenes in food. This method separates Listeria cells from PCR-inhibitory factors present in enrichment broths containing food samples by using magnetic beads coated with specific monoclonal antibodies (MAbs). The separated bacteria were lysed, and the supernatant containing the bacterial DNA was subjected to the PCR. Detection of L. monocytogenes in three naturally contaminated cheese samples with two different MAbs and PCR primers specific for the gene encoding the delayed-hypersensitivity factor showed that with MAb 55 all three samples were positive whereas with MAb A two samples were positive. A further improvement of the method was obtained by using a PCR step based on the listeriolysin O gene. A MIPA employing MAb 55 and the listeriolysin O gene primer set detected L. monocytogenes after 24 h of culture in Listeria Enrichment Broth samples from Port Salut artificially contaminated with 40 CFU/25 g. We could detect 1 CFU of L. monocytogenes per g of cheese after a second enrichment for 24 h in Fraser broth. The analysis time including both enrichments is approximately 55 h.

  5. Comparison of real-time SYBR green dengue assay with real-time taqman RT-PCR dengue assay and the conventional nested PCR for diagnosis of primary and secondary dengue infection

    Directory of Open Access Journals (Sweden)

    Damodar Paudel

    2011-01-01

    Full Text Available Background : Dengue fever and dengue hemorrhagic fever are caused by dengue virus. Dengue infection remains a burning problem of many countries. To diagnose acute dengue in the early phase we improve the low cost, rapid SYBR green real time assay and compared the sensitivity and specificity with real time Taqman® assay and conventional nested PCR assay. Aims: To develop low cost, rapid and reliable real time SYBR green diagnostic dengue assay and compare with Taqman real-time assay and conventional nested PCR (modified Lanciotti. Materials and Methods: Eight cultured virus strains were diluted in tenth dilution down to undetectable level by the PCR to optimize the primer, temperature (annealing, and extension and to detect the limit of detection of the assay. Hundred and ninety three ELISA and PCR proved dengue clinical samples were tested with real time SYBR® Green assay, real time Taqman® assay to compare the sensitivity and specificity. Results: Sensitivity and specificity of real time SYBR® green dengue assay (84% and 66%, respectively was almost comparable to those (81% and 74% of Taqman real time PCR dengue assay. Real time SYBR® green RT-PCR was equally sensitive in primary and secondary infection while real time Taqman was less sensitive in the secondary infection. Sensitivity of real time Taqman on DENV3 (87% was equal to SYBR green real time PCR dengue assay. Conclusion: We developed low cost rapid diagnostic SYBR green dengue assay. Further study is needed to make duplex primer assay for the serotyping of dengue virus.

  6. A PCR-based assay for discriminating Cervus and Rangifer (Cervidae) antlers with mitochondrial DNA polymorphisms.

    Science.gov (United States)

    Kim, Young Hwa; Kim, Eung Soo; Ko, Byong Seob; Oh, Seung-Eun; Ryuk, Jin-Ah; Chae, Seong Wook; Lee, Hye Won; Choi, Go Ya; Seo, Doo Won; Lee, Mi Young

    2012-07-01

    This study describes a method for discriminating Rangifer antlers from true Cervus antlers using agarose gel electrophoresis, capillary electrophoresis, quantitative real-time PCR, and allelic discrimination. Specific primers labeled with fluorescent tags were designed to amplify fragments from the mitochondrial D-loop genes for various Cervus subspecies and Rangifer tarandus differentially. A 466-bp fragment that was observed for both Cervus and Rangifer antlers served as a positive control, while a 270-bp fragment was specifically amplified only from Rangifer antlers. Allelic discrimination was used to differentiate between Cervus and Rangifer antlers, based on the amplification of specific alleles for both types of antlers. These PCR-based assays can be used for forensic and quantitative analyses of Cervus and Rangifer antlers in a single step, without having to obtain any sequence information. In addition, multiple PCR-based assays are more accurate and reproducible than a single assay for species-specific analysis and are especially useful in this study for the identification of original Cervus deer products from fraudulent Rangifer antlers.

  7. Molecular diagnosis of Kingella kingae osteoarticular infections by specific real-time PCR assay.

    Science.gov (United States)

    Cherkaoui, Abdessalam; Ceroni, Dimitri; Emonet, Stéphane; Lefevre, Yan; Schrenzel, Jacques

    2009-01-01

    Kingella kingae is an emerging pathogen that is recognized as a causative agent of septic arthritis and osteomyelitis, primarily in infants and children. The bacterium is best detected by rapid inoculation in blood culture systems or by real-time PCR assays. Pathogenesis of the agent was linked recently to the production of a potent cytotoxin, known as RTX, which is toxic to a variety of human cell types. The locus encoding the RTX toxin is thought to be a putative virulence factor, and is, apparently, essential for inducing cytotoxic effects on respiratory epithelial, synovial and macrophage-like cells. Herein, we describe a novel real-time PCR assay that targets the RTX toxin gene and illustrate its use in two clinical cases. The assay exhibited a sensitivity of 30 c.f.u., which is 10-fold more sensitive than a previously published semi-nested broad-range 16S rRNA gene PCR, and showed no cross-reactivity with several related species and common osteoarticular pathogens.

  8. Detection of mycobacteria, Mycobacterium avium subspecies, and Mycobacterium tuberculosis complex by a novel tetraplex real-time PCR assay.

    Science.gov (United States)

    Sevilla, Iker A; Molina, Elena; Elguezabal, Natalia; Pérez, Valentín; Garrido, Joseba M; Juste, Ramón A

    2015-03-01

    Mycobacterium tuberculosis complex, Mycobacterium avium, and many other nontuberculous mycobacteria are worldwide distributed microorganisms of major medical and veterinary importance. Considering the growing epidemiologic significance of wildlife-livestock-human interrelation, developing rapid detection tools of high specificity and sensitivity is vital to assess their presence and accelerate the process of diagnosing mycobacteriosis. Here we describe the development and evaluation of a novel tetraplex real-time PCR for simultaneous detection of Mycobacterium genus, M. avium subspecies, and M. tuberculosis complex in an internally monitored single assay. The method was evaluated using DNA from mycobacterial (n = 38) and nonmycobacterial (n = 28) strains, tissues spiked with different CFU amounts of three mycobacterial species (n = 57), archival clinical samples (n = 233), and strains isolated from various hosts (n = 147). The minimum detectable DNA amount per reaction was 50 fg for M. bovis BCG and M. kansasii and 5 fg for M. avium subsp. hominissuis. When spiked samples were analyzed, the method consistently detected as few as 100 to 1,000 mycobacterial CFU per gram. The sensitivity and specificity values for the panel of clinical samples were 97.5 and 100% using a verified culture-based method as the reference method. The assays performed on clinical isolates confirmed these results. This PCR was able to identify M. avium and M. tuberculosis complex in the same sample in one reaction. In conclusion, the tetraplex real-time PCR we designed represents a highly specific and sensitive tool for the detection and identification of mycobacteria in routine laboratory diagnosis with potential additional uses.

  9. Is real-time polymerase chain reaction (PCR) more useful than a conventional PCR for the clinical management of leishmaniasis?

    Science.gov (United States)

    Antinori, Spinello; Calattini, Sara; Piolini, Roberta; Longhi, Erika; Bestetti, Giovanna; Cascio, Antonio; Parravicini, Carlo; Corbellino, Mario

    2009-07-01

    It is currently unknown if the use of a real-time polymerase chain reaction (PCR) adds value to the diagnosis and follow-up prognosis of patients affected by leishmaniasis. We performed a study using a real-time PCR directed against the alpha-polymerase gene and a semiquantitative PCR that target the SSU ribosomal RNA (rRNA) gene as control for the diagnosis and quantification of parasites in patients with visceral (VL) and cutaneous (CL) leishmaniasis. Our single copy real-time PCR missed one diagnosis of VL compared with the conventional PCR, whereas both PCR methods were able to detect Leishmania parasites in CL. Under anti-leishmania treatment the kinetics of parasitemia were comparable with the two methods. The real-time PCR directed against alpha-polymerase of Leishmania despite being able to make a more accurate quantification of parasites does not add to the decision-making management compared with a semiquantitative PCR, and it is comparatively expensive.

  10. Fungal granulomatous interstitial nephritis presenting as acute kidney injury diagnosed by renal histology including PCR assay.

    Science.gov (United States)

    Ogura, Makoto; Kagami, Shino; Nakao, Masatsugu; Kono, Midori; Kanetsuna, Yukiko; Hosoya, Tatsuo

    2012-10-01

    We describe two cases of fungal granulomatous interstitial nephritis (GIN) presenting as acute kidney injury (AKI). Increased serum creatinine was detected in Patient 1 after chemotherapy for pharyngeal cancer and in Patient 2 after steroid pulse therapy for bronchial asthma. Renal histology of both patients revealed GIN. Polymerase chain reaction (PCR)-based detection of fungal DNA sequences from kidney tissue demonstrated Trichosporon laibachii and Candida albicans, respectively. When AKI occurs in an immunocompromised host, differential diagnosis of fungal interstitial nephritis should be considered. Furthermore, PCR-based detection of fungal DNA sequences from renal specimens can be useful for rapid diagnosis.

  11. Comparative evaluation of a laboratory developed real-time PCR assay and the RealStar(®) HHV-6 PCR Kit for quantitative detection of human herpesvirus 6.

    Science.gov (United States)

    Yip, Cyril C Y; Sridhar, Siddharth; Cheng, Andrew K W; Fung, Ami M Y; Cheng, Vincent C C; Chan, Kwok-Hung; Yuen, Kwok-Yung

    2017-08-01

    HHV-6 reactivation in immunocompromised patients is common and may be associated with serious morbidity and mortality; therefore, early detection and initiation of therapy might be of benefit. Real-time PCR assays allow for early identification of HHV-6 reactivation to assist in providing a timely response. Thus, we compared the performance of an in-house developed HHV-6 quantitative PCR assay with a commercially available kit, the RealStar(®) HHV-6 PCR Kit. The analytical sensitivity, analytical specificity, linearity, precision and accuracy of the in-house developed HHV-6 qPCR assay were evaluated. The diagnostic performance of the in-house HHV-6 qPCR assay was compared with the RealStar(®) HHV-6 PCR Kit, using 72 clinical specimens and 17 proficiency testing samples. Linear regression analysis of the quantitative results showed a dynamic range from 2 to 10 log10 copies/ml and a coefficient of determination (R(2)) of 0.999 for the in-house assay. A dilution series demonstrated a limit of detection and a limit of quantification of 1.7 log10 and 2 log10 copies/ml, respectively. The precision of the assay was highly reproducible among runs with coefficients of variance (CV) ranging from 0.27% to 4.37%. A comparison of 27 matched samples showed an excellent correlation between the quantitative viral loads measured by the in-house HHV-6 qPCR assay and the RealStar(®) HHV-6 PCR Kit (R(2)=0.926; PPCR Kit. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. RT-qPCR-based microneutralization assay for human cytomegalovirus using fibroblasts and epithelial cells.

    Science.gov (United States)

    Wang, Xiao; Peden, Keith; Murata, Haruhiko

    2015-12-16

    Human cytomegalovirus (HCMV) is a leading cause of congenital infection that can result in serious disabilities in affected children. To facilitate HCMV vaccine development, a microscale neutralization assay based on reverse transcription quantitative PCR (RT-qPCR) was developed to quantify HCMV-neutralizing antibodies. Our approach relies on the generation of crude lysates from virus-infected cells that are amenable to direct analysis by RT-qPCR, thereby circumventing rate-limiting procedures associated with sample RNA extraction and purification. By serial passaging of the laboratory HCMV strain AD169 in epithelial cells (ARPE-19), a revertant virus with restored epithelial cell tropism, designated AD169(wt131), was obtained. AD169 and AD169(wt131) were evaluated in both epithelial cells (ARPE-19) and fibroblasts (MRC-5) by one-step RT-qPCR targeting the immediate-early gene IE1 transcript of HCMV. Expression kinetics indicated that RT-qPCR assessment could be conducted as early as 6h post-infection. Human serum samples (n=30) from healthy donors were tested for HCMV-specific IgG using a commercially available ELISA and for HCMV-neutralizing activity using our RT-qPCR-based neutralization assay. In agreement with the ELISA results, higher neutralizing activity was observed in the HCMV IgG seropositive group when compared with the HCMV IgG seronegative group. In addition, HCMV IgG seropositive human sera exhibited higher neutralizing titers using epithelial cells compared with using fibroblasts (geometric mean titers of 344 and 8 in ARPE-19 cells and MRC-5 cells, respectively). Our assay was robust to variation in input virus dose. In addition, a simple lysis buffer containing a non-ionic detergent was successfully demonstrated to be a less costly alternative to commercial reagents for cell-lysate preparation. Thus, our rapid HCMV neutralization assay may be a straightforward and flexible high-throughput tool for measuring antibody responses induced by vaccination

  13. Development of novel triplex single-step real-time PCR assay for detection of Hepatitis Virus B and C simultaneously.

    Science.gov (United States)

    Prakash, Shantanu; Jain, Amita; Jain, Bhawana

    2016-05-01

    Multiplex RT-PCR assays are widely used tools for detection of hepatitis viruses, but none of them provide quality check of sample. In the present study we developed a single-step triplex real-time polymerase chain reaction (PCR) assay for detection of Hepatitis B Virus (HBV) and Hepatitis C Virus (HCV) with sample quality check, by using β-actin as housekeeping gene. The primers and probes were self-designed and assay was standardized. Assay was also destined to quantitate copy numbers of HBV and HCV. This novel assay was sensitive, specific, and reproducible for detection of HBV and HCV in serum/plasma. The assay also detected all genotypes of HBV and HCV. The detection limit was 60 IU/mL for HBV and 20 IU/mL for HCV. This assay is the first assay developed on single-step platform for nucleic acid detection of HBV and HCV with an extra edge over all other assays by providing inbuilt check for quality of sample.

  14. Development and utility of an internal threshold control (ITC) real-time PCR assay for exogenous DNA detection.

    Science.gov (United States)

    Ni, Weiyi; Le Guiner, Caroline; Moullier, Philippe; Snyder, Richard O

    2012-01-01

    Sensitive and specific tests for detecting exogenous DNA molecules are useful for infectious disease diagnosis, gene therapy clinical trial safety, and gene doping surveillance. Taqman real-time PCR using specific sequence probes provides an effective approach to accurately and quantitatively detect exogenous DNA. However, one of the major challenges in these analyses is to eliminate false positive signals caused by either non-targeted exogenous or endogenous DNA sequences, or false negative signals caused by impurities that inhibit PCR. Although multiplex Taqman PCR assays have been applied to address these problems by adding extra primer-probe sets targeted to endogenous DNA sequences, the differences between targets can lead to different detection efficiencies. To avoid these complications, a Taqman PCR-based approach that incorporates an internal threshold control (ITC) has been developed. In this single reaction format, the target sequence and ITC template are co-amplified by the same primers, but are detected by different probes each with a unique fluorescent dye. Sample DNA, a prescribed number of ITC template molecules set near the limit of sensitivity, a single pair of primers, target probe and ITC probe are added to one reaction. Fluorescence emission signals are obtained simultaneously to determine the cycle thresholds (Ct) for amplification of the target and ITC sequences. The comparison of the target Ct with the ITC Ct indicates if a sample is a true positive for the target (i.e. Ct less than or equal to the ITC Ct) or negative (i.e. Ct greater than the ITC Ct). The utility of this approach was demonstrated in a nonhuman primate model of rAAV vector mediated gene doping in vivo and in human genomic DNA spiked with plasmid DNA.

  15. Comparison between Conventional and Real-Time PCR Assays for Diagnosis of Visceral Leishmaniasis

    Directory of Open Access Journals (Sweden)

    Mariana R. Pereira

    2014-01-01

    Full Text Available The diagnosis of visceral leishmaniasis (VL is a challenging issue and several studies worldwide have evaluated the different tools to reach a diagnostic solution. The polymerase chain reaction (PCR has proven to be effective in detecting the genome of Leishmania species in different biological samples. In this study, we compared the conventional PCR and real-time PCR using the Sybr Green system and their application in molecular diagnosis of visceral leishmaniasis in peripheral blood as a biological sample. The genus-specific conserved region of kinetoplast DNA (kDNA was the target of amplification. We studied 30 samples from patients with suspect of visceral leishmaniasis who were treated by the Medical Clinic of Santa Casa de Belo Horizonte Hospital, Brazil. Among the samples studied, 19 had a confirmed diagnosis for VL by serology and/or by clinical findings. Among these 19 samples, 63% (n=12 presented positive results for serology and 79% (n=15 positive results in both PCR methodologies. This fact suggests that the PCR technique can assist in the diagnosis of visceral leishmaniasis in patients who do not have detectable antibodies by serology but can present the genome of the parasite circulating in whole blood. Also, it was possible to observe that there was conformity between the results of the techniques of cPCR and qPCR using the Sybr Green system in 100% of samples analyzed. These data suggest that both PCR techniques were equally effective for detection of the genome of the parasite in the patient’s blood.

  16. Comparison between Conventional and Real-Time PCR Assays for Diagnosis of Visceral Leishmaniasis

    Science.gov (United States)

    Pereira, Mariana R.; Rocha-Silva, Fabiana; Graciele-Melo, Cidiane; Lafuente, Camila R.; Magalhães, Telcia; Caligiorne, Rachel B.

    2014-01-01

    The diagnosis of visceral leishmaniasis (VL) is a challenging issue and several studies worldwide have evaluated the different tools to reach a diagnostic solution. The polymerase chain reaction (PCR) has proven to be effective in detecting the genome of Leishmania species in different biological samples. In this study, we compared the conventional PCR and real-time PCR using the Sybr Green system and their application in molecular diagnosis of visceral leishmaniasis in peripheral blood as a biological sample. The genus-specific conserved region of kinetoplast DNA (kDNA) was the target of amplification. We studied 30 samples from patients with suspect of visceral leishmaniasis who were treated by the Medical Clinic of Santa Casa de Belo Horizonte Hospital, Brazil. Among the samples studied, 19 had a confirmed diagnosis for VL by serology and/or by clinical findings. Among these 19 samples, 63% (n = 12) presented positive results for serology and 79% (n = 15) positive results in both PCR methodologies. This fact suggests that the PCR technique can assist in the diagnosis of visceral leishmaniasis in patients who do not have detectable antibodies by serology but can present the genome of the parasite circulating in whole blood. Also, it was possible to observe that there was conformity between the results of the techniques of cPCR and qPCR using the Sybr Green system in 100% of samples analyzed. These data suggest that both PCR techniques were equally effective for detection of the genome of the parasite in the patient's blood. PMID:24689047

  17. Cell culture-Taqman PCR assay for evaluation of Cryptosporidium parvum disinfection.

    Science.gov (United States)

    Keegan, Alexandra R; Fanok, Stella; Monis, Paul T; Saint, Christopher P

    2003-05-01

    Cryptosporidium parvum represents a challenge to the water industry and a threat to public health. In this study, we developed a cell culture-quantitative PCR assay to evaluate the inactivation of C. parvum with disinfectants. The assay was validated by using a range of disinfectants in common use in the water industry, including low-pressure UV light (LP-UV), ozone, mixed oxidants (MIOX), and chlorine. The assay was demonstrated to be reliable and sensitive, with a lower detection limit of a single infectious oocyst. Effective oocyst inactivation was achieved (>2 log(10) units) with LP-UV (20 mJ/cm(2)) or 2 mg of ozone/liter (for 10 min). MIOX and chlorine treatments of oocysts resulted in minimal effective disinfection, with disinfection systems for drinking water and recycled water.

  18. Rapid detection of equine influenza virus H3N8 subtype by insulated isothermal RT-PCR (iiRT-PCR) assay using the POCKIT™ Nucleic Acid Analyzer.

    Science.gov (United States)

    Balasuriya, Udeni B R; Lee, Pei-Yu Alison; Tiwari, Ashish; Skillman, Ashley; Nam, Bora; Chambers, Thomas M; Tsai, Yun-Long; Ma, Li-Juan; Yang, Pai-Chun; Chang, Hsiao-Fen Grace; Wang, Hwa-Tang Thomas

    2014-10-01

    Equine influenza (EI) is an acute, highly contagious viral respiratory disease of equids. Currently, equine influenza virus (EIV) subtype H3N8 continues to be the most important respiratory pathogen of horses in many countries around the world. The need to achieve a rapid diagnosis and to implement effective quarantine and movement restrictions is critical in controlling the spread of EIV. In this study, a novel, inexpensive and user-friendly assay based on an insulated isothermal RT-PCR (iiRT-PCR) method on the POCKIT™, a field-deployable device, was described and validated for point-of-need detection of EIV-H3N8 in clinical samples. The newly established iiRT-PCR assay targeting the EIV HA3 gene was evaluated for its sensitivity using in vitro transcribed (IVT) RNA, as well as ten-fold serial dilutions of RNA extracted from the prototype H3N8 strain A/equine/Miami/1/63. Inclusivity and exclusivity panels were tested for specificity evaluation. Published real-time RT-PCR (rRT-PCR) assays targeting the NP and HA3 genes were used as the reference standards for comparison of RNA extracted from field strains and from nasal swab samples collected from experimentally infected horses, respectively. Limit of detection with a 95% probability (LoD95%) was estimated to be 11copies of IVT RNA. Clinical sensitivity analysis using RNA prepared from serial dilutions of a prototype EIV (Miami 1/63/H3N8) showed that the iiRT-PCR assay was about 100-fold more sensitive than the rRT-PCR assay targeting the NP gene of EIV subtype H3N8. The iiRT-PCR assay identified accurately fifteen EIV H3N8 strains and two canine influenza virus (CIV) H3N8 strains, and did not cross-react with H6N2, H7N7, H1N1 subtypes or any other equine respiratory viral pathogens. Finally, 100% agreement was found between the iiRT-PCR assay and the universal influenza virus type A rRT-PCR assay in detecting the EIV A/equine/Kentucky/7/07 strain in 56 nasal swab samples collected from experimentally inoculated

  19. A tissue biopsy-based epigenetic multiplex PCR assay for prostate cancer detection

    Directory of Open Access Journals (Sweden)

    Van Neste Leander

    2012-06-01

    Full Text Available Abstract Background PSA-directed prostate cancer screening leads to a high rate of false positive identifications and an unnecessary biopsy burden. Epigenetic biomarkers have proven useful, exhibiting frequent and abundant inactivation of tumor suppressor genes through such mechanisms. An epigenetic, multiplex PCR test for prostate cancer diagnosis could provide physicians with better tools to help their patients. Biomarkers like GSTP1, APC and RASSF1 have demonstrated involvement with prostate cancer, with the latter two genes playing prominent roles in the field effect. The epigenetic states of these genes can be used to assess the likelihood of cancer presence or absence. Results An initial test cohort of 30 prostate cancer-positive samples and 12 cancer-negative samples was used as basis for the development and optimization of an epigenetic multiplex assay based on the GSTP1, APC and RASSF1 genes, using methylation specific PCR (MSP. The effect of prostate needle core biopsy sample volume and age of formalin-fixed paraffin-embedded (FFPE samples was evaluated on an independent follow-up cohort of 51 cancer-positive patients. Multiplexing affects copy number calculations in a consistent way per assay. Methylation ratios are therefore altered compared to the respective singleplex assays, but the correlation with patient outcome remains equivalent. In addition, tissue-biopsy samples as small as 20 μm can be used to detect methylation in a reliable manner. The age of FFPE-samples does have a negative impact on DNA quality and quantity. Conclusions The developed multiplex assay appears functionally similar to individual singleplex assays, with the benefit of lower tissue requirements, lower cost and decreased signal variation. This assay can be applied to small biopsy specimens, down to 20 microns, widening clinical applicability. Increasing the sample volume can compensate the loss of DNA quality and quantity in older samples.

  20. A probe-free four-tube real-time PCR assay for simultaneous detection of twelve enteric viruses and bacteria.

    Science.gov (United States)

    Zhang, Chen; Niu, Peihua; Hong, Yanying; Wang, Ji; Zhang, Jingyun; Ma, Xuejun

    2015-11-01

    We aim to develop a multiplex real-time PCR assay to detect the most common pathogens causing community outbreaks of diarrhea. Four reaction systems of fluorescence dye-based real-time PCR assay were performed to amplify genes of norovirus, sapovirus, rotavirus, astrovirus, adenovirus, Campylobacter jejuni, Yersinia enterocolitica, Vibrio parahaemolyticus, Salmonella spp., Escherichia coli, and Shigella spp. PCR products of each pathogen were identified by characteristic peaks in melting curves. The assay was able to achieve detection limit of 50 copies/reaction for each individual virus target, and 140-500CFU/mL for each individual bacterium target. A total of 122 clinical specimens from hospitalized children with acute diarrhea were used to evaluate the assay. The clinical sensitivity was very similar to that of reference methods. Norovirus genogroup II revealed the highest detectable rate (45/122, 36.9%). Coinfection was found in 28 out of 122 (23%) clinical specimens. This assay proved to be a cost-effective, sensitive and reliable method for simultaneous detection of enteric viruses and bacteria. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. Noninvasive screening for genital chlamydial infections in asymptomatic men: Strategies and costs using a urine PCR assay

    Science.gov (United States)

    Peeling, Rosanna W; Toye, Baldwin; Jessamine, Peter; Gemmill, Ian

    1998-01-01

    OBJECTIVE: To evaluate cost saving strategies to screen for genital chlamydial infection in men using polymerase chain reaction (PCR) technology. METHODS: Men with no urethral symptoms presenting to a sexually transmitted disease (STD) clinic were recruited. Study participants underwent a questionnaire interview. Urethral swabs were taken to perform a smear for polymorphonuclear leucocytes (PMN) and for the detection of Chlamydia trachomatis by culture and PCR. First-catch urine was collected for a leukocyte esterase test (LET) and PCR. RESULTS: C trachomatis infection was detected in 36 of 463 (7.8%) men. LET and PMN were positive in 10 (28%) and 12 (33%) infected men, respectively. Risk factors for chlamydial infection were younger than age 25 years, LET-positive, PMN-positive and STD contact (P<0.001). The direct cost of genital chlamydial infection in men in Canada has been previously estimated at $381/case. Based on a sensitivity of 90% for urine PCR, the estimated direct cost of testing all participants to detect 32 cases was $453/case. Using risk factors recommended in the Canadian STD Guidelines (age younger than 25 years, new partner, STD contact or unprotected sex), the same number of cases would have been detected by testing only 384 men at $376/case. Using age younger than 25 years or STD contact as the screening criterion, 78% of those infected would have been detected at $259/case, and no new cases would have been detected by adding LET-positive or PMN-positive as risk factors. CONCLUSION: Targeted screening for chlamydial infection using urine PCR assay and risk factors recommended in the Canadian guidelines could substantially reduce the cost of screening at a STD clinic setting. LET and PMN smear did not appear to be useful indicators of chlamydial infection in this population. PMID:22346549

  2. Characterization of ISR region and development of a PCR assay for rapid detection of the fish pathogen Tenacibaculum soleae.

    Science.gov (United States)

    López, Jose R; Hamman-Khalifa, Abdel M; Navas, José I; de la Herran, Roberto

    2011-11-01

    The aims of this work were to characterize the 16S-23S internal spacer region of the fish pathogen Tenacibaculum soleae and to develop a PCR assay for its identification and detection. All T. soleae strains tested displayed a single internal spacer region class, containing tRNA(I) (le) and tRNA(A) (la) genes; nevertheless, a considerable intraspecific heterogeneity was observed. However, this region proved to be useful for differentiation of T. soleae from related and non-related species. Species-specific primers were designed targeting the 16S rRNA gene and the internal spacer region region, yielding a 1555-bp fragment. Detection limit was of 1 pg DNA per reaction (< 30 bacterial cells) when using pure cultures. The detection level in the presence of DNA from fish or other bacteria was lower; however, 10 pg were detected at a target/background ratio of 1 : 10(5) . The PCR assay proved to be more sensitive than agar cultivation for the detection of T. soleae from naturally diseased fish, offering a useful tool for diagnosis and for understanding the epidemiology of this pathogen.

  3. 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.

  4. Reproducibility of Digital PCR Assays for Circulating Tumor DNA Analysis in Advanced Breast Cancer

    Science.gov (United States)

    Hrebien, Sarah; O’Leary, Ben; Beaney, Matthew; Schiavon, Gaia; Fribbens, Charlotte; Bhambra, Amarjit; Johnson, Richard; Turner, Nicholas

    2016-01-01

    Circulating tumor DNA (ctDNA) analysis has the potential to allow non-invasive analysis of tumor mutations in advanced cancer. In this study we assessed the reproducibility of digital PCR (dPCR) assays of circulating tumor DNA in a cohort of patients with advanced breast cancer and assessed delayed plasma processing using cell free DNA preservative tubes. We recruited a cohort of 96 paired samples from 71 women with advanced breast cancer who had paired blood samples processed either immediately or delayed in preservative tubes with processing 48–72 hours after collection. Plasma DNA was analysed with multiplex digital PCR (mdPCR) assays for hotspot mutations in PIK3CA, ESR1 and ERBB2, and for AKT1 E17K. There was 94.8% (91/96) agreement in mutation calling between immediate and delayed processed tubes, kappa 0.88 95% CI 0.77–0.98). Discordance in mutation calling resulted from low allele frequency and likely stochastic effects. In concordant samples there was high correlation in mutant copies per ml plasma (r2 = 0.98; pprocessed tubes, although overall quantification of total cell free plasma DNA had similar prognostic effects in immediate (HR 3.6) and delayed (HR 3.0) tubes. There was moderate agreement in changes in allele fraction between sequential samples in quantitative mutation tracking (r = 0.84, p = 0.0002). Delayed processing of samples using preservative tubes allows for centralized ctDNA digital PCR mutation screening in advanced breast cancer. The potential of preservative tubes in quantitative mutation tracking requires further research. PMID:27760227

  5. 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.

  6. Evaluation of Four Endogenous Reference Genes and Their Real-Time PCR Assays for Common Wheat Quantification in GMOs Detection

    Science.gov (United States)

    Huang, Huali; Cheng, Fang; Wang, Ruoan; Zhang, Dabing; Yang, Litao

    2013-01-01

    Proper selection of endogenous reference genes and their real-time PCR assays is quite important in genetically modified organisms (GMOs) detection. To find a suitable endogenous reference gene and its real-time PCR assay for common wheat (Triticum aestivum L.) DNA content or copy number quantification, four previously reported wheat endogenous reference genes and their real-time PCR assays were comprehensively evaluated for the target gene sequence variation and their real-time PCR performance among 37 common wheat lines. Three SNPs were observed in the PKABA1 and ALMT1 genes, and these SNPs significantly decreased the efficiency of real-time PCR amplification. GeNorm analysis of the real-time PCR performance of each gene among common wheat lines showed that the Waxy-D1 assay had the lowest M values with the best stability among all tested lines. All results indicated that the Waxy-D1 gene and its real-time PCR assay were most suitable to be used as an endogenous reference gene for common wheat DNA content quantification. The validated Waxy-D1 gene assay will be useful in establishing accurate and creditable qualitative and quantitative PCR analysis of GM wheat. PMID:24098735

  7. Evaluation of four endogenous reference genes and their real-time PCR assays for common wheat quantification in GMOs detection.

    Directory of Open Access Journals (Sweden)

    Huali Huang

    Full Text Available Proper selection of endogenous reference genes and their real-time PCR assays is quite important in genetically modified organisms (GMOs detection. To find a suitable endogenous reference gene and its real-time PCR assay for common wheat (Triticum aestivum L. DNA content or copy number quantification, four previously reported wheat endogenous reference genes and their real-time PCR assays were comprehensively evaluated for the target gene sequence variation and their real-time PCR performance among 37 common wheat lines. Three SNPs were observed in the PKABA1 and ALMT1 genes, and these SNPs significantly decreased the efficiency of real-time PCR amplification. GeNorm analysis of the real-time PCR performance of each gene among common wheat lines showed that the Waxy-D1 assay had the lowest M values with the best stability among all tested lines. All results indicated that the Waxy-D1 gene and its real-time PCR assay were most suitable to be used as an endogenous reference gene for common wheat DNA content quantification. The validated Waxy-D1 gene assay will be useful in establishing accurate and creditable qualitative and quantitative PCR analysis of GM wheat.

  8. Evaluation of four endogenous reference genes and their real-time PCR assays for common wheat quantification in GMOs detection.

    Science.gov (United States)

    Huang, Huali; Cheng, Fang; Wang, Ruoan; Zhang, Dabing; Yang, Litao

    2013-01-01

    Proper selection of endogenous reference genes and their real-time PCR assays is quite important in genetically modified organisms (GMOs) detection. To find a suitable endogenous reference gene and its real-time PCR assay for common wheat (Triticum aestivum L.) DNA content or copy number quantification, four previously reported wheat endogenous reference genes and their real-time PCR assays were comprehensively evaluated for the target gene sequence variation and their real-time PCR performance among 37 common wheat lines. Three SNPs were observed in the PKABA1 and ALMT1 genes, and these SNPs significantly decreased the efficiency of real-time PCR amplification. GeNorm analysis of the real-time PCR performance of each gene among common wheat lines showed that the Waxy-D1 assay had the lowest M values with the best stability among all tested lines. All results indicated that the Waxy-D1 gene and its real-time PCR assay were most suitable to be used as an endogenous reference gene for common wheat DNA content quantification. The validated Waxy-D1 gene assay will be useful in establishing accurate and creditable qualitative and quantitative PCR analysis of GM wheat.

  9. Sensitive Detection of Giardia Cysts by Polymerase Chain Reaction (PCR

    Directory of Open Access Journals (Sweden)

    M Nikaeen

    2003-07-01

    Full Text Available Giardia is one of the most common human parasites and causes a lengthly course of nonbacterial diarrhea. Disease outbreaks due to Giardia infection are often attributed to contaminated water supplies. A major problem associated with detection for this organism is the lack of sensitive and reliable methods. PCR has the potential to address many of the limitations.We have performed a PCR-based method for sensitive detection of Giardia cysts. Because the sensitivity of PCR is a function of the efficiency of DNA extraction from cysts, we have also compared some different methods for DNA extraction from the cysts. Giardia cysts were collected from infected human, partially purified and serially diluted samples were prepared. DNA was extracted by 3 different methods and we found that simple repeated freezing and thawing was the best method for extraction of DNA from the cysts. A 163 bp conserved fragment related to the giardial heat shock protein (HSP70 gene was used as the target for PCR amplification. We were able to detect as few as 5 cysts in the samples. The results suggest the potential utilities of PCR for sensitive detection of Giardia in water sources.

  10. Evaluation of a novel real-time fluorescent polymerase chain reaction assay for high-risk human papilloma virus DNA genotypes in cytological cervical screening

    OpenAIRE

    Cheng, Jiaoying; BIAN, MEILU; CONG, XIAO; SUN, AIPING; Li, Min; Ma, Li; Chen, Ying; Liu,Jun

    2012-01-01

    It has been confirmed that detection of high-risk human papillomavirus (HR HPV) DNA is useful in cervical cancer (CC) screening. Recently, a new real-time fluorescent polymerase chain reaction (PCR) assay was developed to detect HR HPV. This assay can synchronize nucleic acid amplification and testing using specific primers for 13 types of HR HPV genomes, combined with specific TaqMan fluorescent marker probe techniques through the fluorescence automatic PCR instrument. Furthermore, it uses T...

  11. Utility of a single nasal polymerase chain reaction assay in predicting absence of skin and environmental contamination in hospitalized patients with past methicillin-resistant Staphylococcus aureus.

    Science.gov (United States)

    Guerrero, Dubert M; Wagner, Matthew; Carson, Grace; Hanish, Christine; Thompson, Jody; Orr, Megan; Roth, Felix; Carson, Paul J

    2016-06-01

    We evaluated hospitalized patients with a history of methicillin-resistant Staphylococcus aureus (MRSA) for persistent colonization and need for contact precautions. Up to 3 daily cultures of nares, skin, and any present wounds were compared with a single nasal polymerase chain reaction (PCR) assay. Most patients (76.2%) were no longer colonized with MRSA. A single PCR assay was sufficient to exclude persistent colonization and environmental contamination and remove the contact precautions.

  12. Allele Specific Locked Nucleic Acid Quantitative PCR (ASLNAqPCR): An Accurate and Cost-Effective Assay to Diagnose and Quantify KRAS and BRAF Mutation

    Science.gov (United States)

    Morandi, Luca; de Biase, Dario; Visani, Michela; Cesari, Valentina; De Maglio, Giovanna; Pizzolitto, Stefano; Pession, Annalisa; Tallini, Giovanni

    2012-01-01

    The use of tyrosine kinase inhibitors (TKIs) requires the testing for hot spot mutations of the molecular effectors downstream the membrane-bound tyrosine kinases since their wild type status is expected for response to TKI therapy. We report a novel assay that we have called Allele Specific Locked Nucleic Acid quantitative PCR (ASLNAqPCR). The assay uses LNA-modified allele specific primers and LNA-modified beacon probes to increase sensitivity, specificity and to accurately quantify mutations. We designed primers specific for codon 12/13 KRAS mutations and BRAF V600E, and validated the assay with 300 routine samples from a variety of sources, including cytology specimens. All were analyzed by ASLNAqPCR and Sanger sequencing. Discordant cases were pyrosequenced. ASLNAqPCR correctly identified BRAF and KRAS mutations in all discordant cases and all had a mutated/wild type DNA ratio below the analytical sensitivity of the Sanger method. ASLNAqPCR was 100% specific with greater accuracy, positive and negative predictive values compared with Sanger sequencing. The analytical sensitivity of ASLNAqPCR is 0.1%, allowing quantification of mutated DNA in small neoplastic cell clones. ASLNAqPCR can be performed in any laboratory with real-time PCR equipment, is very cost-effective and can easily be adapted to detect hot spot mutations in other oncogenes. PMID:22558339

  13. Comparison of agar gel immunodiffusion test, enzyme-linked immunosorbent assay and PCR in diagnostics of enzootic bovine leukosis

    Directory of Open Access Journals (Sweden)

    Malovrh Tadej

    2005-01-01

    Full Text Available Bovine leukaemia virus (BLV is a retrovirus that induces a chronic infection in cattle. Once infected, cattle remain virus carriers for life and start to show an antibody response within a few weeks after infection. Eradication and control of the disease are based on early diagnostics and segregation of the carriers. The choice of a diagnostic method depends on the eradication programme, money resources and characteristics of the herd to be analysed. The agar gel immunodiffusion (AGID test has been the serological test of choice for routine diagnosis of serum samples. Nevertheless, in more recent years, the enzyme-linked immunosorbent assay (ELISA has replaced the AGID for large scale testing. For this purpose, commercially available BLV-ELISA kits were compared to the AGID and to the polymerase chain reaction (PCR method performed with two sets of primers, amplifying env region. The ELISA kit based on the p24 core protein was found to be less specific and served as a screening test. The ELISA kit based on the envelope glycoprotein (gpSI served as a verification test and gave a good correlation with the AGID test and PCR method. However, ELISA showed a higher sensitivity than AGID. The p24 based ELiSA was useful for screening a large number of samples, whereas gp51 based ELISA, AGID and PCR were more important for detecting the antibody response against the individual BLV-proteins and therefore for verification of the infection with BLV.

  14. Development of a real-time PCR assay for Penicillium expansum quantification and patulin estimation in apples.

    Science.gov (United States)

    Tannous, Joanna; Atoui, Ali; El Khoury, André; Kantar, Sally; Chdid, Nader; Oswald, Isabelle P; Puel, Olivier; Lteif, Roger

    2015-09-01

    Due to the occurrence and spread of the fungal contaminants in food and the difficulties to remove their resulting mycotoxins, rapid and accurate methods are needed for early detection of these mycotoxigenic fungi. The polymerase chain reaction and the real time PCR have been widely used for this purpose. Apples are suitable substrates for fungal colonization mostly caused by Penicillium expansum, which produces the mycotoxin patulin during fruit infection. This study describes the development of a real-time PCR assay incorporating an internal amplification control (IAC) to specifically detect and quantify P. expansum. A specific primer pair was designed from the patF gene, involved in patulin biosynthesis. The selected primer set showed a high specificity for P. expansum and was successfully employed in a standardized real-time PCR for the direct quantification of this fungus in apples. Using the developed system, twenty eight apples were analyzed for their DNA content. Apples were also analyzed for patulin content by HPLC. Interestingly, a positive correlation (R(2) = 0.701) was found between P. expansum DNA content and patulin concentration. This work offers an alternative to conventional methods of patulin quantification and mycological detection of P. expansum and could be very useful for the screening of patulin in fruits through the application of industrial quality control.

  15. Detection of infectious bursal disease virus in various lymphoid tissues of experimentally infected specific pathogen free chickens by different reverse transcription polymerase chain reaction assays

    DEFF Research Database (Denmark)

    Kabell, Susanne; Handberg, Kurt; Kusk, Mette;

    2005-01-01

    transcription polymerase chain reaction (RT-PCR) assays, including two recently developed strain-specific assays, were employed for detection of ribonucleic acid (RNA) from three different IBDV strains in bursa tissue samples from experimentally infected specific pathogen free chickens. The virus strains...

  16. Development and use of a real-time polymerase chain reaction assay for the detection of Ophidiomyces ophiodiicola in snakes.

    Science.gov (United States)

    Allender, Matthew C; Bunick, David; Dzhaman, Elena; Burrus, Lucienne; Maddox, Carol

    2015-03-01

    Fungal pathogens threatening the conservation of wildlife are becoming increasingly common. Since 2008, free-ranging snakes across North America have been experiencing a marked increase in the prevalence of snake fungal disease associated with Ophidiomyces ophiodiicola. Diagnosis has historically relied on histology, microbiology, and conventional polymerase chain reaction (PCR). More sensitive methods are needed to adequately characterize the epidemiology. The current study describes the development of a real-time PCR (qPCR) assay for detecting a segment of the internal transcribed spacer 1 region between the 18S and 5.8S ribosomal RNA gene. The assay was able to detect as few as 1.05 × 10(1) gene copies per reaction. An additional 4 positive cases were detected when comparing a conventional PCR (n = 3) and the qPCR (n = 7) when used on swab samples from 47 eastern massasauga rattlesnakes. The newly developed assay is a sensitive and specific tool for surveillance and monitoring in the conservation of free-ranging snakes.

  17. Sensitivity of PCR assays for murine gammaretroviruses and mouse contamination in human blood samples.

    Directory of Open Access Journals (Sweden)

    Li Ling Lee

    Full Text Available Gammaretroviruses related to murine leukemia virus (MLV have variously been reported to be present or absent in blood from chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME patients and healthy controls. Using subjects from New York State, we have investigated by PCR methods whether MLV-related sequences can be identified in nucleic acids isolated from whole blood or from peripheral blood mononuclear cells (PBMCs or following PBMC culture. We have also passaged the prostate cancer cell line LNCaP following incubation with plasma from patients and controls and assayed nucleic acids for viral sequences. We have used 15 sets of primers that can effectively amplify conserved regions of murine endogenous and exogenous retrovirus sequences. We demonstrate that our PCR assays for MLV-related gag sequences and for mouse DNA contamination are extremely sensitive. While we have identified MLV-like gag sequences following PCR on human DNA preparations, we are unable to conclude that these sequences originated in the blood samples.

  18. Novel Luminex Assay for Telomere Repeat Mass Does Not Show Well Position Effects Like qPCR.

    Directory of Open Access Journals (Sweden)

    Muhammad G Kibriya

    Full Text Available Telomere length is a potential biomarker of aging and risk for age-related diseases. For measurement of relative telomere repeat mass (TRM, qPCR is typically used primarily due to its low cost and low DNA input. But the position of the sample on a plate often impacts the qPCR-based TRM measurement. Recently we developed a novel, probe-based Luminex assay for TRM that requires ~50ng DNA and involves no DNA amplification. Here we report, for the first time, a comparison among TRM measurements obtained from (a two singleplex qPCR assays (using two different primer sets, (b a multiplex qPCR assay, and (c our novel Luminex assay. Our comparison is focused on characterizing the effects of sample positioning on TRM measurement. For qPCR, DNA samples from two individuals (K and F were placed in 48 wells of a 96-well plate. For each singleplex qPCR assay, we used two plates (one for Telomere and one for Reference gene. For the multiplex qPCR and the Luminex assay, the telomere and the reference genes were assayed from the same well. The coefficient of variation (CV of the TRM for Luminex (7.2 to 8.4% was consistently lower than singleplex qPCR (11.4 to 14.9% and multiplex qPCR (19.7 to 24.3%. In all three qPCR assays the DNA samples in the left- and right-most columns showed significantly lower TRM than the samples towards the center, which was not the case for the Luminex assay (p = 0.83. For singleplex qPCR, 30.5% of the variation in TL was explained by column-to-column variation and 0.82 to 27.9% was explained by sample-to-sample variation. In contrast, only 5.8% of the variation in TRM for the Luminex assay was explained by column-to column variation and 50.4% was explained by sample-to-sample variation. Our novel Luminex assay for TRM had good precision and did not show the well position effects of the sample that were seen in all three of the qPCR assays that were tested.

  19. A locked nucleic acid (LNA-based real-time PCR assay for the rapid detection of multiple bacterial antibiotic resistance genes directly from positive blood culture.

    Directory of Open Access Journals (Sweden)

    Lingxiang Zhu

    Full Text Available Bacterial strains resistant to various antibiotic drugs are frequently encountered in clinical infections, and the rapid identification of drug-resistant strains is highly essential for clinical treatment. We developed a locked nucleic acid (LNA-based quantitative real-time PCR (LNA-qPCR method for the rapid detection of 13 antibiotic resistance genes and successfully used it to distinguish drug-resistant bacterial strains from positive blood culture samples. A sequence-specific primer-probe set was designed, and the specificity of the assays was assessed using 27 ATCC bacterial strains and 77 negative blood culture samples. No cross-reaction was identified among bacterial strains and in negative samples, indicating 100% specificity. The sensitivity of the assays was determined by spiking each bacterial strain into negative blood samples, and the detection limit was 1-10 colony forming units (CFU per reaction. The LNA-qPCR assays were first applied to 72 clinical bacterial isolates for the identification of known drug resistance genes, and the results were verified by the direct sequencing of PCR products. Finally, the LNA-qPCR assays were used for the detection in 47 positive blood culture samples, 19 of which (40.4% were positive for antibiotic resistance genes, showing 91.5% consistency with phenotypic susceptibility results. In conclusion, LNA-qPCR is a reliable method for the rapid detection of bacterial antibiotic resistance genes and can be used as a supplement to phenotypic susceptibility testing for the early detection of antimicrobial resistance to allow the selection of appropriate antimicrobial treatment and to prevent the spread of resistant isolates.

  20. A Pan-Lyssavirus Taqman Real-Time RT-PCR Assay for the Detection of Highly Variable Rabies virus and Other Lyssaviruses.

    Directory of Open Access Journals (Sweden)

    Ashutosh Wadhwa

    2017-01-01

    Full Text Available Rabies, resulting from infection by Rabies virus (RABV and related lyssaviruses, is one of the most deadly zoonotic diseases and is responsible for up to 70,000 estimated human deaths worldwide each year. Rapid and accurate laboratory diagnosis of rabies is essential for timely administration of post-exposure prophylaxis in humans and control of the disease in animals. Currently, only the direct fluorescent antibody (DFA test is recommended for routine rabies diagnosis. Reverse-transcription polymerase chain reaction (RT-PCR based diagnostic methods have been widely adapted for the diagnosis of other viral pathogens, but there is currently no widely accepted rapid real-time RT-PCR assay for the detection of all lyssaviruses. In this study, we demonstrate the validation of a newly developed multiplex real-time RT-PCR assay named LN34, which uses a combination of degenerate primers and probes along with probe modifications to achieve superior coverage of the Lyssavirus genus while maintaining sensitivity and specificity. The primers and probes of the LN34 assay target the highly conserved non-coding leader region and part of the nucleoprotein (N coding sequence of the Lyssavirus genome to maintain assay robustness. The probes were further modified by locked nucleotides to increase their melting temperature to meet the requirements for an optimal real-time RT-PCR assay. The LN34 assay was able to detect all RABV variants and other lyssaviruses in a validation panel that included representative RABV isolates from most regions of the world as well as representatives of 13 additional Lyssavirus species. The LN34 assay was successfully used for both ante-mortem and post-mortem diagnosis of over 200 clinical samples as well as field derived surveillance samples. This assay represents a major improvement over previously published rabies specific RT-PCR and real-time RT-PCR assays because of its ability to universally detect RABV and other lyssaviruses

  1. Utility of Pooled HIV RNA RT-PCR Assay in Diagnosing Acute HIV Infections

    Institute of Scientific and Technical Information of China (English)

    张麒; 蒋岩; 刘全忠

    2004-01-01

    Abstract: The P24 antigen test, HIV RNA PCR test,HIV isolation/culture and fourth-generation HIV uniform Ag/Ab assay are being utilized in diagnosing acute HIV infection in different labs. Many factors limit the use of screening for acute HIV in high-risk populations, in blood donors and during voluntary HIV testing, including, cost, technique, sensitivity and specificity. In this review we explore a new NAAT method which involves HIV RNA RT-PCR on pooled samples. This technique is able to screen for acute infections in a large testing volume and may he used as a screening method in high-risk populations and blood donors.

  2. Detection of Legionella by quantitative-polymerase chain reaction (qPCR) for monitoring and risk assessment

    DEFF Research Database (Denmark)

    Krøjgaard, Louise H.; Krogfelt, Karen A.; Albrechtsen, Hans-Jorgen

    2011-01-01

    Background: Culture and quantitative polymerase chain reaction (qPCR) assays for the detection of Legionella were compared on samples from a residential area before and after two interventions. A total of 84 samples were collected from shower hoses and taps as first flush samples and at constant...... temperature. Samples were grouped according to the origin of the sample, a) circulation water b) water from empty apartments c) water from shower hoses. The aims were to investigate the usefulness of qPCR compared to culture for monitoring remedial actions for elimination of Legionella bacteria and as a tool...... for risk assessment. Results: In water collected from the apartments Legionella spp were detected by qPCR in the concentration range from LOQ to 9.6* 10(5)GU/L while L. pneumophila were detected in a range from LOQ to 6.8*10(5) GU/L. By culturing, the legionellae were detected in the range from below...

  3. Avian haemosporidian parasites (Haemosporida): A comparative analysis of different polymerase chain reaction assays in detection of mixed infections.

    Science.gov (United States)

    Bernotienė, Rasa; Palinauskas, Vaidas; Iezhova, Tatjana; Murauskaitė, Dovilė; Valkiūnas, Gediminas

    2016-04-01

    Mixed infections of different species and genetic lineages of haemosporidian parasites (Haemosporida) predominate in wildlife, and such infections are particularly virulent. However, currently used polymerase chain reaction (PCR)-based detection methods often do not read mixed infections. Sensitivity of different PCR assays in detection of mixed infections has been insufficiently tested, but this knowledge is essential in studies addressing parasite diversity in wildlife. Here, we applied five different PCR assays, which are broadly used in wildlife avian haemosporidian research, and compared their sensitivity in detection of experimentally designed mixed infections of Haemoproteus and Plasmodium parasites. Three of these PCR assays use primer sets that amplify fragments of cytochrome b gene (cyt b), one of cytochrome oxidase subunit I (COI) gene, and one target apicoplast genome. We collected blood from wild-caught birds and, using microscopic and PCR-based methods applied in parallel, identified single infections of ten haemosporidian species with similar parasitemia. Then, we prepared 15 experimental mixes of different haemosporidian parasites, which often are present simultaneously in wild birds. Similar concentration of total DNA was used in each parasite lineage during preparation of mixes. Positive amplifications were sequenced, and the presence of mixed infections was reported by visualising double-base calling in sequence electropherograms. This study shows that the use of each single PCR assay markedly underestimates biodiversity of haemosporidian parasites. The application of at least 3 PCR assays in parallel detected the majority, but still not all lineages present in mixed infections. We determined preferences of different primers in detection of parasites belonging to different genera of haemosporidians during mixed infections.

  4. Development of a new ultra sensitive real-time PCR assay (ultra sensitive RTQ-PCR for the quantification of HBV-DNA

    Directory of Open Access Journals (Sweden)

    Varaklioti Agoritsa

    2010-03-01

    Full Text Available Abstract Background Improved sensitivity of HBV-DNA tests is of critical importance for the management of HBV infection. Our aim was to develop and assess a new ultra sensitive in-house real-time PCR assay for HBV-DNA quantification (ultra sensitive RTQ-PCR. Results Previously used HBV-DNA standards were calibrated against the WHO 1st International Standard for HBV-DNA (OptiQuant® HBV-DNA Quantification Panel, Accrometrix Europe B.V.. The 95% and 50% HBV-DNA detection end-point of the assay were 22.2 and 8.4 IU/mL. According to the calibration results, 1 IU/mL equals 2.8 copies/mL. Importantly the clinical performance of the ultra sensitive real-time PCR was tested similar (67% to the Procleix Ultrio discriminatory HBV test (dHBV (70% in low-titer samples from patients with occult Hepatitis B. Finally, in the comparison of ultra sensitive RTQ-PCR with the commercially available COBAS TaqMan HBV Test, the in-house assay identified 94.7% of the 94 specimens as positive versus 90.4% identified by TaqMan, while the quantitative results that were positive by both assay were strongly correlated (r = 0.979. Conclusions We report a new ultra sensitive real time PCR molecular beacon based assay with remarkable analytical and clinical sensitivity, calibrated against the WHO 1st International standard.

  5. Comparison of three different commercial PCR assays for the detection of pathogens in critically ill sepsis patients.

    Science.gov (United States)

    Schreiber, J; Nierhaus, A; Braune, S A; de Heer, G; Kluge, S

    2013-05-01

    The high mortality rate associated with sepsis necessitates a timely identification of the causative organism in order to optimize antimicrobial therapy. PCR assays are increasingly being used for this purpose. The aim of this study was to compare three commercially available PCR systems for the diagnosis of systemic infections. In a prospective observational study, a broad-range (SepsiTest®; Molzym, Bremen, Germany) and two multiplex PCR assays (VYOO®; SIRS-Lab, Jena, Germany and LightCycler® SeptiFast; Roche, Mannheim, Germany) were compared to blood cultures with respect to the clinical course of 50 critically ill patients with sepsis, severe sepsis or septic shock. Pathogens were detected by PCR in 12 % (SepsiTest®), 10 % (VYOO®) and 14 % (LightCycler® SeptiFast) of samples and in 26 % by blood culture. Negative results were obtained using all four methods in 32 samples (64 %) and 3 (6 %) samples were positive in all tests. Upon consideration of additional diagnostic findings and the clinical course, eight (16 %) of the positive blood culture results were deemed clinically relevant. All three PCR assays could also identify the causative organism (or a specific gene thereof) in three of these eight positive blood cultures, whereas for five of the eight, all three PCR assays were negative. In one patient with a negative blood culture, the SepsiTest®, VYOO® and LightCycler® SeptiFast assays were positive for Streptococcus species. The PCR assays appeared to be less susceptible than blood cultures to false-positive results arising from contamination with coagulase-negative staphylococcal organisms. There was some variability between the three PCR assays tested and the corresponding blood cultures with regards to the type of pathogen detected. The three PCR assays appeared to be less susceptible to false-positive results than blood cultures.

  6. Real-Time PCR Assay Using Fine-Needle Aspirates and Tissue Biopsy Specimens for Rapid Diagnosis of Mycobacterial Lymphadenitis in Children

    Science.gov (United States)

    van Coppenraet, E. S. Bruijnesteijn; Lindeboom, J. A.; Prins, J. M.; Peeters, M. F.; Claas, E. C. J.; Kuijper, E. J.

    2004-01-01

    A real-time PCR assay was developed to diagnose and identify the causative agents of suspected mycobacterial lymphadenitis. Primers and probes for the real-time PCR were designed on the basis of the internal transcribed spacer sequence, enabling the recognition of the genus Mycobacterium and the species Mycobacterium avium and M. tuberculosis. The detection limit for the assay was established at 1,100 CFU/ml of pus, and the specificity tests showed no false-positive reaction with other mycobacterial species and other pathogens causing lymphadenitis. From 67 children with suspected mycobacterial lymphadenitis based on a positive mycobacterial skin test, 102 samples (58 fine-needle aspirates [FNA] and 44 tissue specimens) were obtained. The real-time PCR assay detected a mycobacterial infection in 48 patients (71.6%), whereas auramine staining and culturing were positive for 31 (46.3%) and 28 (41.8%) of the patients. The addition of the real-time PCR assay to conventional diagnostic tests resulted in the recognition of 13 more patients with mycobacterial disease. These results indicate that the real-time PCR is more sensitive than conventional staining and culturing techniques (P = 0.006). The M. avium-specific real-time PCR was positive for 38 patients, and the M. tuberculosis-specific real-time PCR was positive for 1 patient. Analysis of 27 patients from whom FNA and tissue biopsy specimens were collected revealed significantly more positive real-time PCR results for FNA than for tissue biopsy specimens (P = 0.003). Samples from an age-matched control group of 50 patients with PCR-proven cat scratch disease were all found to be negative by the real-time PCR. We conclude that this real-time PCR assay with a sensitivity of 72% for patients with lymphadenitis and a specificity of 100% for the detection of atypical mycobacteria can provide excellent support for clinical decision making in children with lymphadenitis. PMID:15184446

  7. A type-specific nested PCR assay established and applied for investigation of HBV genotype and subgenotype in Chinese patients with chronic HBV infection

    OpenAIRE

    Nie Jing-Jing; Sun Kui-Xia; Li Jie; Wang Jie; Jin Hui; Wang Ling; Lu Feng-Min; Li Tong; Yan Ling; Yang Jing-Xian; Sun Mi-Shu; Zhuang Hui

    2012-01-01

    Abstract Background Many studies have suggested that hepatitis B virus (HBV) genotypes show not only geographical distribution and race specificity, but also are associated with disease progression and response to interferon treatment. The objective of this study was to develop a nested polymerase chain reaction (nPCR) assay for genotypes A-D and subgenotypes B1, B2, C1 and C2 of hepatitis B virus (HBV) and to investigate the distribution characteristics of HBV genotypes/subgenotype in China....

  8. Multiplexed, rapid detection of H5N1 using a PCR-free nanoparticle-based genomic microarray assay

    Directory of Open Access Journals (Sweden)

    Ragupathy Viswanath

    2010-10-01

    Full Text Available Abstract Background For more than a decade there has been increasing interest in the use of nanotechnology and microarray platforms for diagnostic applications. In this report, we describe a rapid and simple gold nanoparticle (NP-based genomic microarray assay for specific identification of avian influenza virus H5N1 and its discrimination from other major influenza A virus strains (H1N1, H3N2. Results Capture and intermediate oligonucleotides were designed based on the consensus sequences of the matrix (M gene of H1N1, H3N2 and H5N1 viruses, and sequences specific for the hemaglutinin (HA and neuraminidase (NA genes of the H5N1 virus. Viral RNA was detected within 2.5 hours using capture-target-intermediate oligonucleotide hybridization and gold NP-mediated silver staining in the absence of RNA fragmentation, target amplification, and enzymatic reactions. The lower limit of detection (LOD of the assay was less than 100 fM for purified PCR fragments and 103 TCID50 units for H5N1 viral RNA. Conclusions The NP-based microarray assay was able to detect and distinguish H5N1 sequences from those of major influenza A viruses (H1N1, H3N2. The new method described here may be useful for simultaneous detection and subtyping of major influenza A viruses.

  9. A survey of polymerase chain reaction (PCR) amplification studies of unicellular protists using single-cell PCR.

    Science.gov (United States)

    Lynn, Denis H; Pinheiro, Marcel

    2009-01-01

    We surveyed a variety of studies that have used single-cell polymerase chain reaction (SC-PCR) to examine the gene sequences of a diversity of unicellular protists. Representatives of all the Super-Groups of eukaryotes have been subjected to SC-PCR with ciliates and dinoflagellates being most commonly examined. The SC-PCR was carried out either by directly amplifying a single lysed cell or by first extracting DNA and following this with amplification of the DNA extract. Cell lysis methods included heating, freezing, mechanical rupture, and enzyme digestion. Cells fixed or preserved with ethanol, methanol, and Lugol's have also been used successfully. Heminested or seminested PCR might follow the initial PCR, whose products were then directly sequenced or cloned and then sequenced. The methods are not complicated. This should encourage protistologists to use SC-PCR in the description of new or revised taxa, especially rare and unculturable forms, and it should also enable the probing of gene expression in relation to life history stages.

  10. Detection and characterization of verocytotoxin-producing Escherichia coli by automated 5 ' nuclease PCR assay

    DEFF Research Database (Denmark)

    Nielsen, Eva Møller; Andersen, Marianne Thorup

    2003-01-01

    In recent years increased attention has been focused on infections caused by isolates of verocytotoxin-producing Escherichia coli (VTEC) serotypes other than O157. These non-O157 VTEC isolates are commonly present in food and food production animals. Easy detection, isolation, and characterization...... and enteropathogenic E. coli strains) and VTEC strains isolated from calves were tested to validate the PCR assays. The expected virulence profiles were detected for all reference strains. In addition, new information on the subtypes of LEE genes was obtained. For reference strains as well as bovine isolates...

  11. 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.

  12. A novel integrated strategy for detection of human bocavirus based on a heminested PCR assay combined with boiling lysis method of samples in human specimens.

    Science.gov (United States)

    Chen, Long; Yao, Qing; Ma, Jing; Li, Jianning; Zhang, Qian; Yang, Yi; Li, Fang; Sun, Yuning

    2014-07-01

    Human bocavirus (HBoV) has been shown to be associated with acute respiratory tract infection in children. The aim of the work was to develop a novel integrated strategy for human bocavirus detection: heminested PCR assay combined with boiling lysis method of samples. The detection limit of the heminested PCR assay was 1.2 copies of a recombinant DNA plasmid, and no cross-reaction with other respiratory viruses or bacteria was observed. By using the integrated strategy, a total of 202 secretions of the lower respiratory tract of children with acute respiratory diseases were collected and tested. The samples were treated and lysed in boiling lysis buffer rather than extracting viral DNA from secretions, then these sample lysates could be templates and tested by heminested PCR assay, and the amplification of HBoV DNA was detected by using agarose gel electrophoresis. The results showed that, only 7 samples were found to be positive by conventional single-round PCR; importantly, the other new 41 samples were positive by heminested PCR assay. Additionally, the genomic viral DNA was extracted from all positive and some negative specimens, amplified, and sequenced. The results were perfectly consistent with those of the integrated strategy. Taken together, these results suggest that the novel integrated strategy (heminested PCR assay combined with boiling lysis method of samples) is a convenient, sensitive, cost-effective and reliable detective method for HBoV detection and will have broad application prospects in clinical diagnosis. Copyright © 2014 Elsevier B.V. All rights reserved.

  13. A Systematic Investigation of Parameters Influencing Droplet Rain in the Listeria monocytogenes prfA Assay - Reduction of Ambiguous Results in ddPCR

    Science.gov (United States)

    Witte, Anna Kristina; Mester, Patrick; Fister, Susanne; Witte, Matthias; Schoder, Dagmar; Rossmanith, Peter

    2016-01-01

    The droplet digital polymerase chain reaction (ddPCR) determines DNA amounts based upon the pattern of positive and negative droplets, according to Poisson distribution, without the use of external standards. However, division into positive and negative droplets is often not clear because a part of the droplets has intermediate fluorescence values, appearing as “rain” in the plot. Despite the droplet rain, absolute quantification with ddPCR is possible, as shown previously for the prfA assay in quantifying Listeria monocytogenes. Nevertheless, reducing the rain, and thus ambiguous results, promotes the accuracy and credibility of ddPCR. In this study, we extensively investigated chemical and physical parameters for optimizing the prfA assay for ddPCR. While differences in the concentration of all chemicals and the dye, quencher and supplier of the probe did not alter the droplet pattern, changes in the PCR cycling program, such as prolonged times and increased cycle numbers, improved the assay. PMID:27992475

  14. Design and analysis of Q-RT-PCR assays for haematological malignancies using mixed effects models

    DEFF Research Database (Denmark)

    Bøgsted, Martin; Mandrup, Charlotte; Petersen, Anders;

    research use and needs qualit control for accuracy and precision. Especially the identification of experimental variations and statistical analysis has recently created discussions. The standard analytical technique is to use the Delta-Delta-Ct method. Although this method accounts for sample specific...... variations such as RNA purification, it does not account for other experimental effects as variations in cDNA synthesis, amplification efficiency and assay variations. To obtain an assessment of the accuracy and precision of the assays a novel approach for the statistical analysis of Q-RT-PCR has been...... developed based on a linear mixed effects model for factorial designs. The model consists of an analysis of variance where the variation of each fixed effect of interest and identified experimental and biological nuisance variations are split. Hereby it accounts for varying efficiency, inhomogeneous...

  15. Real-Time PCR Assay for the Identification of the Brown Marmorated Stink Bug (Halyomorpha halys

    Directory of Open Access Journals (Sweden)

    Manpreet K Dhami

    2016-02-01

    Full Text Available The brown marmorated stink bug, Halyomorpha halys (Hemiptera: Pentatomidae, is a gregarious crop pest that has rapidly spread across the world in the last two decades. It is an excellent hitchhiker species, especially as an over-wintering adult. During this period it is often associated with non-biological commodities such as shipping containers and machinery that travel long distances. Inadequate identification keys and similarity to common species has assisted its spread across Europe, while accurate identification from immature stages or eggs is not possible. We developed a real-time TaqMan PCR assay for the accurate and sensitive detection of the brown marmorated stink bug from all life stages. The assay performance against required diagnostic criterion and within a quarantine framework are described.

  16. Ready to use dry-reagent PCR assays for the four common bacterial pathogens using switchable lanthanide luminescence probe system.

    Science.gov (United States)

    Lehmusvuori, A; Soikkeli, M; Tuunainen, E; Seppä, T; Spangar, A; Rantakokko-Jalava, K; von Lode, P; Karhunen, U; Soukka, T; Wittfooth, S

    2015-11-01

    Ready to use dry-reagent PCR assays for Escherichia coli, Staphylococcus aureus, Streptococcus pneumoniae, Pseudomonas spp. and for broad-range bacteria detection were developed. The assays were based on novel switchable lanthanide probes that provide sensitive target DNA detection with exceptionally high signal-to-background ratio, thus enabling clear discrimination between positive and negative results. For example, sensitivity of three S. aureus and two S. pneumonia bacteria (colony forming units) per PCR assay was measured with fluorescence signal more than 30 times over the background signal level. The rapid and easy-to-use assays are suitable for routine clinical diagnostics without molecular biology expertise and facilities.

  17. Development and laboratory evaluation of a real-time PCR assay for detecting viruses and bacteria of relevance for community-acquired pneumonia.

    Science.gov (United States)

    Edin, Alicia; Granholm, Susanne; Koskiniemi, Satu; Allard, Annika; Sjöstedt, Anders; Johansson, Anders

    2015-05-01

    Community-acquired pneumonia may present with similar clinical symptoms, regardless of viral or bacterial cause. Diagnostic assays are needed to rapidly discriminate between causes, because this will guide decisions on appropriate treatment. Therefore, a quantitative real-time PCR (qPCR) assay with duplex reactions targeting eight bacteria and six viruses was developed. Technical performance was examined with linear plasmids. Upper and lower respiratory tract specimens were used to compare the qPCR assay with standard microbiological methods. The limit of detection was 5 to 20 DNA template copies with approximately 1000-fold differences in concentrations of the two competing templates. SDs for positive controls were 95% for M. pneumoniae, Streptococcus pyogenes, respiratory syncytial virus, and influenza A virus; whereas it was only 56% for Haemophilus influenzae. Multiple microbial agents were identified in 19 of 44 sputum and 19 of 50 nasopharynx specimens. We conclude that in parallel qPCR detection of the targeted respiratory bacteria and viruses is feasible. The results indicate good technical performance of the assay in clinical specimens.

  18. Generation of HIV-1 and Internal Control Transcripts as Standards for an In-House Quantitative Competitive RT-PCR Assay to Determine HIV-1 Viral Load

    Directory of Open Access Journals (Sweden)

    Anny Armas Cayarga

    2011-01-01

    Full Text Available Human immunodeficiency virus type-1 (HIV-1 viral load is useful for monitoring disease progression in HIV-infected individuals. We generated RNA standards of HIV-1 and internal control (IC by in vitro transcription and evaluated its performance in a quantitative reverse transcription polymerase chain reaction (qRT-PCR assay. HIV-1 and IC standards were obtained at high RNA concentrations, without DNA contamination. When these transcripts were included as standards in a qRT-PCR assay, it was obtained a good accuracy (±0.5 log10 unit of the expected results in the quantification of the HIV-1 RNA international standard and controls. The lower limit detection achieved using these standards was 511.0 IU/mL. A high correlation (=0.925 was obtained between the in-house qRT-PCR assay and the NucliSens easyQ HIV-1 test (bioMerieux for HIV-1 RNA quantitation with clinical samples (=14. HIV-1 and IC RNA transcripts, generated in this study, proved to be useful as standards in an in-house qRT-PCR assay for determination of HIV-1 viral load.

  19. Development and evaluation of a SYBR Green real-time RT-PCR assay for evaluation of cytokine gene expression in horse.

    Science.gov (United States)

    Sánchez-Matamoros, A; Kukielka, D; De las Heras, A I; Sánchez-Vizcaíno, J M

    2013-01-01

    Cytokine secretion is one of the main mechanisms by which the immune system is regulated in response to pathogens. Therefore, the measurement of cytokine expression is fundamental to characterizing the immune response to infections. Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) is widely used to measure cytokine mRNA levels, but assay conditions should be properly evaluated before analyzing important equine infections through relative quantification of gene expression. The aim of this study was to develop and evaluate a set of RT-qPCR assays for a panel of the most common cytokines in horses involved in innate and adaptive immune responses. Eight cytokines (interleukin (IL)-1β, IL-2, IL-4, IL-10, IL-12, TNFα, IFNβ and IFNγ) and a housekeeping gene (β-actin) were detected and amplified with the same annealing temperature in a SYBR Green RT-qPCR assay of samples of mitogen-stimulated peripheral blood mononuclear cells from a healthy horse and whole blood from a horse infected with African horse sickness virus. The method gave good efficiency for all genes tested, allowing quantification of relative expression levels. These SYBR Green RT-qPCR assays may be useful for examining cytokine gene expression in horses in response to exposure to economically important pathogens.

  20. Generation of HIV-1 and Internal Control Transcripts as Standards for an In-House Quantitative Competitive RT-PCR Assay to Determine HIV-1 Viral Load

    Science.gov (United States)

    Armas Cayarga, Anny; Perea Hernández, Yenitse; González González, Yaimé J.; Dueñas Carrera, Santiago; González Pérez, Idania; Robaina Álvarez, René

    2011-01-01

    Human immunodeficiency virus type-1 (HIV-1) viral load is useful for monitoring disease progression in HIV-infected individuals. We generated RNA standards of HIV-1 and internal control (IC) by in vitro transcription and evaluated its performance in a quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay. HIV-1 and IC standards were obtained at high RNA concentrations, without DNA contamination. When these transcripts were included as standards in a qRT-PCR assay, it was obtained a good accuracy (±0.5 log10 unit of the expected results) in the quantification of the HIV-1 RNA international standard and controls. The lower limit detection achieved using these standards was 511.0 IU/mL. A high correlation (r = 0.925) was obtained between the in-house qRT-PCR assay and the NucliSens easyQ HIV-1 test (bioMerieux) for HIV-1 RNA quantitation with clinical samples (N = 14). HIV-1 and IC RNA transcripts, generated in this study, proved to be useful as standards in an in-house qRT-PCR assay for determination of HIV-1 viral load. PMID:21766036

  1. Diagnosis of visceral Leishmaniasis in asymptomatic dogs by the KDNA PCR-hybridization assay using noninvasive samples

    Energy Technology Data Exchange (ETDEWEB)

    Leite, Rodrigo Souza; Andrade, Antero Silva Ribeiro de [Centro de Desenvolvimento da Tecnologia Nuclear (CDTN/CNEN-MG), Belo Horizonte, MG (Brazil). Lab. de Radiobiologia], e-mail: rleite2005@gmail.com; Ferreira, Sydney de Almeida; Ituassu, Leonardo Trindade; Melo, Maria Norma de [Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG (Brazil). Centro de Ciencias Biologicas. Dept. de Parasitologia], e-mail: saninoalmeida@gmail.com

    2009-07-01

    The visceral leishmaniasis (VL) in Brazil is caused by Leishmania (Leishmania) chagasi and the asymptomatic dogs may transmit the parasite to sand flies vectors. The VL epidemiological control in Brazil involves the elimination of seropositive dogs, insecticide treatment and systematic treatment of human cases. Therefore, the accurate diagnosis is important in order to avoid the disease transmission or unnecessary culling of dogs. Serological tests are used for screening of dogs. However, these techniques present limitations. The Polymerase Chain Reaction (PCR) is an attractive alternative to the diagnosis in this context; but non-invasive samplings have great importance because they are simpler, painless and less resisted by dog-owners. This study aimed at evaluating conjunctival swab (CS) for canine VL diagnosis. In this methodology a sterile cotton swab is used to sampling the dog conjunctiva in both eyes. Thirty asymptomatic seropositive dogs were used. The samples were analyzed by the kDNA PCR-hybridization procedure in which the PCR products are hybridized with cloned kDNA mini-circles labeled with {sup 32}P[]dCTP. In addition, blood (B) was collected from each animal. L. chagasi was identified in 90% of CS samples and 13,6% of B samples. The high sensitivity obtained with asymptomatic dogs, in which the diagnosis is more difficult due the low number of parasites in the samples, allow concluding that the conjunctival swab associated to the kDNA PCR-hybridization assay provides a valuable alternative tool for the direct diagnosis of canine leishmaniasis. (author)

  2. In vitro adhesion assay of lactic acid bacteria, Escherichia coli and Salmonella sp. by microbiological and PCR methods

    Directory of Open Access Journals (Sweden)

    Didier Montet

    2006-03-01

    Full Text Available In vitro adhesion assay using Lactobacillus reuteri KUB-AC5 as a test strain has been studied by applying simple PCR reaction together with image analysis and plate count techniques. Critical factor affecting the PCR method was quality and quantity of DNA. The cell lysis technique was modified to optimize this method. Thus, lysozyme and proteinase K were added to lyse the cells, followed by SDS solution to obtain a complete cell lysis. Only PCR products from total cells (TC were obtained, with low consistency, but none from cells bound to mucus (BC at either 0.1 or 0.5 mg/mL concentration. It was hypothesized that the attached cells might not be extracted into the cell suspension. Therefore, 1% SDS solution and 0.1M NaOH were used directly in the extraction. As expected, PCR products were observed when both TC and BC were used as a DNA template. Adhesion appeared at a wide range of 0-45%, with low consistency. Therefore, a simple microbiological method (plate count was used. The extraction of bound cells into cell suspension was critical in this method. Extraction times of 20, 60, 120 and 150 min were tried. Results showed that maximum cell number was obtained with 120 min extraction. L. reuteri KUB-AC5, L. reuteri KUB-AC16, L. reuteri KUB-AC20, L. salivarius KUB-AC21, L. acidophilus KV-1, Escherichia coli E010, Salmonella sp. S003, E. coli ATCC8739, and S. typhimurium ATCC 13311 exhibited adhesion activity of 21.6%, 0.8%, 5.7%, 1.1%, 23.1%, 10.7%, 10.3%, 4.4% and 3.2%, respectively. Among the 9 types of microorganisms tested L. acidophilus KV-1 and L. reuteri KUB-AC5 showed higher adhesion activity than the others.

  3. an overview on the application of polymerase chain reaction (pcr)

    African Journals Online (AJOL)

    DR. AMINU

    Bayero Journal of Pure and Applied Sciences, 2(1): 109 - 114 ... Keywords: Polymerase chain reaction, Diagnosis, Bacteria, Infections .... A brain abscess is a localized pyogenic bacterial ... as encephalitis and skin rash. ... Streptococcus.

  4. Development of a polymerase chain reaction (PCR) test for the detection of virulent forms of Vibrio parahaemolyticus.

    Science.gov (United States)

    Kadhim, H M; Miah, A; Munn, C B; Gilpin, M L

    2012-04-01

    Vibrio parahaemolyticus is a marine bacterium and some strains cause gastroenteritis in humans. Clinical isolates are thought to possess virulence factors that are absent from the majority of environmental isolates. Use of randomly amplified polymorphic DNA (RAPD)-PCR produced a unique 600 bp amplicon (band Y) in the majority of clinical isolates and rarely in environmental isolates tested. The DNA from band Y was cloned and sequenced and found to code for an outer membrane protein (OMP). Two polymerase chain reaction (PCR) primers were designed to specifically amplify a 200 bp unique sequence from presumptive virulent strains (PCR-OMP). The virulence of 23 clinical and 32 environmental isolates was assessed in cytotoxicity tests by treatment of Caco-2 cells with extracellular products (ECPs). All but two of the clinical isolates (91%) were positive for the 200 bp PCR-OMP and their ECPs produced a significantly higher (p PCR-OMP is strongly correlated with virulence, as determined by the cytotoxicity assay, and identified virulent forms better than current PCR tests for tdh, trh or T3SS2.

  5. Development of a Polymerase Chain Reaction Assay for Detection of Burkholderia mallei, a Potent Biological Warfare Agent

    Directory of Open Access Journals (Sweden)

    Vijai Pal

    2016-09-01

    Full Text Available Burkholderia mallei is the etiological agent of glanders, primarily a disease of equines. B. mallei is closely related to B. pseudomallei, the causative agent of melioidosis. Therefore, detection of B. mallei and its differentiation from B. pseudomallei, has always been troublesome. In present investigation, a B. mallei specific DNA sequence was identified by performing BLASTn search using ~3000 ORFs of B. mallei NCTC 10229. A polymerase chain reaction (PCR assay with internal amplification control (IAC was developed for detection of B. mallei and its differentiation from B. pseudomallei. The PCR assay could amplify a specific 224-bp fragment from all the six B. mallei strains used in the study, whereas other closely related organisms were tested negative. The detection limit of the assay was found to be 10 pg of purified DNA of B. mallei. Incorporation of IAC in the assay makes the results reliable as false negative results which may arise due to presence of PCR inhibitors, can be avoided. For validation, the assay was tested on tap water, Bengal gram and grass artificially spiked with B. mallei. The developed assay can be used as a simple and rapid tool for detection of B. mallei.

  6. Study comparing human papillomavirus (HPV) real-time multiplex PCR and Hybrid Capture II INNO-LiPA v2 HPV genotyping PCR assays

    DEFF Research Database (Denmark)

    Iftner, Thomas; Germ, Liesje; Swoyer, Ryan

    2009-01-01

    Human papillomavirus (HPV) DNA genotyping is an essential test to establish efficacy in HPV vaccine clinical trials and HPV prevalence in natural history studies. A number of HPV DNA genotyping methods have been cited in the literature, but the comparability of the outcomes from the different...... methods has not been well characterized. Clinically, cytology is used to establish possible HPV infection. We evaluated the sensitivity and specificity of HPV multiplex PCR assays compared to those of the testing scheme of the Hybrid Capture II (HCII) assay followed by an HPV PCR/line hybridization assay...... (HCII-LiPA v2). SurePath residual samples were split into two aliquots. One aliquot was subjected to HCII testing followed by DNA extraction and LiPA v2 genotyping. The second aliquot was shipped to a second laboratory, where DNA was extracted and HPV multiplex PCR testing was performed. Comparisons...

  7. 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.

  8. Novel wide-range quantitative nested real-time PCR assay for Mycobacterium tuberculosis DNA: development and methodology.

    Science.gov (United States)

    Takahashi, Teruyuki; Tamura, Masato; Asami, Yukihiro; Kitamura, Eiko; Saito, Kosuke; Suzuki, Tsukasa; Takahashi, Sachiko Nonaka; Matsumoto, Koichi; Sawada, Shigemasa; Yokoyama, Eise; Takasu, Toshiaki

    2008-05-01

    Previously, we designed an internally controlled quantitative nested real-time (QNRT) PCR assay for Mycobacterium tuberculosis DNA in order to rapidly diagnose tuberculous meningitis. This technique combined the high sensitivity of nested PCR with the accurate quantification of real-time PCR. In this study, we attempted to improve the original QNRT-PCR assay and newly developed the wide-range QNRT-PCR (WR-QNRT-PCR) assay, which is more accurate and has a wider detection range. For use as an internal-control "calibrator" to measure the copy number of M. tuberculosis DNA, an original new-mutation plasmid (NM-plasmid) was developed. It had artificial random nucleotides in five regions annealing specific primers and probes. The NM-plasmid demonstrated statistically uniform amplifications (F = 1.086, P = 0.774) against a range (1 to 10(5)) of copy numbers of mimic M. tuberculosis DNA and was regarded as appropriate for use as a new internal control in the WR-QNRT-PSR assay. In addition, by the optimization of assay conditions in WR-QNRT-PCR, two-step amplification of target DNA was completely consistent with the standard curve of this assay. Due to the development of the NM-plasmid as the new internal control, significantly improved quantitative accuracy and a wider detection range were realized with the WR-QNRT-PCR assay. In the next study, we will try to use this novel assay method with actual clinical samples and examine its clinical usefulness.

  9. Detection of Campylobacter jejuni and Campylobacter coli in Environmental Waters by PCR Enzyme-Linked Immunosorbent Assay

    OpenAIRE

    Sails, A. D.; Bolton, F. J.; Fox, A. J.; Wareing, D. R. A.; Greenway, D. L. A.

    2002-01-01

    A PCR enzyme-linked immunosorbent assay (ELISA) assay was applied to the detection of Campylobacter jejuni and Campylobacter coli in environmental water samples after enrichment culture. Bacterial cells were concentrated from 69 environmental water samples by using filtration, and the filtrates were cultured in Campylobacter blood-free broth. After enrichment culture, DNA was extracted from the samples by using a rapid-boiling method, and the DNA extracts were used as a template in a PCR ELIS...

  10. Reduce microRNA RT-qPCR Assay Costs by More Than 10-fold Without Compromising Results

    DEFF Research Database (Denmark)

    Goldrick, Marianna; Busk, Peter Kamp; Lepovitz, Lance

    2013-01-01

    This white paper describes a detailed protocol for carrying out qPCR-based microRNA analysis for only ~$0.39 per assay, a cost-savings of >90% compared to commonly used alternative methods.......This white paper describes a detailed protocol for carrying out qPCR-based microRNA analysis for only ~$0.39 per assay, a cost-savings of >90% compared to commonly used alternative methods....

  11. Pre-Clinical Testing of Real-Time PCR Assays for Diarrheal Disease Agents of Genera Escherichia and Shigella

    Science.gov (United States)

    2014-05-16

    FOR DIARRHEAL DISEASE AGENTS OF GENERA ESCHERICHIA AND SHIGELLA May 16, 2014 Reporting Period: October 1, 2010 to September 30, 2013...10-2010 - 30-09-2013 PRE-CLINICAL TESTING OF REAL-TIME PCR ASSAYS FOR DIARRHEAL DISEASE AGENTS OF GENERA ESCHERICHIA AND SHIGELLA ...Texas (MOA 2007 - 2013. Agreement No.: DODI 4000.19; AFI 25-201). Pre-clinical test results qualify ETEC and Shigella real-time PCR assays as lead

  12. 9 CFR 147.30 - Laboratory procedure recommended for the polymerase chain reaction (PCR) test for Mycoplasma...

    Science.gov (United States)

    2010-01-01

    ... the polymerase chain reaction (PCR) test for Mycoplasma gallisepticum and M. synoviae. 147.30 Section... Examination Procedures § 147.30 Laboratory procedure recommended for the polymerase chain reaction (PCR) test... sample (100 to 2000 ng/5 μl) with one of the following 45 μl PCR cocktails: (i) 5 μl 10x PCR buffer, 1...

  13. Multiplex polymerase chain reaction assay for the detection of minute virus of mice and mouse parvovirus infections in laboratory mice.

    Science.gov (United States)

    Wang, K W; Chueh, L L; Wang, M H; Huang, Y T; Fang, B H; Chang, C Y; Fang, M C; Chou, J Y; Hsieh, S C; Wan, C H

    2013-04-01

    Mouse parvoviruses are among the most prevalent infectious pathogens in contemporary mouse colonies. To improve the efficiency of routine screening for mouse parvovirus infections, a multiplex polymerase chain reaction (PCR) assay targeting the VP gene was developed. The assay detected minute virus of mice (MVM), mouse parvovirus (MPV) and a mouse housekeeping gene (α-actin) and was able to specifically detect MVM and MPV at levels as low as 50 copies. Co-infection with the two viruses with up to 200-fold differences in viral concentrations can easily be detected. The multiplex PCR assay developed here could be a useful tool for monitoring mouse health and the viral contamination of biological materials.

  14. Designing Polymerase Chain Reaction (PCR) Primer Multiplexes in the Forensic Laboratory

    Science.gov (United States)

    Elkins, Kelly M.

    2011-01-01

    The polymerase chain reaction (PCR) is a common experiment in upper-level undergraduate biochemistry, molecular biology, and forensic laboratory courses as reagents and thermocyclers have become more affordable for institutions. Typically, instructors design PCR primers to amplify the region of interest and the students prepare their samples for…

  15. Designing Polymerase Chain Reaction (PCR) Primer Multiplexes in the Forensic Laboratory

    Science.gov (United States)

    Elkins, Kelly M.

    2011-01-01

    The polymerase chain reaction (PCR) is a common experiment in upper-level undergraduate biochemistry, molecular biology, and forensic laboratory courses as reagents and thermocyclers have become more affordable for institutions. Typically, instructors design PCR primers to amplify the region of interest and the students prepare their samples for…

  16. Development and validation of a real-time PCR assay for the detection of anguillid herpesvirus 1.

    Science.gov (United States)

    van Beurden, S J; Voorbergen-Laarman, M A; Roozenburg, I; van Tellingen, J; Haenen, O L M; Engelsma, M Y

    2016-01-01

    Anguillid herpesvirus 1 (AngHV1) causes a haemorrhagic disease with increased mortality in wild and farmed European eel, Anguilla anguilla (L.) and Japanese eel Anguilla japonica, Temminck & Schlegel). Detection of AngHV1 is currently based on virus isolation in cell culture, antibody-based typing assays or conventional PCR. We developed, optimized and concisely validated a diagnostic TaqMan probe based real-time PCR assay for the detection of AngHV1. The primers and probe target AngHV1 open reading frame 57, encoding the capsid protease and scaffold protein. Compared to conventional PCR, the developed real-time PCR is faster, less labour-intensive and has a reduced risk of cross-contamination. The real-time PCR assay was shown to be analytically sensitive and specific and has a high repeatability, efficiency and r(2) -value. The diagnostic performance of the assay was determined by testing 10% w/v organ suspensions and virus cultures from wild and farmed European eels from the Netherlands by conventional and real-time PCR. The developed real-time PCR assay is a useful tool for the rapid and sensitive detection of AngHV1 in 10% w/v organ suspensions from wild and farmed European eels.

  17. Determining the analytical specificity of PCR-based assays for the diagnosis of IA: What is Aspergillus?

    Science.gov (United States)

    Morton, C Oliver; White, P Lewis; Barnes, Rosemary A; Klingspor, Lena; Cuenca-Estrella, Manuel; Lagrou, Katrien; Bretagne, Stéphane; Melchers, Willem; Mengoli, Carlo; Caliendo, Angela M; Cogliati, Massimo; Debets-Ossenkopp, Yvette; Gorton, Rebecca; Hagen, Ferry; Halliday, Catriona; Hamal, Petr; Harvey-Wood, Kathleen; Jaton, Katia; Johnson, Gemma; Kidd, Sarah; Lengerova, Martina; Lass-Florl, Cornelia; Linton, Chris; Millon, Laurence; Morrissey, C Orla; Paholcsek, Melinda; Talento, Alida Fe; Ruhnke, Markus; Willinger, Birgit; Donnelly, J Peter; Loeffler, Juergen

    2017-06-01

    A wide array of PCR tests has been developed to aid the diagnosis of invasive aspergillosis (IA), providing technical diversity but limiting standardisation and acceptance. Methodological recommendations for testing blood samples using PCR exist, based on achieving optimal assay sensitivity to help exclude IA. Conversely, when testing more invasive samples (BAL, biopsy, CSF) emphasis is placed on confirming disease, so analytical specificity is paramount. This multicenter study examined the analytical specificity of PCR methods for detecting IA by blind testing a panel of DNA extracted from a various fungal species to explore the range of Aspergillus species that could be detected, but also potential cross reactivity with other fungal species. Positivity rates were calculated and regression analysis was performed to determine any associations between technical specifications and performance. The accuracy of Aspergillus genus specific assays was 71.8%, significantly greater (P PCR assay targeting the rRNA genes (P = .0011). Conversely, there was a significant association between rRNA PCR targets and false positivity (P = .0032). To conclude current Aspergillus PCR assays are better suited for detecting A. fumigatus, with inferior detection of most other Aspergillus species. The use of an Aspergillus genus specific PCR assay targeting the rRNA genes is preferential. © The Author 2016. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  18. Sources of blood meals of sylvatic Triatoma guasayana near Zurima, Bolivia, assayed with qPCR and 12S cloning.

    Science.gov (United States)

    Lucero, David E; Ribera, Wilma; Pizarro, Juan Carlos; Plaza, Carlos; Gordon, Levi W; Peña, Reynaldo; Morrissey, Leslie A; Rizzo, Donna M; Stevens, Lori

    2014-12-01

    In this study we compared the utility of two molecular biology techniques, cloning of the mitochondrial 12S ribosomal RNA gene and hydrolysis probe-based qPCR, to identify blood meal sources of sylvatic Chagas disease insect vectors collected with live-bait mouse traps (also known as Noireau traps). Fourteen T. guasayana were collected from six georeferenced trap locations in the Andean highlands of the department of Chuquisaca, Bolivia. We detected four blood meals sources with the cloning assay: seven samples were positive for human (Homo sapiens), five for chicken (Gallus gallus) and unicolored blackbird (Agelasticus cyanopus), and one for opossum (Monodelphis domestica). Using the qPCR assay we detected chicken (13 vectors), and human (14 vectors) blood meals as well as an additional blood meal source, Canis sp. (4 vectors). We show that cloning of 12S PCR products, which avoids bias associated with developing primers based on a priori knowledge, detected blood meal sources not previously considered and that species-specific qPCR is more sensitive. All samples identified as positive for a specific blood meal source by the cloning assay were also positive by qPCR. However, not all samples positive by qPCR were positive by cloning. We show the power of combining the cloning assay with the highly sensitive hydrolysis probe-based qPCR assay provides a more complete picture of blood meal sources for insect disease vectors.

  19. Sources of blood meals of sylvatic Triatoma guasayana near Zurima, Bolivia, assayed with qPCR and 12S cloning.

    Directory of Open Access Journals (Sweden)

    David E Lucero

    2014-12-01

    Full Text Available In this study we compared the utility of two molecular biology techniques, cloning of the mitochondrial 12S ribosomal RNA gene and hydrolysis probe-based qPCR, to identify blood meal sources of sylvatic Chagas disease insect vectors collected with live-bait mouse traps (also known as Noireau traps. Fourteen T. guasayana were collected from six georeferenced trap locations in the Andean highlands of the department of Chuquisaca, Bolivia.We detected four blood meals sources with the cloning assay: seven samples were positive for human (Homo sapiens, five for chicken (Gallus gallus and unicolored blackbird (Agelasticus cyanopus, and one for opossum (Monodelphis domestica. Using the qPCR assay we detected chicken (13 vectors, and human (14 vectors blood meals as well as an additional blood meal source, Canis sp. (4 vectors.We show that cloning of 12S PCR products, which avoids bias associated with developing primers based on a priori knowledge, detected blood meal sources not previously considered and that species-specific qPCR is more sensitive. All samples identified as positive for a specific blood meal source by the cloning assay were also positive by qPCR. However, not all samples positive by qPCR were positive by cloning. We show the power of combining the cloning assay with the highly sensitive hydrolysis probe-based qPCR assay provides a more complete picture of blood meal sources for insect disease vectors.

  20. A Multiplex PCR Assay for the Detection of Pathogenic Genes of EPEC, ETEC and EIEC

    Institute of Scientific and Technical Information of China (English)

    ZHANG Tienan; LI Jichang; LU Chengwu; HUO Guicheng

    2006-01-01

    A multiplex polymerase chain reaction (PCR) was developed to detect three pathogenic genes of enteropathogenic, enterotocigenic and enteroinvasive Escherichia coli.. In this study three different sets of oligonucleotide primer were simultaneously used, and in this way, specific fragments of 880, 600, 150 bp for EPEC eaeA,EIEC ipaH and ETEC ST genes were amplified, respectively. The best condition of the multiplex PCR was: after an initial heat denaturation step at 95℃ for 5 min, followed by 30 cycles of denaturation at 94 ℃ for 40 s, primer annealing at 51.3 ℃ for 40 s and extension at 72 ℃ for 1 min, final extension at 72 ℃ for 10 min. The detection limit of tively. It may be a good way for the detection and identification of Diarrhea-causing E. coli..

  1. Pentaplex PCR as screening assay for jellyfish species identification in food products.

    Science.gov (United States)

    Armani, Andrea; Giusti, Alice; Castigliego, Lorenzo; Rossi, Aurelio; Tinacci, Lara; Gianfaldoni, Daniela; Guidi, Alessandra

    2014-12-17

    Salted jellyfish, a traditional food in Asian Countries, is nowadays spreading on the Western markets. In this work, we developed a Pentaplex PCR for the identification of five edible species (Nemopilema nomurai, Rhopilema esculentum, Rhizostoma pulmo, Pelagia noctiluca, and Cotylorhiza tuberculata), which cannot be identified by a mere visual inspection in jellyfish products sold as food. A common degenerated forward primer and five specie-specific reverse primers were designed to amplify COI gene regions of different lengths. Another primer pair targeted the 28SrRNA gene and was intended as common positive reaction control. Considering the high level of degradation in the DNA extracted from acidified and salted products, the maximum length of the amplicons was set at 200 bp. The PCR was developed using 66 reference DNA samples. It gave successful amplifications in 85.4% of 48 ready to eat products (REs) and in 60% of 30 classical salted products (CPs) collected on the market.

  2. Audit and improve! Evaluation of a real-time probe-based PCR assay with internal control for the direct detection of Mycobacterium tuberculosis complex.

    Science.gov (United States)

    Inoue, M; Tang, W Y; Wee, S Y; Barkham, T

    2011-01-01

    We retrospectively audited the performance of the commercial kit in use in our laboratory for the detection of Mycobacterium tuberculosis complex (MTBC) and found the sensitivity to be unacceptably low at 69% (52/75). We developed an in-house end-point polymerase chain reaction (PCR) detecting IS6110, an IS-like element of MTBC, and achieved a sensitivity of 90% (66/73) with the same DNA samples, re-emphasising the poor performance of the commercial kit. In order to avoid specificity issues surrounding gel-based PCR, we developed a probe-based real-time PCR assay with an internal control and achieved a sensitivity of 84%, specificity of 97% and diagnostic odds ratio (DOR) of 207. The evaluation was performed on clinically requested samples, so we expect the performance of the assay in real life to match the data from this evaluation. Centers for Disease Control and Prevention (CDC) guidelines recommending nucleic acid tests for the investigation of possible cases of tuberculosis are expected to promote the use of molecular assays. It is important that clinical laboratories do not assume that assays, in-house or commercial, will perform well or that they will continue to perform well. Audit at regular intervals is necessary to maintain confidence and to demonstrate that the assay works to specification in the real test population.

  3. Diagnostic RAS mutation analysis by polymerase chain reaction (PCR

    Directory of Open Access Journals (Sweden)

    Ian A. Cree

    2016-06-01

    Full Text Available RAS mutation analysis is an important companion diagnostic test. Treatment of colorectal cancer with anti-Epidermal Growth Factor Receptor (EGFR therapy requires demonstration of RAS mutation status (both KRAS and NRAS, and it is good practice to include BRAF. In Non-Small Cell Lung Cancer (NSCLC and melanoma, assessment of RAS mutation status can be helpful in triaging patient samples for more extensive testing. This mini-review will discuss the role of PCR methods in providing rapid diagnostic information for cancer patients.

  4. Comprehensive multiplex one-step real-time TaqMan qRT-PCR assays for detection and quantification of hemorrhagic fever viruses.

    Directory of Open Access Journals (Sweden)

    Zheng Pang

    Full Text Available BACKGROUND: Viral hemorrhagic fevers (VHFs are a group of animal and human illnesses that are mostly caused by several distinct families of viruses including bunyaviruses, flaviviruses, filoviruses and arenaviruses. Although specific signs and symptoms vary by the type of VHF, initial signs and symptoms are very similar. Therefore rapid immunologic and molecular tools for differential diagnosis of hemorrhagic fever viruses (HFVs are important for effective case management and control of the spread of VHFs. Real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR assay is one of the reliable and desirable methods for specific detection and quantification of virus load. Multiplex PCR assay has the potential to produce considerable savings in time and resources in the laboratory detection. RESULTS: Primers/probe sets were designed based on appropriate specific genes for each of 28 HFVs which nearly covered all the HFVs, and identified with good specificity and sensitivity using monoplex assays. Seven groups of multiplex one-step real-time qRT-PCR assays in a universal experimental system were then developed by combining all primers/probe sets into 4-plex reactions and evaluated with serial dilutions of synthesized viral RNAs. For all the multiplex assays, no cross-reactivity with other HFVs was observed, and the limits of detection were mainly between 45 and 150 copies/PCR. The reproducibility was satisfactory, since the coefficient of variation of Ct values were all less than 5% in each dilution of synthesized viral RNAs for both intra-assays and inter-assays. Evaluation of the method with available clinical serum samples collected from HFRS patients, SFTS patients and Dengue fever patients showed high sensitivity and specificity of the related multiplex assays on the clinical specimens. CONCLUSIONS: Overall, the comprehensive multiplex one-step real-time qRT-PCR assays were established in this study, and proved to be

  5. Rapid semi-automated quantitative multiplex tandem PCR (MT-PCR assays for the differential diagnosis of influenza-like illness

    Directory of Open Access Journals (Sweden)

    Dwyer Dominic E

    2010-05-01

    Full Text Available Abstract Background Influenza A, including avian influenza, is a major public health threat in developed and developing countries. Rapid and accurate detection is a key component of strategies to contain spread of infection, and the efficient diagnosis of influenza-like-illness is essential to protect health infrastructure in the event of a major influenza outbreak. Methods We developed a multiplexed PCR (MT-PCR assay for the simultaneous diagnosis of respiratory viruses causing influenza-like illness, including the specific recognition of influenza A haemagglutinin subtypes H1, H3, and H5. We tested several hundred clinical specimens in two diagnostic reference laboratories and compared the results with standard techniques. Results The sensitivity and specificity of these assays was higher than individual assays based on direct antigen detection and standard PCR against a range of control templates and in several hundred clinical specimens. The MT-PCR assays provided differential diagnoses as well as potentially useful quantitation of virus in clinical samples. Conclusions MT-PCR is a potentially powerful tool for the differential diagnosis of influenza-like illness in the clinical diagnostic laboratory.

  6. Validation of a sensitive PCR assay for the detection of Chelonid fibropapilloma-associated herpesvirus in latent turtle infections

    DEFF Research Database (Denmark)

    Alfaro Nuñez, Luis Alonso; Gilbert, M Thomas P

    2014-01-01

    clinically healthy (non exhibiting fibropapilloma tumours) turtles, thus representing presumably latent infections of the pathogen. Given that template copy numbers of viruses in latent infections can be very low, extremely sensitive PCR assays are needed to optimize detection efficiency. In this study......, efficiency of several PCR assays designed for CFPHV detection is explored and compared to a method published previously. The results show that adoption of a triplet set of singleplex PCR assays outperforms other methods, with an approximately 3-fold increase in detection success in comparison to the standard...... assay. Thus, a new assay for the detection of CFPHV DNA markers is presented, and adoption of its methodology is recommended in future CFPHV screens among sea turtles....

  7. Multiplex SYBR® green-real time PCR (qPCR) assay for the detection and differentiation of Bartonella henselae and Bartonella clarridgeiae in cats.

    Science.gov (United States)

    Staggemeier, Rodrigo; Pilger, Diogo André; Spilki, Fernando Rosado; Cantarelli, Vlademir Vicente

    2014-01-01

    A novel SYBR® green-real time polymerase chain reaction (qPCR) was developed to detect two Bartonella species, B. henselae and B. clarridgeiae, directly from blood samples. The test was used in blood samples obtained from cats living in animal shelters in Southern Brazil. Results were compared with those obtained by conventional PCR targeting Bartonella spp. Among the 47 samples analyzed, eight were positive using the conventional PCR and 12 were positive using qPCR. Importantly, the new qPCR detected the presence of both B. henselae and B. clarridgeiae in two samples. The results show that the qPCR described here may be a reliable tool for the screening and differentiation of two important Bartonella species.

  8. MULTIPLEX SYBR® GREEN-REAL TIME PCR (qPCR ASSAY FOR THE DETECTION AND DIFFERENTIATION OF Bartonella henselae AND Bartonella clarridgeiae IN CATS

    Directory of Open Access Journals (Sweden)

    Rodrigo Staggemeier

    2014-04-01

    Full Text Available A novel SYBR® green-real time polymerase chain reaction (qPCR was developed to detect two Bartonella species, B. henselae and B. clarridgeiae, directly from blood samples. The test was used in blood samples obtained from cats living in animal shelters in Southern Brazil. Results were compared with those obtained by conventional PCR targeting Bartonella spp. Among the 47 samples analyzed, eight were positive using the conventional PCR and 12 were positive using qPCR. Importantly, the new qPCR detected the presence of both B. henselae and B. clarridgeiae in two samples. The results show that the qPCR described here may be a reliable tool for the screening and differentiation of two important Bartonella species.

  9. Comparison of a commercial real-time PCR assay, RealCycler® PJIR kit, progenie molecular, to an in-house real-time PCR assay for the diagnosis of Pneumocystis jirovecii infections.

    Science.gov (United States)

    Guillaud-Saumur, Thibaud; Nevez, Gilles; Bazire, Amélie; Virmaux, Michèle; Papon, Nicolas; Le Gal, Solène

    2017-04-01

    We compared the RealCycler® PJIR kit (Progenie Molecular), available in Europe, to an in-house real-time PCR assay for the diagnosis of Pneumocystis jirovecii infections. Excellent agreement was found (concordance rate, 97.4%; Cohen's kappa, 0.918>0.8) showing that this commercial assay represents an alternative method for the diagnosis of P. jirovecii infections.

  10. Duplex Quantitative PCR Assay for Detection of Haemophilus influenzae That Distinguishes Fucose- and Protein D-Negative Strains.

    Science.gov (United States)

    de Gier, Camilla; Pickering, Janessa L; Richmond, Peter C; Thornton, Ruth B; Kirkham, Lea-Ann S

    2016-09-01

    We have developed a specific Haemophilus influenzae quantitative PCR (qPCR) that also identifies fucose-negative and protein D-negative strains. Analysis of 100 H. influenzae isolates, 28 Haemophilus haemolyticus isolates, and 14 other bacterial species revealed 100% sensitivity (95% confidence interval [CI], 96% to 100%) and 100% specificity (95% CI, 92% to 100%) for this assay. The evaluation of 80 clinical specimens demonstrated a strong correlation between semiquantitative culture and the qPCR (P < 0.001).

  11. Two novel nonradioactive polymerase chain reaction-based assays of dried blood spots, genomic DNA, or whole cells for fast, reliable detection of Z and S mutations in the alpha 1-antitrypsin gene

    DEFF Research Database (Denmark)

    Andresen, B S; Knudsen, I; Jensen, P K;

    1992-01-01

    Two new nonradioactive polymerase chain reaction (PCR)-based assays for the Z and S mutations in the alpha 1-antitrypsin gene are presented. The assays take advantage of PCR-mediated mutagenesis, creating new diagnostic restriction enzyme sites for unambiguous discrimination between test samples...

  12. Development of a PCR Assay to detect Papillomavirus Infection in the Snow Leopard

    Directory of Open Access Journals (Sweden)

    Eng Curtis

    2011-07-01

    Full Text Available Abstract Background Papillomaviruses (PVs are a group of small, non-encapsulated, species-specific DNA viruses that have been detected in a variety of mammalian and avian species including humans, canines and felines. PVs cause lesions in the skin and mucous membranes of the host and after persistent infection, a subset of PVs can cause tumors such as cervical malignancies and head and neck squamous cell carcinoma in humans. PVs from several species have been isolated and their genomes have been sequenced, thereby increasing our understanding of the mechanism of viral oncogenesis and allowing for the development of molecular assays for the detection of PV infection. In humans, molecular testing for PV DNA is used to identify patients with persistent infections at risk for developing cervical cancer. In felids, PVs have been isolated and sequenced from oral papillomatous lesions of several wild species including bobcats, Asian lions and snow leopards. Since a number of wild felids are endangered, PV associated disease is a concern and there is a need for molecular tools that can be used to further study papillomavirus in these species. Results We used the sequence of the snow leopard papillomavirus UuPV1 to develop a PCR strategy to amplify viral DNA from samples obtained from captive animals. We designed primer pairs that flank the E6 and E7 viral oncogenes and amplify two DNA fragments encompassing these genes. We detected viral DNA for E6 and E7 in genomic DNA isolated from saliva, but not in paired blood samples from snow leopards. We verified the identity of these PCR products by restriction digest and DNA sequencing. The sequences of the PCR products were 100% identical to the published UuPV1 genome sequence. Conclusions We developed a PCR assay to detect papillomavirus in snow leopards and amplified viral DNA encompassing the E6 and E7 oncogenes specifically in the saliva of animals. This assay could be utilized for the molecular

  13. Development of a multiplex real-time PCR assay for phylogenetic analysis of Uropathogenic Escherichia coli.

    Science.gov (United States)

    Hasanpour, Mojtaba; Najafi, Akram

    2017-03-28

    Uropathogenic Escherichia coli (UPEC) is among major pathogens causing 80-90% of all episodes of urinary tract infections (UTIs). Recently, E. coli strains are divided into eight main phylogenetic groups including A, B1, B2, C, D, E, F, and clade I. This study was aimed to develop a rapid, sensitive, and specific multiplex real time PCR method capable of detecting phylogenetic groups of E. coli strains. This study was carried out on E. coli strains (isolated from the patient with UTI) in which the presence of all seven target genes had been confirmed in our previous phylogenetic study. An EvaGreen-based singleplex and multiplex real-time PCR with melting curve analysis was designed for simultaneous detection and differentiation of these genes. The primers were selected mainly based on the production of amplicons with melting temperatures (Tm) ranging from 82°C to 93°C and temperature difference of more than 1.5°C between each peak.The multiplex real-time PCR assays that have been developed in the present study were successful in detecting the eight main phylogenetic groups. Seven distinct melting peaks were discriminated, with Tm value of 93±0.8 for arpA, 89.2±0.1for chuA, 86.5±0.1 for yjaA, 82.3±0.2 for TspE4C2, 87.8±0.1for trpAgpC, 85.4±0.6 for arpAgpE genes, and 91±0.5 for the internal control. To our knowledge, this study is the first melting curve-based real-time PCR assay developed for simultaneous and discrete detection of these seven target genes. Our findings showed that this assay has the potential to be a rapid, reliable and cost-effective alternative for routine phylotyping of E. coli strains.

  14. Study comparing human papillomavirus (HPV) real-time multiplex PCR and Hybrid Capture II INNO-LiPA v2 HPV genotyping PCR assays.

    Science.gov (United States)

    Iftner, Thomas; Germ, Liesje; Swoyer, Ryan; Kjaer, Susanne Kruger; Breugelmans, J Gabrielle; Munk, Christian; Stubenrauch, Frank; Antonello, Joseph; Bryan, Janine T; Taddeo, Frank J

    2009-07-01

    Human papillomavirus (HPV) DNA genotyping is an essential test to establish efficacy in HPV vaccine clinical trials and HPV prevalence in natural history studies. A number of HPV DNA genotyping methods have been cited in the literature, but the comparability of the outcomes from the different methods has not been well characterized. Clinically, cytology is used to establish possible HPV infection. We evaluated the sensitivity and specificity of HPV multiplex PCR assays compared to those of the testing scheme of the Hybrid Capture II (HCII) assay followed by an HPV PCR/line hybridization assay (HCII-LiPA v2). SurePath residual samples were split into two aliquots. One aliquot was subjected to HCII testing followed by DNA extraction and LiPA v2 genotyping. The second aliquot was shipped to a second laboratory, where DNA was extracted and HPV multiplex PCR testing was performed. Comparisons were evaluated for 15 HPV types common in both assays. A slightly higher proportion of samples tested positive by the HPV multiplex PCR than by the HCII-LiPA v2 assay. The sensitivities of the multiplex PCR assay relative to those of the HCII-LiPA v2 assay for HPV types 6, 11, 16, and 18, for example, were 0.806, 0.646, 0.920, and 0.860, respectively; the specificities were 0.986, 0.998, 0.960, and 0.986, respectively. The overall comparability of detection of the 15 HPV types was quite high. Analyses of DNA genotype testing compared to cytology results demonstrated a significant discordance between cytology-negative (normal) and HPV DNA-positive results. This demonstrates the challenges of cytological diagnosis and the possibility that a significant number of HPV-infected cells may appear cytologically normal.

  15. Rapid Detection/pathotyping of Newcastle disease virus isolates in clinical samples using real time polymerase chain reaction assay

    OpenAIRE

    sprotocols

    2014-01-01

    Authors: Abdul Wajid, Muhammad Wasim, Tahir Yaqub, Shafqat F Rehmani, Tasra Bibi, Nadia Mukhtar, Javed Muhammad, Umar Bacha, Suliman Qadir Afridi, Muhammad Nauman Zahid, Zia u ddin, Muhammad Zubair Shabbir, Kamran Abbas & Muneer Ahmad ### Abstract In the present protocol we describe the real time reverse transcription polymerase chain reaction (rRT-PCR) assay for the rapid detection/pathotyping of Newcastle disease virus (NDV) isoaltes in clinical samples. Fusion gene and matrix ...

  16. Development of a Polymerase Chain Reaction Assay for Detection of Burkholderia mallei, a Potent Biological Warfare Agent

    OpenAIRE

    2016-01-01

    Burkholderia mallei is the etiological agent of glanders, primarily a disease of equines. B. mallei is closely related to B. pseudomallei, the causative agent of melioidosis. Therefore, detection of B. mallei and its differentiation from B. pseudomallei, has always been troublesome. In present investigation, a B. mallei specific DNA sequence was identified by performing BLASTn search using ~3000 ORFs of B. mallei NCTC 10229. A polymerase chain reaction (PCR) assay with internal amplification ...

  17. Identification of Klebsiella oxytoca using a specific PCR assay targeting the polygalacturonase pehX gene.

    Science.gov (United States)

    Kovtunovych, Gennadiy; Lytvynenko, Tetyana; Negrutska, Valentyna; Lar, Olena; Brisse, Sylvain; Kozyrovska, Natalia

    2003-10-01

    Bacteria of the genus Klebsiella are important opportunistic pathogens responsible for nosocomial infections that are increasingly resistant to antimicrobial agents. Distinctive identification of the species K. oxytoca, K. pneumoniae, K. planticola, K. ornithinolytica and K. terrigena is difficult based on phenotypic tests and misidentifications are frequent in routine clinical microbiology. We developed a specific method to discriminate K. oxytoca from the other species of the genus Klebsiella, based on the PCR amplification of the polygalacturonase (pehX) gene. A PCR amplicon of 344 bp was obtained in all 35 K. oxytoca strains tested, but in none of the 29 K. pneumoniae, 12 K. planticola/K. ornithinolytica and 7 K. terrigena strains tested. The test was also negative for polygalacturonate-degrading species of the genus Erwinia. Analysis of 24 strains designated as K. pneumoniae from international collections (NCTC, PZH) revealed previous misidentification of six K. oxytoca strains. Key biochemical tests fully confirmed the pehX PCR results. The new K. oxytoca identification assay should be useful for both clinical and ecological monitoring of K. oxytoca strains, as well as for controlling the previous identification of collection strains.

  18. Physical lysis only (PLO) methods suitable as rapid sample pretreatment for qPCR assay.

    Science.gov (United States)

    Wang, Xiaofang; Lee, Byung-Tae; Son, Ahjeong

    2014-10-01

    Quantitative PCR (qPCR) enables rapid and sensitive gene quantification and is widely used in genomics, such as biological, medical, environmental, and food sciences. However, sample pretreatment requires the use of conventional DNA extraction kits which are time-consuming and labor intensive. In this study, we investigated four physical lysis only (PLO) methods which are rapid and could serve as alternatives to conventional DNA extraction kits. These PLO methods are bead mill, heating, sonication, and freeze-thaw. Using ethidium bromide-based assay, their performance was evaluated and compared. The effects of cell debris and its removal were also investigated. Bead mill method without cell debris removal appeared to yield the best qPCR results among the four PLO methods. In addition, bead mill method also performed better than conventional DNA extraction kits. It is probably due to the substantial loss of DNA material during the extensive purification of the conventional DNA extraction kits. The bead mill method has been demonstrated to successfully quantify 10(2) to 10(7) copies of the PAH-RHDα gene of Pseudomonas putida.

  19. Multiplex PCR assays for the detection of Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio vulnificus, and Vibrio cholerae with an internal amplification control.

    Science.gov (United States)

    Wei, Shuang; Zhao, Hui; Xian, Yuyin; Hussain, Malik A; Wu, Xiyang

    2014-06-01

    A multiplex polymerase chain reaction (PCR) assay that can simultaneously detect 4 major Vibrio spp., Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio vulnificus, and Vibrio cholerae, in the presence of an internal amplification control (IAC) was developed. Species-specific PCR primers were designed based on the gyrB gene for V. alginolyticus, the collagenase gene for V. parahaemolyticus, the vvhA gene for V. vulnificus, and the ompW gene for V. cholerae. Additionally, an IAC primer pair was designed in conserved regions of the bacterial 16S rRNA gene that is used to indicate false-negative results. A multiplex PCR method was developed after optimization of the reaction conditions. The specificity of the PCR was validated by using 83 Vibrio strains and 10 other non-Vibrio bacterial species. The detection limit of the PCR was 10 CFU per tube for V. alginolyticus, V. parahaemolyticus, V. vulnificus, and 10(5) CFU per tube for V. cholerae in mixed conditions. This method was used to identify 69 suspicious Vibrio isolates, and the results were consistent with physiological and biochemical tests. This multiplex PCR method proved to be rapid, sensitive, and specific. The existence of IAC could successfully eliminate false-negative results for the detection of V. alginolyticus, V. parahaemolyticus, V. vulnificus, and V. cholerae. Copyright © 2014 Elsevier Inc. All rights reserved.

  20. Real-time PCR assay in differentiating Entamoeba histolytica, Entamoeba dispar, and Entamoeba moshkovskii infections in Orang Asli settlements in Malaysia.

    Science.gov (United States)

    Lau, Yee Ling; Anthony, Claudia; Fakhrurrazi, Siti Aminah; Ibrahim, Jamaiah; Ithoi, Init; Mahmud, Rohela

    2013-08-28

    Amebiasis caused by Entamoeba histolytica is the third leading cause of death worldwide. This pathogenic amoeba is morphologically indistinguishable from E. dispar and E. moshkovskii, the non-pathogenic species. Polymerase chain reaction is the current method of choice approved by World Health Organization. Real-time PCR is another attractive molecular method for diagnosis of infectious diseases as post-PCR analyses are eliminated and turnaround times are shorter. The present work aimed to compare the results of Entamoeba species identification using the real-time assay against the established nested PCR method. In this study, a total of 334 human faecal samples were collected from different Orang Asli settlements. Faecal samples were processed by direct wet smear and formalin ethyl acetate concentration methods followed by iodine staining and was microscopically examined for Entamoeba species and other intestinal parasites. Microscopically positive samples were then subject to nested PCR and real-time PCR. The overall prevalence of Entamoeba infection was 19.5% (65/334). SK Posh Piah recorded highest Entamoeba prevalence (63.3%) while Kampung Kemensah had the lowest prevalence (3.7%) of Entamoeba. Microscopically positive samples were then tested by real-time PCR and nested PCR for the presence of Entamoeba histolytica, Entamoeba dispar, and Entamoeba moshkovskii infection. Real-time PCR showed higher Entamoeba detection (86.2%) compared to nested PCR (80%), although the McNemar test value showed no significant difference between the two methods (p = 0.221). This study is the first in Malaysia to report the use of real-time PCR in identifying and differentiating the three Entamoeba infections. It is also proven to be more effective compared to the conventional nested PCR molecular method.

  1. Development of Nested PCR, Multiplex PCR, and Loop-Mediated Isothermal Amplification Assays for Rapid Detection of Cylindrocladium scoparium on Eucalyptus.

    Science.gov (United States)

    Qiao, Tian-Min; Zhang, Jing; Li, Shu-Jiang; Han, Shan; Zhu, Tian-Hui

    2016-10-01

    Eucalyptus dieback disease, caused by Cylindrocladium scoparium, has occurred in last few years in large Eucalyptus planting areas in China and other countries. Rapid, simple, and reliable diagnostic techniques are desired for the early detection of Eucalyptus dieback of C. scoparium prior to formulation of efficient control plan. For this purpose, three PCR-based methods of nested PCR, multiplex PCR, loop-mediated isothermal amplification (LAMP) were developed for detection of C. scoparium based on factor 1-alpha (tef1) and beta-tubulin gene in this study. All of the three methods showed highly specific to C. scoparium. The sensitivities of the nested PCR and LAMP were much higher than the multiplex PCR. The sensitivity of multiplex PCR was also higher than regular PCR. C. scoparium could be detected within 60 min from infected Eucalyptus plants by LAMP, while at least 2 h was needed by the rest two methods. Using different Eucalyptus tissues as samples for C. scoparium detection, all of the three PCR-based methods showed much better detection results than regular PCR. Base on the results from this study, we concluded that any of the three PCR-based methods could be used as diagnostic technology for the development of efficient strategies of Eucalyptus dieback disease control. Particularly, LAMP was the most practical method in field application because of its one-step and rapid reaction, simple operation, single-tube utilization, and simple visualization of amplification products.

  2. The development and application of the two real-time RT-PCR assays to detect the pathogen of HFMD.

    Directory of Open Access Journals (Sweden)

    Aili Cui

    Full Text Available Large-scale Hand, Foot, and Mouth Disease (HFMD outbreaks have frequently occurred in China since 2008, affecting more than one million children and causing several hundred children deaths every year. The pathogens of HFMD are mainly human enteroviruses (HEVs. Among them, human enterovirus 71 (HEV71 and coxsackievirus A16 (CVA16 are the most common pathogens of HFMD. However, other HEVs could also cause HFMD. To rapidly detect HEV71 and CVA16, and ensure detection of all HEVs causing HFMD, two real-time hybridization probe-based RT-PCR assays were developed in this study. One is a multiplex real-time RT-PCR assay, which was developed to detect and differentiate HEV71 specifically from CVA16 directly from clinical specimens within 1-2 h, and the other is a broad-spectrum real-time RT-PCR assay, which targeted almost all HEVs. The experiments confirmed that the two assays have high sensitivity and specificity, and the sensitivity was up to 0.1 TCID50/ml for detection of HEVs, HEV71, and CVA16, respectively. A total of 213 clinical specimens were simultaneously detected by three kinds of assays, including the two real-time RT-PCR assays, direct conventional RT-PCR assay, and virus isolation assay on human rhabdomyosarcoma cells (RD cells. The total positive rate of both HEV71 and CVA16 was 69.48% with real-time RT-PCR assay, 47.42% with RT-PCR assay, and 34.58% with virus isolation assay. One HFMD clinical specimen was positive for HEV, but negative for HEV71 or CVA16, which was identified as Echovirus 11 (Echo11 by virus isolation, RT-PCR, and sequencing for the VP1 gene. The two real-time RT-PCR assays had been applied in 31 provincial HFMD labs to detect the pathogens of HFMD, which has contributed to the rapid identification of the pathogens in the early stages of HFMD outbreaks, and helped to clarify the etiologic agents of HFMD in China.

  3. Development of a caseinase assay for PCR independent detection of esp gene carriage among enterococci

    Science.gov (United States)

    Dada, Ayokunle Christopher; Asmat, Ahmad; Lee, Yook Heng; Usup, Gires

    2013-11-01

    Currently, there is no known relationship between caseinase and carriage of esp gene. Also, no breakpoints exist for phenotypic assays that are used to infer virulence characteristics among Enterococci. In the present study, caseinase activity was measured by a radial diffusion assay for 113 enterococci isolates. A standard curve with predictive r2 value of 0.939 was produced by dispensing several doubling dilutions of proteinase K into 3% skimmed milk agar wells. Caseinase activity for all tested enterococci was subsequently converted into proteinase K activity, using the obtained chart. Caseinase activity ranged from 1.74 × 10-8 to 4.47 × 10-7ug/ml and 6.37 × 10-8 to 8.82 × 10-8 ug/ml per colony of environmental and clinical enterocococci tested, proportionate to proteinase K activity. Caseinase activity among environmental strains was five-fold higher than was observed among clinical strains. Fishers exact test revealed significant associations between esp gene carriage and caseinase activity (diameter on skimmed milk, z=8 to 13mm) at p<0.1. However, the probability of association was strongest at z=13 mm (p=0.033) suggesting a range of diameter cut-offs that was exclusive to and may be used to predict the presence of environmental enterococci strains harbouring esp gene. Results obtained from sensitivity analysis showed increasing assay sensitivity from cut-off of 9 mm (61.54%) up to 84.62% (13 mm). Specificity of the caseinase assay slightly decreased from 50% to 42.86% as cut-off increased from 9 to 13 mm. The caseinase assay described here potentially proves useful in preliminary PCR independent screening of environmental enterococci isolates for the detection of strains which carry the esp gene known to increase the severity of enterococcal infections.

  4. 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

  5. Detection of deoxyribonucleic acid (DNA) targets using polymerase chain reaction (PCR) and paper surface-enhanced Raman spectroscopy (SERS) chromatography.

    Science.gov (United States)

    Hoppmann, Eric P; Yu, Wei W; White, Ian M

    2014-01-01

    Surface-enhanced Raman spectroscopy (SERS) enables multiplex detection of analytes using simple, portable equipment consisting of a single excitation source and detector. Thus, in theory, SERS is ideally suited to replace fluorescence in assays that screen for numerous deoxyribonucleic acid (DNA) targets, but in practice, SERS-based assays have suffered from complexity and elaborate processing steps. Here, we report an assay in which a simple inkjet-fabricated plasmonic paper device enables SERS-based detection of multiple DNA targets within a single polymerase chain reaction (PCR). In prior work, we demonstrated the principles of chromatographic separation and SERS-based detection on inkjet-fabricated plasmonic paper. The present work extends that capability for post-PCR gene sequence detection. In this design, hydrolysis DNA probes with 5' Raman labels are utilized; if the target is present, the probe is hydrolyzed during PCR, freeing the reporter. After applying the PCR sample to a paper SERS device, an on-device chromatographic separation and concentration is conducted to discriminate between hydrolyzed and intact probes. SERS is then used to detect the reporter released by the hydrolyzed probes. This simple separation and detection on paper eliminates the need for complex sample processing steps. In this work, we simultaneously detect the methicillin-resistant Staphylococcus aureus genes mecA and femB to illustrate the concept. We envision that this approach could contribute to the development of multiplex DNA diagnostic tests enabling screening for several target sequences within a single reaction, which is necessary for cases in which sample volume and resources are limited.

  6. Establishment and Application of a TaqMan Real-Time Quantitative Reverse Transcription-Polymerase Chain Reaction Assay for Rubella Virus RNA

    Institute of Scientific and Technical Information of China (English)

    Li-Hong ZHAO; Yu-Yan MA; Hong WANG; Shu-Ping ZHAO; Wei-Ming ZHAO; Hua LI; Lei-Yi WANG

    2006-01-01

    The aim of this study was to establish and apply a real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) for rubella virus (RV) RNA. First, the primer and TaqMan probe concentrations, as well as reaction temperatures were optimized to establish an efficient real-time quantitative RT-PCR assay for RV RNA. Next, an RV-specific PCR amplicon was made as an external standard to estimate the linearity, amplification efficiency, analytical sensitivity and reproducibility of the real time quantitative assay. Finally, the assay was applied to quantify RVRNA in clinical samples for rubella diagnosis.The RV-specific PCR amplicon was prepared for evaluation of the assay at 503 bp, and its original concentration was 2.75×109 copies/μl. The real time quantitative assay was shown to have good linearity (R2=0.9920), high amplification efficiency (E=1.91), high sensitivity (275 copies/ml), and high reproducibility (variation coefficient range, from 1.25% to 3.58%). Compared with the gold standard, the specificity and sensitivity of the assay in clinical samples was 96.4% and 86.4%, respectively. Therefore, the established quantitative RT-PCR method is a simple, rapid, less-labored, quantitative, highly specific and sensitive assay for RV RNA.

  7. Development of molecular approach based on PCR assay for detection of histamine producing bacteria.

    Science.gov (United States)

    Wongsariya, Karn; Bunyapraphatsara, Nuntavan; Yasawong, Montri; Chomnawang, Mullika Traidej

    2016-01-01

    Histamine fish poisoning becomes highly concern not only in public health but also economic aspect. Histamine is produced from histidine in fish muscles by bacterial decarboxylase enzyme. Several techniques have been developed to determine the level of histamine in fish and their products but the effective method for detecting histamine producing bacteria is still required. This study was attempted to detect histamine producing bacteria by newly developed PCR condition. Histamine producing bacteria were isolated from scombroid fish and determined the ability to produce histamine of isolated bacteria by biochemical and TLC assays. PCR method was developed to target the histidine decarboxylase gene (hdc). The result showed that fifteen histamine producing bacterial isolates and three standard strains produced an amplicon at the expected size of 571 bp after amplified by PCR using Hdc_2F/2R primers. Fifteen isolates of histamine producing bacteria were classified as M. morganii, E. aerogenes, and A. baumannii. The lowest detection levels of M. morganii and E. aerogenes were 10(2) and 10(5) Cfu/mL in culture media and 10(3) and 10(6) Cfu/mL in fish homogenates, respectively. The limit of detection by this method was clearly shown to be sensitive because the primers could detect the presence of M. morganii and E. aerogenes before the histamine level reached the regulation level at 50 ppm. Therefore, this PCR method exhibited the potential efficiency for detecting the hdc gene from histamine producing bacteria and could be used to prevent the proliferation of histamine producing bacteria in fish and fish products.

  8. Development of a qPCR assay for tracking the ecological niches of genetic sub-populations within Pseudo-nitzschia pungens (Bacillariophyceae).

    Science.gov (United States)

    Kim, Jin Ho; Kim, Joo-Hwan; Park, Bum Soo; Wang, Pengbin; Patidar, Shailesh Kumar; Han, Myung-Soo

    2017-03-01

    Three genetic sub-populations (clade I, II and III) of Pseudo-nitzschia pungens, the potential toxic marine diatom, are known to have distinguishable growth characteristics under different culture conditions and distinct distributed patterns in the world. However, to date their exact eco-physiological traits are unrevealed in fields due to lack of the method to detect and/or measure abundances of each sub-populations, hence, the qPCR (quantitative real-time polymerase chain reaction) assay was developed to detect and quantify the P. pungens cells of each clade. Designed two specific primer sets, Pcla12F/R (for clade I and II) and Pcla3F/R (for clade III) only could amplify each target genomic DNA. The, significant linear relationships (R(2)>0.998) was established between Ct (threshold cycle) value and the log of cell abundance for each clade. Through the melting curve analysis, comparisons for gene copy numbers among the three clades and spike test for field study, our qPCR assay was reliable to quantify the cell numbers of each clade. There was strong linear correlation (R(2)>0.990) between cell abundances as estimated by qPCR assay and direct counting via light microscope in spike test, and 0.24 (clade I), 0.25 (clade II) and 0.33 (clade III) P. pungens cells per mL were detected markedly upon the use of specific two-primer set. Finally, developed qPCR assay was applied on field samples successfully. Our study implicate that our qPCR assay is an accurate and sensitive technique to estimate the cell abundances of each clade of P. pungens in field works.

  9. Lab-on-a-Chip-Based PCR-RFLP Assay for the Detection of Malayan Box Turtle (Cuora amboinensis) in the Food Chain and Traditional Chinese Medicines.

    Science.gov (United States)

    Asing; Ali, Md Eaqub; Abd Hamid, Sharifah Bee; Hossain, M A Motalib; Mustafa, Shuhaimi; Kader, Md Abdul; Zaidul, I S M

    2016-01-01

    The Malayan box turtle (Cuora amboinensis) (MBT) is a vulnerable and protected turtle species, but it is a lucrative item in the illegal wildlife trade because of its great appeal as an exotic food item and in traditional medicine. Although several polymerase chain reaction (PCR) assays to identify MBT by various routes have been documented, their applicability for forensic authentication remains inconclusive due to the long length of the amplicon targets, which are easily broken down by natural decomposition, environmental stresses or physiochemical treatments during food processing. To address this research gap, we developed, for the first time, a species-specific PCR-restriction fragment length polymorphism (RFLP) assay with a very short target length (120 bp) to detect MBT in the food chain; this authentication ensured better security and reliability through molecular fingerprints. The PCR-amplified product was digested with Bfa1 endonuclease, and distinctive restriction fingerprints (72, 43 and 5 bp) for MBT were found upon separation in a microfluidic chip-based automated electrophoresis system, which enhances the resolution of short oligos. The chances of any false negative identifications were eliminated through the use of a universal endogenous control for eukaryotes, and the limit of detection was 0.0001 ng DNA or 0.01% of the meat under admixed states. Finally, the optimized PCR-RFLP assay was validated for the screening of raw and processed commercial meatballs, burgers and frankfurters, which are very popular in most countries. The optimized PCR-RFLP assay was further used to screen MBT materials in 153 traditional Chinese medicines of 17 different brands and 62 of them were found MBT positive; wherein the ingredients were not declared in product labels. Overall, the novel assay demonstrated sufficient merit for use in any forensic and/or archaeological authentication of MBT, even under a state of decomposition.

  10. Lab-on-a-Chip-Based PCR-RFLP Assay for the Detection of Malayan Box Turtle (Cuora amboinensis) in the Food Chain and Traditional Chinese Medicines

    Science.gov (United States)

    Asing; Ali, Md. Eaqub; Abd Hamid, Sharifah Bee; Hossain, M. A. Motalib; Mustafa, Shuhaimi; Kader, Md. Abdul; Zaidul, I. S. M.

    2016-01-01

    The Malayan box turtle (Cuora amboinensis) (MBT) is a vulnerable and protected turtle species, but it is a lucrative item in the illegal wildlife trade because of its great appeal as an exotic food item and in traditional medicine. Although several polymerase chain reaction (PCR) assays to identify MBT by various routes have been documented, their applicability for forensic authentication remains inconclusive due to the long length of the amplicon targets, which are easily broken down by natural decomposition, environmental stresses or physiochemical treatments during food processing. To address this research gap, we developed, for the first time, a species-specific PCR-restriction fragment length polymorphism (RFLP) assay with a very short target length (120 bp) to detect MBT in the food chain; this authentication ensured better security and reliability through molecular fingerprints. The PCR-amplified product was digested with Bfa1 endonuclease, and distinctive restriction fingerprints (72, 43 and 5 bp) for MBT were found upon separation in a microfluidic chip-based automated electrophoresis system, which enhances the resolution of short oligos. The chances of any false negative identifications were eliminated through the use of a universal endogenous control for eukaryotes, and the limit of detection was 0.0001 ng DNA or 0.01% of the meat under admixed states. Finally, the optimized PCR-RFLP assay was validated for the screening of raw and processed commercial meatballs, burgers and frankfurters, which are very popular in most countries. The optimized PCR-RFLP assay was further used to screen MBT materials in 153 traditional Chinese medicines of 17 different brands and 62 of them were found MBT positive; wherein the ingredients were not declared in product labels. Overall, the novel assay demonstrated sufficient merit for use in any forensic and/or archaeological authentication of MBT, even under a state of decomposition. PMID:27716792

  11. Detection of Zika Virus in Desiccated Mosquitoes by Real-Time Reverse Transcription PCR and Plaque Assay

    Science.gov (United States)

    Savage, Harry M.

    2017-01-01

    We assayed Zika virus–infected mosquitoes stored at room temperature for <30 days for live virus by using plaque assay and virus RNA by using real-time reverse transcription PCR. Viable virus was detected in samples stored <10 days, and virus RNA was detected in samples held for 30 days. PMID:28075325

  12. In silico and in vitro evaluation of PCR-based assays for the detection of Bacillus anthracis chromosomal signature sequences

    NARCIS (Netherlands)

    Agren, J.; Hamidjaja, R.A.; Hansen, T.; Ruuls, R.C.; Thierry, S.; Vigre, H.; Janse, I.; Sundström, A.; Segerman, B.; Koene, M.G.J.; Löfström, Ch.; Rotterdam, van B.; Derzelle, S.

    2013-01-01

    Bacillus anthracis, the causative agent of anthrax, is a zoonotic pathogen that is relatively common throughout the world and may cause life threatening diseases in animals and humans. There are many PCR-based assays in use for the detection of B. anthracis. While most of the developed assays rely o

  13. A molecular-beacon-based asymmetric PCR assay for easy visualization of amplicons in the diagnosis of trichomoniasis.

    Science.gov (United States)

    Sonkar, Subash C; Sachdev, Divya; Mishra, Prashant K; Kumar, Anita; Mittal, Pratima; Saluja, Daman

    2016-12-15

    The currently available nucleic acid amplification tests (NAATs) for trichomoniasis are accurate, quick and confirmative with superior sensitivity than traditional culture-based microbiology assays. However, these assays are associated with problems of carry over contamination, false positive results, requirement of technical expertise for performance and detection of end product. Hence, a diagnostic assay with easy visualization of the amplified product will be profitable. An in-house, rapid, sensitive, specific molecular-beacon-based PCR assay, using primers against pfoB gene of Trichomonas vaginalis, was developed and evaluated using dry ectocervical swabs (n=392) from symptomatic females with vaginal discharge. Total DNA was isolated and used as template for the PCR assays. The performance and reproducibility of PCR assay was evaluated by composite reference standard (CRS). For easy visualization of the amplified product, molecular-beacon was designed and amplicons were visualized directly using fluorescent handheld dark reader or by Micro-Plate Reader. Molecular-beacons are single-stranded hairpin shaped nucleic acid probes composed of a stem, with fluorophore/quencher pair and a loop region complementary to the desired DNA. The beacon-based PCR assay designed in the present study is highly specific as confirmed by competition experiments and extremely sensitive with detection limit of 20fg of genomic DNA (3-4 pathogens). The minimum infrastructure requirement and ease to perform the assay makes this method highly useful for resource poor countries for better disease management. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. Triplex PCR assay for the rapid identification of 3 major Vibrio species, Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio fluvialis.

    Science.gov (United States)

    Vinothkumar, Kittappa; Bhardwaj, Ashima Kushwaha; Ramamurthy, Thandavarayan; Niyogi, Swapan Kumar

    2013-08-01

    A triplex PCR assay was developed for the identification of 3 major Vibrio spp., Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio fluvialis by targeting their haemolysin, haem-utilizing, and central regulatory genes, respectively. This simple, rapid, sensitive, and specific assay using cell lysates from 227 samples established its usefulness in research and epidemiology. Copyright © 2013 Elsevier Inc. All rights reserved.

  15. Evaluation of a novel PCR-based assay for detection and identification of Chlamydia trachomatis serovars in cervical specimens.

    NARCIS (Netherlands)

    Quint, K.D.; Porras, C.; Safaeian, M.; Gonzalez, P.; Hildesheim, A.; Quint, W.G.V.; Doorn, L.J. van; Silva, S.; Melchers, W.J.G.; Schiffman, M.; Rodriguez, A.C.; Wacholder, S.; Freer, E.; Cortes, B.; Herrero, R.

    2007-01-01

    The aims of this study were to compare a novel PCR-based Chlamydia trachomatis detection and genotyping (Ct-DT) assay with the FDA-approved, commercially available C. trachomatis detection Hybrid Capture 2 (HC2) assay and to investigate the C. trachomatis serovar distribution among young women in a

  16. Development of a Rapid Real-Time PCR Assay for Quantitation of Pneumocystis carinii f. sp. Carinii

    DEFF Research Database (Denmark)

    Larsen, Hans Henrik; Kovacs, Joseph A; Stock, Frida

    2002-01-01

    . In conclusion, a rapid, sensitive, and reproducible quantitative PCR assay for P. carinii f. sp. carinii has been developed and is applicable to in vivo as well as in vitro systems. The assay should prove useful for conducting studies in which quantification of organism burden or growth assessment is critical...

  17. Development and validation of two SYBR green PCR assays and a multiplex real-time PCR for the detection of Shiga toxin-producing Escherichia coli in meat.

    Science.gov (United States)

    Brusa, Victoria; Galli, Lucía; Linares, Luciano H; Ortega, Emanuel E; Lirón, Juan P; Leotta, Gerardo A

    2015-12-01

    Shiga toxin-producing Escherichia coli (STEC) are recognized as food-borne pathogens. We developed and validated two SYBR green PCR (SYBR-PCR) and a real-time multiplex PCR (RT-PCR) to detect stx1 and stx2 genes in meat samples, and compared these techniques in ground beef samples from retail stores. One set of primers and one hydrolysis probe were designed for each stx gene. For RT-PCR, an internal amplification control (IAC) was used. All PCR intra-laboratory validations were performed using pure strains and artificially contaminated ground beef samples. A total of 50 STEC and 30 non-STEC strains were used. Naturally contaminated ground beef samples (n=103) were obtained from retail stores and screened with SYBR-PCR and RT-PCR, and stx-positive samples were processed for STEC isolation. In the intra-laboratory validation, each PCR obtained a 1×10(2) CFU mL(-1) limit of detection and 100% inclusivity and exclusivity. The same results were obtained when different laboratory analysts in alternate days performed the assay. The level of agreement obtained with SYBR-PCR and RT-PCR was kappa=0.758 and 0.801 (P<0.001) for stx1 and stx2 gene detection, respectively. Two PCR strategies were developed and validated, and excellent performance with artificially contaminated ground beef samples was obtained. However, the efforts made to isolate STEC from retail store samples were not enough. Only 11 STEC strains were isolated from 35 stx-positive ground beef samples identically detected by all PCRs. The combination of molecular approaches based on the identification of a virulence genotypic profile of STEC must be considered to improve isolation.

  18. Isolation, identification and differentiation of Campylobacter spp. using multiplex PCR assay from goats in Khartoum State, Sudan.

    Science.gov (United States)

    Elbrissi, Atif; Sabeil, Y A; Khalifa, Khalda A; Enan, Khalid; Khair, Osama M; El Hussein, A M

    2017-03-01

    The aim of this study was to identify and characterize thermophilic Campylobacter species in faecal samples from goats in Khartoum State, Sudan, by application of multiplex polymerase chain reaction. Campylobacteriosis is a zoonotic disease of global concern, and the organisms can be transmitted to human via food, water and through contact with farm animals and pets. There are five clinically related Campylobacter species: Campylobacter jejuni (C. jejuni). Campylobacter coli, Campylobacter lari, Campylobacter upsaliensis and Campylobacter fetus. Conventional cultural methods to diagnose campylobacteriosis are tedious and time consuming. Wide ranges of genes have been reported to be used for PCR-based identification of Campylobacter spp. We used a multiplex PCR assay to simultaneously detect genes from the major five clinically significant Campylobacter spp. The genes selected were hipO (hippuricase) and 23S rRNA from glyA (serine hydroxymethyl transferase) from each of C. jejuni. C. coli, C. lari, and C. upsaliensis; and sapB2 (surface layer protein) from C. fetus subsp. fetus. The assay was used to identify Campylobacter isolates recovered from 336 cultured faecal samples from goats in three localities in Khartoum State. C. coli was the most predominant isolate (234; 69.6%), followed by C. jejuni (19; 5.7%), C. upsaliensis (13; 3.9%), C. fetus subsp. fetus (7; 2.1%) and C. lari (6; 1.8%). Twenty-nine goats showed mixed infection with Campylobacter spp., 21 of which harbored two Campylobacter spp., while eight animals were infected with three species. Ten out of twelve goats that displayed diarrhea harbored C. coli only. C. coli, C. jejuni and C. upsaliensis showed significant variation with localities. The prevalence of C. coli was significantly higher (87; 25.9%) in goats from Omdurman, whereas C. jejuni and C. upsaliensis were significantly higher (11; 3.3%, 9; 2.7%) in goats from Khartoum. The multiplex PCR assay was found to be rapid and easy to perform and

  19. Detection of the rs10250202 polymorphism in protection of telomeres 1 gene through introducing a new restriction enzyme site for PCR-RFLP assay.

    Science.gov (United States)

    Wang, Sihua; Duan, Xiaoran; Wang, Tuanwei; Feng, Xiaolei; Wang, Pengpeng; Yao, Wu; Wu, Yongjun; Wu, Yiming; Yan, Zhen; Feng, Feifei; Yu, Songcheng; Wang, Wei

    2016-01-01

    Human protection of telomeres 1 (POT1) gene is a single stranded telomere binding proteins with a critical role in ensuring chromosome stability. There have been variants of POT1 gene, and the polymorphisms of POT1 gene were associated with some diseases. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) is a traditional method to detect the single nucleotide polymorphism (SNP), and it can be used to detect the polymorphism of rs10250202. But the restriction enzymes required for the detection of the polymorphism of rs10250202 are expensive. So we designed a novel PCR-RFLP assay for genotyping the POT1 rs10250202 SNP. In the study, a new restriction enzyme cutting site was created by created restriction site PCR (CRS-PCR), and the restriction enzyme BclI for CRS-PCR was cheaper than other enzymes. After detecting Han Chinese workers, Allele frequencies were found to be 51.54 % for allele A and 48.46 % for allele C respectively. The PCR results were confirmed by DNA sequencing. CRS-PCR provides a simple, low-cost, practical, and reproducible method.

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

    Directory of Open Access Journals (Sweden)

    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.

  1. Detection of tick blood parasites in Egypt using PCR assay II- Borrelia burgdorferi sensu lato.

    Science.gov (United States)

    Adham, Fatma K; El-Samie-Abd, Emtithal M; Gabre, Refaat M; El Hussein, Hala

    2010-12-01

    The prevalence of Borrelia burgdorferi sensu lato (s.l.), the etiologic agent of Lyme borrelosis (LB), was determined for the first time in Egypt by using polymerase chain reaction (PCR). Questing 5243 hard and soft ticks were collected from animal farms throughout Giza Governorate. DNA from 500 individual tick species was extracted and PCR was performed. Primers verified from the sequence of German strain Pko of Borrelia afzelii were used. Fragments of 642 bp were generated and sequenced. The prevalence of B. burgdorferi sensu lato (s.l.) was 28% of examined soft and hard ticks. High infection rate (66%) of B. burgdorferi s.l. was observed in both nymph and adult soft ticks Ornithodoros savignyi. Beside, the role of hard ticks as potential vectors of Lyme disease in Egypt, where the infection rate was between 0.0-50.0%. Sequence analysis of PCR product of Borrelia burgdorferi sensu lato shares high degree of similarity in sequence compared to similar species in GenBank.

  2. Development of Nested-PCR Assay to Detect Acidovorax citrulli, a Causal Agent of Bacterial Fruit Blotch at Cucurbitaceae

    Directory of Open Access Journals (Sweden)

    Young-Tak Kim

    2015-06-01

    Full Text Available The specific and sensitive nested-PCR method to detect Acidovorax citrulli, a causal agent of bacterial fruit blotch on cucurbitaceae, was developed. PCR primers were designed from the draft genome sequence which was obtained with the Next Generation Sequencing of A. citrulli KACC10651, and the nested-PCR primer set (Ac-ORF 21F/Ac-ORF 21R were selected by checking of specificity to A. citrulli with PCR assays. The selected nested-PCR primer amplified the 140 bp DNA only from A. citrulli strains, and detection sensitivity of the nested PCR increased 10,000 times of 1st PCR detection limit (10 ng genomic DNA/PCR. The nested PCR detected A. citrulli from the all samples of seed surface wash (external seed detection of the artificially inoculated watermelon seeds with 101 cfu/ml and above population of A. citrulli while the nested PCR could not detected A. citrulli from the mashed seed suspension (internal seed detection of the all artificially inoculated watermelon seeds. When the naturally infested watermelon seeds (10% seed infested rate with grow-out test used, the nested PCR detected A. citrulli from 2 seed samples out of 10 replication samples externally and 5 seed samples out of 10 replication samples internally. We believe that the nested-PCR developed in this study will be useful method to detect A. citrulli from the Cucurbitaceae seeds.

  3. Development of a SYBR Green quantitative polymerase chain reaction assay for rapid detection and quantification of infectious laryngotracheitis virus.

    Science.gov (United States)

    Mahmoudian, Alireza; Kirkpatrick, Naomi C; Coppo, Mauricio; Lee, Sang-Won; Devlin, Joanne M; Markham, Philip F; Browning, Glenn F; Noormohammadi, Amir H

    2011-06-01

    Infectious laryngotracheitis is an acute viral respiratory disease of chickens with a worldwide distribution. Sensitive detection of the causative herpesvirus is particularly important because it can persist in the host at a very low copy number and be transmitted to other birds. Quantification of viral genome copy number is also useful for clinical investigations and experimental studies. In the study presented here, a quantitative polymerase chain reaction (qPCR) assay was developed using SYBR Green chemistry and the viral gene UL15a to detect and quantify infectious laryngotracheitis virus (ILTV) in ILTV-inoculated chicken embryos or naturally infected birds. The specificity of the assay was confirmed using a panel of viral and bacterial pathogens of poultry. The sensitivity of the assay was compared with two conventional PCR assays, virus titration and an antigen-detecting enzyme-linked immunosorbent assay. The qPCR developed in this study was highly sensitive and specific, and has potential for quantification of ILTV in tissues from naturally and experimentally infected birds and embryos.

  4. Comparison of PCR-ELISA and LightCycler real-time PCR assays for detecting Salmonella spp. in milk and meat samples

    DEFF Research Database (Denmark)

    Perelle, Sylvie; Dilasser, Françoise; Malorny, Burkhard

    2004-01-01

    In a previous study, we reported the performance of a PCR assay amplifying 285-bp of the invA gene of Salmonella spp. through an international ring-trial involving four participating laboratories [Int. J. Food Microbiol. 89 (2003) 241]. Based on the validated set of primers and recent advancement...

  5. A PCR-RFLP assay to detect and type cytolethal distending toxin (cdt) genes in Campylobacter hyointestinalis

    Science.gov (United States)

    HATANAKA, Noritoshi; KAMEI, Kazumasa; SOMROOP, Srinuan; AWASTHI, Sharda Prasad; ASAKURA, Masahiro; MISAWA, Naoaki; HINENOYA, Atsushi; YAMASAKI, Shinji

    2016-01-01

    Campylobacter hyointestinalis is considered as an emerging zoonotic pathogen. We have recently identified two types of cytolethal distending toxin (cdt) gene in C. hyointestinalis and designated them as Chcdt-I and Chcdt-II. In this study, we developed a PCR-restriction fragment length polymorphism (RFLP) assay that can differentiate Chcdt-I from Chcdt-II. When the PCR-RFLP assay was applied to 17 other Campylobacter strains and 25 non-Campylobacter strains, PCR products were not obtained irrespective of their cdt gene-possession, indicating that the specificity of the PCR-RFLP assay was 100%. In contrast, when the PCR-RFLP assay was applied to 35 C. hyointestinalis strains including 23 analyzed in the previous study and 12 newly isolated from pigs and bovines, all of them showed the presence of cdt genes. Furthermore, a restriction digest by EcoT14-I revealed that 29 strains contained both Chcdt-I and Chcdt-II and 6 strains contained only Chcdt-II, showing 100% sensitivity. Unexpectedly, however, PCR products obtained from 7 C. hyointestinalis strains were not completely digested by EcoT14-I. Nucleotide sequence analysis revealed that the undigested PCR product was homologous to cdtB but not to Chcdt-IB or Chcdt-IIB, indicating the presence of another cdt gene-variant. Then, we further digested the PCR products with DdeI in addition to EcoT14-I, showing that all three cdt genes, including a possible new Chcdt variant, could be clearly differentiated. Thus, the PCR-RFLP assay developed in this study is a valuable tool for evaluating the Chcdt gene-profile of bacteria. PMID:27916784

  6. Validation of a quantitative real-time PCR assay for HTLV-1 proviral load in peripheral blood mononuclear cells.

    Science.gov (United States)

    Rosadas, Carolina; Cabral-Castro, Mauro Jorge; Vicente, Ana Carolina Paulo; Peralta, José Mauro; Puccioni-Sohler, Marzia

    2013-11-01

    The objective of this study was to validate a TaqMan real-time PCR assay for HTLV-1 proviral load detection in peripheral blood mononuclear cells. TARL-2 cells were used to generate a standard curve. Peripheral blood mononuclear cell gDNA from 27 seropositive and 23 seronegative samples was analyzed. The sensitivity, specificity, accuracy, precision, dynamic range of the standard curve and qPCR efficiency were evaluated. All of the positive samples amplified the target gene. All of the negative samples amplified only the control gene (β-actin). The assay presented 100% specificity and sensibility. The intra- and inter-assay variability was 2.4% and 2.2%, respectively. The qPCR efficiency, slope and correlation coefficients (r2) were all acceptable. The limit of detection was 1 copy/rxn. This assay can reliably quantify HTLV-1 proviral load.

  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. 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

  9. NanoPCR observation: different levels of DNA replication fidelity in nanoparticle-enhanced polymerase chain reactions

    Science.gov (United States)

    Shen, Cenchao; Yang, Wenjuan; Ji, Qiaoli; Maki, Hisaji; Dong, Anjie; Zhang, Zhizhou

    2009-11-01

    Nanoparticle-assisted PCR (polymerase chain reaction) technology is getting more and more attention recently. It is believed that some of the DNA recombinant technologies will be upgraded by nanotechnology in the near future, among which DNA replication is one of the core manipulation techniques. So whether or not the DNA replication fidelity is compromised in nanoparticle-assisted PCR is a question. In this study, a total of 16 different metallic and non-metallic nanoparticles (NPs) were tested for their effects on DNA replication fidelity in vitro and in vivo. Sixteen types of nanomaterials were distinctly different in enhancing the PCR efficiency, and their relative capacity to retain DNA replication fidelity was largely different from each other based on rpsL gene mutation assay. Generally speaking, metallic nanoparticles induced larger error rates in DNA replication fidelity than non-metallic nanoparticles, and non-metallic nanomaterials such as carbon nanopowder or nanotubes were still safe as PCR enhancers because they did not compromise the DNA replication fidelity in the Taq DNA polymerase-based PCR system.

  10. Evaluation of a gp63-PCR based assay as a molecular diagnosis tool in canine leishmaniasis in Tunisia.

    Directory of Open Access Journals (Sweden)

    Souheila Guerbouj

    Full Text Available A gp63PCR method was evaluated for the detection and characterization of Leishmania (Leishmania (L. parasites in canine lymph node aspirates. This tool was tested and compared to other PCRs based on the amplification of 18S ribosomal genes, a L. infantum specific repetitive sequence and kinetoplastic DNA minicircles, and to classical parasitological (smear examination and/or culture or serological (IFAT techniques on a sample of 40 dogs, originating from different L. infantum endemic regions in Tunisia. Sensitivity and specificity of all the PCR assays were evaluated on parasitologically confirmed dogs within this sample (N = 18 and control dogs (N = 45 originating from non-endemic countries in northern Europe and Australia. The gp63 PCR had 83.5% sensitivity and 100% specificity, a performance comparable to the kinetoplast PCR assay and better than the other assays. These assays had comparable results when the gels were southern transferred and hybridized with a radioactive probe. As different infection rates were found according to the technique, concordance of the results was estimated by (κ test. Best concordance values were between the gp63PCR and parasitological methods (74.6%, 95% confidence intervals CI: 58.8-95.4% or serology IFAT technique (47.4%, 95% CI: 23.5-71.3%. However, taken together Gp63 and Rib assays covered most of the samples found positive making of them a good alternative for determination of infection rates. Potential of the gp63PCR-RFLP assay for analysis of parasite genetic diversity within samples was also evaluated using 5 restriction enzymes. RFLP analysis confirmed assignment of the parasites infecting the dogs to L. infantum species and illustrated occurrence of multiple variants in the different endemic foci. Gp63 PCR assay thus constitutes a useful tool in molecular diagnosis of L. infantum infections in dogs in Tunisia.

  11. Evaluation of a gp63–PCR Based Assay as a Molecular Diagnosis Tool in Canine Leishmaniasis in Tunisia

    Science.gov (United States)

    Guerbouj, Souheila; Djilani, Fattouma; Bettaieb, Jihene; Lambson, Bronwen; Diouani, Mohamed Fethi; Ben Salah, Afif; Ben Ismail, Riadh; Guizani, Ikram

    2014-01-01

    A gp63PCR method was evaluated for the detection and characterization of Leishmania (Leishmania) (L.) parasites in canine lymph node aspirates. This tool was tested and compared to other PCRs based on the amplification of 18S ribosomal genes, a L. infantum specific repetitive sequence and kinetoplastic DNA minicircles, and to classical parasitological (smear examination and/or culture) or serological (IFAT) techniques on a sample of 40 dogs, originating from different L. infantum endemic regions in Tunisia. Sensitivity and specificity of all the PCR assays were evaluated on parasitologically confirmed dogs within this sample (N = 18) and control dogs (N = 45) originating from non–endemic countries in northern Europe and Australia. The gp63 PCR had 83.5% sensitivity and 100% specificity, a performance comparable to the kinetoplast PCR assay and better than the other assays. These assays had comparable results when the gels were southern transferred and hybridized with a radioactive probe. As different infection rates were found according to the technique, concordance of the results was estimated by (κ) test. Best concordance values were between the gp63PCR and parasitological methods (74.6%, 95% confidence intervals CI: 58.8–95.4%) or serology IFAT technique (47.4%, 95% CI: 23.5–71.3%). However, taken together Gp63 and Rib assays covered most of the samples found positive making of them a good alternative for determination of infection rates. Potential of the gp63PCR-RFLP assay for analysis of parasite genetic diversity within samples was also evaluated using 5 restriction enzymes. RFLP analysis confirmed assignment of the parasites infecting the dogs to L. infantum species and illustrated occurrence of multiple variants in the different endemic foci. Gp63 PCR assay thus constitutes a useful tool in molecular diagnosis of L. infantum infections in dogs in Tunisia. PMID:25153833

  12. Development and validation of a real-time PCR assay for the detection of Toxoplasma gondii DNA in animal and meat samples.

    Science.gov (United States)

    Marino, Anna Maria Fausta; Percipalle, Maurizio; Giunta, Renato Paolo; Salvaggio, Antonio; Caracappa, Giulia; Alfonzetti, Tiziana; Aparo, Alessandra; Reale, Stefano

    2017-03-01

    We report a rapid and reliable method for the detection of Toxoplasma gondii in meat and animal tissues based on real-time polymerase chain reaction (PCR). Samples were collected from cattle, small ruminants, horses, and pigs raised or imported into Sicily, Italy. All DNA preparations were assayed by real-time PCR tests targeted to a 98-bp long fragment in the AF 529-bp repeat element and to the B1 gene using specific primers. Diagnostic sensitivity (100%), diagnostic specificity (100%), limit of detection (0.01 pg), efficiency (92-109%), and precision (mean coefficient of variation = 0.60%), repeatability (100%), reproducibility (100%), and robustness were evaluated using 240 DNA extracted samples (120 positives and 120 negative as per the OIE nested PCR method) from different matrices. Positive results were confirmed by the repetition of both real-time and nested PCR assays. Our study demonstrates the viability of a reliable, rapid, and specific real-time PCR on a large scale to monitor contamination with Toxoplasma cysts in meat and animal specimens. This validated method can be used for postmortem detection in domestic and wild animals and for food safety purposes.

  13. Simultaneous Detection of Six Diarrhea-Causing Bacterial Pathogens with an In-House PCR-Luminex Assay

    Science.gov (United States)

    Gratz, Jean; Maro, Athanasia; Kumburu, Happy; Kibiki, Gibson; Taniuchi, Mami; Howlader, Arif Mahmud; Sobuz, Shihab U.; Haque, Rashidul; Talukder, Kaisar A.; Qureshi, Shahida; Zaidi, Anita; Haverstick, Doris M.; Houpt, Eric R.

    2012-01-01

    Diarrhea can be caused by a range of pathogens, including several bacteria. Conventional diagnostic methods, such as culture, biochemical tests, and enzyme-linked immunosorbent assay (ELISA), are laborious. We developed a 7-plex PCR-Luminex assay to simultaneously screen for several of the major diarrhea-causing bacteria directly in fecal specimens, including pathogenic Aeromonas, Campylobacter jejuni, Campylobacter coli, Salmonella, Shigella, enteroinvasive Escherichia coli (EIEC), Vibrio, and Yersinia. We included an extrinsic control to verify extraction and amplification. The assay was first validated with reference strains or isolates and exhibited a limit of detection of 103 to 105 CFU/g of stool for each pathogen as well as quantitative detection up to 109 CFU/g. A total of 205 clinical fecal specimens from individuals with diarrhea, previously cultured for enteric pathogens and tested for Campylobacter by ELISA, were evaluated. Using these predicate methods as standards, sensitivities and specificities of the PCR-Luminex assay were 89% and 94% for Aeromonas, 89% and 93% for Campylobacter, 96% and 95% for Salmonella, 94% and 94% for Shigella, 92% and 97% for Vibrio, and 100% and 100% for Yersinia, respectively. All discrepant results were further examined by singleplex real-time PCR assays targeting different gene regions, which revealed 89% (55/62 results) concordance with the PCR-Luminex assay. The fluorescent signals obtained with this approach exhibited a statistically significant correlation with the cycle threshold (CT) values from the cognate real-time PCR assays (P < 0.05). This multiplex PCR-Luminex assay enables sensitive, specific, and quantitative detection of the major bacterial causes of gastroenteritis. PMID:22075596

  14. Validation of a newly developed hexaplex real-time PCR assay for screening for presence of GMOs in food, feed and seed.

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    Bahrdt, C; Krech, A B; Wurz, A; Wulff, D

    2010-03-01

    For years, an increasing number and diversity of genetically modified plants has been grown on a commercial scale. The need for detection and identification of these genetically modified organisms (GMOs) calls for broad and at the same time flexible high throughput testing methods. Here we describe the development and validation of a hexaplex real-time polymerase chain reaction (PCR) screening assay covering more than 100 approved GMOs containing at least one of the GMO targets of the assay. The assay comprises detection systems for Cauliflower Mosaic Virus 35S promoter, Agrobacterium tumefaciens NOS terminator, Figwort Mosaic Virus 34S promoter and two construct-specific sequences present in novel genetically modified soybean and maize that lack common screening elements. Additionally a detection system for an internal positive control (IPC) indicating the presence or absence of PCR inhibiting substances was included. The six real-time PCR systems were allocated to five detection channels showing no significant crosstalk between the detection channels. As part of an extensive validation, a limit of detection (LOD(abs)) GMO detection system was still shown in highly asymmetric target situations in the presence of 1,000 copies of all other GMO targets of each detection channel. Furthermore, the applicability to a broad sample spectrum and reliable indication of inhibition by the IPC system was demonstrated. The presented hexaplex assay offers sensitive and reliable detection of GMOs in processed and unprocessed food, feed and seed samples with high efficiency.

  15. 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.

  16. Evaluation of IFN-γ polymorphism+874 T/A in patients with recurrent tonsillitis by PCR real time mismatch amplification mutation assay (MAMA real time PCR).

    Science.gov (United States)

    Bergallo, Massimiliano; Gambarino, Stefano; Loiacono, Elisa; Vergano, Luca; Galliano, Ilaria; Montanari, Paola; Astegiano, Sara; Tavormina, Paolo; Tovo, Pier-Angelo

    2015-02-01

    Interferon gamma (IFN-γ) is an important cytokine that plays a crucial role in the balance between normal and pathological immune response. Defect of IFN-γ can give a predisposition to infectious disease, autoimmune pathologies and tumours. Different polymorphisms in this gene have been described, in particular the single nucleotide polymorphism (SNP)+874∗T/A that may affect IFN-γ gene expression. Several techniques can be used for the detection of SNPs. In this work two PCR Real Time assays were developed, an Amplification Refractory Mutation System (ARMS) and a Mismatch Amplification Mutation Assay (MAMA). Twenty-seven samples from patients (tonsillectomy) and 85 from donor's blood bank were considered. As a result, 78/85 controls (91.7%) and 25/27 patients (92.6%) were heterozygosis, considering the ARMS-PCR; 55/85 (64.7%) and 14/27 (51.9%) were heterozygosis using MAMA-PCR assay. Fourteen of 85 (16.5%) and 8/27 (29.6%) were homozygosis A, 16/85 (18.8%) and 5/27 (18.5%) presented homozygosis T, taking into account the MAMA-PCR. There are statistically difference between the two assay with p<0.0001 at Chi-square test. Our preliminary data suggest that tonsillectomy patients had a statistical trend to possess the low IFN-γ polymorphism when compared with control subject (p=0.3) but is not statistically significant. In conclusion the Real time MAMA-PCR assay has several advantages over other SNP identification techniques such as rapidity, reliability, easily to perform in one working day and applicable in clinical molecular diagnostic laboratories, although sequencing remains the gold standard.

  17. Performance of a real-time PCR assay for the rapid identification of Mycobacterium species.

    Science.gov (United States)

    Wang, Hye-young; Kim, Hyunjung; Kim, Sunghyun; Kim, Do-kyoon; Cho, Sang-Nae; Lee, Hyeyoung

    2015-01-01

    Mycobacteria cause a variety of illnesses that differ in severity and public health implications. The differentiation of Mycobacterium tuberculosis (MTB) from nontuberculous mycobacteria (NTM) is of primary importance for infection control and choice of antimicrobial therapy. The diagnosis of diseases caused by NTM is difficult because NTM species are prevalent in the environment and because they have fastidious properties. In the present study, we evaluated 279 clinical isolates grown in liquid culture provided by The Catholic University of Korea, St. Vincent's Hospital using real-time PCR based on mycobacterial rpoB gene sequences. The positive rate of real-time PCR assay accurately discriminated 100% (195/195) and 100% (84/84) between MTB and NTM species. Comparison of isolates identified using the MolecuTech REBA Myco-ID(®) and Real Myco-ID® were completely concordant except for two samples. Two cases that were identified as mixed infection (M. intracellulare-M. massiliense and M. avium-M. massiliense co-infection) by PCRREBA assay were only detected using M. abscessus-specific probes by Real Myco-ID(®). Among a total of 84 cases, the most frequently identified NTM species were M. intracellulare (n=38, 45.2%), M. avium (n=18, 23.7%), M. massiliense (n=10, 13.2%), M. fortuitum (n=5, 6%), M. abscessus (n=3, 3.9%), M. gordonae (n=3, 3.9%), M. kansasii (n=2, 2.4%), M. mucogenicum (n=2, 2.4%), and M. chelonae (n= 1, 1.2%). Real Myco-ID(®) is an efficient tool for the rapid detection of NTM species as well as MTB and sensitive and specific and comparable to conventional methods.

  18. A duplex PCR assay for the detection of Ralstonia solanacearum phylotype II strains in Musa spp.

    Directory of Open Access Journals (Sweden)

    Gilles Cellier

    Full Text Available Banana wilt outbreaks that are attributable to Moko disease-causing strains of the pathogen Ralstonia solanacearum (Rs remain a social and economic burden for both multinational corporations and subsistence farmers. All known Moko strains belong to the phylotype II lineage, which has been previously recognized for its broad genetic basis. Moko strains are paraphyletic and are distributed among seven related but distinct phylogenetic clusters (sequevars that are potentially major threats to Musaceae, Solanaceae, and ornamental crops in many countries. Although clustered within the Moko IIB-4 sequevar, strains of the epidemiologically variant IIB-4NPB do not cause wilt on Cavendish or plantain bananas; instead, they establish a latent infection in the vascular tissues of plantains and demonstrate an expanded host range and high aggressiveness toward Solanaceae and Cucurbitaceae. Although most molecular diagnostic methods focus on strains that wilt Solanaceae (particularly potato, no relevant protocol has been described that universally detects strains of the Musaceae-infecting Rs phylotype II. Thus, a duplex PCR assay targeting Moko and IIB-4NPB variant strains was developed, and its performance was assessed using an extensive collection of 111 strains representing the known diversity of Rs Moko-related strains and IIB-4NPB variant strains along with certain related strains and families. The proposed diagnostic protocol demonstrated both high accuracy (inclusivity and exclusivity and high repeatability, detected targets on either pure culture or spiked plant extracts. Although they did not belong to the Moko clusters described at the time of the study, recently discovered banana-infecting strains from Brazil were also detected. According to our comprehensive evaluation, this duplex PCR assay appears suitable for both research and diagnostic laboratories and provides reliable detection of phylotype II Rs strains that infect Musaceae.

  19. Biofunctionalization of Polyoxometalates with DNA Primers, Their Use in the Polymerase Chain Reaction (PCR) and Electrochemical Detection of PCR Products.

    Science.gov (United States)

    Debela, Ahmed M; Ortiz, Mayreli; Beni, Valerio; Thorimbert, Serge; Lesage, Denis; Cole, Richard B; O'Sullivan, Ciara K; Hasenknopf, Bernold

    2015-12-01

    The bioconjugation of polyoxometalates (POMs), which are inorganic metal oxido clusters, to DNA strands to obtain functional labeled DNA primers and their potential use in electrochemical detection have been investigated. Activated monooxoacylated polyoxotungstates [SiW11 O39 {Sn(CH2 )2 CO}](8-) and [P2 W17 O61 {Sn(CH2 )2 CO}](6-) have been used to link to a 5'-NH2 terminated 21-mer DNA forward primer through amide coupling. The functionalized primer was characterized by using a battery of techniques, including electrophoresis, mass spectrometry, as well as IR and Raman spectroscopy. The functionality of the POM-labeled primers was demonstrated through hybridization with a surface-immobilized probe. Finally, the labeled primers were successfully used in the polymerase chain reaction (PCR) and the PCR products were characterized by using electrophoresis.

  20. Lyophilized standards for the calibration of real time PCR assay for hepatitis C virus RNA

    Institute of Scientific and Technical Information of China (English)

    WANG Lu-nan; WU Jian-min; DENG Wei; SHEN Zi-yu; CHEN Wen-xiang; LI Jin-ming

    2006-01-01

    Background Since October 1997, an international standard for hepatitis C virus (HCV) nucleic acid amplification technology assay, 96/790, has been available. We compared a series of lyophilized standards with known HCV RNA concentrations against the international standard in fluorescence quantitative PCR detection.Methods A series of lyophilized sera were calibrated by ROCHE COBAS AMPLICOR HCV Monitor test against the international standard and sent to various manufacturers to analyse the samples using their own kits.Then calibration curves from the series were compared with that obtained from the external standard calibration curve with the manufacture's series.Results The standard calibration curve with the series of lyophilized serum showed an excellent correlation(R2>0.98), slope and intercept that were similar to those from the manufacture's series. When the standard calibration curve from the series of lyophilized standards were used to define the values of the given sample,lower coefficients of variation between kits from different manufactures were obtained.Conclusion The results showed that the lyophilized standards could be used to setup the standard calibration curve for clinical HCV RNA quantitative PCR detection.

  1. A real time PCR assay on blood for diagnosis of invasive candidiasis in immunocompromised patient

    Directory of Open Access Journals (Sweden)

    Mohsen Ashrafi

    2015-01-01

    Results: From 2009 to 2011, 72 patients with hematologic malignancies and bone marrow transplant recipients were evaluated for IC. The female to male ratio was 27:45; the mean age was 32.1 years. The most common malignancy in this patient was acute myeloid leukemia (AML (27.8% and acute lymphoblastic leukemia (ALL (26.4%. Out of 72 patients, 11 patients (15.3% had positive real time PCR /probe results. Based on the melting temperature (Tm analysis, 5 (45.4% C. krusei, 3 (27.2% C. tropicalis, 2 (18.1% C. parapsilosis and 1 C. albicans (9% were identified. According to the revised EORTC / MSG, 1 patient (9% and 10 patients (91% were defined as proven and possible groups of IC, respectively. The mortality rate in proven and possible IC patient was found 54.5%. Conclusion: The established Real-time PCR/FRET probe assay is an appropriate diagnostic tool for the detection of Candida species DNA and the management of patients suffering from hematologic malignancies and bone marrow recipient are at risk for IC.

  2. Identification of cytoplasm types in rapeseed (Brassica napus L.) accessions by a multiplex PCR assay.

    Science.gov (United States)

    Zhao, H X; Li, Z J; Hu, S W; Sun, G L; Chang, J J; Zhang, Z H

    2010-08-01

    Cytoplasmic male sterility (CMS) has widely been used as an efficient pollination control system in rapeseed hybrid production. Identification of cytoplasm type of rapeseed accessions is becoming the most important basic work for hybrid-rapeseed breeding. In this study, we report a simple multiplex PCR method to distinguish the existing common cytoplasm resources, Pol, Nap, Cam, Ogu and Ogu-NWSUAF cytoplasm, in rapeseed. Cytoplasm type of 35 F(1) hybrids and 140 rapeseed open pollinated varieties or breeding lines in our rapeseed breeding programme were tested by this method. The results indicated that 10 of 35 F(1) hybrids are the Nap, and 25 the Pol cytoplasm type, which is consistent with the information provided by the breeders. Out of 140 accessions tested, 100 (71.4%), 21 (15%) and 19 (13.6%) accessions possess Nap, Cam and Pol cytoplasm, respectively. All 19 accessions with Pol cytoplasm are from China. Pedigree analysis indicated that these accessions with Pol cytoplasm were either restorers for Pol CMS, including Shaan 2C, Huiyehui, 220, etc. or derived from hybrids with Pol CMS as female parent. Our molecular results are consistent with those of the classical testcross, suggesting the reliability of this method. The multiplex PCR assay method can be applied to CMS "three-line" breeding, selection and validation of hybrid rapeseed.

  3. CRISPR is an optimal target for the design of specific PCR assays for salmonella enterica serotypes Typhi and Paratyphi A.

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    Laetitia Fabre

    Full Text Available BACKGROUND: Serotype-specific PCR assays targeting Salmonella enterica serotypes Typhi and Paratyphi A, the causal agents of typhoid and paratyphoid fevers, are required to accelerate formal diagnosis and to overcome the lack of typing sera and, in some situations, the need for culture. However, the sensitivity and specificity of such assays must be demonstrated on large collections of strains representative of the targeted serotypes and all other bacterial populations producing similar clinical symptoms. METHODOLOGY: Using a new family of repeated DNA sequences, CRISPR (clustered regularly interspaced short palindromic repeats, as a serotype-specific target, we developed a conventional multiplex PCR assay for the detection and differentiation of serotypes Typhi and Paratyphi A from cultured isolates. We also developed EvaGreen-based real-time singleplex PCR assays with the same two sets of primers. PRINCIPAL FINDINGS: We achieved 100% sensitivity and specificity for each protocol after validation of the assays on 188 serotype Typhi and 74 serotype Paratyphi A strains from diverse genetic groups, geographic origins and time periods and on 70 strains of bacteria frequently encountered in bloodstream infections, including 29 other Salmonella serotypes and 42 strains from 38 other bacterial species. CONCLUSIONS: The performance and convenience of our serotype-specific PCR assays should facilitate the rapid and accurate identification of these two major serotypes in a large range of clinical and public health laboratories with access to PCR technology. These assays were developed for use with DNA from cultured isolates, but with modifications to the assay, the CRISPR targets could be used in the development of assays for use with clinical and other samples.

  4. Improved PCR-Based Detection of Soil Transmitted Helminth Infections Using a Next-Generation Sequencing Approach to Assay Design

    Science.gov (United States)

    Pilotte, Nils; Papaiakovou, Marina; Grant, Jessica R.; Bierwert, Lou Ann; Llewellyn, Stacey; McCarthy, James S.; Williams, Steven A.

    2016-01-01

    Background The soil transmitted helminths are a group of parasitic worms responsible for extensive morbidity in many of the world’s most economically depressed locations. With growing emphasis on disease mapping and eradication, the availability of accurate and cost-effective diagnostic measures is of paramount importance to global control and elimination efforts. While real-time PCR-based molecular detection assays have shown great promise, to date, these assays have utilized sub-optimal targets. By performing next-generation sequencing-based repeat analyses, we have identified high copy-number, non-coding DNA sequences from a series of soil transmitted pathogens. We have used these repetitive DNA elements as targets in the development of novel, multi-parallel, PCR-based diagnostic assays. Methodology/Principal Findings Utilizing next-generation sequencing and the Galaxy-based RepeatExplorer web server, we performed repeat DNA analysis on five species of soil transmitted helminths (Necator americanus, Ancylostoma duodenale, Trichuris trichiura, Ascaris lumbricoides, and Strongyloides stercoralis). Employing high copy-number, non-coding repeat DNA sequences as targets, novel real-time PCR assays were designed, and assays were tested against established molecular detection methods. Each assay provided consistent detection of genomic DNA at quantities of 2 fg or less, demonstrated species-specificity, and showed an improved limit of detection over the existing, proven PCR-based assay. Conclusions/Significance The utilization of next-generation sequencing-based repeat DNA analysis methodologies for the identification of molecular diagnostic targets has the ability to improve assay species-specificity and limits of detection. By exploiting such high copy-number repeat sequences, the assays described here will facilitate soil transmitted helminth diagnostic efforts. We recommend similar analyses when designing PCR-based diagnostic tests for the detection of other

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

    Science.gov (United States)

    Xie, Zhixun; Xie, Zhiqin; Deng, Xianwen; Xie, Liji; Huang, Li; Luo, Sisi; Huang, Jiaoling; Zhang, Yanfang; Zeng, Tingting; Wang, Sheng; Liu, Jiabo; Pang, Yaoshan

    2017-01-01

    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 of clinical

  6. Utility of IgM ELISA, TaqMan real-time PCR, reverse transcription PCR, and RT-LAMP assay for the diagnosis of Chikungunya fever.

    Science.gov (United States)

    Reddy, Vijayalakshmi; Ravi, Vasanthapuram; Desai, Anita; Parida, Manmohan; Powers, Ann M; Johnson, Barbara W

    2012-11-01

    Chikungunya fever a re-emerging infection with expanding geographical boundaries, can mimic symptoms of other infections like dengue, malaria which makes the definitive diagnosis of the infection important. The present study compares the utility of four laboratory diagnostic methods viz. IgM capture ELISA, an in house reverse transcription PCR for the diagnosis of Chikungunya fever, TaqMan real-time PCR, and a one step reverse transcription-loop mediated isothermal amplification assay (RT-LAMP). Out of the 70 serum samples tested, 29 (41%) were positive for Chikungunya IgM antibody by ELISA and 50 (71%) samples were positive by one of the three molecular assays. CHIKV specific nucleic acid was detected in 33/70 (47%) by reverse transcription PCR, 46/70 (66%) by TaqMan real-time PCR, and 43/70 (62%) by RT-LAMP assay. A majority of the samples (62/70; 89%) were positive by at least one of the four assays used in the study. The molecular assays were more sensitive for diagnosis in the early stages of illness (2-5 days post onset) when antibodies were not detectable. In the later stages of illness, the IgM ELISA is a more sensitive diagnostic test. In conclusion we recommend that the IgM ELISA be used as an initial screening test followed one of the molecular assays in samples that are collected in the early phase of illness and negative for CHIKV IgM antibodies. Such as approach would enable rapid confirmation of the diagnosis and implementation of public health measures especially during outbreaks. Copyright © 2012 Wiley Periodicals, Inc.

  7. A multiplex nested PCR assay for simultaneous detection of Corchorus golden mosaic virus and a phytoplasma in white jute (Corchorus capsularis L.).

    Science.gov (United States)

    Biswas, C; Dey, P; Satpathy, S

    2013-05-01

    A multiplex nested PCR assay was developed by optimizing reaction components and reaction cycling parameters for simultaneous detection of Corchorus golden mosaic virus (CoGMV) and a phytoplasma (Group 16Sr V-C) causing little leaf and bunchy top in white jute (Corchorus capsularis). Three sets of specific primers viz. a CoGMV specific (DNA-A region) primer, a 16S rDNA universal primer pair P1/P7 and nested primer pair R16F2n/R2 for phytoplasmas were used. The concentrations of the PCR components such as primers, MgCl2 , Taq DNA polymerase, dNTPs and PCR conditions including annealing temperature and amplification cycles were examined and optimized. Expected fragments of 1 kb (CoGMV), 674 bp (phytoplasma) and 370 bp (nested R16F2n/R2) were successfully amplified by this multiplex nested PCR system ensuring simultaneous, sensitive and specific detection of the phytoplasma and the virus. The multiplex nested PCR provides a sensitive, rapid and low-cost method for simultaneous detection of jute little leaf phytoplasma and CoGMV. Based on BLASTn analyses, the phytoplasma was found to belong to the Group 16Sr V-C. © 2013 The Society for Applied Microbiology.

  8. PEMERIKSAAN BAKTERI LEPTOSPIRA PADA SAMPEL DARAH MANUSIA SUSPECT LEPTOSPIROSIS MENGGUNAKAN METODE PCR (POLYMERASE CHAIN REACTION

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    Sefrita Tri Utami

    2014-01-01

    Full Text Available ABSTRACTLeptospirosis is a zoonotic disease, which is caused by leptospira. Leptospirosis cases often show no specificclinical symptoms and is difficult to diagnose without testing samples in the laboratory. Testing using PCR(Polymerase Chain Reaction is considered more accurate than the other methods. Components required in theexamination Leptospira bacteria in human blood samples using PCR method is DNA template, DNA polymeraseenzyme, forward primer (PU1 and SU1 and reverse primer (Lep R1, nuclease free water, Mg 2 +, and dNTPs.Examination of Leptospira bacteria in human blood samples include sampling, DNA isolation, examination byPCR, and electrophoresis running.Key words: leptospirosis, Leptospira, PCR methodsABSTRAKLeptospirosis adalah penyakit zoonosis yang disebabkan oleh bakteri Leptospira. Kasus leptospirosis seringtidak menunjukkan gejala klinis yang spesifik dan sulit didiagnosis tanpa pengujian sampel di laboratorium.Pengujian dengan menggunakan metode PCR (Polymerase Chain Reaction dinilai lebih akurat dibandingkandengan metode yang lain. Komponen-komponen yang dibutuhkan dalam pemeriksaan bakteri Leptospira padasampel darah manusia menggunakan metode PCR adalah DNA template, enzim polymerase, Primer PU 1 danPrimer SU 1, Primer Lep R1, air, Mg2+ , dan dNTP. Pemeriksaan bakteri Leptospira pada sampel darah manusiameliputi pengambilan sampel, isolasi DNA, pemeriksaan dengan metode PCR, dan running elektroforesis.Kata kunci: leptospirosis, Leptospira, metode PCR

  9. Droplet digital polymerase chain reaction (PCR) outperforms real-time PCR in the detection of environmental DNA from an invasive fish species.

    Science.gov (United States)

    Doi, Hideyuki; Takahara, Teruhiko; Minamoto, Toshifumi; Matsuhashi, Saeko; Uchii, Kimiko; Yamanaka, Hiroki

    2015-05-01

    Environmental DNA (eDNA) has been used to investigate species distributions in aquatic ecosystems. Most of these studies use real-time polymerase chain reaction (PCR) to detect eDNA in water; however, PCR amplification is often inhibited by the presence of organic and inorganic matter. In droplet digital PCR (ddPCR), the sample is partitioned into thousands of nanoliter droplets, and PCR inhibition may be reduced by the detection of the end-point of PCR amplification in each droplet, independent of the amplification efficiency. In addition, real-time PCR reagents can affect PCR amplification and consequently alter detection rates. We compared the effectiveness of ddPCR and real-time PCR using two different PCR reagents for the detection of the eDNA from invasive bluegill sunfish, Lepomis macrochirus, in ponds. We found that ddPCR had higher detection rates of bluegill eDNA in pond water than real-time PCR with either of the PCR reagents, especially at low DNA concentrations. Limits of DNA detection, which were tested by spiking the bluegill DNA to DNA extracts from the ponds containing natural inhibitors, found that ddPCR had higher detection rate than real-time PCR. Our results suggest that ddPCR is more resistant to the presence of PCR inhibitors in field samples than real-time PCR. Thus, ddPCR outperforms real-time PCR methods for detecting eDNA to document species distributions in natural habitats, especially in habitats with high concentrations of PCR inhibitors.

  10. Multiplex Real-Time PCR Assay with High-Resolution Melting Analysis for Characterization of Antimicrobial Resistance in Neisseria gonorrhoeae.

    Science.gov (United States)

    Donà, Valentina; Kasraian, Sara; Lupo, Agnese; Guilarte, Yuvia N; Hauser, Christoph; Furrer, Hansjakob; Unemo, Magnus; Low, Nicola; Endimiani, Andrea

    2016-08-01

    Resistance to antibiotics used against Neisseria gonorrhoeae infections is a major public health concern. Antimicrobial resistance (AMR) testing relies on time-consuming culture-based methods. Development of rapid molecular tests for detection of AMR determinants could provide valuable tools for surveillance and epidemiological studies and for informing individual case management. We developed a fast (<1.5-h) SYBR green-based real-time PCR method with high-resolution melting (HRM) analysis. One triplex and three duplex reactions included two sequences for N. gonorrhoeae identification and seven determinants of resistance to extended-spectrum cephalosporins (ESCs), azithromycin, ciprofloxacin, and spectinomycin. The method was validated by testing 39 previously fully characterized N. gonorrhoeae strains, 19 commensal Neisseria species strains, and an additional panel of 193 gonococcal isolates. Results were compared with results of culture-based AMR determination. The assay correctly identified N. gonorrhoeae and the presence or absence of the seven AMR determinants. There was some cross-reactivity with nongonococcal Neisseria species, and the detection limit was 10(3) to 10(4) genomic DNA (gDNA) copies/reaction. Overall, the platform accurately detected resistance to ciprofloxacin (sensitivity and specificity, 100%), ceftriaxone (sensitivity, 100%; specificity, 90%), cefixime (sensitivity, 92%; specificity, 94%), azithromycin (sensitivity and specificity, 100%), and spectinomycin (sensitivity and specificity, 100%). In conclusion, our methodology accurately detects mutations that generate resistance to antibiotics used to treat gonorrhea. Low assay sensitivity prevents direct diagnostic testing of clinical specimens, but this method can be used to screen collections of gonococcal isolates for AMR more quickly than current culture-based AMR testing.

  11. Development and evaluation of multiplex PCR assays for rapid detection of virulence-associated genes in Arcobacter species.

    Science.gov (United States)

    Whiteduck-Léveillée, Jenni; Cloutier, Michel; Topp, Edward; Lapen, David R; Talbot, Guylaine; Villemur, Richard; Khan, Izhar U H

    2016-02-01

    As the pathogenicity of Arcobacter species might be associated with various virulence factors, this study was aimed to develop and optimize three single-tube multiplex PCR (mPCR) assays that can efficiently detect multiple virulence-associated genes (VAGs) in Arcobacter spp. including the Arcobacter butzleri, Arcobacter cryaerophilus and Arcobacter skirrowii, respectively. The recognized target virulence factors used in the study were fibronectin binding protein (cj1349), filamentous hemagglutinin (hecA), hemolysin activation protein (hecB), hemolysin (tlyA), integral membrane protein virulence factor (mviN), invasin (ciaB), outer membrane protein (irgA) and phospholipase (pldA). Identical results were obtained between singleplex PCR and mPCR assays and no cross- and/or non-specific amplification products were obtained when tested against other closely related bacterial species. The sensitivities of these three mPCR assays were ranging from 1ngμL(-1) to 100ngμL(-1) DNA. The developed assays with combinations of duplex or triplex PCR primer pairs of VAGs were further evaluated and validated by applying them to isolates of the A. butzleri, A. cryaerophilus and A. skirrowii recovered from fecal samples of human and animal origins. The findings revealed that the distribution of the ciaB (90%), mviN (70%), tlyA (50%) and pldA (45%) genes among these target species was significantly higher than the hecA (16%), hecB (10%) and each of irgA and cj1349 (6%) genes, respectively. The newly developed mPCR assays can be used as rapid technique and useful markers for the detection, prevalence and profiling of VAGs in the Arcobacter spp. Moreover, these assays can easily be performed with a high throughput to give a presumptive identification of the causal pathogen in epidemiological investigation of human infections.

  12. Detection of Campylobacter jejuni and Campylobacter coli in environmental waters by PCR enzyme-linked immunosorbent assay.

    Science.gov (United States)

    Sails, A D; Bolton, F J; Fox, A J; Wareing, D R A; Greenway, D L A

    2002-03-01

    A PCR enzyme-linked immunosorbent assay (ELISA) assay was applied to the detection of Campylobacter jejuni and Campylobacter coli in environmental water samples after enrichment culture. Bacterial cells were concentrated from 69 environmental water samples by using filtration, and the filtrates were cultured in Campylobacter blood-free broth. After enrichment culture, DNA was extracted from the samples by using a rapid-boiling method, and the DNA extracts were used as a template in a PCR ELISA assay. A total of 51 samples were positive by either PCR ELISA or culture; of these, 43 were found to be positive by PCR ELISA and 43 were found to be positive by culture. Overall, including positive and negative results, 59 samples were concordant in both methods. Several samples were positive in the PCR ELISA assay but were culture negative; therefore, this assay may be able to detect sublethally damaged or viable nonculturable forms of campylobacters. The method is rapid and sensitive, and it significantly reduces the time needed for the detection of these important pathogens by 2 to 3 days.

  13. Development of a rapid PCR assay specific for Staphylococcus saprophyticus and application to direct detection from urine samples.

    Science.gov (United States)

    Martineau, F; Picard, F J; Ménard, C; Roy, P H; Ouellette, M; Bergeron, M G

    2000-09-01

    Staphylococcus saprophyticus is one of the most frequently encountered microorganisms associated with acute urinary tract infections (UTIs) in young, sexually active female outpatients. Conventional identification methods based on biochemical characteristics can efficiently identify S. saprophyticus, but the rapidities of these methods need to be improved. Rapid and direct identification of this bacterium from urine samples would be useful to improve time required for the diagnosis of S. saprophyticus infections in the clinical microbiology laboratory. We have developed a PCR-based assay for the specific detection of S. saprophyticus. An arbitrarily primed PCR amplification product of 380 bp specific for S. saprophyticus was sequenced and used to design a set of S. saprophyticus-specific PCR amplification primers. The PCR assay was specific for S. saprophyticus when tested with DNA from 49 gram-positive and 31 gram-negative bacterial species. This assay was also able to amplify efficiently DNA from all 60 strains of S. saprophyticus from various origins tested. This assay was adapted for direct detection from urine samples. The sensitivity levels achieved with urine samples was 19 CFU with 30 cycles of amplification and 0.5 CFU with 40 cycles of amplification. This PCR assay for the specific detection of S. saprophyticus is simple and rapid (approximately 90 min, including the time for urine specimen preparation).

  14. Duplex Real-Time RT-PCR Assays for the Detection and Typing of Epizootic Haemorrhagic Disease Virus

    Science.gov (United States)

    Viarouge, Cyril; Breard, Emmanuel; Zientara, Stephan; Vitour, Damien; Sailleau, Corinne

    2015-01-01

    Epizootic haemorrhagic disease virus (EHDV) may cause severe clinical episodes in some species of deer and sometimes in cattle. Laboratory diagnosis provides a basis for the design and timely implementation of disease control measures. There are seven distinct EHDV serotypes, VP2 coding segment 2 being the target for serotype specificity. This paper reports the development and validation of eight duplex real-time RT-PCR assays to simultaneously amplify the EHDV target (S9 for the pan-EHDV real-time RT-PCR assay and S2 for the serotyping assays) and endogenous control gene Beta-actin. Analytical and diagnostic sensitivity and specificity, inter- and intra-assay variation and efficiency were evaluated for each assay. All were shown to be highly specific and sensitive. PMID:26161784

  15. Improved diagnostic PCR assay for Actinobacillus pleuropneumoniae based on the nucleotide sequence of an outer membrane lipoprotein

    DEFF Research Database (Denmark)

    Gram, Trine; Ahrens, Peter

    1998-01-01

    The gene (omlA) coding for an outer membrane protein of Actinobacillus pleuropneumoniae serotypes 1 and 5 has been described earlier and has formed the basis for development of a specific PCR assay, The corresponding regions of all 12 A. pleuropneumoniae reference strains of biovar 1 were sequenc...... and sensitivity of this PCR compared to those of culture suggest the use of this PCR for routine identification of A. pleuropneumoniae.......The gene (omlA) coding for an outer membrane protein of Actinobacillus pleuropneumoniae serotypes 1 and 5 has been described earlier and has formed the basis for development of a specific PCR assay, The corresponding regions of all 12 A. pleuropneumoniae reference strains of biovar 1 were sequenced...... species related to A. pleuropneumoniae or isolated from pigs were assayed. They were all found negative in the PCR, as were tonsil cultures from 50 pigs of an A. pleuropneumoniae-negative herd. The sensitivity assessed by agarose gel analysis of the PCR product was 10(2) CFU/PCR test tube. The specificity...

  16. Real time RT-PCR assay for detection of different serotypes of FMDV in Egypt

    Directory of Open Access Journals (Sweden)

    Laila El-Shehawy

    Full Text Available Aim: The present study indicated that rRT-PCR could be provided for the detection of FMDV in infected, contact and carrier cattle and also provide a rapid sensitive tool aiming to aid in rapid disease detection and control. Foot and Mouth disease virus serotypes O and A still existing in Egypt. In January 2012, sever outbreaks struck the animal population in most Egyptian 1 governorates. The causative virus was identified as FMDV SAT2. Material and Methods: Five samples of tongue epithelium (ET and five oesophageal-pharyngeal (OP fluid samples were collected from FMD suspected cattle in infected farm at El-Fayoum and 20 OP samples from in-contact cattle at the same farm in addition to 30 OP samples from apparently healthy cattle at three different localities in El-Fayoum governorate (12 from Fayoum; 9 from Sinoras and 9 from Edsa in order to detect carrier cattle. All of these samples were collected during November and December 2011 and January 2012. Results: All the ET and OP samples were inoculated on BHK cell culture and baby mice. The obtained results were identified using complement fixation test in addition to real-time reverse transcriptase polymerase chain reaction (rRT-PCR. In the infected farm at El-Fayoum FMDV type SAT2 was detected in cattle which are considered as the first introduction of this type while FMDV type O and SAT2 were detected in the in-contact cattle in the same farm. The sensitivity of rRT-PCR was cleared in the in-contact cattle as 13 out of 20 OP samples were positive to FMDV by rRT-PCR while 11 out of 20 OP samples were positive to FMDV by CFT. The OP samples collected from apparently healthy cattle from Fayoum, Sinoras and Edsa localities in Fayoum governorate demonstrate the circulation of the FMDV type A, O and the recent SAT2 in carrier cattle which threaten cattle population in Fayoum governorate. Also the sensitivity of real time RT-PCR over the CFT in detection of FMDV carrier cattle was clearly noticed in

  17. Comparative evaluation of a competitive polymerase chain reaction (PCR) and a SYBR Green-based real-time PCR to quantify Porcine circovirus-2 DNA in swine tissue samples.

    Science.gov (United States)

    Dezen, Diogenes; Rijsewijk, Franciscus A M; Teixeira, Thais F; Holz, Carine L; Varela, Ana P; Cibulski, Samuel P; Gregianini, Tatiane Shäffer; Batista, Helena B C R; Franco, Ana C; Roehe, Paulo M

    2011-11-01

    Porcine circovirus-2 (PCV-2) is considered the major etiological agent of post-weaning multisystemic wasting syndrome (PMWS) in pigs. The clinical manifestations of the disease are correlated with moderate to high amounts of PCV-2 DNA in biological samples of affected pigs. A threshold of 10(7) DNA copies/ml is suggested as the trigger factor for symptoms. A comparative study was conducted to determine which quantitative method would be more suitable to estimate the PCV-2 DNA load. Two polymerase chain reaction (PCR) assays were developed: a competitive PCR (cPCR) and a SYBR Green-based real-time PCR. The assays were compared for their capacity to detect PCV-2 in DNA samples extracted from liver, lung, spleen, mesenteric lymph nodes, and kidney of PMWS-affected (n = 23) or non-PMWS-affected pigs (n = 9). Both assays could successfully quantify PCV-2 DNA in all tissue samples and were able to detect significant differences between the numbers of PCV-2 DNA copies found in tissues of PMWS-affected and non-PMWS-affected pigs (≥ 10(2.5)). The highest mean viral loads were detected by the SYBR Green real-time PCR, up to 10(7.0 ± 1.5) copies/100 ng of total DNA sample, while the cPCR detected up to 10(4.8 ± 1.5). A mean difference of 10(1.8) was found between the amounts of PCV-2 DNA detected, using the SYBR Green real-time PCR and the cPCR, suggesting that the viral load threshold for PMWS should be determined for each particular assay.

  18. Development of a nested PCR assay to detect the pathogenic free-living amoeba Naegleria fowleri.

    Science.gov (United States)

    Réveiller, Fabienne L; Cabanes, Pierre-André; Marciano-Cabral, Francine

    2002-05-01

    Naegleria fowleri is the causative agent of primary amoebic meningoencephalitis, a fatal disease of the central nervous system that is acquired while swimming or diving in freshwater. A cDNA clone designated Mp2C15 obtained from N. fowleri was used as a probe to distinguish N. fowleri from other free-living amoebae. The Mp2C15 probe hybridized to genomic DNA from pathogenic N. fowleri and antigenically related non-pathogenic N. lovaniensis. Mp2C15 was digested with the restriction enzyme XbaI, resulting in two fragments, Mp2C15.G and Mp2C15.P. Four species of Naegleria and four species of Acanthamoeba were examined for reactivity with Mp2C15.P. Mp2C15.P was specific for N. fowleri and was used in the development of a nested PCR assay which is capable of detecting as little as 5 pg of N. fowleri DNA or five intact N. fowleri amoebae. In summary, a rapid, sensitive, and specific assay for the detection of N. fowleri was developed.

  19. Locked Nucleic Acid Probe-Based Real-Time PCR Assay for the Rapid Detection of Rifampin-Resistant Mycobacterium tuberculosis.

    Directory of Open Access Journals (Sweden)

    Yong Zhao

    Full Text Available Drug-resistant Mycobacterium tuberculosis can be rapidly diagnosed through nucleic acid amplification techniques by analyzing the variations in the associated gene sequences. In the present study, a locked nucleic acid (LNA probe-based real-time PCR assay was developed to identify the mutations in the rpoB gene associated with rifampin (RFP resistance in M. tuberculosis. Six LNA probes with the discrimination capability of one-base mismatch were designed to monitor the 23 most frequent rpoB mutations. The target mutations were identified using the probes in a "probe dropout" manner (quantification cycle = 0; thus, the proposed technique exhibited superiority in mutation detection. The LNA probe-based real-time PCR assay was developed in a two-tube format with three LNA probes and one internal amplification control probe in each tube. The assay showed excellent specificity to M. tuberculosis with or without RFP resistance by evaluating 12 strains of common non-tuberculosis mycobacteria. The limit of detection of M. tuberculosis was 10 genomic equivalents (GE/reaction by further introducing a nested PCR method. In a blind validation of 154 clinical mycobacterium isolates, 142/142 (100% were correctly detected through the assay. Of these isolates, 88/88 (100% were determined as RFP susceptible and 52/54 (96.3% were characterized as RFP resistant. Two unrecognized RFP-resistant strains were sequenced and were found to contain mutations outside the range of the 23 mutation targets. In conclusion, this study established a sensitive, accurate, and low-cost LNA probe-based assay suitable for a four-multiplexing real-time PCR instrument. The proposed method can be used to diagnose RFP-resistant tuberculosis in clinical laboratories.

  20. Locked Nucleic Acid Probe-Based Real-Time PCR Assay for the Rapid Detection of Rifampin-Resistant Mycobacterium tuberculosis.

    Science.gov (United States)

    Zhao, Yong; Li, Guilian; Sun, Chongyun; Li, Chao; Wang, Xiaochen; Liu, Haican; Zhang, Pingping; Zhao, Xiuqin; Wang, Xinrui; Jiang, Yi; Yang, Ruifu; Wan, Kanglin; Zhou, Lei

    2015-01-01

    Drug-resistant Mycobacterium tuberculosis can be rapidly diagnosed through nucleic acid amplification techniques by analyzing the variations in the associated gene sequences. In the present study, a locked nucleic acid (LNA) probe-based real-time PCR assay was developed to identify the mutations in the rpoB gene associated with rifampin (RFP) resistance in M. tuberculosis. Six LNA probes with the discrimination capability of one-base mismatch were designed to monitor the 23 most frequent rpoB mutations. The target mutations were identified using the probes in a "probe dropout" manner (quantification cycle = 0); thus, the proposed technique exhibited superiority in mutation detection. The LNA probe-based real-time PCR assay was developed in a two-tube format with three LNA probes and one internal amplification control probe in each tube. The assay showed excellent specificity to M. tuberculosis with or without RFP resistance by evaluating 12 strains of common non-tuberculosis mycobacteria. The limit of detection of M. tuberculosis was 10 genomic equivalents (GE)/reaction by further introducing a nested PCR method. In a blind validation of 154 clinical mycobacterium isolates, 142/142 (100%) were correctly detected through the assay. Of these isolates, 88/88 (100%) were determined as RFP susceptible and 52/54 (96.3%) were characterized as RFP resistant. Two unrecognized RFP-resistant strains were sequenced and were found to contain mutations outside the range of the 23 mutation targets. In conclusion, this study established a sensitive, accurate, and low-cost LNA probe-based assay suitable for a four-multiplexing real-time PCR instrument. The proposed method can be used to diagnose RFP-resistant tuberculosis in clinical laboratories.

  1. Development of field-based real-time reverse transcription-polymerase chain reaction assays for detection of Chikungunya and O'nyong-nyong viruses in mosquitoes.

    Science.gov (United States)

    Smith, Darci R; Lee, John S; Jahrling, Jordan; Kulesh, David A; Turell, Michael J; Groebner, Jennifer L; O'Guinn, Monica L

    2009-10-01

    Chikungunya (CHIK) and O'nyong-nyong (ONN) are important emerging arthropod-borne diseases. Molecular diagnosis of these two viruses in mosquitoes has not been evaluated, and the effects of extraneous mosquito tissue on assay performance have not been tested. Additionally, no real-time reverse transcription-polymerase chain reaction (RT-PCR) assay exists for detecting ONN virus (ONNV) RNA. We describe the development of sensitive and specific real-time RT-PCR assays for detecting CHIK and ONN viral RNA in mosquitoes, which have application for field use. In addition, we compared three methods for primer/probe design for assay development by evaluating their sensitivity and specificity. This comparison resulted in development of virus-specific assays that could detect less than one plaque-forming unit equivalent of each of the viruses in mosquitoes. The use of these assays will aid in arthropod-borne disease surveillance and in the control of the associated diseases.

  2. Development of a neutralization assay for influenza virus using an endpoint assessment based on quantitative reverse-transcription PCR.

    Directory of Open Access Journals (Sweden)

    Belete Teferedegne

    Full Text Available A microneutralization assay using an ELISA-based endpoint assessment (ELISA-MN is widely used to measure the serological response to influenza virus infection and vaccination. We have developed an alternative microneutralization assay for influenza virus using a quantitative reverse transcription PCR-based endpoint assessment (qPCR-MN in order to improve upon technical limitations associated with ELISA-MN. For qPCR-MN, infected MDCK-London cells in 96-well cell-culture plates are processed with minimal steps such that resulting samples are amenable to high-throughput analysis by downstream one-step quantitative reverse transcription PCR (qRT-PCR; SYBR Green chemistry with primers targeting a conserved region of the M1 gene of influenza A viruses. The growth curves of three recent vaccine strains demonstrated that the qRT-PCR signal detected at 6 hours post-infection reflected an amplification of at least 100-fold over input. Using ferret antisera, we have established the feasibility of measuring virus neutralization at 6 hours post-infection, a duration likely confined to a single virus-replication cycle. The neutralization titer for qPCR-MN was defined as the highest reciprocal serum dilution necessary to achieve a 90% inhibition of the qRT-PCR signal; this endpoint was found to be in agreement with ELISA-MN using the same critical reagents in each assay. qPCR-MN was robust with respect to assay duration (6 hours vs. 12 hours. In addition, qPCR-MN appeared to be compliant with the Percentage Law (i.e., virus neutralization results appear to be consistent over an input virus dose ranging from 500 to 12,000 TCID(50. Compared with ELISA-MN, qPCR-MN might have inherent properties conducive to reducing intra- and inter-laboratory variability while affording suitability for automation and high-throughput uses. Finally, our qRT-PCR-based approach may be broadly applicable to the development of neutralization assays for a wide variety of viruses.

  3. Detection of Human Papillomavirus DNA in Cervical Samples: Analysis of the New PGMY-PCR Compared To the Hybrid Capture II and MY-PCR Assays and a Two-Step Nested PCR Assay

    OpenAIRE

    Giovannelli, Lucia; Lama, Anna; Capra, Giuseppina; Giordano, Viviana; Aricò, Pietro; Ammatuna, Pietro

    2004-01-01

    The PGMY-PCR for human papillomavirus (HPV) was evaluated, in parallel with nested PCR (nPCR), in samples with noted Hybrid Capture II (HCII) and MY-PCR results. PGMY-PCR detected HPV DNA in 2.5% of HCII-negative-MY-PCR-negative samples and in 71.7% of HCII-positive-MY-PCR-negative samples; also, it detected the MY-PCR-negative-nPCR-negative types HPV-42, HPV-44, HPV-51, HPV-87, and HPV-89.

  4. Quantitative Analysis of Periodontal Pathogens Using Real-Time Polymerase Chain Reaction (PCR).

    Science.gov (United States)

    Marin, Mª José; Figuero, Elena; Herrera, David; Sanz, Mariano

    2017-01-01

    The quantitative polymerase chain reaction (qPCR) is a variant of PCR aimed to detect and quantify a targeted DNA molecule through the addition of probes labeled with fluorescent molecules that emit fluorescence within each amplification cycle, what results in fluorescence values proportional to the amount of accumulated PCR product. This chapter presents the detailed procedures for quantification of different periodontal pathogens (Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Tannerella forsythia, Campylobacter rectus, and Fusobacterium spp.) using qPCR. It also includes the description of the most frequent problems encountered and how to solve them. In addition, a detailed protocol for multiplex qPCR to detect and quantify P. gingivalis and A. actinomycetemcomitans is included.

  5. Development of a quantitative PCR assay for rapid detection of Lactobacillus plantarum and Lactobacillus fermentum in cocoa bean fermentation.

    Science.gov (United States)

    Schwendimann, Livia; Kauf, Peter; Fieseler, Lars; Gantenbein-Demarchi, Corinne; Miescher Schwenninger, Susanne

    2015-08-01

    To monitor dominant species of lactic acid bacteria during cocoa bean fermentation, i.e. Lactobacillus plantarum and Lactobacillus fermentum, a fast and reliable culture-independent qPCR assay was developed. A modified DNA isolation procedure using a commercial kit followed by two species-specific qPCR assays resulted in 100% sensitivity for L. plantarum and L. fermentum. Kruskal-Wallis and post-hoc analyses of data obtained from experiments with cocoa beans that were artificially spiked with decimal concentrations of L. plantarum and L. fermentum strains allowed the calculation of a regression line suitable for the estimation of both species with a detection limit of 3 to 4 Log cells/g cocoa beans. This process was successfully tested for efficacy through the analyses of samples from laboratory-scale cocoa bean fermentations with both the qPCR assay and a culture-dependent method which resulted in comparable results.

  6. Preclinical detection of porcine circovirus type 2 infection using an ultrasensitive nanoparticle DNA probe-based PCR assay.

    Directory of Open Access Journals (Sweden)

    Yong Huang

    Full Text Available Porcine circovirus type 2 (PCV2 has emerged as one of the most important pathogens affecting swine production globally. Preclinical identification of PCV2 is very important for effective prophylaxis of PCV2-associated diseases. In this study, we developed an ultrasensitive nanoparticle DNA probe-based PCR assay (UNDP-PCR for PCV2 detection. Magnetic microparticles coated with PCV2 specific DNA probes were used to enrich PCV2 DNA from samples, then gold nanoparticles coated with PCV2 specific oligonucleotides were added to form a sandwich nucleic acid-complex. After the complex was formed, the oligonucleotides were released and characterized by PCR. This assay exhibited about 500-fold more sensitive than conventional PCR, with a detection limit of 2 copies of purified PCV2 genomic DNA and 10 viral copies of PCV2 in serum. The assay has a wide detection range for all of PCV2 genotypes with reliable reproducibility. No cross-reactivity was observed from the samples of other related viruses including porcine circovirus type 1, porcine parvovirus, porcine pseudorabies virus, porcine reproductive and respiratory syndrome virus and classical swine fever virus. The positive detection rate of PCV2 specific UNDP-PCR in 40 preclinical field samples was 27.5%, which appeared greater than that by conventional and real-time PCR and appeared application potency in evaluation of the viral loads levels of preclinical infection samples. The UNDP-PCR assay reported here can reliably rule out false negative results from antibody-based assays, provide a nucleic acid extraction free, specific, ultrasensitive, economic and rapid diagnosis method for preclinical PCV2 infection in field, which may help prevent large-scale outbreaks.

  7. Multi-Fluorescence Real-Time PCR Assay for Detection of RIF & INH Resistance of M. tuberculosis

    Directory of Open Access Journals (Sweden)

    Jingfu ePeng

    2016-04-01

    Full Text Available Background: Failure to early detect multidrug-resistant tuberculosis (MDR-TB results in treatment failure and poor clinical outcomes, and highlights the need to rapidly detect resistance to rifampicin (RIF and isoniazid (INHMethods: In Multi-Fluorescence quantitative Real-Time PCR (MF-qRT-PCR assay, 10 probes labeled with 4 kinds of fluorophores were designed to detect the mutations in regions of rpoB, katG, mabA-inhA, oxyR-ahpC and rrs. The efficiency of MF-qRT-PCR assay was tested using 261 bacterial isolates and 33 clinical sputum specimens. Among these samples, 227 Mycobacterium tuberculosis isolates were analyzed using drug susceptibility testing (DST, DNA sequencing and MF-qRT-PCR assay.Results: Compared with DST, MF-qRT-PCR sensitivity and specificity for RIF-resistance were 94.6% and 100%, respectively. And the detection sensitivity and specificity for INH-resistance were 85.9% and 95.3%, respectively. Compared with DNA sequencing, the sensitivity and specificity of our assay were 97.2% and 100% for RIF-resistance and 97.9% and 96.4% for INH-resistance. Compared with Phenotypic strain identification, MF-qRT-PCR can distinguish 227 Mycobacterium tuberculosis complexes (MTC from 34 Non-tuberculous mycobacteria (NTM isolates with 100% accuracy rate.Conclusions: MF-qRT-PCR assay was an efficient, accurate, reliable and easy-operated method for detection of RIF and INH-resistance, and distinction of MTC and NTM of clinical isolates.

  8. Application of polymerase chain reaction (PCR) for diagnosis of equine herpes virus-1 (EHV-1).

    Science.gov (United States)

    Gupta, A K; Singh, B K; Yadav, M P

    1996-11-01

    Fifty aborted foetus samples were diagnosed for the presence of equine herpes virus-1 (EHV-1) by polymerase chain reaction (PCR) technique. Specific primer pair for amplification of a particular segment of EHV-1 DNA in gc region having 3 Hae III restriction endonuclease sites was used. A 409 base pair segment obtained as PCR amplification product in 9 samples was digested with Hae III to confirm the presence of EHV-1 as the infectious agent in aborted tissues. It was observed that PCR technique was more sensitive, specific and rapid than the conventional virological diagnostic methods.

  9. On-Chip integration of sample pretreatment and Multiplex polymerase chain reaction (PCR) for DNA analysis

    DEFF Research Database (Denmark)

    Brivio, Monica; Snakenborg, Detlef; Søgaard, E.

    2008-01-01

    In this paper we present a modular lab-on-a-chip system for integrated sample pre-treatment (PT) by magnetophoresis and DNA amplification by polymerase chain reaction (PCR). It consists of a polymer-based microfluidic chip mounted on a custom-made thermocycler (Figure 1) and includes a simple...... and efficient method for switching the liquid flow between the PT and PCR chamber. Purification of human genomic DNA from EDTA-treated blood and multiplex PCR were successfully carried out on-chip using the developed lab-on-a-chip system....

  10. A duplex real-time PCR assay for the quantitative detection of Naegleria fowleri in water samples.

    Science.gov (United States)

    Behets, Jonas; Declerck, Priscilla; Delaedt, Yasmine; Verelst, Lieve; Ollevier, Frans

    2007-01-01

    A fast and accurate duplex real-time PCR (qPCR) was developed to detect and quantify the human pathogenic amoeba Naegleria fowleri in water samples. In this study, primers and probe based on the Mp2Cl5 gene were designed to amplify and quantify N. fowleri DNA in a single duplex reaction. The qPCR detection limit (DL) corresponds to the minimum DNA quantity showing significant fluorescence with at least 90% of the positive controls in a duplex reaction. Using fluorescent Taqman technology the qPCR was found to be 100% specific for N. fowleri with a DL of 3 N. fowleri cell equivalents and a PCR efficiency of 99%. The quantification limit (QL) was 16 N. fowleri cell equivalents (corresponded with 320 N. fowleri cell equivalents l(-1) water sample) in a duplex qPCR reaction and corresponds to the lowest DNA quantity amplifiable with a coefficient of variation less than 25%. To detect inhibition an exogenous internal positive control (IPC) was included in each PCR reaction preventing false negative results. Comparison of qPCR and most probable number (MPN) culture results confirms that the developed qPCR is well suited for rapid and quantitative detection of this human pathogen in real water samples. Nevertheless 'low contamination levels' of water samples (fowleri cells l(-1)) still require culture method analyses. When other thermophilic Naegleria are very dominant, the MPN culture method could result in an underestimation in the real number of N. fowleri and some caution is necessary to interpret the data. The N. fowleri qPCR could be a useful tool to study further competitive phenomena between thermophilic Naegleria strains.

  11. Detection of respiratory viruses using a multiplex real-time PCR assay in Germany, 2009/10.

    Science.gov (United States)

    Bierbaum, Sibylle; Forster, Johannes; Berner, Reinhard; Rücker, Gerta; Rohde, Gernot; Neumann-Haefelin, Dieter; Panning, Marcus

    2014-04-01

    The aim of this study was to determine the prevalence of respiratory viruses and to prospectively evaluate the performance of the fast-track diagnostics (FTD) respiratory pathogens multiplex PCR assay shortly after the 2009/10 influenza pandemic. Highly sensitive monoplex real-time PCR assays served as references. Discrepant results were further analyzed by the xTAG RVP Fast assay. A total of 369 respiratory samples from children and adults were collected prospectively in Germany from December 2009 until June 2010. The sensitivity and specificity of the FTD assay after resolution of discrepant results was 92.2 % and 99.5 %, respectively. Lowest specificity of the FTD assay was observed for human bocavirus. Multiple detections were recorded in 33/369 (8.9 %) of the samples by monoplex PCR and in 43/369 (11.7 %) using the FTD assay. The most prevalent viruses were respiratory syncytial virus and human metapneumovirus. Only pandemic influenza virus A/H1N1 (2009), and not seasonal influenza virus, was detected. Viruses other than influenza virus accounted for the majority of acute respiratory infections. The FTD assay can be easily implemented in general diagnostic laboratories and facilitate the optimization of patient-management schemes.

  12. Multiplex Real-Time PCR Assay Targeting Eight Parasites Customized to the Korean Population: Potential Use for Detection in Diarrheal Stool Samples from Gastroenteritis Patients

    Science.gov (United States)

    Won, Eun Jeong; Kim, Soo Hyun; Kee, Seung Jung; Shin, Jong Hee; Suh, Soon Pal; Chai, Jong Yil; Ryang, Dong Wook; Shin, Myung Geun

    2016-01-01

    Intestinal parasitic diseases occur worldwide and can cause diarrhea or gastroenteritis; however, their diagnosis is quite difficult, especially in low-endemism countries. We developed a multiplex real-time PCR assay for detection of eight intestinal parasites and prospectively evaluated it for patients with gastroenteritis. The assay targeted Cryptosporidium parvum, Giardia lamblia, Entamoeba histolytica, Blastocystis hominis, Dientamoeba fragilis, Clonorchis sinensis, Metagonimus yokogawai, and Gymnophalloides seoi. Performance characteristics were evaluated based on recovery after DNA extraction, analytical sensitivity, specificity, reproducibility, cross-reactivity, and interference characteristics. Clinical performance was validated against microscopy on 123 diarrheal samples. The assay demonstrated strong correlations between DNA concentrations and Ct values (R2, 0.9924–0.9998), and had a high PCR efficiency (83.3%–109.5%). Polymerase chain reactions detected as few as 10–30 copies of genomic DNA, and coefficient of variance was 0–7%. There was no cross-reactivity to the other 54 microorganisms tested. Interference occurred only in presence of high concentrations of erythrocytes or leukocytes. This assay had a higher correct identification rate (100.0% vs. 90.2%) and lower incorrect ID rate (0.0% vs. 9.8%) when compared to microscopy. Overall, this assay showed a higher sensitivity (100.0%; 95% confidence interval [CI] of 80.5–100.0) than microscopy (29.4%; 95% CI 10.31–55.96), and the specificity levels were comparable for both methods (100.0%; 95% CI 96.58–100.0). This newly developed multiplex real-time PCR assay offers a potential use for detecting intestinal parasitic pathogens customized to the Korean population. PMID:27861635

  13. Development of an internal amplification control system for a real-time PCR assay for detection of Neisseria meningitidis in CSF and EDTA blood.

    Science.gov (United States)

    McIver, Christopher J; Bell, Sydney M; Er, Noel

    2014-06-01

    The aim of this study was to assemble and assess a non-competitive internal amplification control (IAC) system targeting the Escherichia coli alanine racemase (alr) gene to include in a real-time polymerase chain reaction (PCR) assay for Neisseria meningitidis. Primers and hybridisation probes specific for the IAC were designed and assessed for specificity. Amplification efficiency and limit of detection for the assembled assay was extrapolated using standard curves constructed with serial dilutions of N. meningitidis in saline, pooled cerebrospinal fluid (CSF) and EDTA blood. The 95% confidence limits (CI) were calculated for IAC crossing-points recorded for assays for N. meningitidis ctrA in saline (negative blank), and N. meningitides-negative samples of CSF and EDTA blood. These limits served as a reference range against which the IAC crossing-points recorded for prospective assays are compared to detect sample inhibition. This system was used in testing consecutive EDTA blood samples from two cases of meningococcal disease. The IAC system is specific for Escherichia coli and Shigella species. The amplification efficiency of the assembled assay for N. meningitidis and ability to detect low target DNA levels was not compromised with the inclusion of the IAC system. The IAC crossing-points varied in clinical samples of CSF and EDTA blood. The elucidated reference range for EDTA blood was used to detect sample inhibition in one of the two clinical cases investigated.The IAC system monitors the performance of all processes in the assembled assay for N. meningitidis. Measuring IAC crossing-points serves as an indicator of sample stability and inhibitory properties when testing single or multiple samples from the same patient. Specificity for E. coli and Shigella species enables inclusion in assays of different targets within the same laboratory. Reporting PCR assay results in the context of the IAC crossing-points and reference ranges validates against sample

  14. Development of A Real-Time PCR Assay for Plasmodiophora brassicae and Its Detection in Soil Samples

    Institute of Scientific and Technical Information of China (English)

    LI Jin-ping; LI Yan; SHI Yan-xia; XIE Xue-wen; Chai A-li; LI Bao-ju

    2013-01-01

    A SYBR Green I real-time PCR assay was developed to detect and quantify Plasmodiophora brassicae ribosomal DNA (rDNA) and internal transcribed spacer (ITS). A pair of primers PBF1/PBR1 was designed based on the conservative region of rDNA-ITS of P. brassicae. The positive plasmid pB12 was obtained and used as the template to create standard curve. The specificity, sensitivity, and reproducibility of real-time PCR were evaluated respectively. Naturally and artificially infested soil samples containing different concentrations of P. brassicae were detected. The results demonstrated that standard curve established by recombinant plasmid was shown a fine linear relationship between threshold cycle and template concentration. The melting curve was specific with the correlation coefficient of 0.995 and that the amplification efficiency was 93.8%. The detection limit of P. brassicae genomic DNA was approximately 40 copies per 25μL. The sensitivity of the assay was at least 100-fold higher than conventional PCR. Only DNA from P. brassicae could be amplified and detected using this assay, suggesting the highly specific of this assay. The coefficient of variation was less than 3%, indicating the PCR method revealed high reproducibility. The detection limit in soil samples corresponded to 1 000 resting spores g-1 soil. Bait plants were used to validate the real-time PCR assay. This developed real-time PCR assay allows for fast and sensitive detection of P. brassicae in soil and should be useful in disease management and pest interception so as to prevent further spread of P. brassicae.

  15. Prevalence of Chlamydia infection among women visiting a gynaecology outpatient department: evaluation of an in-house PCR assay for detection of Chlamydia trachomatis

    Directory of Open Access Journals (Sweden)

    Patel Achchhe L

    2010-09-01

    Full Text Available Abstract Background Screening women for Chlamydia trachomatis infection in developing countries is highly desirable because of asymptomatic infection. The existing diagnostic methods in developing countries are not effective and their sensitivity fall below 45.0% which leads to further spread of infection. There is an urgent need for improved and cost effective diagnostic tests that will reduce the burden of sexually transmitted infections in the developing world. Methods Prevalence of C. trachomatis infection among women visiting gynaecology department of Hindu Rao hospital in Delhi, India was determined using Roche Amplicor Multi Well Plate kit (MWP as well as using in-house PCR assay. We used 593 endocervical swabs for clinical evaluation of the in-house developed assay against Direct Fluorescence Assay (DFA; Group I n = 274 and Roche Amplicor MWP kit (Group II, n = 319 samples and determined the sensitivity, specificity, positive predictive value (PPV, negative predictive value (NPV of the in-house developed assay. Results We detected 23.0% positive cases and there was a higher representation of women aged 18-33 in this group. An in-house PCR assay was developed and evaluated by targeting unique sequence within the gyrA gene of C. trachomatis. Specificity of the reaction was confirmed by using genomic DNA of human and other STI related microorganisms as template. Assay is highly sensitive and can detect as low as 10 fg of C. trachomatis DNA. The resolved sensitivity of in-house PCR