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

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

A multiplex PCR assay for the detection of five human pathogenic Vibrio species and Plesiomonas

A multiplex PCR (mPCR) assay was established to detect five pathogenic Vibrio species and Plesiomonas shigelloides. Twelve genes were included: ompW, ctxA, rfbN, and wbfR from V. cholerae; tl, tdh, and trh from V. parahaemolyticus; toxR and vmhA from V. mimicus; toxR from V. fluvialis; vvhA from V. vulnificus; and the 23S rRNA gene from P. shigelloides. The specificity of the mPCR assay was 100% for the detection of 136 strains and the limits of detection (LoD) were 12.5–50 pg/reaction. The assay exhibited higher sensitivity than cultivation methods in the detection of APW cultures of 113 diarrhea samples. In the analysis of 369 suspected Vibrio populations from estuarine water samples, the specificity of the mPCR for V. cholerae and V. parahaemolyticus was 100% for both, while the sensitivities were 100% and 96.1%, respectively. The assay can be applied to screen enrichment cultures and suspected colonies from environmental and clinical samples.     

Establishment of a duplex SYBR green I-based real-time polymerase chain reaction assay for the rapid detection of canine circovirus and canine astrovirus

The similar clinical characteristics of canine circovirus (CaCV) and canine astrovirus (CaAstV) infections and high frequency of co-infection make diagnosis difficult. In this study, a duplex SYBR Green I-based real-time polymerase chain reaction (PCR) assay was established for the rapid, simultaneous detection of CaCV and CaAstV. Two pairs of specific primers were designed based on the Rep gene of CaCV and the Cap gene of CaAstV. By using the real-time PCR assay method, the two viruses can be distinguished by the difference in melting temperatures, 79 °C and 86 °C for CaCV and CaAstV, respectively. This assay had high specificity, showing no cross-reaction with other common canine viruses, as well as high sensitivity, with minimum detection limits of 9.25 × 10¹ copies/μL and 6.15 × 10¹ copies/μL for CaCV and CaAstV, respectively. Based on the mean coefficient of variation, the method had good reproducibility and reliability. In a clinical test of 57 fecal samples, the rates of positive detection by real-time PCR were 14.04% (8/57) and 12.28% (7/57) for CaCV and CaAstV, respectively, and the rate of co-infection was 8.77% (5/57). In conclusion, the newly established duplex SYBR Green I-based real-time PCR assay is sensitive, specific, reliable, and rapid and is an effective tool for the detection of co-infections with CaCV and CaAstV.     

A multiplex PCR/LDR assay for simultaneous detection and identification of the NIAID category B bacterial food and water-borne pathogens

Enteric pathogens that cause gastroenteritis remain a major global health concern. The goal of this study was to develop a multiplex PCR/ligation detection reaction (LDR) assay for the detection of all NIAID category B bacterial food and water-borne pathogens directly from stool specimens. To validate the PCR/LDR assay, clinical isolates of Campylobacter spp., Vibrio spp., Shigella spp., Salmonella spp., Listeria monocytogenes, Yersinia enterocolitica, and diarrheagenic Escherichia coli were tested. The sensitivity and specificity of the assay were assessed using a large number of seeded culture-negative stool specimens and a smaller set of clinical specimens from Haiti. The overall sensitivity ranged from 91% to 100% (median 100%) depending on the species. For the majority of organisms, the sensitivity was 100%. The overall specificity based on initial testing ranged from 98% to 100% depending on the species. After additional testing of discordant samples, the lowest specificity was 99.4%. PCR/LDR detected additional category B agents (particularly diarrheagenic E. coli) in 11/40 specimens from Haiti that were culture-positive for V. cholerae and in approximately 1% of routine culture-negative stool specimens from a hospital in New York. This study demonstrated the ability of the PCR/LDR assay to detect a large comprehensive panel of category B enteric bacterial pathogens as well as mixed infections. This type of assay has the potential to provide earlier warnings of possible public health threats and more accurate surveillance of food and water-borne pathogens.     

Evaluation of Seeplex® STD6 ACE Detection kit for the diagnosis of six bacterial sexually transmitted infections

Traditionally, the diagnosis of bacterial sexually transmitted infection (STI) has been dependent on the isolation of the causative pathogens by culturing endocervical or urethral swab specimens on selective media. While such procedures typically provide excellent diagnostic accuracy, they are often time-consuming and expensive. A multiplex polymerase chain reaction (PCR) assay, based on a semi-automated detection system, was evaluated for the detection of six STI causative organisms. The Seeplex^® STD6 ACE (auto-capillary electrophoresis) Detection assay employed six pairs of dual priming oligonucleotide (DPO?) primers specifically targeted to unique genes of Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, Ureaplasma urealyticum, Mycoplasma hominis, and Trichomonas vaginalis. A total of 739 specimens (304 cervical swabs and 435 urine samples) collected for 4 months were tested, and results were compared to those obtained with a combined monoplex PCR. The concordance between the multiplex PCR and monoplex PCR assay was 100% for both sensitivity and specificity. We also tested for the presence of two pathogenic bacteria (C. trachomatis and N. gonorrhoeae) and compared the results obtained with the multiplex PCR and BD ProbeTec duplex strand displacement amplification (SDA). The results of the multiplex PCR and duplex SDA were 99.7% concordant for C. trachomatis and 100% concordant for N. gonorrhoeae. The multiplex PCR assay using the Seeplex^® STD6 ACE Detection kit proved to be a novel cost-effective and fast diagnostic tool with high sensitivity and specificity for the simultaneous detection of six STI pathogens.     

Comparing the performance of conventional PCR,RTQ-PCR,and droplet digital PCR assays in detection of Shigella

The incidence of foodborne infections caused by Shigella spp. is still very high in every year, which poses a great potential threat to public health. Conventional quantification methods based on culture techniques, biochemical, and serological identification are time-consuming and labor-intensive. To develop a more rapid and efficient detection method of Shigella spp., we compared the sensitivity and specificity of three different polymerase chain reaction (PCR) methods, including conventional PCR, quantitative real-time PCR (RTQ-PCR), and droplet digital PCR (ddPCR). Our results indicated that ddPCR method exhibited higher sensitivity, and the limit of detection was 10⁻⁵ ng/μl for genomic DNA templates, 10⁻¹ cfu/ml for Shigella bacteria culture. In addition, we found that ddPCR was a time-saving method, which required a shorter pre-culturing time. Collectively, ddPCR assay was a reliable method for rapid and effective detection of Shigella spp.     

Distribution and pathogenic relationship of virulence associated genes among Vibrio alginolyticus from the mariculture systems

Vibrio alginolyticus has been confirmed as an important pathogen for aquatic animals. However, the pathogenic mechanism of V. alginolyticus is not completely understood. A total of 31 isolates of V. alginolyticus from sea water, fish and shrimp on the mariculture systems were fingerprinted by pulsed-field gel electrophoresis. The pathogenicity of these isolates was tested by challenge and the 21 genes associated with the virulence of Vibrio cholerae or Vibrio parahaemolyticus were examined in V. alginolyticus using PCR. The results showed that the 31 V. alginolyticus isolates belonged to 26 PFGE genotypes and the isolates from different source had different genotypes. Nine of the 31 isolates were confirmed as pathogenic strains by challenge. Moreover, 12 vibrio virulence genes were detected in this study. Of the detected genes, VCtoxR, VCtoxS, hlyA, VPtoxR and tlh were found in both pathogenic and non-pathogenic isolates. However, the other 7 virulence genes, ctxB, zot, tagA, stn, sto, tdh and trh, were only present in pathogenic isolates. Analysis of the relationship between virulence associated genes and pathogenicity of V. alginolyticus provides a possible explanation that the pathogenic mechanism of V. alginolyticus might be similar to that of V. parahaemolyticus instead of V. cholerae.     

A real-time PCR assay for detection of emerging infectious Elizabethkingia miricola

Elizabethkingia miricola, a Gram-negative bacillus, is emerging as a life-threatening pathogen in both humans and animals. However, no specific and rapid diagnostic method exists to detect E. miricola. Here, we established a real-time PCR assay for the rapid, sensitive, and specific detection of E. miricola with a wide dynamic range of 10⁸ copies/μL to 10² copies/μL. The detection limit of the real-time assay was 145 copies/μL, which was 100 times more sensitive than conventional PCR. All clinical isolates E. miricola from different host species yield very close Tm (80.25 ± 0.25 °C). Additionally, no cross-reaction or false positives were observed in the assay for non-target bacterial species. The performance of this assay was primarily assessed by testing frog tissue samples. Overall, our study provided a real-time PCR assay, which is a rapid, sensitive, and specific diagnostic method that could be used for early diagnosis and epidemiological investigation of E. miricola.     

Comparison of real-time polymerase chain reaction and conventional biochemical methods for identification of Mycobacterium chelonae–Mycobacterium abscessus group to the species level

The Mycobacterium chelonae–Mycobacterium abscessus group (MCAG) is the most common cause of infections because of rapidly growing mycobacteria. Rapid identification of MCAG to the species level is essential for choosing empiric antibiotic treatment and for public health measures. In this study, we compared the performance of a single-tube multiplex, real-time polymerase chain reaction (PCR) assay to 3 biochemical tests for species-level identification of 46 MCAG isolates. We show that real-time PCR provides the most accurate results for rapid species-level identification of MCAG.     

Multiplex PCR assays for the detection of Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio vulnificus, and Vibrio cholerae with an internal amplification control

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 10⁵ 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.     

Real-time PCR using SYBR Green for the detection of Shigella spp. in food and stool samples

Shigella spp are exquisitely fastidious Gram negative organisms that frequently get missed in the detection by traditional culture methods. For this reason, this work has adapted a classical PCR for detection of Shigella in food and stool specimens to real-time PCR using the SYBR Green format. This method follows a melting curve analysis to be more rapid and provide both qualitative and quantitative data about the targeted pathogen.A total of 117 stool samples with diarrhea and 102 food samples were analyzed in Public Health Regional Laboratory of Nabeul by traditional culture methods and real-time PCR. To validate the real-time PCR assay, an experiment was conducted with both spiked and naturally contaminated stool samples. All Shigella strains tested were ipaH positive and all non-Shigella strains yielded no amplification products. The melting temperature (T_m = 81.5 ± 0.5 °C) was consistently specific for the amplicon. Correlation coefficients of standard curves constructed using the quantification cycle (C_q) versus copy numbers of Shigella showed good linearity (R² = 0.995; slope = 2.952) and the minimum level of detection was 1.5 × 10³ CFU/g feces. All food samples analyzed were negative for Shigella by standard culture methods, whereas ipaH was detected in 8.8% culture negative food products. Moreover, the ipaH specific PCR system increased the detection rate over that by culture alone from 1.7% to 11.1% among patients with diarrhea.The data presented here shows that the SYBR Green I was suitable for use in the real-time PCR assay, which provided a specific, sensitive and efficient method for the detection and quantification of Shigella spp in food and stool samples.     

Utilization of a real-time PCR approach for Haemophilus influenzae serotype determination as an alternative to the slide agglutination test

Our laboratory has developed a simple two-step multiplex real-time PCR for use on isolates of Haemophilus influenzae for molecular serotype identification and the detection of capsular gene targets. The assay consists of a 2-plex real-time PCR targeting the capsule transport gene (bexA), and serotype b specific gene (bcsB), and a 5-plex real-time PCR detecting serotypes a, c, d, e, and f targeting Region II serotype-specific genes. Both real-time PCR assays are highly sensitive (<8 CFU) for all serotypes and 100% specific when tested by a panel of more than 40 bacterial organisms. A retrospective study of 214 isolates received between 1998 and 2011 were tested and compared against the traditional slide agglutination test (SAT) resulting in 100% concordance. We demonstrate that this two-step real-time PCR approach is more sensitive than previously published PCR assays and provides a simple alternative to the SAT. Reliable, rapid and sensitive H. influenzae serotyping is critical for identifying new emerging strains for epidemiological surveillance.     

Standardization non-invasive fetal RHD and SRY determination into clinical routine using a new multiplex RT-PCR assay for fetal cell-free DNA in pregnant women plasma: results in clinical benefits and cost saving

IntroductionAmong negative RhD mothers it is essential to know the fetal RhD status in order to avoid the possibility of hemolytic disease of the newborn. In this regard, the detection of fetal DNA in maternal plasma might become a new diagnostic tool. In the current study, we have evaluated the standardization of a Multiplex-PCR targeted towards two exons of the RHD and one SRY gene to monitor RhD negative women. The current study addresses questions concerning feasibility and applicability of this approach into the clinical practice.Materials and methodsBoth single and multiplex real-time PCRs targeting RHD exons 5 and 7 and SRY were applied for the detection of fetal-specific RHD sequences and sex in maternal plasma. A large cohort of 2127 women was studied between 10 and 28 weeks of pregnancy. 134 of them were used for single TaqMan PCR studies and 1993 were evaluated using Multiplex TaqMan PCR studies. All of them were serologically typed as RhD negative according to Spanish guidelines. Single and multiplex real-time PCR results were compared with postnatal serology and sex identification.ResultsThere was a 100% concordance between results obtained with single and multiplex real-time PCR assays. At present, 1012 of the 1993 pregnant women studied gave birth and the results of RHD status obtained with the multiplex TaqMan PCR assay were confirmed postpartum by serological methods showing that sensitivity, specificity, and accuracy of the multiplex assay were 100, 98.6, and 99.3%, respectively. This procedure improved the speed of the assay, avoided over-treatment among RhD negative pregnant women bearing RhD negative fetus, and reduced the requirements for clinical and biological monitoring, resulting in a clinical benefit and cost saving.ConclusionsThe routine determination of fetal RHD status and SRY in maternal plasma, using multiplex real-time PCR, is feasible. The use of multiplex real-time PCR allows improving the response of the laboratory, saving time and reagent costs, opening the door to a complete automatization of the process.     

Development of a serogroup-specific multiplex PCR assay to detect a set of Escherichia coli serogroups based on the identification of their O-antigen gene clusters

The Escherichia coli serogroups O115, O126, O137, O158, O165, and O173 are pathogenic strains associated with diarrhea. Molecular approaches such as PCR have been proven to be rapid, inexpensive, and accurate. The sequences of the O-antigen-processing genes wzx and wzy are specific for different O antigens and are generally used as the target genes for the detection and identification of E. coli strains belonging to different O serogroups. In this report, the O-antigen gene clusters of these 6 O serogroups were sequenced, and genes were identified on the basis of homology. By screening these sequences against all 186 E. coli and Shigella strains, we found that the sequences of the wzx and wzy genes were serogroup-specific, and 2 specific primer pairs for each serogroup were screened out. A multiplex PCR assay targeting all 6 serogroups was developed. Twenty-nine strains were used to validate the specificity of the assay. The detection sensitivity was 1 ng genomic DNA. As the assay was shown to be accurate and sensitive, it can be used for the identification and detection of strains belonging to these serogroups in stool and other environmental samples after being isolated by culture.     

Multiplex real-time polymerase chain reaction for rapid detection of β-lactamase–negative,ampicillin-resistant Haemophilus influenzae

We evaluated a multiplex real-time quantitative polymerase chain reaction (PCR) method for quantification of Haemophilus influenzae and rapid detection of β-lactam–resistant strains. We designed 5 PCR primer sets to simultaneously detect the β-lactam–resistant genes and quantify the pathogen. To demonstrate the validity of this assay, we used 191 clinical isolates, including 141 H. influenzae strains, and 100 purulent sputum samples, including 30 samples from which H. influenzae had been isolated. This assay showed 92.9% sensitivity and 91.8% specificity for detecting β-lactam–resistant genes, relative to the conventional phenotypic method, and this assay correlated well with conventional quantitative culture counts. By using this assay, we could quantify H. influenzae and identify β-lactam susceptibility in only 3 h and with only one tube. This method will be helpful for the rapid detection of H. influenzae infections and the selection of appropriate antibiotics.     

Development of multiplex real-time quantitative PCR for simultaneous detection of Chlamydia trachomatis and Ureaplasma parvum

ObjectivesChlamydia trachomatis and Ureaplasma urealyticum are common pathogens of sexually transmitted diseases. The majority of human ureaplasma isolates belong to the new species U. parvum. Clinically, C. trachomatis and U. parvum usually double infect in the nongonococcal urethritis patients. A novel method for simultaneous detection of C. trachomatis and U. parvum was set up in the present work.Design and methodsMultiple real-time quantitative PCR was developed to allow for rapid, sensitive, specific and quantitative detection of C. trachomatis and U. parvum, simultaneously. To evaluate the applicability of the multiplex real-time quantitative PCR assay to clinical specimens, 64 samples of cervical swabs collected were studied.ResultsCompared to the results obtained from single real-time quantitative PCR of C. trachomatis and U. parvum, the specificity, sensitivity and quantitative detection results of multiple real-time quantitative PCR are approximately identical with those of the former.ConclusionsThis assay will be of great value in the simultaneous and rapid diagnosis of C. trachomatis and U. parvum in the future.     

Development of an oligonucleotide-based microarray to detect multiple foodborne pathogens

Escherichia coli O157:H7, Salmonella enterica, Listeria monocytogenes and Campylobacter jejuni are considered important pathogens causing the most food-related human illnesses worldwide. Current methods for pathogen detection have limitations in the effectiveness of identifying multiple foodborne pathogens. In this study, a pathogen detection microarray was developed using various 70-mer oligonucleotides specifically targeting the above pathogens. To reduce the cost of detection, each microarray chip was designed and fabricated to accommodate 12 identical arrays which could be used for screening up to 12 different samples. To achieve high detection sensitivity and specificity, target-specific DNA amplification instead of whole genome random amplification was used prior to microarray analysis. Combined with 14-plex PCR amplification of target sequences, the microarray unambiguously distinguished all 4 pathogens with a detection sensitivity of 1 × 10^?4 ng (approximately 20 copies) of each genomic DNA. Applied the assay to 39 fresh meat samples, 16 samples were found to be contaminated by either 1 or 2 of these pathogens. The co-occurrences of Salmonella and E. coli O157:H7, Salmonella and L. monocytogenes in the same meat samples were also observed. Overall, the microarray combined with multiplex PCR method was able to effectively screen single or multiple pathogens in food samples and to provide important genotypic information related to pathogen virulence.     

Multiplex real-time PCR assay combined with rolling circle amplification (MPRP) using universal primers for non-invasive detection of tumor-related mutations

With the continuous development and application of targeted drugs, it is particularly desirable to find a non-invasive diagnostic approach to screen patients for precision treatment. Specifically, detection of multiple cancer-related mutations is very important for targeted therapy and prediction of drug resistance. Although numerous advanced PCR methods have been developed to discriminate single nucleotide polymorphisms, their drawbacks significantly limit their application, such as low sensitivity and throughput, complicated operations, and expensive costs. In order to overcome these challenges, in this study, we developed a method combining multiplex and sensitive real-time PCR assay with rolling circle amplification. This allows specific and sensitive discrimination of the single nucleotide mutation and provides convenient multiplex detection by real-time PCR assay. The clinical potential of the MPRP assay was further demonstrated by comparing samples from 8 patients with a digital PCR assay. The coincident results between these two methods indicated that the MPRP assay can provide a specific, sensitive, and convenient method for multiplex detection of cancer-related mutations.

The MPRP system for SNP discrimination was developed, which showed high specificity and sensitivity for multiplex detection of tumor-related mutations.     

模拟粪便标本单增李斯特菌和伊氏李斯特菌TaqMan双重实时荧光定量-聚合酶链反应检测方法

目的建立一种TaqMan双重实时荧光定量-聚合酶链反应(real-time PCR),用于模拟粪便标本中单增李斯特菌和伊氏李斯特菌的快速检测。方法以单增李斯特菌特异基因hly和伊氏李斯特菌特异基因smcL作为靶基因,合成2对引物和其相应的荧光探针,制作标准曲线,建立从模拟粪便标本中直接检测单增李斯特菌和伊氏李斯特菌的TaqMan双重real-time PCR。利用李斯特菌属中其他种李斯特菌及常见致病菌验证引物、探针的特异性;通过制备模拟粪便标本并对其进行二次增菌后,分别提取DNA,采用TaqMan双重real-time PCR进行检测,以达到快速检测单增李斯特菌和伊氏李斯特菌的目的。结果采用本研究建立的TaqMan双重real-time PCR对模拟粪便标本的检测结果显示特异性良好,与其他种李斯特菌和其他病原菌均无交叉反应。模拟粪便标本中单增李斯特菌和伊氏李斯特菌的检测下限分别为2.45×103cfu/g和2.92×103cfu/g;模拟粪便标本在3 h内可得出检测结果,当模拟粪便标本中单增李斯特菌含量为6 cfu/g和伊氏李斯特菌含量为5 cfu/g时,经过增菌后使用双重real-time PCR可检测出阳性结果。结论本研究建立了以单增李斯特菌的特异基因hly和伊氏李斯特菌的特异基因smcL为靶基因的TaqMan双重realtime PCR检测方法,该方法具有特异性好、敏感性高、快速易操作等优点,可用于临床、食品及环境标本的快速诊断,以及我国人群中单增李斯特菌和伊氏李斯特菌的携带或感染状况的调查分析。