Comparing multiplex and multiplex real-time polymerase chain reaction with traditional blood culture in bacterial detection among patients with septicemia

Aims@#This study was aimed to test the specificity of primers and probes with target genes by using multiplex PCR and multiplex real-time PCR methods. These methods were compared with traditional blood culture methods in detecting five bacteria causing sepsis, including Acinetorbacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus.@*Methodology and results@#A total of 587 blood samples from patients diagnosed with sepsis and septic shock were collected at Thanh Nhan Hospital, Hanoi, Vietnam. Each sample was divided into three parts for bacterial culture, multiplex PCR and multiplex real-time PCR to detect the similarity of the two PCR methods with the bacterial culture method. Conditions in multiplex PCR and multiplex real-time PCR were optimized to ensure the successful amplification of target genes. Results showed that the primers and probes were tested completely specific to the target genes and using multiplex PCR and multiplex real-time PCR techniques could detect five pathogens causing sepsis, including A. baumannii, K. pneumoniae, P. aeruginosa, E. coli and S. aureus.@*Conclusion, significance and impact of study@#Both multiplex PCR and multiplex real-time PCR methods have high similarities with the culture method, showing potential in the application of bacteria detection in sepsis.

Multiplex real-time PCR revealed very high prevalence of soil-transmitted helminth infections among aborigines in Peninsular Malaysia

Objective: To determine the true prevalence of soil-transmitted helminth infections in the Malaysian aborigines using real-time PCR. Methods: A total of 122 aborigines from seven tribes were recruited from settlements and nearby hospitals which served the communities, located in four states in Peninsular Malaysia. The stool samples were examined for the presence of soil-transmitted helminth using real-time PCR and microscopy. The latter included the direct wet mount and formalin-ether concentration technique (FECT). The infection load in FECT-positive samples was determined by the Kato-Katz method. Rotorgene real-time analyzer detected five helminth species using two sets of assays. Results: The real-time PCR detected soil-transmitted helminth in 98.4% samples (n=122), which were 1.56 times higher than by microscopy. Ascaris lumbricoides and Trichuris trichiura were detected in more than 90% of the samples, while hookworm was detected in 46.7% (Necator americanus) and 13.9% (Ancylostoma sp.) of the samples. Comparison with previous reports on the Malaysian aborigines showed that the real-time PCR markedly improved the detection of Ascaris lumbricoides, hookworm and Strongyloides stercoralis. The real-time PCR detected poly-helminths in 92.6% of the samples compared to 28.7% by microscopy. In addition, 27 samples (22.1%) showed amplification of Strongyloides stercoralis DNA. Conclusions: The real-time PCR showed very high prevalence rates of soil-transmitted helminth infections in the aborigines and is the recommended method for epidemiological investigation of soil-transmitted helminth infections in this population.

Multiplex real-time PCR revealed very high prevalence of soil-transmitted helminth infections among aborigines in Peninsular Malaysia

Objective: To determine the true prevalence of soil-transmitted helminth infections in the Malaysian aborigines using real-time PCR. Methods: A total of 122 aborigines from seven tribes were recruited from settlements and nearby hospitals which served the communities, located in four states in Peninsular Malaysia. The stool samples were examined for the presence of soil-transmitted helminth using real-time PCR and microscopy. The latter included the direct wet mount and formalin-ether concentration technique (FECT). The infection load in FECT-positive samples was determined by the Kato-Katz method. Rotorgene real-time analyzer detected five helminth species using two sets of assays. Results: The real-time PCR detected soil-transmitted helminth in 98.4% samples (n=122), which were 1.56 times higher than by microscopy. Ascaris lumbricoides and Trichuris trichiura were detected in more than 90% of the samples, while hookworm was detected in 46.7% (Necator americanus) and 13.9% (Ancylostoma sp.) of the samples. Comparison with previous reports on the Malaysian aborigines showed that the real-time PCR markedly improved the detection of Ascaris lumbricoides, hookworm and Strongyloides stercoralis. The real-time PCR detected poly-helminths in 92.6% of the samples compared to 28.7% by microscopy. In addition, 27 samples (22.1%) showed amplification of Strongyloides stercoralis DNA. Conclusions: The real-time PCR showed very high prevalence rates of soil-transmitted helminth infections in the aborigines and is the recommended method for epidemiological investigation of soil-transmitted helminth infections in this population.

Evaluation of the Real-Q RV Detection Kit for the Identification of Viruses That Result in Respiratory Infections

Viral respiratory infections are one of the most common infections worldwide. It is important to detect the virus early and precisely. In this study, we evaluated the limit of detection (LoD) and usefulness of the Real-Q RV Detection kit (BioSewoom, Seoul, Korea). We measured the LoD of the Real-Q RV Detection kit using 10 strains of standard viruses. We then compared the detection results by the Allplex Respiratory Panel Assay kit (Seegene, Seoul, Korea) using 123 clinical specimens. The discrepant results were confirmed by sequencing. Among the 10 standard viruses, the LoD of human rhinovirus (HRV) was the lowest and that of parainfluenza virus 2 and 3 was relatively high as detected by Real-Q RV Detection kit. Agreements of the two kits ranged from 95.9% to 100%. Three specimens detected negative by the Allplex Respiratory Panel kit were detected as adenovirus (AdV) by the Real-Q RV Detection kit and were confirmed by sequencing. Similarly, a specimen detected negative by the Allplex Respiratory Panel kit was detected as HRV by the Real-Q RV Detection kit and was confirmed by sequencing. A specimen detected as human enterovirus by the Allplex Respiratory Panel kit was detected as HRV by the Real-Q RV Detection kit and was confirmed by sequencing. Real-Q RV Detection kit showed good diagnostic performance and can be useful for detecting major viruses that cause respiratory infections.

Comparison of the Luminex xTAG Respiratory Viral Panel Fast v2 Assay With Anyplex II RV16 Detection Kit and AdvanSure RV Real-Time RT-PCR Assay for the Detection of Respiratory Viruses

BACKGROUND: The accurate and rapid identification of the causative viruses is important for the timely diagnosis and management of respiratory infections. Multiplex molecular diagnostic techniques have been widely adopted to detect respiratory viruses. We compared the results of a newly upgraded, multiplex, molecular bead-based respiratory viral panel (RVP) assay with the results of Anyplex II RV16 detection kit and AdvanSure RV real-time RT-PCR assay. METHODS: We tested 254 respiratory specimens and cultured viral strains using the Luminex xTAG RVP Fast v2 assay (Luminex Molecular Diagnostics, Canada) and Anyplex II RV16 detection kit and compared the results. Specimens showing discordant results between the two assays were tested with a AdvanSure RV real-time RT-PCR assay. RESULTS: Of the 254 respiratory specimens, there was total agreement in the results between the xTAG RVP Fast v2 assay and the other real-time PCR assay in 94.1–100% of the specimens. The agreement levels were relatively low (94.1–97.6%) for specimens of adenovirus, coronavirus NL63, and parainfluenza type 3. In comparison to the other assay, the xTAG RVP Fast v2 assay detected a higher number of parainfluenza type 3 (4 cases) and metapneumovirus (9 cases). CONCLUSIONS: The xTAG RVP Fast v2 assay showed comparable capabilities compared with the other assays; it will be useful for identifying respiratory viral infections in patients with respiratory symptoms. Clinicians should be aware of the characteristics of the assays they use, since different assays show different detectability for each virus.

Establishment of a multiplex real-time PCR method for quantitative detection of Campylobacter jejuni, Salmonella and Shigella in tree shrews / 中国比较医学杂志

Objective To establish a rapid, simple, sensitive, and specific multiplex real-time PCR method for quantitative detection of Campylobacter jejuni, Salmonella and Shigella in tree shrews.Methods Specific primers and probes were designed, according to the HipO gene of Campylobacter jejuni, inV gene of Salmonella and ipaH gene of Shigella.The primers were confirmed by single pathogen quantitative PCR, and the sensitivity and specificity of the multiplex PCR were analyzed.Finall, the samples of experimental tree shrews were detected by this multiplex PCR method.Results The PCR element of TaqMan-MGB real-time PCR assay was able to quantitatively amplify the Campylobacter jejuni, Salmonella or Shigella.Appropriate standard amplification curves of Campylobacter jejuni, Salmonella and Shigella in the multiplex quantitative PCR were obtained.The sensitivity of this method was 1×103 ng/μL.There was no false positive detection from other bacterial strains.Conclusions This multiplex quantitative real-time PCR method has good application and development prospects in the detection of microorganisms in tree shrews.

In-house standardization and validation of a multiplex RT-PCR assay for the detection of 13 respiratory viruses / Estandarización interna y validación de un ensayo RT-PRC múltiple para la detección de 13 virus respiratorios

Background. Multiplex real time PCR is increasingly used to diagnose respiratory viruses and has shown to be superior to traditional methods, such as culture and antigen detection. Objective. Standardization and validation of a multiplex real-time PCR assay for the detection of 13 respiratory viruses. Methods. The assay was validated using RNA control targets and comparing results to single-target PCR's. Results. Using RNA controls the multiplex format was found to be as sensitive and specific as the single-target PCRs, and no competition was observed between targets. The efficiencies for most of the reactions were approximately 90%, but a lower efficiency was found for Parainfluenza 2 with a rate of amplification in each cycle of 86.63%. On the other hand, a higher efficiency was observed in respiratory syncytial virus A and respiratory syncytial virus B ((93.07% each). Conclusion: This multiplex RT-PCR format shows an adequate efficiency, demonstrating an excellent sensitivity, specificity and repeatability for all the studied respiratory viruses.

Antecedentes. PRC múltiple en tiempo real es usada cada vez más para el diagnóstico de virus respiratorios y ha mostrado ser superior a metodos tradicionales, como cultivo y detección de antígeno. Objetivo. Estandarizar y validar una PRC múltiple en tiempo real para la detección de 13 virus respiratorios. Metodos. El ensayo fue realizado usando blanco de RNA control y comparando los resultados a blancos únicos de PCR. Resultados. Usando el RNA control, el formato de multiplex era tan sensible y específico como la PCR. Las eficiencias para la mayoría de las reacciones de aproximadamente el 90%, pero una eficiencia baja fue encontrada para influenza 2 con una tasa de amplificación en cada ciclo de 86.63%. Por otra parte, una mayor eficiencia fue observada en virus sincitial respitario A y B (93, 67% cada uno). Conclusión. Este formato RT-PCR múltiple muestra una adecuada eficiencia, demostrando un excelente especificidad y reproducibilidad para todos los virus respiratorios estudiados.

Multiplex Real-Time PCR Method for Simultaneous Identification and Toxigenic Type Characterization of Clostridium difficile From Stool Samples

BACKGROUND: The aim of this study was to develop and validate a multiplex real-time PCR assay for simultaneous identification and toxigenic type characterization of Clostridium difficile. METHODS: The multiplex real-time PCR assay targeted and simultaneously detected triose phosphate isomerase (tpi) and binary toxin (cdtA) genes, and toxin A (tcdA) and B (tcdB) genes in the first and sec tubes, respectively. The results of multiplex real-time PCR were compared to those of the BD GeneOhm Cdiff assay, targeting the tcdB gene alone. The toxigenic culture was used as the reference, where toxin genes were detected by multiplex real-time PCR. RESULTS: A total of 351 stool samples from consecutive patients were included in the study. Fifty-five stool samples (15.6%) were determined to be positive for the presence of C. difficile by using multiplex real-time PCR. Of these, 48 (87.2%) were toxigenic (46 tcdA and tcdB-positive, two positive for only tcdB) and 11 (22.9%) were cdtA-positive. The sensitivity, specificity, negative predictive value (NPV), and positive predictive value (PPV) of the multiplex real-time PCR compared with the toxigenic culture were 95.6%, 98.6%, 91.6%, and 99.3%, respectively. The analytical sensitivity of the multiplex real-time PCR assay was determined to be 103colonyforming unit (CFU)/g spiked stool sample and 0.0625 pg genomic DNA from culture. Analytical specificity determined by using 15 enteric and non-clostridial reference strains was 100%. CONCLUSIONS: The multiplex real-time PCR assay accurately detected C. difficile isolates from diarrheal stool samples and characterized its toxin genes in a single PCR run.

Molecular Differentiation of Opisthorchis viverrini and Clonorchis sinensis Eggs by Multiplex Real-Time PCR with High Resolution Melting Analysis

Opisthorchis viverrini and Clonorchis sinensis are parasites known to be carcinogenic and causative agents of cholangiocarcinoma in Asia. The standard method for diagnosis for those parasite infections is stool examination to detect parasite eggs. However, the method has low sensitivity, and eggs of O. viverrini and C. sinensis are difficult to distinguish from each other and from those of some other trematodes. Here, we report a multiplex real-time PCR coupled with high resolution melting (HRM) analysis for the differentiation of O. viverrini and C. sinensis eggs in fecal samples. Using 2 pairs of species-specific primers, DNA sequences from a portion of the mitochondrial NADH dehydrogenase subunit 2 (nad 2) gene, were amplified to generate 209 and 165 bp products for O. viverrini and C. sinensis, respectively. The distinct characteristics of HRM patterns were analyzed, and the melting temperatures peaked at 82.4+/-0.09degrees C and 85.9+/-0.08degrees C for O. viverrini and C. sinensis, respectively. This technique was able to detect as few as 1 egg of O. viverrini and 2 eggs of C. sinensis in a 150 mg fecal sample, which is equivalent to 7 and 14 eggs per gram of feces, respectively. The method is species-specific, rapid, simple, and does not require fluorescent probes or post-PCR processing for discrimination of eggs of the 2 species. It offers a new tool for differentiation and detection of Asian liver fluke infections in stool specimens.

Evaluation of a Multiplex Real-time PCR Assay for the Detection of Respiratory Viruses in Clinical Specimens

BACKGROUND: In this study, we evaluated the analytical performance and clinical potential of a one-step multiplex real-time PCR assay for the simultaneous detection of 14 types of respiratory viruses using the AdvanSure RV real-time PCR Kit (LG Life Sciences, Korea). METHODS: Three hundred and twenty clinical specimens were tested with the AdvanSure RV real-time PCR Kit and conventional multiplex reverse transcription (RT)-PCR assay. The assay results were analyzed and the one-step AdvanSure RV real-time PCR Kit was compared with the conventional multiplex RT-PCR assay with respect to the sensitivity and specificity of the detection of respiratory viruses. RESULTS: The limit of detection (LOD) was 1.31 plaque-forming units (PFU)/mL for human rhinoviruses (hRVs), 4.93 PFU/mL for human coronavirus HCoV-229E/NL63, 2.67 PFU/mL for human coronavirus HCoV-OC43, 18.20 PFU/mL for parainfluenza virus 1 (PIV)-1, 24.57 PFU/mL for PIV-2, 1.73 PFU/mL for PIV-3, 1.79 PFU/mL for influenza virus group (Flu) A, 59.51 PFU/mL for FluB, 5.46 PFU/mL for human respiratory syncytial virus (hRSV)-A, 17.23 PFU/mL for hRSV-B, 9.99 PFU/mL for human adenovirus (ADVs). The cross-reactivity test for this assay against 23 types of non-respiratory viruses showed negative results for all viruses tested. The agreement between the one-step AdvanSure multiplex real-time PCR assay and the conventional multiplex RT-PCR assay was 98%. CONCLUSIONS: The one-step AdvanSure RV multiplex real-time PCR assay is a simple assay with high potential for specific, rapid and sensitive laboratory diagnosis of respiratory viruses compared to conventional multiplex RT-PCR.

Detection of alpha-thalassemia-1 Southeast Asian and Thai Type Deletions and beta-thalassemia 3.5-kb Deletion by Single-tube Multiplex Real-time PCR with SYBR Green1 and High-resolution Melting Analysis / 대한진단검사의학회지

BACKGROUND: Prevention and control of thalassemia requires simple, rapid, and accurate screening tests for carrier couples who are at risk of conceiving fetuses with severe thalassemia. METHODS: Single-tube multiplex real-time PCR with SYBR Green1 and high-resolution melting (HRM) analysis were used for the identification of alpha-thalassemia-1 Southeast Asian (SEA) and Thai type deletions and beta-thalassemia 3.5-kb gene deletion. The results were compared with those obtained using conventional gap-PCR. DNA samples were derived from 28 normal individuals, 11 individuals with alpha-thalassemia-1 SEA type deletion, 2 with alpha-thalassemia-1 Thai type deletion, and 2 with heterozygous beta-thalassemia 3.5-kb gene deletion. RESULTS: HRM analysis indicated that the amplified fragments from alpha-thalassemia-1 SEA type deletion, alpha-thalassemia-1 Thai type deletion, beta-thalassemia 3.5-kb gene deletion, and the wild-type beta-globin gene had specific peak heights at mean melting temperature (Tm) values of 86.89degrees C, 85.66degrees C, 77.24degrees C, and 74.92degrees C, respectively. The results obtained using single-tube multiplex real-time PCR with SYBR Green1 and HRM analysis showed 100% consistency with those obtained using conventional gap-PCR. CONCLUSIONS: Single-tube multiplex real-time PCR with SYBR Green1 and HRM analysis is a potential alternative for routine clinical screening of the common types of alpha- and beta-thalassemia large gene deletions, since it is simple, cost-effective, and highly accurate.

Detection of alpha-thalassemia-1 Southeast Asian and Thai Type Deletions and beta-thalassemia 3.5-kb Deletion by Single-tube Multiplex Real-time PCR with SYBR Green1 and High-resolution Melting Analysis / 대한진단검사의학회지

BACKGROUND: Prevention and control of thalassemia requires simple, rapid, and accurate screening tests for carrier couples who are at risk of conceiving fetuses with severe thalassemia. METHODS: Single-tube multiplex real-time PCR with SYBR Green1 and high-resolution melting (HRM) analysis were used for the identification of alpha-thalassemia-1 Southeast Asian (SEA) and Thai type deletions and beta-thalassemia 3.5-kb gene deletion. The results were compared with those obtained using conventional gap-PCR. DNA samples were derived from 28 normal individuals, 11 individuals with alpha-thalassemia-1 SEA type deletion, 2 with alpha-thalassemia-1 Thai type deletion, and 2 with heterozygous beta-thalassemia 3.5-kb gene deletion. RESULTS: HRM analysis indicated that the amplified fragments from alpha-thalassemia-1 SEA type deletion, alpha-thalassemia-1 Thai type deletion, beta-thalassemia 3.5-kb gene deletion, and the wild-type beta-globin gene had specific peak heights at mean melting temperature (Tm) values of 86.89degrees C, 85.66degrees C, 77.24degrees C, and 74.92degrees C, respectively. The results obtained using single-tube multiplex real-time PCR with SYBR Green1 and HRM analysis showed 100% consistency with those obtained using conventional gap-PCR. CONCLUSIONS: Single-tube multiplex real-time PCR with SYBR Green1 and HRM analysis is a potential alternative for routine clinical screening of the common types of alpha- and beta-thalassemia large gene deletions, since it is simple, cost-effective, and highly accurate.

Evaluation of a Newly Developed Multiplex Real-time PCR Assay for the Detection of Vancomycin-Resistant Enterococci from Rectal Swabs / 대한임상미생물학회지

BACKGROUND: Asymptomatic vancomycin-resistant enterococci (VRE) colonization precedes infection. VRE-colonized patients serve as silent reservoirs of enterococci that go on to colonize other patients. Rapidly identifying colonized patients is crucial to prevent the spread of VRE. The culture-based method of VRE screening is time-consuming. We evaluated the diagnostic performance of a recently developed multiplex real-time PCR for the detection of VRE. METHODS: We obtained 105 rectal swabs from patients who were being monitored for carriage of VRE. After 24 hour incubation of swabs in enterococcosel broth (EB) supplemented with 6 microg/mL vancomycin, multiplex real-time PCR was performed using the Anyplex(TM) VanR Real-time Detection (VanR) kit (Seegene, Inc., Seoul, Korea). The results of multiplex real-time PCR were compared to those of culture. We evaluated the specificity and detection limits of multiplex real-time PCR using VanR for VRE. RESULTS: A total of 96/105 (91.4%) samples were VRE positive according to multiplex real-time PCR with EB while 85/105 (80.9%) samples were positive in culture. Eleven discordant results (10.4%) (multiplex real-time PCR positive, culture negative) were noted. All non-enterococcal bacteria and vancomycin-susceptible enterococci were negative. The DNA detection limits of VanR were 0.035 pg per reaction (3 microL) for Enterococcus faecium and 0.35 pg for Enterococcus faecalis. CONCLUSION: The application of multiplex real-time PCR after EB incubation allows rapid and sensitive detection in 26-28 hours for VRE screening from rectal swabs. This method could facilitate the timely implementation of contact isolation to prevent the spread of VRE.

Detection of Vancomycin-resistant Enterococci using Multiplex Real-time PCR Assay and Melting Curve Analysis / 대한진단검사의학회지

BACKGROUND: We developed and evaluated the utility of a multiplex real-time PCR assay that uses melting curve analysis and allows simultaneous identification of vancomycin-resistant genotypes and clinically relevant enterococci. METHODS: The specificity of the assay was tested using 4 reference strains of vancomycin-resistant enterococci (VRE) and 2 reference strains of vancomycin-susceptible enterococci. Ninety-three clinical isolates of enterococci with different glycopeptide-resistant phenotypes were genotyped and identified using a multiplex real-time PCR assay and melting curve analysis. RESULTS: Representative melting curves were obtained for Enterococcus faecium, Enterococcus faecalis, vanA-containing E. faecium, vanB-containing E. faecalis, Enterococcus gallinarum, and Enterococcus casseliflavus. Phenotypic and genotypic analysis of the isolates revealed same results for 82 enterococcal isolates, while in 4 isolates, the glycopeptide-resistant phenotypes were inconsistent with the glycopeptide-resistant genotypes and in the 4 other isolates, species could not be accurately identified. Three isolates with mixed strains, which were detected by the PCR assay, could not be correctly identified using phenotypic methods. CONCLUSIONS: VRE genotyping and identification of clinically relevant enterococci were rapidly and correctly performed using multiplex real-time PCR assay and melting curve analysis.

Development of multiplex real time PCR methodology for better identification and discrimination of Vibrio cholerae and O139 serotype / 中华流行病学杂志

Objective To develop a rapid, sensitive and specific assay method, based on multiplex real time PCR for identifying Vibrio cholerae and distinguishing Vibrio cholerae O139 serotype from Vibrio cholerae. Methods Cholera toxin A subunit gene (ctxA) and glycosyltransferase gene (LPSgt) were chosen as targets according to Vibrio cholerae and Vibrio cholerae O139 serotype,and then the primers and TaqMan-MGB probe were designed. The 5'end of probes was labeled with FAM and VIC fluoresceins respectively while the 3' end of probes was labeled with MGB. The PCR reaction was optimized systematically. Then the specificity, sensitivity and reproducibility of multiplex real time PCR were estimated. Finally, multiplex real time PCR was applied to detect the clinical specimens. Results Vibrio cholerae was identified by multiplex real time PCR accurately and quickly,which could distinguish Vibrio cholerae O139 serotype from Vibrio cholerae. Vibrio cholerae was identified positive for primer pairs and probes from ctxA gene, and Vibrio cholerae O139 serotype tested was positive for LPSgt gene. Meanwhile, none of other bacteria was identified. Sensitivity of the test was 2 × 102 cfu/ml. When this assay was applied directly to identify 45 clinical specimens, results showed that 10 were positive to Vibrio cholerae, in which 4 clinical specimens were positive to Vibrio cholerae O139 serotype. All the results were the same to the one that had been obtained from the conventional assays. Conclusion Our rsults showed that the multiplex real time PCR was a reliable,accurate and feasible method for identifying Vibrio cholerae that carrying toxin gene and distinguishing Vibrio cholerae O139 serotype from Vibrio cholerae. This method could be applied to the cholera surveillance, prevention and control system for identifying and distinguishing Vibrio cholerae in the labs.

A multiplex real-time PCR for differential detection and quantification of Salmonella spp., Salmonella enterica serovar Typhimurium and Enteritidis in meats

Salmonella (S.) Typhimurium and S. Enteritidis are the major causative agents of food-borne illnesses worldwide. Currently, a rapid detection system using multiplex real-time polymerase chain reaction (PCR) has been applied for other food-borne pathogens such as Escherichia coli, Staphylococcus aureus and Streptococcus spp. A multiplex real-time PCR was developed for the simultaneous detection of Salmonella spp., especially S. Typhimurium and S. Enteritidis, in beef and pork. For the specific and sensitive multiplex real-time PCR, three representative primers and probes were designed based on sequence data from Genbank. Among the three DNA extraction methods (boiling, alkaline lysis, and QIAamp DNA Mini Kit), the QIAamp DNA Mini Kit was the most sensitive in this study. The optimized multiplex real-time PCR was applied to artificially inoculated beef or pork. The detection sensitivity of the multiplex real-time PCR was increased. The specificity of the multiplex real-time PCR assay, using 128 pure-cultured bacteria including 110 Salmonella isolates and 18 non-Salmonella isolates, was 100%, 100% and 99.1% for Salmonella spp., S. Typhimurium and S. Enteritidis, respectively. The sensitivity was 100%, 100% and 91.7% for Salmonella spp., S. Typhimurium and S. Enteritidis, respectively. The multiplex real-time PCR assay developed in this study could detect up to 0.54 +/- 0.09 and 0.65 +/- 0.07 log10 CFU/ml for S. Typhimurium and S. Enteritidis for beef, 1.45 +/- 0.21 and 1.65 +/- 0.07 log10 CFU/ml for S. Typhimurium and S. Enteritidis for pork, respectively, with all conditions optimized. Our results indicated that the multiplex real-time PCR assay developed in this study could sensitively detect Salmonella spp. and specifically differentiate S. Typhimurium from S. Enteritidis in meats.

Detection and identification of Escherichia coli O157:H7 by multiplex real-time PCR / 中华微生物学和免疫学杂志

Objective To develop a rapid, sensitive and specific assay based on multiplex real-time PCR for detecting and identifying Escherichia coli O157: H7. Methods The lipopolysaccharide gene (rJbE) and H7 flagellar antigen gene(fliC) of Escherichia coli O157:H7 was chosen as targets, and then the primers and TaqMan-MGB probe were designed. The 5'end of probes was labeled with FAM and HEX fluo-resceins respectively; the 3'end of probes was labeled with MGB. The PCR reaction was optimized systemati-cally. Then the specificity, sensitivity and reproducibility of multiplex real-time PCR were estimated. Final-ly, multiplex real-time PCR was applied to detected clinical specimens. Results Escherichia coil O157:H7 were detected by multiplex real-time PCR accurately and quickly, which could distinguish Escherichia coli O157:H7 from O157: non-H7. Meanwhile, none of other bacteria could be identified. The sensitivity was 10 CFU/ml in pure culture. The coefficient of variation of intra-assay and inter-assay was less than 5%. When this assay was applied directly to identify 66 clinical specimens, the results showed that t5 were positive to Escherichia coil O157:H7 and 2 were positive to Escherichia coil O157: non-H7, in which 16 was the same to the results obtained from the conventional assays. The coincidence was 98.49%. Conclusion It is showed that multiplex real-time PCR is a reliable, accurate and feasible assay for detecting and identifying Escherich-ia coli Oi57: H7, The assay reported here provided a tool for analysis and diagnosis in the field of detecting clinical pathogens, epidemiologic survey and food safety monitoring.

Development of Multiplex Real-time PCR for Detection of Toxigenic Vibrio cholerae and Virbio parahaemolyticus / 微生物学通报

A multiplex real-time PCR was developed to detect ctxA of Vibrio cholerae, gyrB and tdh of Vibrio parahaemolyticus simultaneously. The multiplex real-time PCR were evalidated by detection for the three genes in 47 toxigenic V. cholerae O1 and O139 strains (ctxA+; O1=3, O139=44), 25 non-toxigenic V. cholerae strains (ctxA-; O1=12, O139=6, non-O1 and non-O139=7), 116 V. parahaemolyticus strains with or without tdh (73 or 43) and 9 other bacteria strains. The specificity and sensitivity of the multiplex real-time PCR in detection for the ctxA and the tdh genes in the strains tested were both 100.0%, compared to the results by routine PCRs. In the detection for V. parahaemolyticus specific gyrB using the multiplex real-time PCR, all of 116 V. parahaemolyticus strains were positive, and 9 other strains and 72 V. cholerae strains were all negative. The multiplex real-time PCR is a sensitive, specific and quick assay not only for detecting virulence genes of V. cholerae and V. parahaemolyticus but also for identifying V. parahaemolyticus at species level. In addition, two real-time PCRs for detection of V. parahaemolyticus virulence genes trh1 and trh2 were also developed.