Multiplex real-time PCR detection of Vibrio cholerae

Cholera is an important enteric disease, which is endemic to different regions of the world and has historically been the cause of severe pandemics. Vibrio cholerae is a natural inhabitant of the aquatic environment and the toxigenic strains are causative agents of potentially life-threatening diarrhoea. A multiplex, real-time detection assay was developed targeting four genes characteristic of potentially toxigenic strains of V. cholerae, encoding: repeat in toxin (rtxA), extracellular secretory protein (epsM), mannose-sensitive pili (mshA) and the toxin coregulated pilus (tcpA). The assay was developed on the Cepheid Smart Cycler using SYBR Green I for detection and the products were differentiated based on melting temperature (Tm) analysis. Validation of the assay was achieved by testing against a range of Vibrio and non-Vibrio species. The detection limit of the assay was determined to be 10(3) CFU using cells from pure culture. This assay was also successful at detecting V. cholerae directly from spiked environmental water samples in the order of 10(4) CFU, except from sea water which inhibited the assay. The incorporation of a simple DNA purification step prior to the addition to the PCR increased the sensitivity 10 fold to 10(3) CFU. This multiplex real-time PCR assay allows for a more reliable, rapid detection and identification of V. cholerae which is considerably faster than current conventional detection assays.     

A highly sensitive and specific multiplex PCR assay for simultaneous detection of Vibrio cholerae,Vibrio parahaemolyticus and Vibrio vulnificus

Aims: To develop an effective multiplex PCR for simultaneous and rapid detection of Vibrio cholerae, Vibrio vulnificus and Vibrio parahaemolyticus, the three most important Vibrio species that can cause devastating health hazards among human. Methods and Results: Species‐specific PCR primers were designed based on toxR gene for V. cholerae and V. parahaemolyticus, and vvhA gene for V. vulnificus. The multiplex PCR was validated with 488 Vibrio strains including 322 V. cholerae, 12 V. vulnificus, and 82 V. parahaemolyticus, 20 other Vibrio species and 17 other bacterial species associated with human diseases. It could detect the three target bacteria without any ambiguity even among closely related species. It showed good efficiency in detection of co‐existing target species in the same sample. The detection limit of all the target species was ten cells per PCR tube. Conclusions: Specificity and sensitivity of the multiplex PCR is 100% each and sufficient for simultaneous detection of these potentially pathogenic Vibrio species in clinical and environmental samples. Significance and Impact of the Study: This simple, rapid and cost‐effective method can be applicable in a prediction system to prevent disease outbreak by these Vibrio species and can be considered as an effective tool for both epidemiologist and ecologist.     

Development of a multiplex single-tube nested PCR (MSTNPCR) assay for Vibrio cholerae O1 detection

A multiplex nested PCR method for detection of Vibrio cholerae O1 using a single tube was developed (MSTNPCR). Firstly, single-tube nested PCR (STNPCR) with primers directed to ctxA gene was standardized, and its detection limit was compared to simple PCR and two-step nested PCR. Secondly, primers directed to rfbN gene were added to the reaction. The detection limit of the multiplex reaction was determined using V. cholerae O1 DNA and V. cholerae O1 grown in alkaline peptone water (APW). STNPCR was shown to be approximately 100-fold more sensitive than simple PCR and 10 times less sensitive than two-step nested PCR. This drawback is compensated by a lower risk of cross-contamination. The addition of a second target did not impair the detection limit of STNPCR (as little as 1 pg of V. cholerae O1 DNA detected). MSTNPCR could specifically detect up to three V. cholerae O1 cells or colony forming units (cfu) directly from the APW growth. A diagnostic kit consisting of a set of microtubes having the inner primers fixed onto the inside of the tube cap and a set of tubes containing the reaction mixture was evaluated for stability, and it proved to be stable for five months at -20 degrees C. Therefore, MSTNPCR would be useful in the detection of V. cholerae O1 directly from environmental waters in cholera endemic areas and in complementing the identification of toxigenic strains isolated by culture.     

A multiplex real-time PCR assay for detection of oseltamivir-resistant strains of influenza virus

Influenza is a contagious disease of humans and animals caused by viruses belonging to the Orthomyxoviridae family. The influenza A virus genome consists of negative sense, single-stranded, segmented RNA. Influenza viruses are classified into subtypes based on two surface antigens known as hemagglutinin (H) and neuraminidase (N). The main problem with influenza A viruses infecting humans is drug resistance, which is caused by antigenic changes. A few antiviral drugs are available, but the most popular is the neuraminidase inhibitor — oseltamivir. The resistance against this drug has probably developed through antigenic drift by a point mutation in one amino acid at position 275 (H275Y). In order to prevent a possible influenza pandemic it is necessary to develop fast diagnostic tests. The aim of this project was to develop a new test for detection of influenza A virus and determination of oseltamivir resistance/sensitivity in humans. Detection and differentiation of oseltamivir resistance/sensitivity of influenza A virus was based on real-time PCR. This test contains two TaqMan probes, which work at different wavelengths. Application of techniques like multiplex real-time PCR has greatly enhanced the capability for surveillance and characterization of influenza viruses. After its potential validation, this test can be used for diagnosis before treatment.     

WSSV和IHHNV二重实时荧光PCR检测方法的建立

根据基因库中对虾白斑综合征病毒WSSV(AF369029)和传染性皮下及造血器官坏死病毒IHHNV(AF218226)基因序列,设计了WSSV和IHHNV的两对特异性引物和两条用不同荧光基团标记的TaqMan探针。对反应条件和试剂浓度进行优化,建立了能够同时检测WSSV和IHHNV的二重实时荧光PCR方法。该方法特异性好,对WSSV和IHHNV的检测敏感性分别达到2和20个模板拷贝数;此外抗干扰能力强,对WSSV和IHHNV不同模板浓度进行组合,仍可有效地同时检测这二个病毒。对保存的30份经常规PCR检测仅为WSSV或IHHNV阳性的样品进行二重实时荧光PCR检测,结果都为阳性,其中1份为WSSV和IHHNV混合感染。本研究建立的二重实时荧光PCR方法用于WSSV和IHHNV的检测具有特异、敏感、快速、定量等优点。     

A novel multiplex PCR for the identification of Vibrio parahaemolyticus, Vibrio cholerae and Vibrio vulnificus

AIM: To establish a simple multiplex polymerase chain reaction (PCR) that will identify Vibrio parahaemolyticus, Vibrio cholerae and Vibrio vulnificus. METHODS AND RESULTS: A total of 429 Vibrio spp. from various origins were tested with the novel primers targeting toxR. The reverse primers were all designed to be species specific, while the forward primer was universal. The primers correctly identified all the V. parahaemolyticus, V. cholerae and V. vulnificus isolates tested. CONCLUSIONS: The toxR multiplex PCR works well when the initial colony morphology is known. If not, Vibrio alginolyticus might represent a diagnostic obstacle. SIGNIFICANCE AND IMPACT OF THE STUDY: The method provides a fast and reliable way of identifying the main Vibrio spp. involved in food-borne disease. The method could prove very useful for laboratories working with identification of these Vibrio spp.     

A MIQE-compliant real-time PCR assay for Aspergillus detection

The polymerase chain reaction (PCR) is widely used as a diagnostic tool in clinical laboratories and is particularly effective for detecting and identifying infectious agents for which routine culture and microscopy methods are inadequate. Invasive fungal disease (IFD) is a major cause of morbidity and mortality in immunosuppressed patients, and optimal diagnostic criteria are contentious. Although PCR-based methods have long been used for the diagnosis of invasive aspergillosis (IA), variable performance in clinical practice has limited their value. This shortcoming is a consequence of differing sample selection, collection and preparation protocols coupled with a lack of standardisation of the PCR itself. Furthermore, it has become clear that the performance of PCR-based assays in general is compromised by the inadequacy of experimental controls, insufficient optimisation of assay performance as well as lack of transparency in reporting experimental details. The recently published "Minimum Information for the publication of real-time Quantitative PCR Experiments" (MIQE) guidelines provide a blueprint for good PCR assay design and unambiguous reporting of experimental detail and results. We report the first real-time quantitative PCR (qPCR) assay targeting Aspergillus species that has been designed, optimised and validated in strict compliance with the MIQE guidelines. The hydrolysis probe-based assay, designed to target the 18S rRNA DNA sequence of Aspergillus species, has an efficiency of 100% (range 95-107%), a dynamic range of at least six orders of magnitude and limits of quantification and detection of 6 and 0.6 Aspergillus fumigatus genomes, respectively. It does not amplify Candida, Scedosporium, Fusarium or Rhizopus species and its clinical sensitivity is demonstrated in histological material from proven IA cases, as well as concordant PCR and galactomannan data in matched broncho-alveolar lavage and blood samples. The robustness, specificity and sensitivity of this assay make it an ideal molecular diagnostic tool for clinical use.     

Development of a multiplex real-time PCR assay with an internal amplification control for the detection of total and pathogenic Vibrio parahaemolyticus bacteria in oysters

Vibrio parahaemolyticus is an estuarine bacterium that is the leading cause of shellfish-associated cases of bacterial gastroenteritis in the United States. Our laboratory developed a real-time multiplex PCR assay for the simultaneous detection of the thermolabile hemolysin (tlh), thermostable direct hemolysin (tdh), and thermostable-related hemolysin (trh) genes of V. parahaemolyticus. The tlh gene is a species-specific marker, while the tdh and trh genes are pathogenicity markers. An internal amplification control (IAC) was incorporated to ensure PCR integrity and eliminate false-negative reporting. The assay was tested for specificity against >150 strains representing eight bacterial species. Only V. parahaemolyticus strains possessing the appropriate target genes generated a fluorescent signal, except for a late tdh signal generated by three strains of V. hollisae. The multiplex assay detected <10 CFU/reaction of pathogenic V. parahaemolyticus in the presence of >10(4) CFU/reaction of total V. parahaemolyticus bacteria. The real-time PCR assay was utilized with a most-probable-number format, and its results were compared to standard V. parahaemolyticus isolation methodology during an environmental survey of Alaskan oysters. The IAC was occasionally inhibited by the oyster matrix, and this usually corresponded to negative results for V. parahaemolyticus targets. V. parahaemolyticus tlh, tdh, and trh were detected in 44, 44, and 52% of the oyster samples, respectively. V. parahaemolyticus was isolated from 33% of the samples, and tdh(+) and trh(+) strains were isolated from 19 and 26%, respectively. These results demonstrate the utility of the real-time PCR assay in environmental surveys and its possible application to outbreak investigations for the detection of total and pathogenic V. parahaemolyticus.     

Simultaneous detection of waterborne viruses by multiplex real-time PCR

Norovirus, Rotavirus group A, the Hepatitis A virus, and Coxsackievirus are all common causes of gastroenteritis. Conventional diagnoses of these causative agents are based on antigen detection and electron microscopy. To improve the diagnostic potential for viral gastroenteritis, internally controlled multiplex real-time polymerase chain reaction (PCR) methods have been recently developed. In this study, individual real-time PCRs were developed and optimized for specific detections of Norovirus genogroup I, Norovirus genogroup II, Rotavirus group A, the Hepatitis A virus, and Coxsackievirus group B1. Subsequently, individual PCRs were combined with multiplex PCR reactions. In general, multiplex real-time PCR assays showed comparable sensitivities and specificities with individual assays. A retrospective clinical evaluation showed increased pathogen detection in 29% of samples using conventional PCR methods. Prospective clinical evaluations were detected in 123 of the 227 (54%) total samples used in the multiplex real-time PCR analysis. The Norovirus genogroup II was found most frequently (23%), followed by Rotavirus (20%), the Hepatitis A virus (4.5%), Coxsackievirus (3.5%), and Norovirus genogroup I (2.6%). Internally controlled multiplex real-time PCR assays for the simultaneous detection of Rotavirus, Coxsackievirus group B, the Hepatitis A virus, and Norovirus genogroups I and II showed significant improvement in the diagnosis of viral gastroenteritis.     

用基于TaqMan探针的Real-time PCR技术定量检测副溶血弧菌

副溶血弧菌是一种引起食源性疾病的重要病原菌,传统的鉴定方法费时费力且容易出现假阴性,建立一种定量检测副溶血弧菌基因的方法尤为重要。根据GenBank公布的副溶血弧菌的gyrB基因序列设计一对引物和TaqMan探针,建立了基于TaqMan探针的RealtimePCR方法。通过对9种细菌(12株菌株)的DNA进行扩增,结果所有4株副溶血弧菌均可产生扩增曲线,其他8株非副溶血弧菌均不产生扩增曲线,证明了引物和探针具有很高的特异性。细菌纯培养物品和人工布菌的检测敏感度分别为1CFUPCR反应体系和10CFUPCR反应体系,相关系数均为0.99(r2=0.99),整个试验可在1h内完成。建立的方法可用于海产品中副溶血弧菌的快速定量检测。     

Fluorescent detection techniques for real-time multiplex strand specific detection of Bacillus anthracis using rapid PCR

Speed is a key area in our development of PCR assays for Bacillus anthracis. We believe that the strand specific detection of amplicons within 10 min is a realistic goal and that this will be achieved through fluorescent in-tube assays. We have used the Idaho LightCycler to study and develop candidate assays for B. anthracis. New strand specific fluorescent methods have been developed and a number of formats have been studied for speed and sensitivity. Internal controls have been developed as a method of improving our assay confidence. In this communication we will introduce the field of rapid PCR whilst discussing previous work in the areas described above, the development of our own rapid assay and a novel internal control system for B. anthracis. This work used PCR assays and hardware that are either commercially available, or have been previously described in open literature publications.     

Thermal factors influencing detection of Vibrio vulnificus using real-time PCR

Five thermal factors, including initial denaturation temperature, cycling denaturation temperature, annealing temperature, extension temperature and the temperature at which the intensity of the fluorescent signal is read, were evaluated for their effects on the detection of Vibrio vulnificus via real-time PCR. Fluorescent signal detection after extension was set between the Tm value of the primer-dimers (79 degrees C) and that of the PCR target amplicons (84 degrees C). This effectively eliminated the overestimation of the yield of PCR amplicons due to the presence of primer-dimers which otherwise led to erroneously lower Ct values (1.91+/-0.22 cycles lower). The annealing and extension steps were combined to convert a three-step PCR to a two-step PCR. This consisted of initial denaturation at 95 degrees C for 3 min, cycling denaturation at 94 degrees C for 15 s and a combined annealing and extension step at 60 degrees C for 5 s in each PCR cycle. One genomic target per real-time PCR reaction was detected with the simplified two-step PCR.     

Analysis of lolB gene sequence and its use in the development of a PCR assay for the detection of Vibrio cholerae

A PCR assay has been developed based on a lolB (hemM) gene, which was found to be highly conserved among the Vibrio cholerae species but non-conserved among the other enteric bacteria. The lolB PCR detected all O1, O139 and non-O1/non-O139 serogroup and biotypes of V. cholerae. The analytical specificity of this assay was 100% while the analytical sensitivity was 10 pg/microL and 10(3) CFU/mL at DNA and bacterial level respectively. The diagnostic sensitivity and specificity was 98.5% and 100% respectively.     

Quantitative multiplex real-time PCR for detection of PLP1 gene duplications in Pelizaeus-Merzbacher patients

Pelizaeus-Merzbacher disease (PMD) is an X-linked recessive disorder of central nervous system (CNS) myelination typically affecting males. A genomic duplication of variable size at Xq22.2, containing the entire proteolipid protein 1 gene (PLP1), is responsible for approximately 60-70% of PMD cases. The aim of this study was to develop a rapid and robust method for determination of PLP1 gene dosage. We optimized two multiplex real-time quantitative PCR (Q-PCR) assays targeting exons 3 and 6 of the PLP1 gene, and then validated these assays by retrospective analysis of a set of genomic DNAs from 67 previously tested patients and 43 normal controls. Samples were analyzed in multiplex PCR reactions using TaqMan chemistry and the ABI Prism 7000 Sequence Detection System. PLP1 dosage was determined by the relative quantitative comparative threshold cycle method (DeltaDeltaCt) using the human serum albumin gene as the endogenous reference gene. Three clearly non-overlapping ranges of results, corresponding to the presence of one, two, or three PLP1 copies, were detected in both assays. The results were completely concordant with gender and previous PLP1 gene dosage testing based on quantitative fluorescent multiplex PCR and analysis of a dinucleotide polymorphism in the first intron of the PLP1 gene. We conclude that multiplex real-time Q-PCR represents a fast and reliable tool for PLP1 duplication testing in PMD families.     

A simple and convenient microtiter plate assay for the detection of bactericidal antibodies to Vibrio cholerae O1 and Vibrio cholerae O139

It is believed that the correlate of protection for cholera can be determined by the serum vibriocidal assay. The currently available vibriocidal assays, based on the conventional agar plating technique, are labor intensive. We developed a simple and convenient microtiter plate assay for the detection of vibriocidal antibodies that is equally as efficient for Vibrio cholerae O1 and for V. cholerae O139. The addition of succinate and neotetrazolium made it possible to measure the growth of surviving bacterial target cells by monitoring a color change. We evaluated assay parameters (target strains, growth of target cells, complement source and concentration) that may affect the reproducibility of the method for V. cholerae O139. The results obtained with the microtiter plate assay were uniformly similar to those obtained with the conventional agar plating assay, when testing both the Inaba and Ogawa serotypes of V. cholerae O1. The microtiter plate assay was also convenient for measuring the activity of animal sera and mouse monoclonal antibodies.