Differentiation of the Gene Clusters Encoding Botulinum Neurotoxin Type A Complexes in Clostridium botulinum Type A, Ab, and A(B) Strains

We describe a strategy to identify the clusters of genes encoding components of the botulinum toxin type A (boNT/A) complexes in 57 strains of Clostridium botulinum types A, Ab, and A(B) isolated in Italy and in the United States from different sources. Specifically, we combined the results of PCR for detecting the ha33 and/or p47 genes with those of boNT/A PCR-restriction fragment length polymorphism analysis. Three different type A toxin gene clusters were revealed; type A1 was predominant among the strains from the United States, whereas type A2 predominated among the Italian strains, suggesting a geographic distinction between strains. By contrast, no relationship between the toxin gene clusters and the clinical or food source of strains was evident. In two C. botulinum type A isolates from the United States, we recognized a third type A toxin gene cluster (designated type A3) which was similar to that previously described only for C. botulinum type A(B) and Ab strains. Total genomic DNA from the strains was subjected to pulsed-filed gel electrophoresis and randomly amplified polymorphic DNA analyses, and the results were consistent with the boNT/A gene clusters obtained.     

Differentiation of the gene clusters encoding botulinum neurotoxin type A complexes in Clostridium botulinum type A, Ab, and A(B) strains

We describe a strategy to identify the clusters of genes encoding components of the botulinum toxin type A (boNT/A) complexes in 57 strains of Clostridium botulinum types A, Ab, and A(B) isolated in Italy and in the United States from different sources. Specifically, we combined the results of PCR for detecting the ha33 and/or p47 genes with those of boNT/A PCR-restriction fragment length polymorphism analysis. Three different type A toxin gene clusters were revealed; type A1 was predominant among the strains from the United States, whereas type A2 predominated among the Italian strains, suggesting a geographic distinction between strains. By contrast, no relationship between the toxin gene clusters and the clinical or food source of strains was evident. In two C. botulinum type A isolates from the United States, we recognized a third type A toxin gene cluster (designated type A3) which was similar to that previously described only for C. botulinum type A(B) and Ab strains. Total genomic DNA from the strains was subjected to pulsed-filed gel electrophoresis and randomly amplified polymorphic DNA analyses, and the results were consistent with the boNT/A gene clusters obtained.     

嗜水气单胞菌菌落多重PCR方法的建立及应用

[背景]嗜水气单胞菌(Aeromonas hydrophila)对水产动物、畜禽和人类均有致病性.基因表达的溶血素、气溶素和肠毒素是重要毒力因子,在致病性嗜水气单胞菌早期检测及防治中尤为重要.目前采用菌落直接提取DNA用于多重PCR研究的相关报道较少.[目的]基于菌落PCR方法建立针对嗜水气单胞菌溶血性基因、肠毒素基因...     

A multiplex nested PCR assay for simultaneous detection of Corchorus golden mosaic virus and a phytoplasma in white jute (Corchorus capsularis L.)

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.

Significance and Impact of the Study

Incidence of phytoplasma diseases is increasing worldwide and particularly in the tropical and subtropical world. Co‐infection of phytoplasma and virus(s) is also common. Therefore, use of single primer PCR in detecting these pathogens would require more time and effort, whereas multiplex PCR involving several pairs of primers saves time and reduces cost. In this study, we have developed a multiplex nested PCR assay that provides more sensitive and specific detection of Corchorus golden mosaic virus (CoGMV) and a phytoplasma in white jute simultaneously. It is the first report of simultaneous detection of CoGMV and a phytoplasma in Corchorus capsularis by multiplex nested PCR.     

Simple Method for Detection of Clostridium botulinum Type A to F Neurotoxin Genes by Ploymerase Chain Reaction

A polymerase chain reaction (PCR)-based method was established to detect each type of neurotoxin genes of Clostridium botulinum types A to F by employing the oligonucleotide primer sets corresponding to special regions of the light chains of the neurotoxins. In this procedure, the PCR products were easily confirmed by restriction enzyme digestion profiles, and as little as 2.5 pg of template DNAs from toxigenic strains could be detected. The specific PCR products were obtained from toxigenic C. botulinum types A to F, a type E toxin-producing C. butyricum strain, and a type F toxin-producing C. baratii strain, but no PCR product was detected in nontoxigenic strains of C. botulinum and other clostridial species. The neurotoxin genes were also detected in food products of a seasoned dry salmon and a fermented fish (Izushi) which had caused type E outbreaks of botulism. Therefore, it is concluded that this PCR-based detection method can be used for the rapid diagnosis of botulism.     

A multiplex PCR method to detect 14 Escherichia coli serogroups associated with urinary tract infections

Urinary tract infections (UTIs) are one of the most common bacterial infections and are predominantly caused by uropathogenic Escherichia coli (UPEC). E. coli strains belonging to 14 serogroups, including O1, O2, O4, O6, O7, O8, O15, O16, O18, O21, O22, O25, O75 and O83, are the most frequently detected UPEC strains in a diverse range of clinical urine specimens. In the current study, the O-antigen gene clusters of E. coli serogroups O1, O2, O18 and O75 were characterized. A multiplex PCR method based on O-antigen-specific genes was developed for the simultaneous detection of all 14 E. coli serogroups. The multiplex PCR method was shown to be highly specific and reproducible when tested against 186 E. coli and Shigella O-serogroup reference strains, 47 E. coli clinical isolates and 10 strains of other bacterial species. The sensitivity of the multiplex PCR method was analyzed and shown to detect O-antigen-specific genes in samples containing 25 ng of genomic DNA or in mock urine specimens containing 40 colony-forming units (CFUs) per ml. Five urine specimens from hospital were examined using this multiplex PCR method, and the result for one sample was verified by the conventional serotyping methods. The multiplex PCR method developed herein can be used for the detection of relevant E. coli strains from clinical and/or environmental samples, and it is particularly useful for epidemiologic analysis of urine specimens from patients with UTIs.     

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.     

Multiplex PCR assay for simultaneous detection and differentiation of Mycobacterium tuberculosis, Mycobacterium avium complexes and other Mycobacterial species directly from clinical specimens

Aims: Polymerase chain reaction (PCR) is the most rapid and sensitive method for diagnosing mycobacterial infections and identifying the aetiological Mycobacterial species in order to administer the appropriate therapy and for better patient management. Methods and Results: Two hundred and thirty‐five samples from 145 clinically suspected cases of tuberculosis were processed for the detection of Mycobacterial infections by ZN (Ziehl Neelsen) smear examination, L‐J & BACTEC^TM MGIT‐960 culture and multiplex PCR tests. The multiplex PCR comprised of genus‐specific primers targeting hsp65 gene, Mycobacterium tuberculosis complex‐specific primer targeting cfp10 (Rv3875, esxB) region and Mycobacterium avium complex‐specific primer pairs targeting 16S–23S Internal Transcribed Spacer sequences. The multiplex PCR developed had an analytical sensitivity of 10 fg (3–4 cells) of mycobacterial DNA. The multiplex PCR test showed the highest (77·24%) detection rate, while ZN smear examination had the lowest (20%) detection rate, which was bettered by L‐J culture (34·4%) and BACTEC^TM MGIT‐960 culture (50·34%) methods. The mean isolation time for M. tuberculosis was 19·03 days in L‐J culture and 8·7 days in BACTEC^TM MGIT‐960 culture. Using the multiplex PCR, we could establish M. tuberculosis + M. avium co‐infection in 1·3% HIV‐negative and 2·9% HIV‐positive patients. The multiplex PCR was also highly useful in diagnosing mycobacteraemia in 38·09% HIV‐positive and 15·38% HIV‐negative cases. Conclusions: The developed in‐house multiplex PCR could identify and differentiate the M. tuberculosis and M. avium complexes from other Mycobacterial species directly from clinical specimens. Significance and Impact of the Study: The triplex PCR developed by us could be used to detect and differentiate M. tuberculosis, M. avium and other mycobacteria in a single reaction tube.     

5种转基因油菜转化体特异性多重PCR检测方法

【目的】全球转基因植物及其产品的数量和种类越来越多,迫切需要可同时精准高效检测多个转化载体的检测方法。【方法】针对RF1、MS8、Topas19/2、Oxy235和RF3等5个转基因油菜品系的侧翼序列及油菜内源基因cruciferin A(Cru A)序列设计多重聚合酶链式反应特异性引物,通过对转基因油菜、转基因大豆、转基因玉米、转基因水稻、转基因棉花等不同作物进行PCR扩增来测试所选择的引物特异性,优化多重PCR反应引物的浓度,用所建立的检测体系对不同混合比例的转基因油菜进行多重PCR扩增来测试所建立的检测方法的灵敏度。【结果】通过测试,仅在含有目标样品中检测出阳性结果,灵敏度达0.05%,表明所建立的6重PCR检测方法可同时精准检测RF1、MS8、Topas19/2、Oxy235和RF3等5种转基因油菜转化载体。【结论】所建立的6重转基因油菜转化体特异性PCR检测方法通量高、特异性好、灵敏度高,符合有关转基因产品检测的要求,可作为转基因油菜检测的有效方法。     

Detection of Microbial Pathogens in Shellfish with Multiplex PCR

Multiplex PCR amplification of uidA, cth, invA, ctx, and tl genes was developed enabling simultaneous detection in shellfish of Escherichia coli, an indicator of fecal contamination and microbial pathogens, Salmonella typhimurium, Vibrio vulnificus, V. cholerae, and V. parahaemolyticus, respectively. Each of the five pairs of oligonucleotide primers was found to support PCR amplifications of only its targeted gene. The optimized multiplex PCR reaction utilized a PCR reaction buffer containing 2.5 mM MgCl₂ and primer annealing temperature of 55°C. Oyster tissue homogenate seeded with these microbial pathogens was subjected to DNA purification by the Chelex™ 100 (BioRad) method. The sensitivity of detection for each of the microbial pathogens was ≤10¹–10² cells following a “double” multiplex PCR amplification approach. Amplified target genes in a multiplex PCR reaction were subjected to a colorimetric GeneComb™ (BioRad) DNA-DNA hybridization assay. This assay was rapid and showed sensitivity of detection comparable to the agarose gel electrophoresis method. The colorimetric GeneComb™ assay avoids use of hazardous materials inherent in conventional gel electrophoresis and radioactive-based hybridization methods. Multiplex PCR amplification, followed by colorimetric GeneComb™ DNA-DNA hybridization, has been shown to be an effective, sensitive, and rapid method to detect microbial pathogens in shellfish. Received: 17 November 1997 / Accepted: 17 February 1998     

Pentaplexed Quantitative Real-Time PCR Assay for the Simultaneous Detection and Quantification of Botulinum Neurotoxin-Producing Clostridia in Food and Clinical Samples

Botulinum neurotoxins are produced by the anaerobic bacterium Clostridium botulinum and are divided into seven distinct serotypes (A to G) known to cause botulism in animals and humans. In this study, a multiplexed quantitative real-time PCR assay for the simultaneous detection of the human pathogenic C. botulinum serotypes A, B, E, and F was developed. Based on the TaqMan chemistry, we used five individual primer-probe sets within one PCR, combining both minor groove binder- and locked nucleic acid-containing probes. Each hydrolysis probe was individually labeled with distinguishable fluorochromes, thus enabling discrimination between the serotypes A, B, E, and F. To avoid false-negative results, we designed an internal amplification control, which was simultaneously amplified with the four target genes, thus yielding a pentaplexed PCR approach with 95% detection probabilities between 7 and 287 genome equivalents per PCR. In addition, we developed six individual singleplex real-time PCR assays based on the TaqMan chemistry for the detection of the C. botulinum serotypes A, B, C, D, E, and F. Upon analysis of 42 C. botulinum and 57 non-C. botulinum strains, the singleplex and multiplex PCR assays showed an excellent specificity. Using spiked food samples we were able to detect between 10³ and 10⁵ CFU/ml, respectively. Furthermore, we were able to detect C. botulinum in samples from several cases of botulism in Germany. Overall, the pentaplexed assay showed high sensitivity and specificity and allowed for the simultaneous screening and differentiation of specimens for C. botulinum A, B, E, and F.Botulinum neurotoxins (BoNTs), the causative agents of botulism, are produced by the anaerobic bacterium Clostridium botulinum and are divided into seven serotypes, A to G. While the botulinum neurotoxins BoNT/A, BoNT/B, BoNT/E, and BoNT/F are known to cause botulism in humans, BoNT/C and BoNT/D are frequently associated with botulism in cattle and birds. Despite its toxicity, BoNT/G has not yet been linked to naturally occurring botulism (26).Botulism is a life-threatening illness caused by food contaminated with BoNT (food-borne botulism), by the uptake and growth of C. botulinum in wounds (wound botulism), or by colonization of the intestinal tract (infant botulism) (14). In addition, C. botulinum and the botulinum neurotoxins are regarded as potential biological warfare agents (8).The gold standard for the detection of BoNTs from food or clinical samples is still the mouse lethality assay, which is highly sensitive but rather time-consuming. In addition to various immunological assays for BoNT detection, several conventional and real-time PCR-based assays for the individual detection of bont genes have been reported (2, 9-12, 15, 20, 23, 27-30). A major improvement is the simultaneous detection of more than one serotype, which results in a reduction of effort and in the materials used. In recent years, both conventional and real-time PCR-based multiplex assays have been developed for the simultaneous detection of C. botulinum serotypes (1, 6, 22, 24). To date, however, no internally controlled multiplex real-time PCR assay for the simultaneous detection and differentiation of all four serotypes relevant for humans has been reported.We describe here a highly specific and sensitive multiplex real-time PCR assay based on the 5′-nuclease TaqMan chemistry (17) for the simultaneous detection of the C. botulinum types A, B, E, and F, including an internal amplification control (IAC). Furthermore, we developed six different singleplex assays based on the TaqMan chemistry for the detection of C. botulinum serotypes A to F. Assays were validated on 42 C. botulinum strains, 57 non-C. botulinum strains, on spiked food samples, and on real samples from cases of botulism in Germany.     

One‐step Multiplex RT‐PCR for Simultaneous Detection of Four Viruses in Tobacco

A one‐step multiplex RT‐PCR method has been developed for the simultaneous detection of four viruses frequently occurring in tobacco (Cucumber mosaic virus, Tobacco mosaic virus, Tobacco etch virus and Potato virus Y). Four sets of specific primers were designed to work with the same reaction reagents and cycling conditions, resulting in four distinguishable amplicons representative of the four viruses independently. This one‐step multiplex RT‐PCR is consistently specific using different combinations of virus RNA as templates, and no non‐specific band was observed. It has high sensitivity compared to single RT‐PCR. Moreover, field samples in China can be tested by this method for virus detection. Our results show that one‐step multiplex RT‐PCR is a high‐throughput, specific, sensitive method for tobacco virus detection.     

Multiplex PCR assay for the detection of enterotoxic <Emphasis Type="Italic">Bacillus cereus</Emphasis> group strains and its application in food matrices

Bacillus cereus, Bacillus thuringiensis and Bacillus anthracis are the major concerns for the food safety in terms of frequency and/or seriousness of the disease. Being members of the same group and sharing DNA homology to a larger extent, they do create problems when their specific detection/identification is attempted from different food and environmental sources. Numerous individual polymerase chain reaction (PCR) and few multiplex PCR (mPCR) methods have been employed to detect these organisms by targeting toxin genes but with lack of internal amplification control (IAC). Therefore, we attempted a mPCR with IAC for the detection of enterotoxic B. cereus group strains by selecting hbl A, nhe A and cyt K genes from B. cereus, indicative of the diarrheal potential and cry I A and pag genes, the plasmid borne phenotypic markers specific to B. thuringiensis and B. anthracis strains, respectively. Multiplex PCR assay validation was performed by simultaneous comparison with the results of single-target PCR assays and correlated to the classical conventional and biochemical identification of the organisms. The mPCR was able to detect as low as 10¹–10² organisms per ml following overnight enrichment of spiked food samples (vegetable biriyani and milk) in buffered peptone water (BPW). The presence of these organisms could also be detected by mPCR in naturally contaminated samples of rice based dishes and milk. The high throughput and cost-effective mPCR method described could provide a powerful tool for simultaneous, rapid and reliable detection of enterotoxic B. cereus group organisms.     

A Simple Allele-Specific PCR Assay for Detecting <Emphasis Type="Italic">FAD2</Emphasis> Alleles in Both A and B Genomes of the Cultivated Peanut for High-Oleate Trait Selection

In cultivated tetraploid peanut (2n = 4x = 40, AABB), the conversion of oleic acid to linoleic acid is mainly catalyzed by the Δ¹² fatty acid desaturase (FAD). Two homoeologous genes (FAD2A and FAD2B) encoding for the desaturase are located on the A and B genomes, respectively. Abolishing or reducing the desaturase activity by gene mutation can significantly increase the oleic acid/linoleic acid ratio. F435-derived high-oleate peanut cultivars contain two key mutations within the Δ¹² fatty acid desaturase gene which include a 1-bp substitution of G:C→A:T in the A genome and a 1-bp insertion of A:T in the B genome. Both of these mutations contribute to abolishing or reducing the desaturase activity, leading to accumulation of oleate versus linoleate. Currently, detection of FAD2 alleles can be achieved by a cleaved amplified polymorphic sequence marker for the A genome and a real-time polymerase chain reaction (PCR) marker for the B genome; however, detection of these key mutations has to use different assay platforms. Therefore, a simple PCR assay for detection of FAD2 alleles on both genomes was developed by designing allele-specific primers and altering PCR annealing temperatures. This assay was successfully used for detecting FAD2 alleles in peanut. Gas chromatography (GC) was used to determine fatty acid composition of PCR-assayed genotypes. The results from the PCR assay and GC analysis were consistent. This PCR assay is quick, reliable, economical, and easy to use. Implementation of this PCR assay will greatly enhance the efficiency of germplasm characterization and marker-assisted selection of high oleate in peanut.     

Detection of Tobacco mosaic virus and Tomato mosaic virus in pepper and tomato by multiplex RT-PCR

Aims: To develop a highly sensitive and rapid protocol for simultaneous detection and differentiation of Tobacco mosaic virus (TMV) and Tomato mosaic virus (ToMV) in pepper and tomato. In this study, we use the multiplex PCR technique to detect dual infection of these two viruses. Methods and Results: A multiplex RT–PCR method consisting of one‐tube reaction with two primer pairs targeted to replicase genes was developed to simultaneously detect TMV and ToMV in seed samples of pepper and tomato. Specific primers were designed from conserved regions of each of the virus genomes, and their specificity was confirmed by sequencing PCR products. RT–PCR detected up to 10^?6 dilution of total RNA extracted from infected leaves. Multiplex RT–PCR revealed the presence of both TMV and ToMV in three of 18 seed samples of tomato and one of 18 seed samples of pepper. Conclusions: The multiplex PCR assay was a cost effective, quick diagnostic technique, which was helpful in differentiating TMV and ToMV accurately. Significance and Impact of the Study: The multiplex PCR assay described in this study is a valuable tool for plant pathology and basic research studies. This method may facilitate better recognition and distinction of TMV and ToMV in both pepper and tomato.     

Development and evaluation of a multiplex PCR for simultaneous detection of five foodborne pathogens

Aims: To develop a rapid multiplex PCR method for simultaneous detection of five major foodborne pathogens (Staphylococcus aureus, Listeria monocytogenes, Escherichia coli O157:H7, Salmonella Enteritidis and Shigella flexneri, respectively). Methods and Results: Amplification by PCR was optimized to obtain high efficiency. Sensitivity and specificity assays were investigated by testing different strains. With a multipathogen enrichment, multiplex PCR assay was able to simultaneously detect all of the five organisms in artificially contaminated pork samples. The developed method was further applied to retail meat samples, of which 80% were found to be positive for one or more of these five organisms. All the samples were confirmed by traditional culture methods for each individual species. Conclusions: This study reported a rapid multiplex PCR assay using five primers sets for detection of multiple pathogens. Higher consistency was obtained between the results of multiplex PCR and traditional culture methods. Significance and Impact of the Study: This work has developed a reliable, useful and cost‐effective multiplex PCR method. The assay performed equally as well as the traditional cultural method and facilitated the sensitive detection both in artificially contaminated and naturally contaminated samples.     

Quick PCR based diagnosis of typhoid using specific genetic markers

A quick multiplex PCR based detection method was developed for early diagnosis of typhoid using specific genetic markers of S. typhi. Primers of tyv gene, flag gene, viaB gene and ratA gene confirmed the specificity and sensitivity of the PCR. The serum samples of the suspected typhoid patients were taken directly for PCR without culturing and isolating genomic DNA. Overall diagnosis required 2 h which is the least time ever reported for a PCR based methods. The sensitivity of the method is up to 5 fg genomic DNA. The genetic markers are specific and the four pairs of primers give selective amplification and differentiate S. typhi from closely related S. typhimurium.     

Method for monitoring deletions in the aflatoxin biosynthesis gene cluster of Aspergillus flavus with multiplex PCR

The report presents a rapid, inexpensive and simple method for monitoring indels with influence on aflatoxin biosynthesis within Aspergillus flavus populations. PCR primers were developed for 32 markers spaced approximately every 5 kb from 20 kb proximal to the aflatoxin biosynthesis gene cluster to the telomere repeat. This region includes gene clusters required for biosynthesis of aflatoxins and cyclopiazonic acid; the resulting data were named cluster amplification patterns (CAPs). CAP markers are amplified in four multiplex PCRs, greatly reducing the cost and time to monitor indels within this region across populations. The method also provides a practical tool for characterizing intraspecific variability in A. flavus not captured with other methods.

Significance and Impact of the Study

Aflatoxins, potent naturally‐occurring carcinogens, cause significant agricultural problems. The most effective method for preventing contamination of crops with aflatoxins is through use of atoxigenic strains of Aspergillus flavus to alter the population structure of this species and reduce incidences of aflatoxin producers. Cluster amplification pattern (CAP) is a rapid multiplex PCR method for identifying and monitoring indels associated with atoxigenicity in A. flavus. Compared to previous techniques, the reported method allows for increased resolution, reduced cost, and greater speed in monitoring the stability of atoxigenic strains, incidences of indel mediated atoxigenicity and the structure of A. flavus populations.     

Development of a haemolysin gene‐based multiplex PCR for simultaneous detection of Vibrio campbellii,Vibrio harveyi and Vibrio parahaemolyticus

Aim: To develop a haemolysin (hly) gene‐based species‐specific multiplex PCR for simple and rapid detection of Vibrio campbellii, V. harveyi and V. parahaemolyticus. Methods and Results: The complete hly genes of three V. campbellii strains isolated from diseased shrimps were sequenced and species‐specific PCR primers were designed based on these sequences and the registered hly gene sequences of Vibrio harveyi and Vibrio parahaemolyticus. Specificity and sensitivity of the multiplex PCR was validated with 27 V. campbellii, 16 V. harveyi, and 69 V. parahaemolyticus, 18 other Vibrio species, one Photobacterium damselae and nine other bacterial species. The detection limits of all the three target species were in between 10 and 100 cells per PCR tube. Conclusions: Specificity and sensitivity of the multiplex PCR is 100% each and sufficient to be considered as an effective tool in a prediction system to prevent potential disease outbreak by these Vibrio species. Significance and Impact of the Study: Because there is lack of simple, rapid and cost‐effective method to differentiate these closely related V. campbellii, V. harveyi and V. parahaemolyticus species, the multiplex PCR developed in this study will be very effective in epidemiological, ecological and economical points of view.     

Rapid detection and differentiation of Erysipelothrix spp. by a novel multiplex real‐time PCR assay

Aim: The aim of this study was to develop a multiplex real‐time PCR assay for the identification and discrimination of Erysipelothrix rhusiopathiae, tonsillarum and Erysipelothrix sp. strain 2 for direct detection of Erysipelothrix spp. from animal specimens. Methods and Results: A primer set and three species‐specific probes with different end labelling were designed from the noncoding region downstream of the 5S rRNA coding region. The sensitivity, specificity and repeatability of the assay were validated by analysing 27 Erysipelothrix spp. reference serotype strains and ten septicemia‐associated non‐Erysipelothrix spp. bacterial isolates. Cross‐reactivity with Erysipelothrix sp. strain 1 was not observed with any of the primer probe combinations. The detection limit was determined to be <10 colony forming units and as low as one genome equivalent per PCR . Further evaluation of the Erysipelothrix spp. multiplex PCR was performed by comparing an enrichment isolation culture method and a conventional differential PCR on 15 samples from pigs experimentally inoculated with Erysipelothrix spp. and 22 samples from pigs with suspected natural infection. Conclusion: The multiplex real‐time PCR assay was found to be simple, rapid, reliable, specific and highly sensitive. Significance and Impact of the Study: The developed real‐time multiplex PCR assay does not require cumbersome and lengthy cultivation steps prior to DNA extraction, obtained comparable results to enrichment isolation, and will be useful in diagnostic laboratories for rapid detection of Erysipelothrix spp.