Multiplex PCR For Simultaneous Detection Research Articles

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

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). 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. 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. 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.

Real-time multiplex PCR for simultaneous detection of Campylobacter jejuni, Salmonella, Shigella and Yersinia species in fecal samples

Diarrheal diseases due to notifiable bacterial infections require rapid diagnosis of the causative pathogens. To facilitate detection, a real-time multiplex PCR was developed that identifies common diarrhea-causing bacteria in fecal samples. On the basis of published sequence data, sets of primers and probes were designed that were specific for Campylobacter jejuni, Salmonella, Shigella/enteroinvasive Escherichia coli EIEC, and Yersinia species, suitable for use in a one-tube PCR assay. The assay was assessed using a list of 137 well-defined intestinal bacterial strains or isolates. Furthermore, 393 routine clinical stool samples were analyzed, and the results of real-time multiplex PCR were compared with those obtained by established microbiological methods. The PCR yielded results within 3 h including DNA purification. No false-positive signals or cross-reactions were observed. The analytical sensitivity was 10 3 cfu mL −1 for Campylobacter jejuni, 10 4 cfu mL −1 for Salmonella, and 10 5 cfu mL −1 for Shigella/EIEC and Yersinia, respectively. Compared with culture, PCR detected 79 out of 81 Campylobacter jejuni (97.5%), 71 out of 74 Salmonella (96%), 8 out of 8 Shigella (100%), and 10 out of 10 Yersinia-positive (100%) clinical samples. In culture-negative samples ( n = 192), PCR additionally detected 2 Shigella, 1 Salmonella, and 5 Campylobacter jejuni infections. Thus, the new real-time multiplex PCR provides reliable results within a short time and might be useful as an additional diagnostic tool whenever time is important in the diagnosis of enteropathogenic bacteria.

Highly sensitive multiplex PCR for simultaneous detection and discrimination of Dirofilaria immitis and Dirofilaria repens in canine peripheral blood

Dirofilaria immitis and Dirofilaria repens are the most common species of filarial nematodes described in the dogs with increasing spread into new geographical areas. The diagnosis of canine dirofilariosis is usually based upon the microscopical detection and identification of circulating microfilariae together with ELISA detection of serum circulating heartworm antigens or antibodies. The identification of the parasite species using the traditional approaches sometimes can be difficult and can lead to misdiagnosis especially on samples from areas where both Dirofilaria are present. In this paper we report a new molecular method based on single-step multiplex PCR to detect and differentiate simultaneously and unequivocally D. immitis and D. repens on DNA extracted from canine peripheral blood. The amplification was performed using a set of primers designed on a portion of the small subunit ribosomal RNA gene of the mitochondrion (12S rDNA). The single-step multiplex PCR here described ensured high (4 mf/ml) sensitivity and specificity with reduced cost and time saving. The multiplex PCR assay represents an additional tool for epidemiological studies and routine disease assessment in areas co-endemic for the two Dirofilaria species.

The development and evaluation of a single step multiplex PCR for simultaneous detection of Anopheles annularis group mosquitoes, human host preference and Plasmodium falciparum sporozoite presence

The Anopheles annularis group mosquitoes, subgenus Cellia Theobald (Diptera: Culicidae), includes five recognized species: An. annularis Van der Wulp, An. nivipes Theobald, An. pallidus Theobald, An. philippinensis Ludlow and An. schueffneri Stanton. From these five, the three most common species found in Orissa were considered for this study because of their remarkable vectorial and behavioral variation and the important role they play in malaria transmission. To identify and understand their role in malaria transmission we developed a single multiplex PCR-based assay. This assay included the detection of human blood feeding habit and Plasmodium falciparum sporozoite presence. Of the 186 An. annularis mosquitoes collected, morphological character-based identification showed that 94 were An. annularis, 54 were An. philippinensis and 38 were An. pallidus. However, the multiplex PCR assay confirmed that 91 were An. annularis, 56 were An. philippinensis and 39 were An. pallidus individuals after adjustments were made for misidentified specimens in the morphological method. Anopheles annularis and An. philippinensis were found positive for human blood, and two samples of An. annularis species were positive for P. falciparum sporozoites. This one-step PCR-based method constitutes a very powerful tool in large surveys of anopheline populations.

Development of a novel hot-start multiplex PCR for simultaneous detection of classical swine fever virus, African swine fever virus, porcine circovirus type 2, porcine reproductive and respiratory syndrome virus and porcine parvovirus

A novel hot-start multiplex PCR (mPCR) assay was developed and subsequently evaluated for its effectiveness in simultaneously detecting multiple viral infections of swine. Specific primers for each of five virus genomes, namely classical swine fever virus (CSFV), African swine fever virus (ASFV), porcine circovirus type 2 (PCV2), porcine reproductive and respiratory syndrome virus (PRRSV) and porcine parvovirus (PPV) were used. Combined nucleic acid purification was carried out using a commercial RNA/DNA extraction kit. The mPCR consisted of a two-step procedure which included reverse transcription and PCR amplification. This mPCR and the corresponding separate assays were evaluated comparatively on serial ten-fold dilutions of each virus. Analysis of the sensitivity in comparison to the corresponding single PCR (sPCR) for the detection of each of the five targets was identical for CSFV, PCV2 and PPV, 1 log lower for PRRSV and 2 logs lower for ASFV. No spurious PCR amplification reactions among all five pathogens were noticed with various amounts of DNA and RNA mixtures. All the uninfected controls were scored negative. The relative efficiency of the mPCR developed in this study compared to performing sPCR for each virus, suggests its potential application for routine molecular diagnostic purposes.

A single‐step multiplex PCR for simultaneous detection of white spot syndrome virus and infectious hypodermal and haematopoietic necrosis virus in penaeid shrimp

White spot syndrome virus (WSSV) and infectious hypodermal and haematopoietic necrosis virus (IHHNV) are the major viral pathogens of penaeid shrimp worldwide (Lightner & Redman 1998). Litopenaeus vannamei was introduced into China from the Americas, and quickly became widely cultured. Following its introduction, both IHHNV and WSSV have become important pathogens of cultured penaeid shrimp and have had a huge impact on the culture industry in China in recent years.

Copro-DNA tests for diagnosis of animal taeniid cestodes

PCR has proven its value for the diagnosis of taeniid cestodes in animal definitive hosts, although only few specific tests are available at the moment ( Echinococcus multilocularis, Echinococcus granulosus ‘sheep strain’). Additional tests with specificities for further taeniids are urgently needed and new tests are currently being developed, e.g. a multiplex PCR for simultaneous detection of E. multilocularis, E. granulosus (all strains) and Taenia spp. (all species). PCR is a technically demanding and expensive technique: DNA isolation from faecal specimens remains a laborious task because of the presence of PCR-inhibitory substances, and special precautions need to be taken to avoid false-positive results due to cross-contamination of amplification reactions. PCR, therefore, is mainly used for confirmative purposes of coproantigen-positive samples or for identification of taeniid eggs recovered from faecal specimens or from environmental samples.

Multiplex PCR for simultaneous detection of five virulence hemolysin genes in Vibrio anguillarum

A multiplex PCR was developed for detection of hemolysin-producing Vibrio anguillarum using primers targeting five hemolysin genes ( vah1, vah2, vah3, vah4 and vah5). This method was successful in amplifying reactions containing as little as 100 fg of genomic template DNA. The direct detection of V. anguillarum in clinical specimens by this multiplex PCR was also successful in reactions containing as few as 10 bacterial cells. This multiplex PCR method can be a rapid and sensitive method for detecting pathogenic V. anguillarum.

Application of a multiplex PCR for the simultaneous detection of Escherichia coli O157:H7, Salmonella and Shigella in raw and ready-to-eat meat products

Escherichia coli O157:H7, Salmonella and Shigella might contaminate similar types of meat products and cause deadly diseases in humans. Traditional microbiological analyses to detect these pathogens are labor-intensive and time-consuming. The objective of this study was to apply a multiplex PCR for simultaneous detection of the pathogenic bacteria in certain raw and ready-to-eat meat matrices. The tested samples had aerobic plate counts ranging from non-detectable, in chicken nuggets and salami, to 8.36 log 10 CFU/g in ground pork. The pH of homogenates spanned from 6.86, in ground beef, to 7.17 in salami. Following a 24-h enrichment, the multiplex PCR assay could concurrently detect the three pathogens at 0.2 log 10 CFU/g in ground beef, roast beef, beef frankfurters, chicken nuggets, salami and turkey ham, and 1.2 log 10 CFU/g in ground pork. This multiplex PCR offers an efficient microbiological tool for presumptive detection of E. coli O157:H7, Salmonella and Shigella in meat.

A multiplex PCR for simultaneous detection and differentiation of North American serotypes of bluetongue and epizootic hemorrhagic disease viruses

In the present study, a multiplex RT-PCR-based assay for simultaneous detection and differentiation of North American serotypes of bluetongue (BT) virus (BTV) and epizootic hemorrhagic disease (EHD) virus (EHDV) in cell culture and clinical samples was developed. Two pairs of primers (B1 and B4) and (E1 and E4) were designed to hybridize to non-structural protein 1 (NS1) genomes of (BTV-11) and (EHDV-1), respectively. The multiplex PCR-based assay utilized a single tube-PCR amplification in which EHDV and BTV primers were used simultaneously in a multiplex format. The BTV primers generated a 790 base pair (bp) specific PCR product from RNA samples of North American BTV serotypes 2, 10, 11, 13 and 17; whereas EHDV serotypes 1 and 2 or total nucleic acid extract from non-infected baby hamster kidney (BHK) cells failed to demonstrate the 790 bp specific BTV PCR product. Likewise, the EHDV primers produced a 387 bp specific PCR product from RNA samples of EHDV serotypes 1 and 2, but not from BTV serotypes 2, 10, 11, 13, 17 or from total nucleic acid extract of BHK cell controls. Two pairs of nested primers (B2 and B3) and (E2 and E3), internal to the annealing sites of primers (B1and B4) and primers (E1 and E4), produced a 520 bp specific BTV and a 224 bp specific EHDV PCR product from BTV and EHDV first amplification products, respectively. These nested amplifications increased the sensitivity of the PCR assay and confirmed the specificity of the first amplified EHDV or BTV PCR products. The described multiplex RT-PCR-based assay could be used to facilitate rapid detection and differentiation of North American BTV and EHDV serotypes and to provide a valuable tool to study the epidemiology of these orbivirus infections in susceptible animal populations.

Multiplex PCR for simultaneous detection of 677 C→T and 1298 A→C polymorphisms in methylenetetrahydrofolate reductase gene for population studies of cancer risk

Methylenetetrahydrofolate reductase (MTHFR) plays a pivotal role in folate metabolism by regulating the diversion of folate metabolites toward DNA methylation or toward DNA synthesis. Because aberrations in both of these pathways can be tumor promoting, the two common polymorphisms in the MTHFR gene, 677 C→T and 1298 A→C, have been implicated as risk factors for several cancers. Homozygosity for the 677 C→T polymorphism and compound heterozygosity for 677 C→T and 1298 A→C polymorphisms both reduce enzyme activity by more than 50% and can promote oncogenic alterations in DNA methylation especially when folate status is low. Thus, rapid identification of both polymorphisms in MTHFR gene would be of importance in understanding the genetics of abnormal folate metabolism as related to human cancer risk. Here we describe a multiplex polymerse chain reaction/restriction fragment length polymorphism procedure in which two sets of primers are used to amplify simultaneously the DNA regions spanning 677 and 1298 loci in one PCR reaction. The amplified products are digested by HinfI or MboII followed by agarose gel electrophoresis for simultaneous detection of the 677 C→T and 1298 A→C polymorphisms in the same gel.

Use of multiplex PCR for simultaneous detection of four bacterial species in middle ear effusions.

A multiplex PCR procedure was developed for the simultaneous detection of Alloiococcus otitidis, Haemophilus influenzae, Moraxella catarrhalis, and Streptococcus pneumoniae in middle ear effusions (MEEs) from patients with chronic otitis media with effusion. The bacterial 16S rRNA gene was chosen as the target, and the procedure used one common lower primer and four species-specific upper primers. The reaction was optimized by changing the primer concentrations to yield equal amounts of amplification products. The specificity of the reaction was verified with various bacterial species found in the nasopharynx. The performance of the procedure was examined with 25 MEE specimens, and the results were compared to those obtained by conventional culture methods. A detection level of 10 bacterial cells/reaction for each of the study organisms was achieved. By conventional culture methods, 8 (32%) of the specimens showed growth of one of the study organisms. In contrast, 21 (84%) of the specimens tested positive by the multiplex PCR. None of the culture-positive specimens were PCR negative, whereas three (12%) of the PCR-positive specimens tested positive for two of the four study organisms. Thus, the multiplex PCR method improves the detection rate significantly compared to that of the conventional culture method.

Application of a nested, multiplex PCR to psittacosis outbreaks.

We developed a nested, multiplex PCR for simultaneous detection of three species of chlamydiae in human and avian specimens. The PCR was designed to increase sensitivity and to circumvent inhibitors of PCR present in clinical specimens. The target sequence was the 16S rRNA gene. The first-step PCR was genus specific, and the second-step PCR was multiplexed (i.e., had multiple primer sets in the same tube) and could discriminate among Chlamydia pneumoniae, Chlamydia psittaci, and Chlamydia trachomatis on the basis of the molecular weight of the amplicon. The limit of detection of each of the two PCR steps was 5 inclusion-forming units. We used PCR and serologic evidence during outbreaks of psittacosis to infer that C. psittaci had been transmitted from birds purchased in pet stores to humans. We also used this method to test both live and dead birds from pet stores for infection with C. psittaci. Compared with culture, the application of PCR to avian specimens increased the rate of C. psittaci detection.