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PubMed2004

Rapid identification and differentiation of Saccharomyces cerevisiae, Saccharomyces bayanus and their hybrids by multiplex PCR.

AIMS: To develop a multiplex PCR assay for the specific identification and differentiation of Saccharomyces cerevisiae, S. bayanus and their hybrids. METHODS AND RESULTS: Two sets of primers with sequences complementary to the region YBR033w were used. A single amplicon of 1710 bp or 329 bp was obtained with species S. cerevisiae and S. bayanus, respectively, while the presence of both bands was observed in S. pastorianus because of its hybrid nature. Both amplification products were also obtained after amplification from DNA of several laboratory S. cerevisiae x S. bayanus hybrid strains. CONCLUSIONS: Multiplex PCR was optimized for the rapid and reliable identification of S. cerevisiae, S. bayanus and their hybrids. SIGNIFICANCE AND IMPACT OF THE STUDY: The procedure may be used for routine detection of the most common Saccharomyces sensu stricto yeasts involved in industrial fermentation processes, overcoming the problems of conventional techniques.

DNA, Fungal↗

PubMed2001

Multiplex PCR for screening of microdeletions on the Y chromosome.

PURPOSE: The aim of this study was to develop a multiplex PCR protocol, which could be suitable for screening of microdeletions in the three azoospermia factor (AZF) regions on the Y chromosome. METHODS: In the screening protocol, 36 known sequence tagged site (STS) primer pairs were first tested in single PCR reactions and thereafter combined in multiplex PCR to test for specificity and sensitivity in order to develop a stable and reliable multiplex PCR assay to detect Y microdeletions. RESULTS: Of the 36 primers tested, 11 turned out not to be specific or produced PCR products that were too weak, and they were therefore not used in the multiplex PCR. The remaining 25 STSs were selected on the basis of their ability to be reproducibly amplified with each other using identical amplification conditions. Five multiplex sets, each consisting of five primer pairs, were established in the multiplex PCR setup. CONCLUSION: The multiplex PCR protocol presented in this study is an easy and reliable method for detection of Y chromosome microdeletions and could be used for screening of infertile men to allow genetic counseling about the risk of transmitting infertility from father to son.

DNA-Directed DNA Polymerase↗

PubMed2001

Capillary electrophoresis as a tool for optimization of multiplex PCR reactions.

Copying multiple regions of a DNA molecule is routinely performed today using the polymerase chain reaction (PCR) in a process commonly referred to as multiplex PCR. The development of a multiplex PCR reaction involves designing primer sets and examining various combinations of those primer sets and different reaction components and/or thermal cycling conditions. The process of optimizing a multiplex PCR reaction in order to obtain a well-balanced set of amplicons can be time-consuming and labor-intensive. The rapid separation and quantitation capabilities of capillary electrophoresis make it an efficient technique to help in the multiplex PCR optimization process.

Alleles↗

PubMed2003

Molecular typing of Salmonella enterica serovar typhi isolates from various countries in Asia by a multiplex PCR assay on variable-number tandem repeats.

A multiplex PCR method incorporating primers flanking three variable-number tandem repeat (VNTR) loci (arbitrarily labeled TR1, TR2, and TR3) in the CT18 strain of Salmonella enterica serovar Typhi has been developed for molecular typing of S. enterica serovar Typhi clinical isolates from several Asian countries, including Singapore, Indonesia, India, Bangladesh, Malaysia, and Nepal. We have demonstrated that the multiplex PCR could be performed on crude cell lysates and that the VNTR banding profiles produced could be easily analyzed by visual inspection after conventional agarose gel electrophoresis. The assay was highly discriminative in identifying 49 distinct VNTR profiles among 59 individual isolates. A high level of VNTR profile heterogeneity was observed in isolates from within the same country and among countries. These VNTR profiles remained stable after the strains were passaged extensively under routine laboratory culture conditions. In contrast to the S. enterica serovar Typhi isolates, an absence of TR3 amplicons and a lack of length polymorphisms in TR1 and TR2 amplicons were observed for other S. enterica serovars, such as Salmonella enterica serovar Typhimurium, Salmonella enterica serovar Enteritidis, and Salmonella enterica serovar Paratyphi A, B, and C. DNA sequencing of the amplified VNTR regions substantiated these results, suggesting the high stability of the multiplex PCR assay. The multiplex-PCR-based VNTR profiling developed in this study provides a simple, rapid, reproducible, and high-resolution molecular tool for the epidemiological analysis of S. enterica serovar Typhi strains.

Alleles↗

PubMed2004

Simultaneous detection of HBV and HCV by multiplex PCR normalization.

AIM: To design and establish a method of multiplex PCR normalization for simultaneously detecting HBV and HCV. METHODS: Two pairs of primers with a 20 bp joint sequence were used to amplify the target genes of HBV and HCV by two rounds of amplification. After the two rounds of amplification all the products had the joint sequence. Then the joint sequence was used as primers to finish the last amplification. Finally multiplex PCR was normalized to a single PCR system to eliminate multiplex factor interference. Four kinds of nucleic acid extraction methods were compared and screened. A multiplex PCR normalization method was established and optimized by orthogonal design of 6 key factors. Then twenty serum samples were detected to evaluate the validity and authenticity of this method. RESULTS: The sensitivity, specificity, diagnostic index and efficiency were 83.3%, 70%, 153.3% and 72.2%, respectively for both HBsAg and anti-HCV positive patients, and were 78.6%, 80%, 258.6% and 79.2%, respectively for HBsAg positive patients, and were 75%, 90%, 165% and 83.3%, respectively for anti-HCV positive patients. CONCLUSION: The multiplex PCR normalization method shows a broad prospect in simultaneous amplification of multiple genes of different sources. It is practical, correct and authentic, and can be used to prevent and control HBV and HCV.

DNA↗

PubMed2007

Improved multiplex-PCR to identify hepatitis B virus genotypes A-F and subgenotypes B1, B2, C1 and C2.

BACKGROUND: There are eight genotypes (A-H) and numerous subgenotypes of hepatitis B virus (HBV). The genotype has been shown to affect the course of HBV infection. OBJECTIVES: To develop an efficient genotyping and subgenotyping method for large-scale epidemiological surveys of HBV infection in countries with high prevalence of HBV B and C such as China. STUDY DESIGN: We designed genotype and subgenotype-specific primer pairs, and adjusted PCR conditions for a multiplex-PCR using common Taq polymerase to identify HBV genotypes A-F in one reaction and for the main subgenotypes B1/B2 and C1/C2 in another reaction. RESULTS: We have developed a multiplex-PCR system, which specifically amplifies DNA of HBV genotypes and the corresponding main subgenotypes B and C from the sera of HBV patients. Our patients were infected with HBV of subgenotypes B2 (n=18), C1 (n=2) and C2 (n=48). Eleven patients were doubly infected and three showed a triple infection with HBV A, B and C. CONCLUSIONS: The low-cost multiplex-PCR for identification of HBV genotypes A-F and main subgenotypes of HBV B and C, is rapid, reliable and sufficient for large-scale epidemiological surveys and clinical studies.

Base Sequence↗

PubMed2004

Simultaneous detection of marine fish pathogens by using multiplex PCR and a DNA microarray.

We coupled multiplex PCR and a DNA microarray to construct an assay suitable for the simultaneous detection of five important marine fish pathogens (Vibrio vulnificus, Listonella anguillarum, Photobacterium damselae subsp. damselae, Aeromonas salmonicida subsp. salmonicida, and Vibrio parahaemolyticus). The array was composed of nine short oligonucleotide probes (25-mer) complementary to seven chromosomal loci (cyt, rpoN, gyrB, toxR, ureC, dly, and vapA) and two plasmid-borne loci (fatA and A.sal). Nine primer sets were designed to amplify short fragments of these loci (100 to 177 bp) in a multiplex PCR. PCR products were subsequently labeled by nick translation and hybridized to the microarray. All strains of the five target species (n = 1 to 21) hybridized to at least one species-specific probe. Assay sensitivities ranged from 100% for seven probes to 83 and 67% for the two remaining probes. Multiplex PCR did not produce any nonspecific amplification products when tested against 23 related species of bacteria (n = 40 strains; 100% specificity). Using purified genomic DNA, we were able to detect PCR products with < 20 fg of genomic DNA per reaction (equivalent to four or five cells), and the array was at least fourfold more sensitive than agarose gel electrophoresis for detecting PCR products. In addition, our method allowed the tentative identification of virulent strains of L. anguillarum serotype O1 based on the presence of the fatA gene (67% sensitivity and 100% specificity). This assay is a sensitive and specific tool for the simultaneous detection of multiple pathogenic bacteria that cause disease in fish and humans.

Animals↗

PubMed1999

[Establishment of a multiplex PCR system to detect plasmodium].

AIM: To establish a simple, rapid and practical multiplex PCR system to detect Plasmodium vivax(P. v) and Plasmodium falciparum(P. f). METHODS: A common upper primer S1 and two species-specific lower primers of P. v and P. f, S2, S3 were designed according to the sequences of the small subunit ribosomal DNA (SSUrDNA) fragments of the two Plasmodium species. Using these three oligonucleotide primers, the two-temperature-point multiplex PCR system was established and applied to detect P. v and P. f in the stock blood samples of clinically confirmed patients. RESULTS: DNA fragments of about 705 and 575 base pairs were successfully amplified by multiplex PCR from the genomic DNA of P. v and P. f, but no fragments were obtained from that of P. knowlesi, P. cynomolgi and blood of healthy persons. By means of restriction endonuclease digestion, the amplified fragments were confirmed to be the SSUrDNA fragments of P. v and P. f as expected. This method was successfully used in detecting parasitemia 2-10 parasite/microliter whole blood. Of 104 samples tested by this system, 81 were coincident with microscopic examination. The multiplex PCR system also found 17 samples of mixed infection, which were not detected microscopically. Another 2 samples detected as P. v by microscopic examination were verified to be P. f infection by the multiplex PCR. CONCLUSION: The ease of operation together with high sensitivity and specificity, particularly the sensitive detection for mixed infection in a single run of amplification suggests that the multiplex PCR system might be a useful tool for malaria diagnosis.

Animals↗

PubMed2005

[Development and application of multiplex-PCR for identification of Actinobacillus pleuropneumoniae].

A multiplex-PCR assay was developed to identify Actinobacillus pleuropneumoniae (App). Two pairs of polymerase chain reaction (PCR) primers were designed for the 16S rRNA and the apxlVA gene, which is specific to all serotypes of App. Two PCR products of 692bp and 363bp were obtained, from the 16S rRNA and the apxlVA gene respectively, for 27 reference A. pleuropneumoniae strains. Only the 692bp fragment was amplified for closely related strains including A. lignieresii. Using the designed primers, the method is capable of detecting A. pleuropneumoniae of as low as 1.3 x 10(3) CFU or 9pg DNA. For 302 suspected isolates, this multiplex-PCR method correctly identified 4 A. pleuropneumoniae strains. The result suggests the use of the multiplex-PCR for routine identification of App.

Actinobacillus pleuropneumoniae↗

PubMed2002

Molecular typing of Helicobacter pylori by virulence-gene based multiplex PCR and RT-PCR analysis.

BACKGROUND: In recent years, numerous PCR amplification typing methods have been developed for the identification of H. pylori specific virulence genes. To reduce the number of PCR amplifications needed we have previously developed virulence gene based multiplex PCR and RT-PCR assays. AIM: The aim of the present study was to characterise Helicobacter pylori strains by means of virulence-gene based multiplex PCR and reverse-transcription PCR. SUBJECTS: Helicobacter pylori clinical reference strains HP-HJM 1-25 originally obtained from routine cultures of clinical gastric biopsy specimens from dyspeptic patients were used in the present study. METHODS: Helicobacter pylori multiplex PCR and RT-PCR assays were carried out using primer pairs targeting the cagA, vacA, ureA, ureI, hspA (hsp60), and sodB genes and their cDNA. RESULTS: Unexpected multiplex PCR and RT-PCR patterns were observed by ethidium bromide staining of agarose gels and Southern blot hybridisation analysis. DNA sequence analysis revealed a complex pattern of point mutations, deletions and insertions in the sodB, cagA and vacA genes, respectively. Mutations occurring in the PCR and hybridisation primer binding sites might explain some of the discrepancies previously observed in the expression of these genes. Furthermore, the present data show that coexpression of cagA and vacA transcripts of different sizes may take place in the same strain. CONCLUSIONS: The multiplex PCR and RT-PCR assay described allows rapid characterisation of H. pylori virulence genes at the DNA and RNA (cDNA) levels. However, extensive DNA sequence analysis seems necessary if one wants to reveal details of mutations occurring in the cagA and vacA genes.

Amino Acid Sequence↗

PubMed1998

Detection of pathogenic Yersinia enterocolitica in enrichment media and pork by a multiplex PCR: a study of sample preparation and PCR-inhibitory components.

A multiplex PCR assay including sample preparation was developed to detect viable pathogenic strains of Yersinia enterocolitica in PCR-inhibitory samples, such as pork and enrichment media. The method developed was used to simultaneously detect the plasmid-borne virulence gene yadA and a Yersinia-specific region of the 16S rRNA gene. According to an auto-agglutination test for virulence-plasmid-bearing strains of Y. enterocolitica, all potential pathogenic strains tested were detected by the assay. A DNA extraction procedure, an aqueous two-phase system composed of polyethylene glycol 4000 and dextran 40 and a buoyant density centrifugation method, based on Percoll, were compared with regard to their efficiency in separating Yersinia enterocolitica from PCR inhibitors originating from enrichment media and pork. Using the density gradient centrifugation method resulted in a detection level of 4.0 x 10(2) CFU Y. enterocolitica per ml enrichment media. To ensure detection of viable bacteria a short enrichment step was included in the sample preparation together with the density gradient centrifugation. When this sample treatment method was evaluated with a selective enrichment medium together with a background flora inoculated with approximately 1.0 x 10(1) CFU per ml of Y. enterocolitica and incubated at 25 degrees C, a positive PCR result was obtained after 6 to 8 h. Our results indicate that selective enrichment followed by buoyant density gradient centrifugation provides a convenient and user-friendly sample preparation method prior to PCR.

Agglutination Tests↗

PubMed1997

Typing of porcine reproductive and respiratory syndrome viruses by a multiplex PCR assay.

A rapid multiplex PCR assay was developed to distinguish between North American and European genotypes of porcine reproductive and respiratory syndrome (PRRS) virus after a portion of the polymerase gene (open reading frame 1b) was sequenced for two North American PRRS virus strains. DNA products with unique sizes characteristic of each genotype were obtained.

Animals↗

PubMed2005

Detection and differentiation of Burkholderia pseudomallei, Burkholderia mallei and Burkholderia thailandensis by multiplex PCR.

Burkholderia pseudomallei, a Gram-negative bacterium that causes melioidosis may be differentiated from closely related species of Burkholderia mallei that causes glanders and non-pathogenic species of Burkholderia thailandensis by multiplex PCR. The multiplex PCR consists of primers that flank a 10-bp repetitive element in B. pseudomallei and B. mallei amplifying PCR fragment of varying sizes between 400-700 bp, a unique sequence in B. thailandensis amplifying a PCR fragment of 308 bp and the metalloprotease gene amplifying a PCR fragment of 245 bp in B. pseudomallei and B. thailandensis. The multiplex PCR not only can differentiate the three Burkholderia species but can also be used for epidemiological typing of B. pseudomallei and B. mallei strains.

Animals↗

PubMed2004

A multiplex PCR for detection of genes encoding exfoliative toxins from Staphylococcus hyicus.

AIMS: To develop a multiplex PCR for detection of genes encoding the exfoliative toxins ExhA, ExhB, ExhC and ExhD from Staphylococcus hyicus and to estimate the prevalence of exfoliative toxins among Staph. hyicus isolates from Danish pig herds with exudative epidermitis (EE). METHODS AND RESULTS: A multiplex PCR employing specific primers for each of the genes encoding four different exfoliative toxins was developed and evaluated using a collection of Staph. hyicus with known toxin type and a number of other staphylococcal species. A total of 314 Staph. hyicus isolates from pigs with EE were screened by multiplex PCR and the combined results of the present and previous investigations showed that ExhA, ExhB, ExhC and ExhD was found in 20, 33, 18 and 22%, respectively, of 60 cases of EE investigated. CONCLUSIONS: This study has provided a new tool for detection of toxigenic Staph. hyicus and a more comprehensive picture of the prevalence of the Staph. hyicus exfoliative toxins in Danish pig herds. SIGNIFICANCE AND IMPACT OF THE STUDY: The multiplex PCR can be used in studies on the prevalence of toxigenic Staph. hyicus elucidating the epidemiology of EE in pigs. The multiplex PCR is currently being used for selection of Staph. hyicus isolates for production of autogenous vaccine.

Animals↗

PubMed1997

Multiplex PCR: critical parameters and step-by-step protocol.

By simultaneously amplifying more than one locus in the same reaction, multiplex PCR is becoming a rapid and convenient screening assay in both the clinical and the research laboratory. While numerous papers and manuals discuss in detail conditions influencing the quality of PCR in general, relatively little has been published about the important experimental factors and the common difficulties frequently encountered with multiplex PCR. We have examined various conditions of the multiplex PCR, using a large number of primer pairs. Especially important for a successful multiplex PCR assay are the relative concentrations of the primers at the various loci, the concentration of the PCR buffer, the cycling temperatures and the balance between the magnesium chloride and deoxynucleotide concentrations. Based on our experience, we propose a protocol for developing a multiplex PCR assay and suggest ways to overcome commonly encountered problems.

Buffers↗

PubMed2006

[Screening for Y chromosomal microdeletions in AZF region with modified multiplex PCR].

OBJECTIVE: Screening for Y chromosomal microdeletions in azoospermia factor (AZF) region with modified multiplex PCR. METHODS: 160 cases with spermatogenetic failure were recruited in the experimental group, while 90 cases of donors in controls. According to the laboratory guidelines supported by European Academy of Andrology (EAA) and European Molecular Genetics Quality Network (EMQN), Y chromosomal microdeletions in AZFa, b, c regions were screened with multiplex PCR. The primers of sequence targeted sites (STSs) and conditions of PCR were modified. RESULTS: Using modified multiplex PCR, 14 (8.75%) cases with Y chromosomal microdeletions were found in the experimental group, while no case in controls. There were 12 cases in AZFc, 1 case in AZFa + b + c, 1 case in AZFb + c. According to statistics, the difference between two groups was significant (P <0.001). Reaction products could be clearly separated with agarose gel and finished in 1 h. CONCLUSION: Modified multiplex PCR protocols supported by EAA and EMQN proved to be very accurate, sensitive and quick, which could be put into screening practice for Y chromosomal microdeletions in AZF region.

Adult↗

PubMed2001

Molecular characterization of Bacillus anthracis using multiplex PCR, ERIC-PCR and RAPD.

AIMS: To investigate the molecular characterization of Bacillus anthracis strains by multiplex PCR, enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR) and random amplification of polymorphic DNA (RAPD). METHODS AND RESULTS: Three primers were used to amplify the cya, cap and cereolysinAB genes in the multiplex PCR. Two distinct ERIC-PCR and RAPD fragments, which separated B. anthracis into two groups, were used as probes in Southern hybridization experiments. The probes hybridized only to the cya+ B. anthracis strains identified by the multiplex PCR. Nucleotide sequence analysis of the two cloned fragments showed they were from the pXO1 plasmid of B. anthracis. CONCLUSION: Multiplex PCR simultaneously identified isolates of the Bacillus cereus group and the B. anthracis virulence factors. ERIC-PCR and RAPD, combined with the Southern hybridization analyses, differentiated B. anthracis strains and separated them from the closely related B. cereus group bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: ERIC-PCR and RAPD assay could be effective in differentiating virulent from avirulent B. anthracis. Our results also show that the amplification of the large plasmids was allowed in the ERIC-PCR and RAPD assay.

Bacillus anthracis↗

PubMed2000

Detection of thermophilic Campylobacter from sparrows by multiplex PCR: the role of sparrows as a source of contamination of broilers with Campylobacter.

The best combination of primers and the annealing temperature of multiplex PCR for Campylobacter jejuni, Campylobacter coli, and Campylobacter lari were examined. The multiplex PCR was able to detect type strains of the three species. All results of identification of wild strains (30 strains of C. jejuni, 20 strains of C. coli, and 4 strains of C. lari) by the multiplex PCR coincided with those of the conventional biochemical identification tests, suggesting that the multiplex PCR can simultaneously differentiate C. jejuni, C. coli, and C. lari from wild strains of campylobacters easily and rapidly. Campylobacters were detected from sparrow feces by the multiplex PCR and antimicrobial sensitivities of the strains were determined to discuss the role of sparrows in contamination of broilers with C. jejuni. Three out of 13 strains of C. jejuni isolated from sparrow feces showed quinolone resistance. From the frequent use of quinolones for treatment of industrial animals like chickens, pigs, and cows, the three strains of quinolone-resistant C. jejuni in sparrows must have been originated from those industrial animals. Sparrows that have quinolone-resistant C. jejuni were considered to have contacted with industrial animals or thier feed. It may be presumed, on the contrary, that C. jejuni in sparrows could be a potential source of contamination of broilers.

Animals↗