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PubMed2004

Sequence-selective recognition of extended-spectrum beta-lactamase GES-2 by a competitive, peptide nucleic acid-based multiplex PCR assay.

Extended-spectrum beta-lactamases (ESBLs) in Pseudomonas aeruginosa, such as GES-2, which compromises the efficacy of imipenem, tend to be geographically restricted. The CC-to-AA base pair substitution at positions 493 and 494 of the bla(GES-2)-coding region distinguishes this ESBL from bla(GES-1) and the bla(IBC)-type genes, making it an ideal target for the development of a novel sequence-specific, peptide nucleic acid (PNA)-based multiplex PCR detection method. By using two primer pairs in conjunction with a PNA probe, this method provided an accurate means of identification of bla(GES-2) compared to standard PCR and gene sequencing techniques when it was used to test 100 P. aeruginosa clinical isolates as well as previously published, well-described control strains encompassing all presently known genes in the bla(GES-IBC) ESBL family. This novel method has the potential to be used in large-scale, cost-effective screening programs for specific or geographically restricted ESBLs.

Cost-Benefit Analysis↗

PubMed2000

The use of multiplex PCR reactions to characterize populations of lactic acid bacteria associated with meat spoilage.

A rapid, systematic and reliable approach for identifying lactic acid bacteria associated with meat was developed, allowing for detection of Carnobacterium spp., Lactobacillus curvatus, Lact. sakei and Leuconostoc spp. Polymerase chain reaction primers specific for Carnobacterium and Leuconostoc were created from 16S rRNA oligonucleotide probes and used in combination with species-specific primers for the 16S/23S rRNA spacer region of Lact. curvatus and Lact. sakei in multiplex PCR reactions. The method was used successfully to characterize lactic acid bacteria isolated from a vacuum-packaged pork loin stored at 2 degrees C. Seventy isolates were selected for identification and 52 were determined to be Lact. sakei, while the remaining 18 isolates were identified as Leuconostoc spp.

Animals↗

PubMed1999

Direct detection of Shiga toxigenic Escherichia coli strains belonging to serogroups O111, O157, and O113 by multiplex PCR.

Shiga toxigenic Escherichia coli (STEC) strains are a diverse group of organisms associated with severe gastrointestinal and systemic diseases in humans. Within the STEC family, eae-positive STEC strains, particularly those belonging to serogroups O157 and O111, appear to have greater virulence for humans. However, in spite of being eae negative, STEC strains belonging to serogroup O113 have frequently been associated with cases of severe STEC disease, including hemolytic-uremic syndrome (HUS). We have developed a modified multiplex PCR assay for detection of STEC strains belonging to these three serogroups in cultures of feces by using primers specific for portions of the genetic loci (rfb) encoding biosynthesis of the respective O antigen. These primers direct amplification of PCR products of 259, 406, and 593 bp for serogroups O157, O111, and O113, respectively. The assay was validated by testing 40 previously characterized STEC strains, with 100% agreement. It also detected STEC strains of the appropriate genotype in primary fecal cultures from 13 patients with HUS or bloody diarrhea. Thirty other primary fecal cultures from patients without evidence of STEC infection were negative.

Bacterial Toxins↗

PubMedDate not supplied

An assessment of a multiplex PCR assay for differentiating clinically important mycobacteria based on pncA gene variation.

The pncA genes in mycobacteria are responsible for the production of pyrazinamidase (PZase). In Mycobacterium tuberculosis, PZase hydrolyses pyrazinamide (PZA) to pyrazonic acid, a compound that possesses bactericidal activity against tubercle bacilli. Nucleotide sequences of pncA genes found within mycobacteria where aligned in an effort to ascertain the significance of any variability in sequence. Three sets of primers (one degenerate and five consensus sequences) were designed and employed in a multiplex PCR assay to amplify the pncA region in seven clinically common mycobacteria. The banding patterns generated from each species in conjunction with PZase activity tests demonstrated that the mycobacterial species examined could be clearly identified and differentiated from one another. Although not yet tested with clinical isolates, the combination of these two assays has provided a promising discriminatory tool for the identification of commonly encountered clinical mycobacteria species.

Amidohydrolases↗

PubMed2005

Development of a multiplex PCR and SHV melting-curve mutation detection system for detection of some SHV and CTX-M beta-lactamases of Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae in Taiwan.

Infection by extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae has been increasing in Taiwan. Accurate identification of the ESBL genes is necessary for surveillance and for epidemiological studies of the mode of transmission in the hospital setting. We describe herein the development of a novel system, which consists of a multiplex PCR to identify bla(SHV), bla(CTX-M-3)-like, and bla(CTX-M-14)-like genes and a modified SHV melting-curve mutation detection method to rapidly distinguish six prevalent bla(SHV) genes (bla(SHV-1), bla(SHV-2), bla(SHV-2a), bla(SHV-5), bla(SHV-11), and bla(SHV-12)) in Taiwan. Sixty-five clinical isolates, which had been characterized by nucleotide sequencing of the bla(SHV) and bla(CTX-M) genes, were identified by the system. The system was then used to genotype the ESBLs from 199 clinical isolates, including 40 Enterobacter cloacae, 68 Escherichia coli, and 91 Klebsiella pneumoniae, collected between August 2002 and March 2003. SHV-12 (80 isolates) was the most prevalent type of ESBL identified, followed in order of frequency by CTX-M-3 (65 isolates) and CTX-M-14 (36 isolates). Seventeen (9%) of the 199 clinical isolates harbored both SHV- and CTX-M-type ESBLs. In contrast to Enterobacter cloacae, the majority of which produced SHV-type ESBLs, E. coli and K. pneumoniae were more likely to possess CTX-M-type ESBLs. Three rare CTX-M types were identified through sequencing of the bla(CTX-M-3)-like (CTX-M-15) and bla(CTX-M-14)-like (CTX-M-9 and CTX-M-13) genes. The system appears to provide an efficient differentiation of ESBLs among E. coli, K. pneumoniae, and Enterobacter cloacae in Taiwan. Moreover, the design of the system can be easily adapted for similar purposes in areas where different ESBLs are prevalent.

Enterobacter cloacae↗

PubMed1999

Rapid and sensitive detection of Escherichia coli O157:H7 in bovine faeces by a multiplex PCR.

Cattle are considered the major reservoir for Escherichia coli O157:H7, one of the newly emerged foodborne human pathogens of animal origin and a leading cause of haemorrhagic colitis in humans. A sensitive test that can accurately and rapidly detect the organism in the food animal production environment is critically needed to monitor the emergence, transmission, and colonization of this pathogen in the animal reservoir. In this study, a novel multiplex polymerase chain reaction (PCR) assay was developed by using 5 sets of primers that specifically amplify segments of the eaeA, slt-I, slt-II, fliC, rfbE genes, which allowed simultaneous identification of serotype O157:H7 and its virulence factors in a single reaction. Analysis of 82 E. coli strains (49 O157:H7 and 33 non-O157:H7) demonstrated that this PCR system successfully distinguished serotype O157:H7 from other serotypes of E. coli and provided accurate profiling of the shiga-like toxins and the intimin adhesin in individual strains. This multiplex PCR assay did not cross-react with the background bacterial flora in bovine faeces and could detect a single O157:H7 organism per gram of faeces when combined with an enrichment step. Together, these results indicate that the multiplex PCR assay can be used for specific identification and profiling of E. coli O157:H7 isolates, and may be applied to rapid and sensitive detection of E. coli O157:H7 in bovine faeces when combined with an enrichment step.

Animals↗

PubMed2002

Detection in Escherichia coli of the genes encoding the major virulence factors, the genes defining the O157:H7 serotype, and components of the type 2 Shiga toxin family by multiplex PCR.

Strains of Shiga toxin-producing Escherichia coli (STEC) have been associated with outbreaks of diarrhea, hemorrhagic colitis, and hemolytic-uremic syndrome in humans. Most clinical signs of disease arise as a consequence of the production of Shiga toxin 1 (Stx1), Stx2 or combinations of these toxins. Other major virulence factors include enterohemorrhagic E. coli hemolysin (EHEC hlyA), and intimin, the product of the eaeA gene that is involved in the attaching and effacing adherence phenotype. In this study, a series of multiplex-PCR assays were developed to detect the eight most-important E. coli genes associated with virulence, two that define the serotype and therefore the identity of the organism, and a built-in gene detection control. Those genes detected were stx(1), stx(2), stx(2c), stx(2d), stx(2e), stx(2f), EHEC hlyA, and eaeA, as well as rfbE, which encodes the E. coli O157 serotype; fliC, which encodes the E. coli flagellum H7 serotype; and the E. coli 16S rRNA, which was included as an internal control. A total of 129 E. coli strains, including 81 that were O157:H7, 10 that were O157:non-H7, and 38 that were non-O157 isolates, were investigated. Among the 129 samples, 101 (78.3%) were stx positive, while 28 (21.7%) were lacked stx. Of these 129 isolates, 92 (71.3%) were EHEC hlyA positive and 96 (74.4%) were eaeA positive. All STEC strains were identified by this procedure. In addition, all Stx2 subtypes, which had been initially identified by PCR-restriction fragment length polymorphism, were identified by this method. A particular strength of the assay was the identification of these 11 genes without the need to use restriction enzyme digestion. The proposed method is a simple, reliable, and rapid procedure that can detect the major virulence factors of E. coli while differentiating O157:H7 from non-O157 isolates.

DNA, Bacterial↗

PubMed2004

Determination of verocytotoxin and eae gene loci by multiplex PCR in Escherichia coli O157:H7 isolated from human faeces in Northern Ireland: a four-year study of trends, 1997-2000.

This study aims to determine the distribution and frequency of verocytotoxin genes in human faecal clinical isolates of Escherichia coli O157 in Northern Ireland during the period 1997-2000, using a special four-target multiplex polymerase chain reaction (PCR) assay. One hundred and thirty two isolates of E. coli O157:H7 cultured during the four-year period (1997 [n=28]; 1998 [n=25]); 1999 (n=43); 2000 [n=36]), representing approximately 79% of total E. coli O157 laboratory isolations throughout N. Ireland, are examined for the presence of verocytotoxin gene loci (VT1, VT2 and eae) using a multiplex PCR assay. These isolates originate from the four Regional Area Health Boards that constitute the healthcare system in N. Ireland as follows: Eastern (53.8%; n=71), Northern (34.1%; n=45), Western (6.8%; n=9) and Southern (5.3%; n=7). Results showed that over 80% of these isolates possessed the VT2 and eae gene loci, with the remainder being predominantly VT1-, VT2- and eae-positive. None possessed the VT1 gene locus alone. Development and adoption of this simple four-target (three virulence and one control gene loci) multiplex PCR assay and subsequent recording of resulting verocytotoxin-typing data in a database, permitted local, rapid determination of carriage of known molecular virulence determinants of E. coli O157 isolates, which may aid in outbreak-related epidemiological investigations or other longitudinal studies.

Adolescent↗

PubMedDate not supplied

Rapid and specific differentiation of enterotoxin-producing Escherichia coli strains from other gram-negative enteric bacteria using multiplex PCR.

Enterotoxigenic Escherichia coli (ETEC) may produce heat-labile (LT) and heat-stable (STI or STII) enterotoxins. Differentiation between ETEC and other pathogenic and non-pathogenic E. coli as well as other Gram-negative bacteria responsible for induction of diarrhoea, requires isolation, biochemical identification and determination of toxins (or their genes--elt, estI, estII). A multiplex polymerase chain reaction (PCR) system for the rapid and specific detection of enterotoxin-gene-positive E. coli was developed. The primers described by other authors, specific for the universal stress protein A (UspA) of E. coli and enterotoxin genes were used and allowed a simultaneous amplification of the E. coli-specific uspA and the respective toxin genes. The specificity of this multiplex PCR system was confirmed by testing ETEC, non-ETEC and other non-E. coli bacteria. The specific 884 bp uspA gene and 280 bp (eltI), 166 bp (estI) or 278 bp (estII) amplification products were generated with the respective ETEC strains whereas no amplification was detected with non-E. coli bacteria. The multiplex PCR developed allowed the rapid and specific identification of enterotoxin-producing E. coli colonies directly grown from faecal samples of pigs with diarrhoea. The test may be used as a method for the determination of ETEC among other pathogenic groups of E. coli and other Gram-negative enteric isolates.

Animals↗

PubMed2005

Development of a rapid identification method for Aeromonas species by multiplex-PCR.

Existing biochemical methods cannot distinguish among some species of Aeromonads, while genetic methods are labor intensive. In this study, primers were developed to three genes of Aeromonas: lipase, elastase, and DNA gyraseB. In addition, six previously described primer sets, five corresponding to species-specific signature regions of the 16S rRNA gene from A. veronii, A. popoffii, A. caviae, A. jandaei, and A. schubertii, respectively, and one corresponding to A. hydrophila specific lipase (hydrolipase), were chosen. The primer sets were combined in a series of multiplex-PCR (mPCR) assays against 38 previously characterized strains. Following PCR, each species was distinguished by the production of a unique combination of amplicons. When the assays were tested using 63 drinking water isolates, there was complete agreement in the species identification (ID) for 59 isolates, with ID established by biochemical assays. Sequencing the gyrB and the 16S rRNA gene from the remaining four strains established that the ID obtained by mPCR was correct for three strains. For only one strain, no consensus ID could be obtained. A rapid and reliable method for identification of different Aeromonas species is proposed that does not require restriction enzyme digestions, thus simplifying and speeding up the process.

Aeromonas↗

PubMed2004

Multiplex PCR with 16S rRNA gene-targeted primers of bifidobacterium spp. to identify sources of fecal pollution.

Bifidobacteria are one of the most common bacterial types found in the intestines of humans and other animals and may be used as indicators of human fecal pollution. The presence of nine human-related Bifidobacterium species was analyzed in human and animal wastewater samples of different origins by using species-specific primers based on 16S rRNA sequences. Only B. adolescentis and B. dentium were found exclusively in human sewage. A multiplex PCR approach with strain-specific primers was developed. The method showed a sensitivity threshold of 10 cells/ml. This new molecular method could provide useful information for the characterization of fecal pollution sources.

Animals↗

PubMed1996

A duplicated PLP gene causing Pelizaeus-Merzbacher disease detected by comparative multiplex PCR.

Pelizaeus-Merzbacher disease (PMD) is an X-linked dysmyelinating disorder caused by abnormalities in the proteolipid protein (PLP) gene, which is essential for oligodendrocyte differentiation and CNS myelin formation. Although linkage analysis has shown the homogeneity at the PLP locus in patients with PMD, exonic mutations in the PLP gene have been identified in only 10%-25% of all cases, which suggests the presence of other genetic aberrations, including gene duplication. In this study, we examined five families with PMD not carrying exonic mutations in PLP gene, using comparative multiplex PCR (CM-PCR) as a semiquantitative assay of gene dosage. PLP gene duplications were identified in four families by CM-PCR and confirmed in three families by densitometric RFLP analysis. Because a homologous myelin protein gene, PMP22, is duplicated in the majority of patients with Charcot-Marie-Tooth 1A, PLP gene overdosage may be a important genetic abnormality in PMD and affect myelin formation.

Base Sequence↗

PubMed1995

Classification of mutations at the human hprt-locus in T-lymphocytes of bus maintenance workers by multiplex-PCR and reverse transcriptase-PCR analysis.

Polymerase chain reaction (PCR)-based screening methods were used to classify mutations arising in vivo at the hypoxanthine guanine phosphoribosyl-transferase (hprt) locus in small samples of human T-lymphocyte clones (< 5 x 10(4) cells) from 29 bus maintenance workers exposed to diesel exhaust, and 14 control individuals. All subjects were healthy, non-smoking males. Among 462 T-cell mutants studied by multiplex-PCR of genomic DNA, only 12 (2.6%) deletions were found: three total deletions, five partial exon deletions and four mutants with one or two exons deleted. Point mutations were classified in 323 mutants using reverse transcriptase-PCR amplification: 74 (22.9%) of these had splice site mutations and 241 (74.6%) had coding errors. Splice mutation was more frequent among the garage workers (24.8%) as compared to the controls (19.5%), possibly reflecting a polycyclic aromatic hydrocarbon-specific mutation induction in these workers. Our results also show that both gene deletion and splice mutation at the hprt-locus in T-cells of healthy non-smokers could be less frequent than previously reported.

Base Sequence↗

PubMed1999

Four common mutations of the cystathionine beta-synthase gene detected by multiplex PCR and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

BACKGROUND: A deficiency of cystathionine beta-synthase (CBS) is the most frequent cause of homocystinuria. The effect of therapy is related to the underlying CBS genotype, which makes early diagnosis of this genetic defect important. Our aim was to develop a fast and reliable method based on matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry for the determination of common mutations of the CBS gene. METHODS: We used MALDI-TOF mass spectrometry to detect four common CBS mutations (G307S, T272M, I278T, and V320A). The method is based on multiplex PCR of exons 7, 8, and 9, followed by single nucleotide extension in the presence of dideoxy NTPs of four primers targeted at the separate mutation sites. The extension products, as well as the 3-hydroxypicolinic acid matrix, were incubated with cation-exchange beads to remove disturbing salt contaminants. RESULTS: The above-mentioned mutations were determined in samples from 12 homocystinuria patients. The MALDI-TOF spectra allowed unambiguous discrimination between primers and extension products (>9 Da) in the mass range between 4500 and 7500 Da. No labeled primers or ddNTPs were required. The genotyping was verified by reference technique. CONCLUSION: Our results demonstrate fast, simple, and unambiguous multiplex genotyping of four common CBS mutations by MALDI-TOF mass spectrometry.

Cystathionine beta-Synthase↗

PubMed2005

Modified multiplex PCR for identification of Bacillus Calmette-Guérin substrain Tokyo among clinical isolates.

When an adverse reaction occurs and a mycobacterial species is isolated from a person vaccinated with Bacillus Calmette-Guérin (BCG) or a patient receiving BCG immunotherapy, it is essential to identify whether the isolate is BCG or another mycobacterial species. However, differentiation of BCG from other members of Mycobacterium tuberculosis complex has been very difficult. Using several specific primer-pairs, Bedwell et al. [Bedwell J, Kairo SK, Behr MA, Bygraves JA. Identification of substrains of BCG vaccine using multiplex PCR. Vaccine 2001; 19: 2146-51] recently reported that they could distinguish BCG substrains. We modified their method to improve differentiation of Tokyo 172 from other members of the M. tuberculosis complex, and examined whether this modified method could be applied to clinical isolates. Our method clearly identified BCG substrain (BCG Tokyo 172) among clinical isolates and easily distinguished between M. tuberculosis and wild-type Mycobacterium bovis.

Adjuvants, Immunologic↗

PubMed1999

Use of multiplex PCR to detect classical and newly described pyrogenic toxin genes in staphylococcal isolates.

Staphylococcus aureus may contain one or more genes that encode a variety of immunomodulatory pyrogenic toxins (PTs), including the staphylococcal enterotoxins and toxic shock syndrome toxin (TSST). The PTs interact with several cellular targets to produce disease, such as food poisoning and toxic shock syndrome. At present, nine serologically distinct enterotoxins and one immunoreactive form of TSST have been identified and characterized. As isolates of S. aureus are further assessed, it is anticipated that this number will increase. To facilitate screening, a multiplex PCR was designed to simultaneously determine which of these 10 currently known PT genes an individual S. aureus isolate possesses. We show here, using S. aureus isolates with characterized PT phenotypes, that this novel PCR technique reliably detects each of the known PTs in a single reaction.

Adult↗

PubMed2005

Fluorescent multiplex PCR--fast method for autosomal dominant spinocerebellar ataxias screening.

Expansion of CAG trinucleotide repeats has been shown to cause a number of autosomal dominant spinocerebellar ataxias such as SCA1, SCA2, SCA3/MJD, SCA6 and SCA7. These disorders are characterized by a wide inter- and intrafamiliar variation in clinical features. The same mutation can result in different phenotypes and the very similar phenotypes can be caused by different mutations. Therefore it is necessary to investigate more SCA genes (according to prevalence) to identify the causal elongation. We developed a fast and efficient screening method based on touchdown multiplex PCR with fluorescent labelled primers for the most common types of SCAs (SCA 1, 2, 3 and 7). It has been reliable in 113 probands tested. Fragment analysis was performed by using 6% denaturing polyacrylamide gel and employing the automated DNA sequencer. This method considerably shortens the process of molecular genetic screening of SCAs and might be used as a tip for designing other SCA screening sets.

Genetic Testing↗

PubMed2001

Differentiation of Lactobacillus plantarum, L. pentosus, and L. paraplantarum by recA gene sequence analysis and multiplex PCR assay with recA gene-derived primers.

In this study, we succeeded in differentiating Lactobacillus plantarum, Lactobacillus pentosus, and Lactobacillus paraplantarum by means of recA gene sequence comparison. Short homologous regions of about 360 bp were amplified by PCR with degenerate consensus primers, sequenced, and analyzed, and 322 bp were considered for the inference of phylogenetic trees. Phylograms, obtained by parsimony, maximum likelihood, and analysis of data matrices with the neighbor-joining model, were coherent and clearly separated the three species. The validity of the recA gene and RecA protein as phylogenetic markers is discussed. Based on the same sequences, species-specific primers were designed, and a multiplex PCR protocol for the simultaneous distinction of these bacteria was optimized. The sizes of the amplicons were 318 bp for L. plantarum, 218 bp for L. pentosus, and 107 bp for L. paraplantarum. This strategy permitted the unambiguous identification of strains belonging to L. plantarum, L. pentosus, and L. paraplantarum in a single reaction, indicating its applicability to the speciation of isolates of the L. plantarum group.

Bacteriocins↗