SEARCH · PubMed Health

Results for “Multiplex PCR”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 469 records · Page 26Linked to original sources

PubMed1995

Reverse transcription multiplex PCR for differentiation between polio- and enteroviruses from clinical and environmental samples.

For the rapid detection of polioviruses and their differentiation from nonpoliovirus enteroviruses, we developed a protocol in which clinical or environmental specimens are first inoculated onto cell cultures in tubes. After overnight incubation, the cultures are subjected to reverse transcription multiplex PCR with a primer pair which detects all enteroviruses (T. Hyypiä, P. Auvinen, and M. Maaronen, J. Gen. Virol. 70:3261-3268 1989) and two newly designed primer pairs specific for all 36 poliovirus strains tested. The PCR products can unequivocally be identified by their lengths in agarose gels, whereas the genetic heterogeneity of the poliovirus strains precludes identification by back-hybridization with internal probes. The proposed protocol is highly insensitive to the inhibitory effects of substances in the sample (stool, sewage). It allows for the detection of polioviruses and for polioviruses to be distinguished from nonpoliovirus enteroviruses within 24 h, and it allows for the concomitant isolation of a viable strain suitable for further typing.

Base Sequence↗

PubMed2000

Performance of a multiplex PCR for the determination of Haemophilus influenzae capsular types in the clinical microbiology laboratory.

To improve tools for the surveillance of invasive H. influenzae in the context of the drastic decrease of type b infections following the implementation of vaccination, a two-step PCR technique was developed to detect the capsule and type specific regions of H. influenzae. The technique of Falla et al (1994) was modified to amplify in a first step the capsule and type b regions by multiplex PCR. For non-b capsulated strains, the type a, c, d, e, and f loci were afterward detected simultaneously by an optimized touch-down PCR technique. An internal control of extraction and amplification (16S rDNA) was included for both PCR techniques. Overall, this technique was shown to perform as efficiently or better than the slide agglutination without risks of interpretation errors. Of the 138 H. influenzae strains tested, seven that had given doubtful results by the agglutination technique were unequivocally typed by PCR.

ATP-Binding Cassette Transporters↗

PubMed1996

Identification and characterization of Bacillus anthracis by multiplex PCR analysis of sequences on plasmids pXO1 and pXO2 and chromosomal DNA.

Bacillus anthracis can be identified on the basis of the detection of virulence factor genes located on two plasmids, pXO1 and pXO2. Thus isolates lacking both pXO1 and pXO2 are indistinguishable from closely related B. cereus group bacteria. We developed a multiplex PCR assay for characterization of B. anthracis isolates, and simultaneous confirmation of the species identity independent of plasmid content. The assay amplifies lef, cya, pag (pXO1) and cap (pXO2) genes, and a B. anthracis specific chromosomal marker, giving an easy-to-read profile. This system unambiguously identified virulent (pXO1+/2+) and avirulent (pXO1+/2-, pXO1-/2+ and pXO1-/2-) strains of B. anthracis and distinguished "anthrax-like' strains from other B. cereus group bacteria.

Bacillus anthracis↗

PubMed2006

Identification of Salmonella spp. isolates from chicken abattoirs by multiplex-PCR.

The present study was carried out to report the occurrence Salmonella spp., Salmonella Enteritidis, and Salmonella Typhimurium in chicken abattoirs. Samples of feces; feathers; scald, evisceration, and chiller water; and rinse water of non-eviscerated, eviscerated, and chilled carcass were collected from six chicken abattoirs. Salmonella isolates were identified by a multiplex-PCR using three sets of primers targeting the invA, pefA, and sefA gene sequences from Salmonella spp., S. Typhimurium and S. Enteritidis, respectively. Salmonella spp. was detected in 10% (29/288) of the samples, whereas serovars Enteritidis and Typhimurium were identified in 62% (7/288), respectively. The results indicate the need to improve hygiene and sanitary standards in poultry slaughter lines, besides the education of food handlers and information to consumers.

Abattoirs↗

PubMed2004

[Development of a subtype screening assay for human immunodeficiency virus type 1 by nested multiplex PCR].

OBJECTIVE: The current available assays for HIV subtyping, such as sequence-based phylogenetic analysis or heteroduplex mobility assay (HMA), are labor-intensive and time-consuming. The authors have just developed a simple and rapid subtype-screening assay for subtypes B, C, and CRF01-AE using a single nested multiplex PCR. METHODS: Proviral DNA from HIV-positive samples was extracted and subjected to first round PCR with universal primers for gag region that can detect HIV-1 M group isolates. In the second round PCR, three pairs of subtype-specific primers, respectively detecting subtype B, C and CRF01-AE, were added into one tube. The PCR products of different subtypes could be distinguished in agarose-gel electrophoresis. Another pair of primers exclusively detecting the prevalent recombinant B/C strains CRF07-BC and CRF08-BC were designed and used. Additionally, all of these samples were sequenced and analyzed phylogenetically. RESULTS: Phylogenetic analysis showed that out of 119 samples, there were 43 subtype B samples (Euro-American B 11, Thailand B 32), 54 subtype C, 17 CRF01-AE, 3 subtype A and 2 subtype D samples. The subtype B, C, and CRF01-AE specific primer sets detected 35 (81.4%), 46 (85.2%), and 13(76.5%) samples with accuracy and specificity. Non-specific bands occasionally appeared but did not interfere with interpretation of the results. The primer pairs for CRF07-BC and CRF08-BC amplified target sequences were confirmed by sequencing and phylogenetic analysis. The specificity of all these subtype-specific primers was found to be 100%. CONCLUSION: A simple and rapid assay was developed for screening subtypes B, C, CRF01-AE, CRF07-BC and CRF08-BC in China. This assay may have potential application in HIV laboratories in China and worldwide.

Acquired Immunodeficiency Syndrome↗

PubMed1996

Rapid detection of trisomies 21 and 18 and sexing by quantitative fluorescent multiplex PCR.

Aneuploidies involving chromosomes 21, 18, 13, X and Y account for over 95% of all chromosomal abnormalities in live-born infants. Prenatal diagnosis of these disorders is usually accomplished by cytogenetic analysis of amniotic or chorionic cells but this is a lengthy procedure requiring great technical expertise. In this paper, we assess the diagnostic value of using a quantitative fluorescent polymerase chain reaction (PCR) suitable for the simultaneous and rapid diagnosis of trisomies 21 and 18 together with the detection of DNA sequences derived from the X and Y chromosomes. Samples of DNA, extracted from amniotic fluid, fetal blood or tissues, and peripheral blood from normal adults were investigated by quantitative fluorescent PCR amplification of polymorphic small tandem repeats (STRs) specific for two loci on each of chromosomes 21 and 18. Quantitative analysis of the amplification products allowed the diagnosis of trisomies 21 and 18, while sexing was performed simultaneously using PCR amplification of DNA sequences derived from the chromosomes X and Y. These results indicate the advantages of using two sets of STR markers for the detection of chromosome 21 trisomies and confirmed the usefulness of quantitative fluorescent multiplex PCR for the rapid prenatal diagnosis of selected chromosomal abnormalities.

Aneuploidy↗

PubMed2002

Selective amplification of tyv (rfbE), prt (rfbS), viaB, and fliC genes by multiplex PCR for identification of Salmonella enterica serovars Typhi and Paratyphi A.

The PCR primers for O, H, and Vi antigen genes, tyv (rfbE), prt (rfbS), fliC-d, fliC-a, and viaB, were designed and used for the rapid identification of Salmonella enterica serovars Typhi and Paratyphi A with multiplex PCR. The results showed that all the clinical isolates examined of Salmonella serovars Typhi and Paratyphi A were accurately identified by this assay.

Antigens, Bacterial↗

PubMed2006

Validation of an internal method for the diagnosis of infections with Chlamydophila abortus and Coxiella burnetii by real-time multiplex PCR.

Chlamydophila abortus and Coxiella burnetii are one of the major pathogens implicated in abortion in cattle. Their characteristic of obligate intracellular bacteria, and of zoonotic agents, makes their culture difficult in diagnostic laboratories, and the traditional tools of diagnosis (detection of sera antibodies by ELISA, Stamp's coloration) encounter specificity, sensitivity and interpretability limits. Individual PCR have recently been developed. Nevertheless, their income/cost is a limiting factor for breeders. As the symptoms are not specific, the request for analysis often concerns the two valences. Consequently, the development and the validation of an internal multiplex PCR appears to be a suitable solution.

Abortion, Veterinary↗

PubMed2001

Specific identification and targeted characterization of Bifidobacterium lactis from different environmental isolates by a combined multiplex-PCR approach.

The species Bifidobacterium lactis, with its main representative strain Bb12 (DSM 10140), is a yoghurt isolate used as a probiotic strain and is commercially applied in different types of yoghurts and infant formulas. In order to ensure the genetic identity and safety of this bacterial isolate, species- and strain-specific molecular tools for genetic fingerprinting must be available to identify isolated bifidobacteria or lactic acid bacteria from, e.g., various clinical environments of relevance in medical microbiology. Two opposing rRNA gene-targeted primers have been developed for specific detection of this microorganism by PCR. The specificity of this approach was evaluated and verified with DNA samples isolated from single and mixed cultures of bifidobacteria and lactobacilli (48 isolates, including the type strains of 29 Bifidobacterium and 9 Lactobacillus species). Furthermore, we performed a Multiplex-PCR using oligonucleotide primers targeting a specific region of the 16S rRNA gene for the genus Bifidobacterium and a conserved eubacterial 16S rDNA sequence. The specificity and sensitivity of this detection with a pure culture of B. lactis were, respectively, 100 bacteria/ml after 25 cycles of PCR and 1 to 10 bacteria/ml after a 50-cycle nested-PCR approach.

Bacterial Typing Techniques↗

PubMed2005

Rapid differential diagnosis of Mycoplasma agalactiae and Mycoplasma bovis based on a multiplex-PCR and a PCR-RFLP.

The membrane-protein 81 gene (mb-mp81) of Mycoplasma bovis was cloned, sequenced and compared to membrane-protein 81 gene (ma-mp81) of Mycoplasma agalactiae. After alignment of both sequences, specific primers pairs were designed from variable or unchanging nucleotide segments. In this study, we describe the development and optimization of a multiplex-PCR (MPCR) for the rapid detection of M. agalactiae and M. bovis strains. In addition, a simple and rapid PCR-restriction fragment length polymorphism (RFLP) assay, using the restriction enzymes AluI, DraI, RsaI and XbaI, is described to distinguish between both species. The results suggest that MPCR and PCR-RFLP assays could be used as an alternative method in routine diagnosis for rapid and specific simultaneous detection of M. agalactiae and M. bovis.

Bacterial Proteins↗

PubMed1998

Development of a multiplex PCR gene fingerprinting method using gyrA and pflA polymorphisms to identify genotypic relatedness within Campylobacter jejuni species.

The two genes gyrA and pflA, whose sequence variability had been previously described, were evaluated separately for their potential value in discriminating strains of Campylobacter jejuni. A single method was then developed by which the two loci were simultaneously amplified using a multiplex PCR procedure, and banding patterns were generated using a pre-selected set of restriction endonuclease enzymes. The method was applied to 18 strains of Camp. jejuni from different poultry sources varying in geographical origin and year of isolation. Results were combined and compared by means of numerical analysis with the classification obtained by flaA-typing and macrorestriction SmaI and KpnI. The usefulness of PCR fingerprinting of the gyrA/pflA genes for rapid ordering of strains by genotypic relatedness and providing additional information for estimating the degree of linkage between strains was demonstrated.

Animals↗

PubMed2006

Rapid identification of nontypeable and serotype b Haemophilus influenzae from nasopharyngeal secretions by the multiplex PCR.

OBJECTIVE: Heamophilus influenzae (H. influenzae) is an important pathogen responsible for both invasive and non-invasive infectious diseases. While encapsulated type b strain recognized as a major cause of severe invasive diseases, nontypeable strains are the major causes of non-invasive infectious diseases. Detection of this pathogen from nasopharyngeal secretions (NPS) is important. METHODS: We developed a multiplex polymerase chain reaction (PCR) for rapid identification of nontypeable and serotype b H. influenzae from nasopharyngeal secretions. RESULTS: A total 25 nasopharyngeal secretions were evaluated in this study. The multiplex PCR provided rapid and unequivocal results for determining either nontypeable or encapsulated typeable especially type b strains including a determination of beta-lactamase productions. CONCLUSION: The multiplex PCR based serotyping provided more reliable results than slide agglutination test (SAT) and is a valuable and expeditious method for identification of H. influenzae with determining capsular serotypes.

Haemophilus influenzae↗

PubMed2004

Multiplex PCR assay for differentiation of Helicobacter felis, H. bizzozeronii, and H. salomonis.

Helicobacter felis, Helicobacter bizzozeronii, and Helicobacter salomonis are frequently found in the gastric mucous membrane of dogs and cats. These large spiral organisms are phylogenetically highly related to each other. Their fastidious nature makes it difficult to cultivate them in vitro, hampering traditional identification methods. We describe here a multiplex PCR test based on the tRNA intergenic spacers and on the urease gene, combined with capillary electrophoresis, that allows discrimination of these three species. In combination with previously described 16S ribosomal DNA-based primers specific for the nonculturable "Candidatus Helicobacter suis," our procedure was shown to be very useful in determining the species identity of "Helicobacter heilmannii"-like organisms observed in human stomachs and will facilitate research concerning their possible zoonotic importance.

Animals↗

PubMed2005

Detection of virulence genes in Malaysian Shigella species by multiplex PCR assay.

BACKGROUND: In Malaysia, Shigella spp. was reported to be the third commonest bacterial agent responsible for childhood diarrhoea. Currently, isolation of the bacterium and confirmation of the disease by microbiological and biochemical methods remain as the "gold standard". This study aimed to detect the prevalence of four Shigella virulence genes present concurrently, in randomly selected Malaysian strains via a rapid multiplex PCR (mPCR) assay. METHODS: A mPCR assay was designed for the simultaneous detection of chromosomal- and plasmid-encoded virulence genes (set1A, set1B, ial and ipaH) in Shigella spp. One hundred and ten Malaysian strains (1997-2000) isolated from patients from various government hospitals were used. Reproducibility and sensitivity of the assay were also evaluated. Applicability of the mPCR in clinical settings was tested with spiked faeces following preincubation in brain heart infusion (BHI) broth. RESULTS: The ipaH sequence was present in all the strains, while each of the set1A, set1B and ial gene was present in 40% of the strains tested. Reproducibility of the mPCR assay was 100% and none of the non-Shigella pathogens tested in this study were amplified. The mPCR could detect 100 colony-forming units (cfu) of shigellae per reaction mixture in spiked faeces following preincubation. CONCLUSIONS: The mPCR system is reproducible, sensitive and is able to identify pathogenic strains of shigellae irrespective of the locality of the virulence genes. It can be easily performed with a high throughput to give a presumptive identification of the causal pathogen.

Antigens, Bacterial↗

PubMed2004

Identification of CYP2B6 sequence variants by use of multiplex PCR with allele-specific genotyping.

BACKGROUND: Cytochrome P450 2B6 (CYP2B6) has a role in the metabolism of many clinically important substances, but the variation within the CYP2B6 gene has not been fully characterized. The aim of the present study was to develop a reliable and robust assay for determining genotypic variants. METHODS: We used a two-stage procedure. An initial multiplex PCR reaction amplified the relevant gene fragments in exonic and regulatory regions to ensure isolation of CYP2B6 from its similar pseudogene (CYP2B7). This product was then genotyped by allele-specific PCR. RESULTS: The assay detected the following published single-nucleotide polymorphisms: C64T (Arg22Cys), C78T, G216C, G516T (Gln172His), C777A (Ser259Arg), A785G (Lys262Arg), and C1459T (Arg487Cys), as well as additional loci found within the single-nucleotide polymorphism (SNP) databases: A1190G, C1268A, C1330T, A1382G, A1402T, and an A/T SNP in intron 2 (A12917T). This approach detected all common, previously reported alleles and identified a new allele (CYP2B6*4C) present in 2.2% of a Caucasian population. Genotypic frequencies obtained were consistent with previously published results. CONCLUSIONS: This method is simple, reliable, rapid, and amenable to automation and could facilitate the large-scale genotypic analysis of CYP2B6.

Alleles↗

PubMed2006

Development of a multiplex PCR assay for polymorphism analysis of Brucella suis biovars causing brucellosis in swine.

Swine brucellosis is caused by the biovars 1, 2 and 3 of Brucella suis the identification of which up to now relies on microbiological tests lacking adequate specificity together with time consuming and expensive molecular procedures. Based on sequence variation of the omp2b gene, we have developed a four primer set multiplex PCR assay that was tested for polymorphism analysis of B. suis biovars causing brucellosis in swine. The assay exploits the single nucleotide polymorphisms found in omp2b gene of B. suis reference biovars which are conserved in 43 B. suis field isolates from different geographic origins and hosts. Three specific amplification patterns (S1, S2 and S3) were obtained for reference strains of B. suis biovars 1, 2 and 3, respectively. However, some B. suis field isolates identified as biovars 2 or 3 according AMOS-PCR, PCR-RFLP of omp31 and omp2 genes and classical bacteriological methods, resulted also in S1 patterns, limiting the typing usefulness of the method.

Amino Acid Sequence↗

PubMedDate not supplied

Multiplex PCR assay for the detection of mycobacterial DNA sequences directly from sputum.

Time consuming classical diagnostic tests and the increasing incidence of tuberculosis epidemics have rendered the need for new more sensitive diagnostic tools, urgent. This study explored the possibility of a direct and rapid method for the identification and characterization of pathogenic typical and atypical species of mycobacteria from sputum, based on a multiplex PCR assay. Gene bank search on the mycobacterial genome revealed specific sequences that fulfilled the above set criteria. Two pairs of primers were used to amplify a 243 bp fragment of the gene encoding the immunogenic protein MPB 64 and a 133 bp fragment of the gene encoding the 65-KDa mycobacterial antigen. The first pair of primers was selected among others, to detect specifically bacteria of the M. tuberculosis complex, whereas the second, to detect in addition to the latter, those of the M. avium-intracellular complex. Our mutiplex PCR assay, detected and identified correctly, all the mycobacterium tuberculosis and M. avium-intracellulare complex strains provided on pure culture as controls with a sensitivity of 10(-3) colony forming units. Furthermore, by performing our assay on 55 sputum samples from patients with positive culture and Ziehl-Neelsen staining, we identified mycobacterial DNA sequences in all--39 samples with M. tuberculosis complex and 16 with M. avium-intracellulare complex. Out of 300 sputum specimens from patients with clinical evidence of tuberculosis, 149 were positive by our method (95 M. tuberculosis and 54 M. avium-intracellulare complex) whereas 157 samples (95 M. tuberculosis complex, 59 M. avium-intracellulare complex, 1 M. xenopi, and 2 that could not be positively identified) were culture positive and only 95 Ziehl Neelsen positive. These findings suggest that the method described can be applied on sputum, and can identify in one step strains of the M. tuberculosis and M. avium-intracellulare complex and has effectivness comparable to culture methodologies.

DNA Primers↗

PubMed1993

Multiplex PCR amplification from the CFTR gene using DNA prepared from buccal brushes/swabs.

Traditionally, DNA used for PCR-based diagnostic analysis has originated from white cells fractionated from whole blood. Although this method yields substantial quantities of DNA, there are some drawbacks to the procedure, including the inconvenience of drawing blood, risk of exposure to blood-borne pathogens, liquid sample handling, and the somewhat involved extraction procedure. Alternatively, DNA for genetic diagnosis has been derived from finger stick blood samples, hair roots, cheek scrapings, and urine samples. Oral saline rinses have also been used extensively as a means of collecting buccal epithelial cells as a DNA source. However, this method still requires liquid sample handling. Herein, we present our results involving the rapid extraction of DNA from buccal cells collected on cytology brushes and swabs for use in PCR reactions, specifically the multiplex amplification of 5 exons within the CFTR gene. The quality of DNA isolated from buccal cells, collected in this manner, has been sufficient to reproducibly support multiplex amplification. Cheek cell samples and the DNA prepared from them as described here are highly stable. The success rate of PCR amplification on DNA prepared from buccal cells is 99%. In a blind study comparing the analysis of 12 mutations responsible for cystic fibrosis in multiplex products amplified with DNA from both blood and buccal cell samples from 464 individuals, there was 100% correlation of results for blood and cheek cell DNA, validating the use of DNA extracted from cheek cells collected on cytology brushes for use in genetic testing.

Base Sequence↗