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Results for “Multiplex PCR”

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At least 433 records · Page 24Linked to original sources

PubMed1995

Comparison of multiplex PCR and culture for detection of Legionellae in cooling tower water samples.

We compared multiplex polymerase chain reaction (PCR) and culture for detecting the presence of Legionella pneumophila and Legionella spp in cooling tower water samples. Multiplex PCR was performed after phenol extraction of DNA from the samples. The set of primers for the PCR assay involved the 5S rRNA (Legionella spp) and the mip (macrophage infectivity potentiator gene, specific for L. pneumophila) genes as target sequences for amplification. Both the sensitivity and the specificity of the PCR assay were 100% when the 5S rRNA gene was used as target sequence. Isolation of Legionellae from the samples was observed only with the PCR-positive samples. We propose that PCR be used as a screening test before attempting to culture Legionellae from cooling tower water samples.

Air Conditioning↗

PubMed2006

Noninvasive genotyping of 9 Y-chromosome specific STR loci using circulatory fetal DNA in maternal plasma by multiplex PCR.

BACKGROUND: Human Y-Chromosome specific STR (Y-STR) has now become a useful loci in casework. However, noninvasive genotyping of multiple Y-STR loci and its application in prenatal genetic diagnosis haven't been reported. The purpose of this study is to develop a Y-STR multiplex PCR amplification system that is suitable for the amplification of short-sized templates of circulatory male fetal DNA and use the established multiplex in noninvasive prenatal genetic diagnosis and its further applications in forensic casework. METHODS: On the basis of the characteristic of circulatory fetal DNA in maternal plasma, we selected 9 Y-STR loci in which the allele size was less than 180 bp in length and developed two multiplexes that allowed fluorescent genotyping of 9 Y-STR loci simultaneously. These Y-STR loci include two trinucleotide repeats (DYS426 and DYS388) and seven tetranucleotide repeats (DYS393, DYS460, H4; DYS391, DYS389 I, DYS456 and DYS458). Sixty-four pairs of plasma DNA samples from pregnant women and genomic DNA samples from their husbands were detected by our method. RESULTS: As a result, an average of 7.3 Y-STR specific alleles was detected in each of the 30 plasma DNA samples from pregnancies with male fetuses. However, none of these 9 Y-STR specific alleles was detected in 34 plasma samples from pregnant women carrying female babies. The chances of detecting Y-STR alleles ranged from 66.7 to 93.3%. Fifty-eight haplotypes were detected in 64 unrelated Chinese male individuals; haplotype diversity was 0.9966. This highly polymorphic Y-STR multiplex has greatly improved the chances of detecting the Y-STR allele. CONCLUSIONS: This assay provides a sensitive, accurate and efficient method for noninvasive prenatal genetic diagnosis and forensic casework.

Alleles↗

PubMed2005

Simplified fluorescent multiplex PCR method for evaluation of the T-cell receptor V beta-chain repertoire.

RATIONALE: Evaluation of the T-cell receptor (TCR) V beta-chain repertoire by PCR-based CDR3 length analysis allows fine resolution of the usage of the TCR V beta repertoire and is a sensitive tool to monitor changes in the T-cell compartment. A multiplex PCR method employing 24 labeled upstream V beta primers instead of the conventionally labeled downstream C beta primer is described. METHOD: RNA was isolated from purified CD4 and CD8 T-cell subsets from umbilical cord blood and clinical samples using TRI reagent followed by reverse transcription using a C beta primer and an Omniscript RT kit. The 24 V beta primers were multiplexed based on compatibility and product sizes into seven reactions. cDNA was amplified using 24 V beta primers (labeled with tetrachloro-6-cardoxyfluorescein, 6-carboxyfluorescein, and hexachloro-6-carboxyfluorescein), an unlabeled C beta primer, and Taqgold polymerase. The fluorescent PCR products were resolved on an automated DNA sequencer and analyzed using the Genotyper 2.1 software. RESULTS: V beta spectratypes of excellent resolution were obtained with RNA amounts of 250 ng using the labeled V beta primers. The resolution was superior to that obtained with the labeled C beta primer assay. Also the numbers of PCRs were reduced to 7 from the 12 required in the C beta labeling method, and the sample processing time was reduced by half. CONCLUSION: The method described for T-cell receptor V beta-chain repertoire analysis eliminates tedious dilutions and results in superior resolution with small amounts of RNA. The fast throughput makes this method suitable for automation and offers the feasibility to perform TCR V beta repertoire analyses in clinical trials.

CD4 Antigens↗

PubMed1996

Mutational scanning of large genes by extensive PCR multiplexing and two-dimensional electrophoresis: application to the RB1 gene.

With the rapid increase in the number of identified human disease genes, the development of accurate and cost-efficient mutation tests has become opportune. Here we present a combination of extensive PCR multiplexing and two-dimensional (2-D) DNA electrophoresis to screen for mutations in 26 exons of the retinoblastoma (RB1) tumor suppressor gene. In 2-D electrophoresis, fragments are separated according to size and base pair sequence in non-denaturing and denaturing gradient gels, respectively. All target fragments, designed to have optimal melting characteristics, were prepared in a two-step PCR (a 6-plex long-PCR pre-amplification and a subsequent 25-plex short-PCR) followed by heteroduplexing. The mixture of PCR amplicons was then subjected to 2-D electrophoresis under a single set of experimental conditions. With this design, 35 previously identified mutations in 18 different exons were detected in 33 bilateral retinoblastoma patients. These results suggest that 2-D electrophoresis in this format provides a generally applicable, practical and fast way to diagnose with high accuracy large genes for a broad spectrum of possible disease-causing mutations.

DNA Mutational Analysis↗

PubMed2005

Multiplex PCR for simultaneous detection of macrolide and tetracycline resistance determinants in streptococci.

Resistance to macrolides and tetracyclines is increasing among streptococci and co-occurs as their resistance determinants are carried on the same mobile element. We developed a multiplex PCR to facilitate simultaneous and specific detection of resistance determinants for both macrolides [erm(A), erm(B), and mef(A/E)] and tetracyclines [tet(M), tet(O), tet(K), and tet(L)] in streptococci.

Drug Resistance, Bacterial↗

PubMed2006

Rapid identification and differentiation of clinical isolates of enteropathogenic Escherichia coli (EPEC), atypical EPEC, and Shiga toxin-producing Escherichia coli by a one-step multiplex PCR method.

Enteropathogenic Escherichia coli (EPEC), atypical enteropathogenic E. coli, and Shiga toxin-producing E. coli differ in their virulence factor profiles, clinical manifestations, and prognosis, and they require different therapeutic measures. We developed and evaluated a robust multiplex PCR to identify these pathogroups based on sequences complementary to escV, bfpB, stx1, and stx2.

DNA, Bacterial↗

PubMed1995

Simultaneous identification of strains of Escherichia coli serotype O157:H7 and their Shiga-like toxin type by mismatch amplification mutation assay-multiplex PCR.

Mismatch amplification mutation assay primers, specific for a unique base substitution in uidA of Escherichia coli O157:H7, was coupled with primers for the Shiga-like toxin I (SLT-I) and SLT-II genes in a multiplex PCR assay. Analysis of 108 bacteria showed that all Escherichia coli serotype O157:H7 strains were identified simultaneously with the SLT types encoded by these strains.

Bacterial Toxins↗

PubMed2005

Cytogenetic changes and loss of heterozygosity in atypical adenomatous hyperplasia, in carcinoma of the prostate and in non-neoplastic prostate tissue using comparative genomic hybridization and multiplex-PCR.

High grade prostatic intraepithelial neoplasia (PIN) and atypical adenomatous hyperplasia (AAH) are discussed to be precursors of prostate cancer (PC). Unlike high grade PIN the relation between AAH and PC is however unclear. In the present study we analyzed AAH, accompanying prostate carcinomas and carcinomas of the transitional zone after microdissection using comparative genomic hybridization (CGH) and multipelx-PCR with 10 microsatellite polymorphic markers. In every case non-neoplastic prostatic tissue was investigated for the same allelic imbalances. Two AAH showed allelic imbalances in multiplex-PCR. These imbalances did not correlate with the corresponding tumours and furthermore were different to the LOH found in the investigated prostate tumours of the transitional zone. One AAH showed loss on chromosome 22q. We found allelic imbalances in over 50% of non-neoplastic tissue adjacent to prostatic carcinoma. Our findings support the idea that AAH does not seem to be linked closely to PC and should not be considered as an obligate premalignant lesion.

Aged↗

PubMed2006

Combined use of MLPA and nonfluorescent multiplex PCR analysis by high performance liquid chromatography for the detection of genomic rearrangements.

Large genomic rearrangements are recognized as playing a pathogenic role in an increasing number of human genetic diseases. It is important to develop efficient methods for the routine detection and confirmation of these germline defects. Multiplex ligation-dependent probe amplification (MLPA) is considered an early step for molecular diagnosis of several genetic disorders. However, artifacts might hamper the interpretation of MLPA analysis, especially when rearrangements involve a single exon. Therefore, rearrangements must be verified by two independent methods. In this study, we developed nonfluorescent multiplex-PCR coupled to high-performance liquid chromatography (NFMP-HPLC) and analyzed whether the use of this method combined with MLPA could be helpful in the detection and confirmation of gross MSH2 and MLH1 genomic rearrangements. A total of nine nonfluorescent multiplex-PCRs were developed to analyze the 16 MSH2 and 19 MLH1 exons. Reliable multiplex amplifications and nonfluorescent peak quantitation were obtained with a limited number of cycles (< or = 25) using a denaturing HPLC (DHPLC) instrument under nondenaturing conditions. The results obtained by NFMP-HPLC were highly reproducible. The combined use of MLPA and NFMP-HPLC identified and independently confirmed putative MLH1 and MSH2 deletions in eight out of 50 unrelated patients with hereditary nonpolyposis colorectal cancer (HNPCC). In five cases, the deletions affected a single exon and in three cases multiple contiguous exons. These results were in agreement with breakpoint and complementary DNA (cDNA) analyses. Considering that MLPA and NFMP-HPLC are unlikely to be affected by the same artifacts, their combined use could also provide a robust and cost-effective strategy for routine screening and confirmation of putative rearrangements in other genes, especially when a single exon is involved or a precise characterization of breakpoints is not achieved.

Adaptor Proteins, Signal Transducing↗

PubMed2001

Identification of human enterovirulent Escherichia coli strains by multiplex PCR.

Some strains of Escherichia coli are involved in enteric infections in both adults and children. However the classical diagnostic methods can not differentiate pathogenic from nonpathogenic E. coli, because of the lack of phenotypic differences. In this study, we developed multiplex PCR in order to amplify fragments of specific virulence genes of the five main E. coli pathotypes. Fragments of the expected size were obtained using previously or newly designed primers and allowed identification of 10 virulence genes in only 5 reactions. This method was applied to the detection of pathogenic E. coli isolated from 90 patients' stools specimens during an 18-month survey. Patients were suffering from diarrhea or hemolytic uremic syndrome and in 13 cases (14.4%), an enterovirulent E. coli strain was detected. This diagnostic method could therefore represent an important technique in clinical laboratories which lack standard tests for these pathogens.

Adolescent↗

PubMed2004

A combined AFLP-multiplex PCR assay for molecular typing of Escherichia coli strains using variable bacterial interspersed mosaic elements.

The original method for molecular typing of E. coli strains was developed using the polymorphism in chromosomal sequences of bacterial interspersed mosaic elements (BIMEs) detected by multiplex PCR and analysed by AFLP assay. The applicability of the method in the epidemiology of E. coli was tested on a group of 524 strains of human and veterinary origin. In the studied group 18 different genotypes were detected. Significant differences were found in the frequencies of the genotypes among various groups of strains, suggesting the method could be a promising tool in the epidemiology of E. coli.

Animals↗

PubMed2006

Rapid multiplex PCR assay for identification of USA300 community-associated methicillin-resistant Staphylococcus aureus isolates.

Recent reports have noted a discernible increase in the number of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infections in patients without traditional risk factors. In the United States, the most prominent CA-MRSA strain encodes Panton-Valentine leukocidin (PVL) cytotoxin genes, belongs to pulsed field gel electrophoresis type USA300 and multilocus sequence type 8, and carries staphylococcal cassette chromosome mec (SCCmec) type IV. At present, molecular characterization of MRSA strains, such as USA300, can be time-consuming and is often beyond the technical capability of many clinical laboratories, making routine identification difficult. We analyzed the chromosomal regions flanking the SCCmec element in 44 USA300 MRSA isolates and identified a signature "AT repeat" sequence within the conserved hypothetical gene SACOL0058 located 1.4 kb downstream of the 3' end of the J1-SCCmec chromosomal junction. Only USA300 isolates tested contained a sequence of > or =6 AT repeats in combination with PVL (e.g., related USA500 or Iberian strains had > or =6 AT repeats but were PVL negative). Using a locked nucleic acid primer specific for > or =6 AT repeats in combination with primers to detect PVL, we developed a multiplex PCR assay specific for the identification of USA300 strains. Multiplex results were 100% concordant with DNA sequencing, suggesting that the method has promise as a means of rapidly identifying USA300 isolates.

Bacterial Proteins↗

PubMed2005

Detection of virulence-related genes by multiplex PCR in multidrug-resistant diarrhoeagenic Escherichia coli isolates from Kenya and Japan.

We compared serotypes, drug susceptibility and presence of virulence-related genes in diarrhoeagenic Escherichia coli isolates from children < 5 years from Kenya (n = 82) and Japan (n = 47). Multiplex PCR was used to detect genes coding for enteroaggregative adherence (aggR), heat-stable toxin (st), heat-labile toxin (It), verotoxin (vt), attaching and effacing mechanism (eaeA), enteroaggregative E. coli heat-stable enterotoxin 1 (astA) and enteroinvasive mechanism (invE). Kenyan E. coli O-serotypes were more diverse than those from Japan (29 vs. 12 serotypes) and exhibited high level multidrug resistance to World Health Organization (WHO) recommended antibiotics. Resistance rates to tetracycline, ampicillin and sulphamethoxazole-trimethoprim were 70.7, 65.9 and 68.3% respectively, but resistance to sulphamethoxazole-trimethoprim among the E. coli isolates from Japan was low (21%). Kenyan isolates harboured virulence-related genes in high frequency (82.9%) compared to those from Japan (25.5%) with aggR and astA being the most frequently detected genes. The presence of multiple virulence genes was associated with multidrug resistance and this merits further investigation.

Anti-Bacterial Agents↗

PubMed2001

Genotyping of Cre-lox mice and detection of tissue-specific recombination by multiplex PCR.

Conditional gene targeting, based on Cre-lox or other systems, requires frequent genotyping of transgenic mouse populations and monitoring of tissue-specific Cre recombinatory efficiency. This is currently achieved by Southern analysis from tail- and tissue-derived DNA. Multiplex PCR amplification of the floxed (flanked by loxP sites) genomic region, combined with the PCR detection of the Cre transgene, simplifies this task. Here, we show that complete genotyping of a floxed locus is possible with three appropriately placed primers and that this triplex PCR can be performed simultaneously with a universal PCR assay for the detection of Cre transgenes. Using this approach, we also determined the ratios of recombined versus non-recombined floxed genomic segments in genomic DNA samples. This allowed us to estimate the efficiency of in vivo conditional inactivation from biopsy material and tissue samples that were too small for Southern analysis. As many new conditional knockouts are spatiotemporally restricted, such assays will become increasingly useful. The proposed PCR strategy is flexible and may be adapted to the structural specificities of any target gene.

Alleles↗

PubMed2001

Multiplex PCR assay for detection and identification of Clostridium botulinum types A, B, E, and F in food and fecal material.

Botulism is diagnosed by detecting botulinum neurotoxin and Clostridium botulinum cells in the patient and in suspected food samples. In this study, a multiplex PCR assay for the detection of Clostridium botulinum types A, B, E, and F in food and fecal material was developed. The method employs four new primer pairs with equal melting temperatures, each being specific to botulinum neurotoxin gene type A, B, E, or F, and enables a simultaneous detection of the four serotypes. A total of 43 C. botulinum strains and 18 strains of other bacterial species were tested. DNA amplification fragments of 782 bp for C. botulinum type A alone, 205 bp for type B alone, 389 bp for type E alone, and 543 bp for type F alone were obtained. Other bacterial species, including C. sporogenes and the nontoxigenic nonproteolytic C. botulinum-like organisms, did not yield a PCR product. Sensitivity of the PCR for types A, E, and F was 10(2) cells and for type B was 10 cells per reaction mixture. With a two-step enrichment, the detection limit in food and fecal samples varied from 10(-2) spore/g for types A, B, and F to 10(-1) spore/g of sample material for type E. Of 72 natural food samples investigated, two were shown to contain C. botulinum type A, two contained type B, and one contained type E. The assay is sensitive and specific and provides a marked improvement in the PCR diagnostics of C. botulinum.

Animals↗

PubMed2005

Polar body diagnosis for hemophilia a using multiplex PCR for linked polymorphic markers.

Preimplantation genetic diagnosis (PGD) is usually performed on blastomeres. In Germany, the only possibility to perform PGD is by analysis of polar bodies. We performed PGD using polar bodies in a woman who is a carrier of hemophilia A. Multiplex PCR followed by nested fluorescent PCR for five linked polymorphic markers was established. From 11 analyzed polar bodies, only 1 showed alleles linked to the mutation. The corresponding oocyte was transferred and no pregnancy was established. As seen in other investigations, the rate of heterozygous first polar bodies is surprisingly high.

Adult↗

PubMed2006

Detection and typing of 46 genital human papillomaviruses by the L1F/L1R primer system based multiplex PCR and hybridization.

There are several genital HPV DNA detection systems described, however most of them present different disadvantages regarding the number of amplified and detected types, sensitivity, type specificity. A new PCR and hybridization based detection method was developed for sensitive and balanced amplification and specific typing of HPV DNA from clinical samples. The technique amplifies and detects 46 HPV types: 2a, 3, 6, 7, 10, 11, 13, 16, 18, 26, 27, 28, 29, 30, 31, 33, 34, 35, 39, 40, 42, 43, 44/55, 45, 51, 52, 53, 54, 56, 57, 58, 59, 61, 66, 67, 68, 70, 72, 73(MM9), 74, 82(MM4), 84(MM8), 89, 90, 91. Key elements of the L1F/L1R PCR and hybridization system are: the special selection of the amplified region, a novel and optimized amplification primer set, circumspectly designed general and type-specific oligonucleotide probes. Detection following a multiplex PCR is based on solid phase hybridization in microtiter plate format using general and type-specific probes at medium stringency, which makes the detection robust in case of small sequence variations. The assay is highly reproducible and suitable for automation. The method was compared to Hybrid Capture II test, and after clarifying conflicting results, the comparison showed an excellent agreement (96.2%).

Cervix Uteri↗

PubMed2000

Development of a differential multiplex PCR assay for equine herpesvirus 1 and 4 as a diagnostic tool.

In this study, a multiplex polymerase chain reaction (PCR) procedure was developed for differentiation of strains and field isolates of equine herpesvirus type 1 (EHV-1) and type 4 (EHV-4). Specific oli-gonucleotide primers were combined to amplify the thymidine kinase (TK) gene region of EHV-1 and EHV-4, which would yield fragments of different lengths for each virus in the same amplification reaction. The specificity of the largest PCR amplicon for EHV-4 was confirmed by restriction digestion with HindIII. The multiplex PCR proved to be a fast and sensitive method for typing EHV-1 and EHV-4 isolates and for detection and differentiation of both viruses in field samples in which infectious virus is no longer available. The sensitivity was improved by combining cycling optimization and visualization of PCR products in ethidium bromide and silver-stained acrylamide gels.

Animals↗