PubMed Health⌕ Search

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 1,801 records · Page 100Linked to original sources
PubMed2003

The prevalence of Arcobacter spp. on chicken carcasses sold in retail markets in Turkey, and identification of the isolates using SDS-PAGE.

In this study, the prevalence of Arcobacter spp. on chicken carcasses sold in various retail markets in Turkey was investigated. The isolates were characterized and identified using various phenotypic and molecular tests. The membrane filtration technique employing 0.45-microm pore size membrane filters laid onto a nonselective blood agar was used after enrichment in Oxoid Arcobacter Enrichment Broth (AEB) to examine a total of 75 chicken carcasses (44 fresh and 31 frozen). Species level identification was performed using SDS-PAGE of whole-cell proteins and a recently developed multiplex-PCR assay. All isolates were identified as Arcobacter butzleri. Of the 44 fresh chicken carcasses examined, 42 (95%) were positive for A. butzleri. A. butzleri was also recovered from seven (23%) of the 31 frozen carcasses examined.

Animals↗
PubMed2001

Molecular characterization of the VP7 gene of Rotavirus isolated from a clinical sample of Calcutta, India.

The gene coding for outer capsid protein, VP7 of rotaviruses detected among the children suffering from watery diarrhea in the eastern part of India was studied. The full length gene, coding for VP7 was synthesized from a local strain, WD33, by combined reverse transcriptase-polymerase chain reaction. A total of 42 fecal RNA out of 93 rotavirus-positive samples hybridized with this DNA, indicating the prevalence of this strain in the community. Comparison of the nucleotide and deduced amino acid sequence with the VP7 gene of other serotypes revealed that the local strain,WD33, resembled closely with G1 serotype, the homology being 94 and 97%, respectively. Multiplex PCR using the specific oligonucleotide primers also amplified the G1 specific DNA fragment. The overall change of 16 amino acids was noticed when compared with G1 specific prototype Wa strain of which nine amino acids are within the neutralization domain. However, the phylogenetic tree constructed by comparing the G1 strains from different countries demonstrated that this strain WD33 clustered as a distinct sub-lineage of lineage IV and not with Wa strain, that clustered with lineage III.

Amino Acid Sequence↗
PubMed2002

Distribution of rotavirus VP7 genotypes among children suffering from watery diarrhea in Kolkata, India.

A combined reverse transcriptase-polymerase chain reaction (RT/PCR) was used to produce cDNA of the VP7 gene of rotavirus present in the stool samples. A total of 150 rotavirus positive stool samples were used in this study. Multiplex PCR, using the type specific primers, revealed the presence of G1 (49/150, 32.7%), G2 (27/150, 18%) and G4 (30/150, 20%) genotypes among the samples collected during 1999-2000 from children suffering from acute watery diarrhea. Eighteen samples (12%) were of mixed genotype and the remaining 16 samples (10.6%) could not be typed. Comparative analysis of the full length genes of the representative strains with corresponding genotypes incorporated in the human-rhesus rotavirus tetravalent vaccine (RRV-TV) formulation demonstrates variations of the circulating G1, G2 and G4 strains with the corresponding G genotypes present in the vaccine strain.

Amino Acid Sequence↗
PubMed2003

A population-based study of glutathione S-transferase M1, T1 and P1 genotypes and risk for lung cancer.

A deletion polymorphism for glutathione S-transferase M1 (GSTM1) has been related to risk for lung cancer among smokers in some studies but not in others. We examined GSTM1, a GSTT1 deletion polymorphism and a common GSTP1 gene variant (iso-->val), as risk factors for lung cancer in a population-based case-control study of men. Cases (N=274) were males identified from 1993 to 1996 through the Fred Hutchinson Cancer Research Center Cancer Surveillance System registry for western Washington State. Male age-matched controls (N=501) were selected by random-digit dialing. Subjects participated in a telephone interview and blood draw. GSTM1 and GSTT1 were genotyped with a multiplex PCR assay using beta-globin as a positive control, and GSTP1 single nucleotide variant determined with PCR-based oligonucleotide ligation assays. GSTM1 absence was associated with a modest elevation in risk among all cases (odds ratio=1.27, 95% CI 0.91-1.77) and among non-small cell cancers (adenocarcinoma OR=1.58, 95% CI 0.99-2.52; squamous cell OR=1.40, 95% CI 0.83-2.34). Risk associated with GSTM1 null was increased two to sixfold among heavy smokers. GSTT1 was not associated with lung cancer risk and GSTP1 val was non-significantly associated with a modest reduction in risk, particularly among heavy smokers. No specific combination of GST genotypes was particularly associated with risk. These results support previous reports that the GSTM1 null genotype is associated with a modest increase in risk for lung cancer, particularly among heavy smokers, suggest no role for GSTT1 and the need for further study of GSTP1.

Adolescent↗
PubMed1999

Differentiation of antitumor-specific cytotoxic T lymphocytes from autologous tumor infiltrating lymphocytes in non-Hodgkin's lymphomas.

The present study describes a new culture protocol allowing the activation and proliferation of autologous tumor infiltrating T lymphocytes (TIL), and the generation of antitumor specific CTL in non-Hodgkin's lymphoma (NHL). Cells from eight patients with indolent NHL were used. We performed 3-week co-cultures of TIL with irradiated autologous malignant B cells in the presence of low doses of IL-1beta, IL-2 and IL-12. The proliferation, phenotype and cytotoxicity, and antitumor specificity of T cells recovered were studied. T-cell clonality was analyzed using TCRgamma gene rearrangement amplification by a multiplex PCR. Under these culture conditions, TIL proliferated, and the CD8+ T lymphocytes that were in a minority at the beginning of the culture increased dramatically in 6 out of 8 cases. In two cases, CD4+ T lymphocytes expanded. We showed that an oligoclonal selection of reactive T cells occurred in culture. Specific cytotoxicity developed against autologous malignant B cells in the 6 cases where there was an expansion of CD8+ T lymphocytes. Inhibition experiments performed with mAb directed against HLA class I and II molecules, CD4, CD8 and TCRgammadelta showed that the cytotoxic effector cells were CD8+ T lymphocytes probably expressing TCRalphabeta+. Cytokine secretion was analyzed in culture medium, and we detected significant levels of IFN-gamma, TNF-alpha, and IL-10 and no IL-4 (except in one case). Our results demonstrate that memory T cells from lymphoma patients can be amplified and differentiated into antitumor cytotoxic cells using a combination of the cytokines IL-1beta, IL-2, and IL-12 in association with non modified tumor cells.

Aged↗
PubMed2000

Mutation detection by pyrosequencing: sequencing of exons 5-8 of the p53 tumor suppressor gene.

The ability to sequence a large number of DNA samples rapidly and accurately for detection of all possible mutations is a critical goal for the future application of DNA sequencing in routine medical diagnostics. Pyrosequencing() is a non-electrophoretic real-time DNA sequencing method that uses the luciferase-luciferin light release as the detection signal for nucleotide incorporation into target DNA. For pyrosequencing of the human p53 gene, a nested multiplex PCR method for amplification of exons 5-8 was prepared. In order to investigate the use of pyrosequencing in mutation detection, DNA samples from skin-cancer patients were used. Two forms of nucleotide dispensation strategy were used, cyclic and programmed. Bi-directional pyrosequencing was performed and the overlapping sequence data produced were assembled to determine the sequence of the gene. Reliable sequencing data were obtained with both dispensation strategies, but some advantages were obtained using the programmed nucleotide dispensation approach, such as longer and faster reads, and fewer out-of-phase problems. The accuracy of pyrosequencing for detection of p53 mutations and allele distribution was demonstrated.

Base Sequence↗
PubMed2001

High-performance liquid chromatography multiplex detection of two single nucleotide mutations associated with hereditary hemochromatosis.

High-performance liquid chromatography (HPLC) has been applied to the multiplex detection of the two single nucleotide mutations commonly found in hereditary hemochromatosis (HH). HH is associated with a major G to A transition at position 845 (mutation Cys282Tyr) and a minor C to G transition at position 187 (mutation His63Asp) in the cDNA of the HFE gene. Two detection assays were developed based on HPLC analysis of restriction fragment length polymorphism (RFLP) or single nucleotide extension (SNE) products following multiplex PCR amplification. RFLP genotypes the two sites as dsDNA fragments of different lengths generated by restriction enzymes Rsa I/Bcl I. SNE extends primers 5'-adjacent to the sites of interest with a dideoxynucleotide triphosphate (ddNTP) to generate extended ssDNA. The identity of the added ddNTP reveals the identity of the original possible mutation site(s). Application of these methods with HPLC analysis provides simple and reliable genotyping for HH and can be applied to other single nucleotide polymorphism studies.

Chromatography, High Pressure Liquid↗
PubMed2001

Sequence structure of 12 novel Y chromosome microsatellites and PCR amplification strategies.

In this work, we present sequencing data for 12 recently reported Y STR loci (DYS434, DYS435, DYS436, DYS437, DYS438, DYS439, GATA A10, GATA 7.1, GATA 7.2, GATA C4, GATA H4, GATA A4), as well as the PCR multiplex strategies we implemented for their detection. Sequenced allelic ladders were constructed and a nomenclature for these new systems is proposed based on the sequence structure and following ISFG recommendations.GATA A4 and DYS439 are likely the same STR. They have the same STR structure and the alleles are always the same in the same individuals.Sequence polymorphisms were observed in the GATA C4 and DYS437 STRs. The variation in DYS437 was associated with a specific population group and is very interesting not only for forensic genetics but also for anthropological studies.

Alleles↗
PubMed1998

Fluorescent multiplex analysis of nine STR loci: Spanish population data.

A total of 171 Caucasians living in Andalucia (southern Spain) have been typed for nine short tandem repeat (STR) loci by multiplex PCR amplification using a commercially available kit (Profiler Plus; Perkin-Elmer, Norwalk, CT, USA) and semi-automatic electrophoresis (ABI Prism 377 DNA Sequencer, Applied Biosystems, Foster City, CA, USA). The kit enables typing of the STR loci D3S1358, VWA, FGA, D5S818, D13S317, D7S820, D8S1179, D21S11, and D18S51. All loci, except D7S820, meet Hardy-Weinberg equilibrium. Because of the large number of loci that can be analyzed, the power of discrimination (PD) is greater than 0.99999, and the probability of exclusion (PE) reaches 0.99991 in our population sample.

Alleles↗
PubMed1999

Population genetic studies on the tetrameric short tandem repeat loci D3S1358, VWA, FGA, D8S1179, D21S11, D18S51, D5S818, D13S317 and D7S820 in Egypt.

The short tandem repeat loci (STRs) D3S1358, VWA, FGA, D8S1179, D21S11, D18S51, D5S818, D13S317, D7S820 and a locus allowing for sex-discrimination (amelogenin) can be co-amplified by the polymerase chain reaction using a commercially available kit (AmpFlSTR Profiler plus, Perkin-Elmer Biosystems, San Jose, CA) and subsequently typed using capillary electrophoresis (ABI Prism 310 Genetic analyzer, Perkin Elmer Applied Biosystems, San Jose CA). To establish databases for these loci for an Arab population sample from Egypt, 140 unrelated persons were typed. Analysis of these data revealed that all loci except for VWA were in Hardy-Weinberg equilibrium, that the combined mean paternity exclusion chance (MEC) was 0.999875 and that the combined discriminating power (DP) was 2.635 x 10(-11). The allelic distributions found in the Egyptian sample were significantly different at four loci from those found for an Austrian Caucasian population, at all nine loci from an African-American sample and at six of six loci from a Chinese sample. No evidence of linkage equilibrium between any of the co-amplified loci was found. Our results support that the combination of multiplex PCR and capillary electrophoresis can both save time and yield excellent results for paternity testing and stain analysis.

Alleles↗
PubMed2006

Chromosomal abnormality and Y chromosome microdeletion in Chinese patients with azoospermia or severe oligozoospermia.

Chromosomal abnormality and Y chromosome microdeletion are regarded as two frequent genetic causes associated with spermatogenic failure in Caucasian population. To investigate the distribution of the two genetic defects in Chinese patients with azoospermia or severe oligozoospermia, karyotype analysis by G-banding was carried out in 358 idiopathic infertile men, including 256 patients with azoospermia and 102 patients with severe oligozoospermia, and screening of AZF region microdeletion of Y chromosome by multiplex PCR was performed in those patients without detectable chromosomal abnormality and 100 fertile controls. Of 358 patients, 39(10.9%) were found to have chromosomal abnormalities in which Klinefelter's syndrome (47, XXY) was the most common chromosomal aberration. The incidence of sex chromosomal abnormality in patients with azoospermia was significantly higher than that in patients with severe oligozoospermia (12.1% vs 1%). Among the rest of the 319 patients with normal karyotype, 46 (14.4%) were found to have microdeletions in AZF region. The prevalence rates of AZF microdeletion was 15% and 13.1% in patients with azoospermia and severe oligozoospermia respectively. The microdeletion in AZFc was the most frequent deletion and all the microdeletions in AZFa were found in azoospermic patients. No microdeletion in AZF region was detected in fertile controls. In conclusion, chromosomal abnormality and AZF region microdeletion of Y chromosome might account for about 25% of Chinese infertile patients with azoospermia or severe oligozoospermia, suggesting the two abnormalities are important genetic etiology of spermatogenic failure in Chinese population and it is essential to screen them during diagnosis of male infertility before in vitro assisted fertilization by introcytoplasmic sperm injection.

Adult↗
PubMed2001

Direct detection of mecA, nuc and 16S rRNA genes in BacT/Alert blood culture bottles.

A benzyl alcohol-guanidine hydrochloride extraction method was used to remove sodium polyanetholesulfonate present in BacT/Alert blood culture bottles. Multiplex PCR using touchdown annealing was used to detect the mecA, nuc, and 16S rRNA genes in bottles growing staphylococci. This direct PCR assay demonstrated excellent sensitivity, specificity and improved accuracy compared to routine phenotypic methods for determination of methicillin resistance in coagulase negative staphylococci (CoNS). However, with this PCR assay, bottles that contained both methicillin-resistant CoNS and methicillin-susceptible Staphylococcus aureus would be misidentified as containing methicillin-resistant S. aureus.

Bacterial Proteins↗
PubMed2002

Training costs and investment payback of implementing molecular diagnostics for identification of vancomycin resistant enterococci in a clinical microbiology laboratory.

Molecular diagnostics may be a more efficient method to manage resources; but most Microbiology laboratories have not introduced them into routine use due to the specialized training required. Using vancomycin resistant enterococci (VRE) screening during a comparison of a multiplex PCR (MPCR) and conventional biochemical testing (CBT) we studied 3 objectives: 1) to develop a molecular diagnostics in-house training program, 2) to assess the training program outcomes for competency and confidence, and 3) to determine laboratory payback. A training program for 14 technologists using multiple adult learning methods was implemented. Methods to minimize technical errors were introduced and included: use of a calibrated loop to deliver sample; prealiquotting reagents; increasing volume of specimen; addition of gel loading dye directly into reaction tubes; and establishment of an equivocal zone. In our laboratory MPCR costs $7.06 less than CBT, therefore the payback period for training and implementation would be approximately 3 years.

Cost-Benefit Analysis↗
PubMed2002

Oxacillin susceptibility testing of coagulase-negative staphylococci using the disk diffusion method and the Vitek GPS-105 card.

One hundred and ninety-three isolates of coagulase-negative staphylococci (CoNS) were tested against oxacillin by agar dilution, disk diffusion, and Vitek (GPS-105 card), and the presence of the mecA gene determined by multiplex PCR. The results obtained by all testing methods were in agreement for 190 isolates. Two mecA-negative isolates (S. lugdunensis and S. haemolyticus) had MICs of < or = 0.25 microg/ml by agar dilution and Vitek but were resistant by disk diffusion. One mecA-positive isolate was resistant by Vitek and disk diffusion but had an agar dilution MIC of < or = 0.25 microg/ml. For the species of CoNS tested, oxacillin susceptibility results obtained with the Vitek GPS-105 card and disk diffusion correlated well with results obtained by National Committee for Clinical Laboratory Standards agar dilution and with the presence of the mecA gene.

Bacterial Proteins↗
PubMed2003

Shiga toxin- and enterotoxin-producing Escherichia coli isolated from subjects with bloody and nonbloody diarrhea in Bangkok, Thailand.

A total of 314 stool samples collected from 92 subjects with bloody diarrhea, 119 subjects with non-bloody diarrhea and 103 normal subjects in Bangkok, Thailand, were investigated for the presence of Shiga toxin-producing Escherichia coli (STEC) and enterotoxin-producing E. coli (ETEC) by multiplex PCR assay. Virulence genes and cytotoxic effect to Vero cells of STEC were also determined. STEC (5 isolates) and ETEC (18 isolates) were detected in 3 and 14 subjects, respectively. Among subjects containing ETEC, only one person belonged to normal control group. The detected STEC included two isolates (serotypes O26:H(-) and O111:H(-)) of Shiga toxin type 1 (Stx1-only) STEC from a child with non-bloody diarrhea, two isolates (Stx1-Stx2 STEC and Stx1-only STEC) from an adult with bloody diarrhea, and one isolate of Stx1-Stx2v STEC (O157:H7) from normal child. Only Stx1-Stx2 STEC isolate was found to exhibit toxicity to Vero cells and carry hlyA gene. The intimin encoding gene locus eaeA was not detected in any isolate. These results indicate that most of STEC isolates in Thailand were low virulent.

Adult↗

Refine your search to explore more results.