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Results for “Multiplex PCR”

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At least 991 records · Page 55Linked to original sources

PubMed2002

Rapid and reliable method for cytochrome P450 2D6 genotyping.

BACKGROUND: Single-nucleotide polymorphisms and single-base deletions within the cytochrome P450 2D6 (CYP2D6) gene have been associated with a poor metabolizer (PM) phenotype and display a frequency of 7-10% in the Caucasian population. METHODS: We developed a reliable and rapid procedure to identify five major PM-associated mutations (CYP2D6*4, *7, and *8) and deletions (CYP2D6*3 and *6) by real-time PCR with subsequent fluorometric melting point analysis of the PCR product. These polymorphisms within the CYP2D6 gene were detected by use of two primer pairs and five different pairs of hybridization probes. DNA extracted from whole blood of 323 individuals was analyzed, and results were compared with genotypes obtained by allele-specific multiplex PCR. In case of uncertain results, additional sequence analysis was performed. RESULTS: Genotyping results by real-time PCR were 100% reliable, whereas conventional allele-specific multiplex PCR produced uncertain results for 12.1% of samples, as confirmed by sequence analysis. Costs for reagents and consumables were considerably higher for the real-time PCR technology, but labor time was reduced by 2 h compared with allele-specific PCR. The allele frequencies in the population investigated were 0.186 for allele *4, 0.026 for allele *5, 0.009 for allele *3, 0.031 for allele *6, and 0.002 for allele *8. The defective CYP2D6*7 allele was not found. In addition, three additional mutations were detected, one of them displaying a PM genotype. CONCLUSION: Genotyping of CYP2D6 by real-time PCR with fluorometric melting point analysis is a rapid and reliable method.

Cytochrome P-450 CYP2D6↗

PubMed1996

Multiplex sets for the amplification of polymorphic short tandem repeat loci--silver stain and fluorescence detection.

Multiplex PCR amplification systems were developed using well-characterized, polymorphic short tandem repeat (STR) loci. Eight loci utilized in the multiplex amplifications included HUMCSF1PO, HUMTPOX, HUMTH01, HUMVWFA31, HUMF13A01, HUMFESFPS, HUMBFXIII and HUMLIPOL. From this list, three or four non-overlapping loci were simultaneously amplified, separated by denaturing polyacrylamide gel electrophoresis and visualized using silver stain or fluorescence detection. The multiplex PCR amplification systems offer a non-isotopic method for rapid, simple and accurate analysis of STR loci. This high-throughput method for DNA identification has immediate and valuable application in forensic analysis, paternity determination, tissue culture strain identification and bone marrow transplantation studies.

DNA↗

PubMed1997

Nature of mutation in the human hprt gene following in vivo exposure to ionizing radiation of cesium-137.

The current study comprises the analysis of mutations in 10 individuals accidentally exposed to cesium-137 during the 1987 radiological accident in Goiânia, Brazil. Their exposures were among the highest experienced, ranging from 1 to 7 Gy. Peripheral T-lymphocyte samples were obtained 3.3 years after the original exposure and mutation was studied at the hprt locus using the 6-thioguanine-resistance selection assay. The mutational spectrum for the exposed population is comprised of 90 independent mutants. Based on T-cell receptor analysis, only 5% (5/95) were clonally related. Mutants were initially studied using RT-PCR and directly sequenced using an automated laser fluorescent DNA sequencer. Mutants that repeatedly failed to produce cDNAs were studied using a multiplex PCR assay with genomic DNA. Missense mutations were the most frequent event recovered, comprising 40% (23/57) of the spectral sample. An excess of events involving A:T base pairs was observed, exhibiting a significant difference (chi 2 = 12.7, P = 0.0004) when compared to the spontaneous spectrum. This finding may reflect the effect of ionizing radiation-induced damage, suggesting a potential similarity to radiation effects in prokaryotes. At the genomic level, 36.7% (33/90) of the mutants exhibited gross structural alterations, as detected by multiplex PCR. Deletion events were over-represented in our spectral sample, displaying a twofold increase when compared to the frequency observed in the spontaneous mutation database.

Adolescent↗

PubMedDate not supplied

Evaluation of DAZ microdeletions in 34 infertile men.

Microdeletions in Yq11 are a common molecular cause of spermatogenic failure in men and are recurrently detected in about 10-15% of idiopathic azoospermia and severe oligozoospermia. Screening for AZF microdeletions is often performed by multiplex PCR. AZFc deletions, involving the DAZ gene, form the majority of these deletions. The aim of this study was to evaluate in a group of 34 Tunisian infertile patients (16 oligozoospermic and 18 azoospermic men) the prevalence of DAZ microdeletions using a rapid molecular strategy: the PCR-DGGE method based on the high degree of homology between the DAZ gene and its autosomally equivalent DAZLA gene. DAZ microdeletions were detected in 8.8% of patients. The three deleted patients have a 46, XY karyotype. Two of them were azoospermic and the other had an extreme oligo-asthenoteratozoospermia with a predominant abnormality: small round head spermatozoa (Y46). Our findings suggest that PCR-DGGE method, for detection of DAZ gene deletion, could be particularly useful as a first step in the diagnosis workup of nonobstructive azoospermia and severe oligozoospermia for three reasons. First, it is a simple and fast system; second, DAZ microdeletions are the most common Y deletions; and third, partial DAZ microdeletions and mosaicism may be recognized by PCR-DGGE while only deletions removing the whole DAZ gene cluster can be detected by STS-PCR [211]. Nevertheless, this procedure has limitations because other deletions of AZFa and AZFb may go undetected. Therefore, molecular investigation by multiplex PCR must be conducted in a second step according to European guidelines for the molecular diagnosis of Y chromosome microdeletions, particularly before ICSI procedures.

Chromosome Deletion↗

PubMed2005

Low density DNA microarray for detection of most frequent TP53 missense point mutations.

BACKGROUND: We have developed an oligonucleotide microarray (genosensor) utilizing a double tandem hybridization technique to search for 9 point mutations located in the most frequently altered codons of the TP53 gene. Isolated and multiplexed PCR products, 108 and 92 bp long, from exons 7 and 8, respectively, were obtained from 24 different samples. Single-stranded target DNA was then prepared from isolated or multiplexed PCR products, through cyclic DNA synthesis. Independent ssDNA's were annealed with the corresponding pairs of labeled stacking oligonucleotides to create partially duplex DNA having a 7-nt gap, which contains the sequence that will be interrogated by the capture probes forming double tandem hybridization. In the case of multiplexed ssPCR products, only two stacking oligonucleotides were added per target, therefore the gap for the PCR products having two consecutive codons to be interrogated in exon 7 was 12 nt long, so only single tandem hybridization was produced with these respective probes. RESULTS: 18 codon substitutions were found by DNA sequencing. In 13 of them a perfect correlation with the pattern of hybridization was seen (In 5 no signal was seen with the wt probe while a new signal was seen with the appropriate mutant probe, and in 8 more, as expected, no signal was seen with any probe due to the absence of the corresponding probe in the array). In 3 other cases a mutation was falsely suggested by the combination of the absence of the wild type signal along with a false signal in the other probe. In the other 2 cases the presence of the mutation was not detected due to the production of a false hybridization signal with the wild type probe. In both cases (false mutation or no mutation detected) relatively stable mismatched target/probe duplexes should be formed. These problems could be avoided by the addition of probes to improve the performance of the array. CONCLUSION: Our results demonstrate that a simple TP53 microarray employing short (7-mer) probes, used in combination with single or double tandem hybridization approach and a simple or multiplex target preparation method, can identify common TP53 missense mutations from a variety of DNA sources with good specificity.

Base Sequence↗

PubMed2001

A PCR assay for detecting clonal rearrangement of the TCR-gamma gene.

BACKGROUND: The T-cell receptor gamma chain (TCR-gamma) gene has 11 functional variable (V) exons that can be organized into four subfamilies and four functional joining (J) exons that can be divided into two subfamilies. METHOD AND RESULTS: Three multiplex PCR reactions amplifying the TCR-gamma gene were developed. Primer combinations for multiplex PCR were chosen so that the V-region subfamily and J-region subfamily involved in a clonal band could be identified. Control primers from the protease inhibitor (PI) gene were also included in each reaction to verify the presence of amplifiable DNA. Fifty-six archived samples that had been tested by Southern blot for clonal rearrangement of the TCR-beta gene were analyzed with the TCR-gamma PCR protocol. Twenty-one of 56 samples were TCR-beta positive by Southern blot and thus expected to be positive with TCR-gamma PCR. Thirty-five of 56 samples were TCR-beta negative by Southern blot. Of these, 14 samples showed clonal rearrangement of the immunoglobulin heavy chain gene. The TCR-gamma PCR protocol showed a diagnostic sensitivity for detecting T-lineage clonality of 90%, with a diagnostic specificity for detecting T-cell lineage of only 74%. CONCLUSION: The PCR protocol described here performed well in comparison with a TCR-beta Southern protocol.

Blotting, Southern↗

PubMed2006

[Identification of adenovirus associated with conjunctivitis by molecular methodology].

OBJECTIVE: To identify adenovirus in patients with conjunctivitis by molecular methods such as the Polymerase Chain Reaction (PCR) and DNA sequencing. METHODS: Samples of scrapings from the inferior fornix of 51 patients (39 diagnosed with Follicular Conjunctivitis and 12 diagnosed with Vernal Conjunctivitis) were processed by generic PCR to identify adenovirus. All the samples that were PCR positive were cultured on VERO cells for virus isolation, with this being demonstrated by immunofluorescence. For the identification of the isolated serotype, the multiplex PCR was utilized and DNA automated sequencing was employed to identify the genetic variants. RESULTS: Twenty-eight of the individuals diagnosed with Follicular Conjunctivitis and six of those diagnosed with Vernal Conjunctivitis, had positive results to adenovirus (67%). The cultures in VERO cells allowed the isolation of eight samples. Only three of the isolated viruses (one Ad1 and two Ad2) were identified by the multiplex PCR used to identify the subgenus C adenovirus. An Ad1 genetic variant was identified by automated sequencing while the Ad2 serotypes were identical to the ones reported by Genbank. CONCLUSIONS: The Polymerase Chain Reaction and DNA sequencing are useful tools to identify and characterize microorganisms responsible for diseases such as conjunctivitis caused by adenoviruses.

Adenoviridae↗

PubMed2001

A powerful and rapid approach to human genome scanning using small quantities of genomic DNA.

Dense maps of short-tandem-repeat polymorphisms (STRPs) have allowed genome-wide searches for genes involved in a great variety of diseases with genetic influences, including common complex diseases. Generally for this purpose, marker sets with a 10 cM spacing are genotyped in hundreds of individuals. We have performed power simulations to estimate the maximum possible intermarker distance that still allows for sufficient power. In this paper we further report on modifications of previously published protocols, resulting in a powerful screening set containing 229 STRPs with an average spacing of 18.3 cM. A complete genome scan using our protocol requires only 80 multiplex PCR reactions which are all carried out using one set of conditions and which do not contain overlapping marker allele sizes. The multiplex PCR reactions are grouped by sets of chromosomes, which enables on-line statistical analysis of a set of chromosomes, as sets of chromosomes are being genotyped. A genome scan following this modified protocol can be performed using a maximum amount of 2.5 micrograms of genomic DNA per individual, isolated from either blood or from mouth swabs.

Alleles↗

PubMed2001

Assessment of male CVID patients for mutations in the Btk gene: how many have been misdiagnosed?

The presentation of hypogammaglobulinaemia in young males without a family history of immunodeficiency can pose a diagnostic problem. In the past, the presence of B-cells has suggested a diagnosis of common variable immunodeficiency (CVID), although genotypic analysis has now clarified that individuals with B cells may have mutations in their Btk gene. In order to address the issue of how many male individuals with a clinical diagnosis of CVID do in fact have mutations in the Btk gene, we analysed a group of 24 male patients. Single-strand conformation polymorphism (SSCP) analysis was used to screen the patient cohort for mutations in the Btk gene. Given the size of the Btk gene, the number of patients in the cohort and the amount of available DNA, multiplex PCR reactions were utilized to span the 19 exons and promoter region of the gene. Where abnormal migration patterns were observed with multiplex PCR reactions, in nine of the 24 patients, the individual Btk gene fragments were re-amplified and analysed again by SSCP. Following this analysis, four patients continued to demonstrate abnormal SSCP migration patterns. However, direct sequencing of the relevant Btk gene fragments for these four CVID patients revealed a mutation in only one patient. The mutation was the previously described polymorphism at position 2031 of Btk gene within exon 18. These results indicate that caution should be taken with the application of SSCP analysis to mutation detection. While it has a role to play in screening large patient cohorts, direct sequencing is a necessary adjunct to such analysis. Finally, the clinical diagnosis of CVID in this cohort successfully excluded males with Btk mutations.

Adult↗

PubMed2003

Genetic damage induced by in vitro irradiation of human G0 lymphocytes with low-energy protons (28 keV/microm): HPRT mutations and chromosome aberrations.

Cell survival, mutations and chromosomal effects were studied in primary human lymphocytes exposed in G0 phase to a proton beam with an incident energy of 0.88 MeV (incident LET of 28 keV/microm) in the dose range 0.125-2 Gy. The curves for survival and mutations at the hypoxanthine-guanine phosphoribosyl transferase locus were obtained by fitting the experimental data to linear and linear-quadratic equations, respectively. In the dose interval 0-1.5 Gy, the alpha parameters of the curves were 0.42/Gy and 3.6 x 10(-6) mutants/Gy, respectively. The mutation types at the HPRT locus were analyzed by multiplex-PCR in 94 irradiated and 41 nonirradiated clones derived from T lymphocytes from five healthy donors. All clones showed a normal multiplex-PCR pattern and were classified as point mutations. Chromosome aberration data were fitted as a linear function of dose (alpha = 0.62 aberrations per cell Gy(-1)). By irradiating G0 lymphocytes from a single subject with 28 keV/microm protons and gamma rays, an RBE of 6.07 was obtained for chromosome aberrations. An overinvolvement of chromosome 9 relative to chromosome 7 was found in chromosome breaks after chromosome painting analysis.

Cell Survival↗

PubMed2006

Molecular analysis of the subgingival microbiota in health and disease.

This investigation provides molecular analyses of the periodontal microbiota in health and disease. Subgingival samples from 47 volunteers with healthy gingivae or clinically diagnosed chronic periodontitis were characterized by PCR-denaturing gradient gel electrophoresis (DGGE) with primers specific for the V2-V3 region of the eubacterial 16S rRNA gene. A hierarchical dendrogram was constructed from band patterns. All unique PCR amplicons (DGGE bands) were sequenced for identity. Samples were also analyzed for the presence of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and Tannerella forsythensis by multiplex PCR. Associations of patient age, gender, and smoking status together with the presence of each unique band and putative periodontal pathogens with disease were assessed by logistic regression. Periodontal pockets were colonized by complex eubacterial communities (10 to 40 distinct DGGE bands) with substantial individual variation in the community profile. Species diversity in health and disease was determined by the Shannon-Weaver index of diversity and compared by the Mann-Whitney U test. Sequence analyses of DGGE amplicons indicated the occurrence of many nontypical oral species and eubacteria previously associated with this environment. With the exception of T. forsythensis, the putative pathogens were not detected by DGGE. Multiplex PCR, however, detected T. forsythensis, A. actinomycetemcomitans, and P. gingivalis in 9% 16%, and 29% of the patients with disease, respectively. The presence of A. actinomycetemcomitans was significantly associated with disease (P < 0.01). Statistical analyses indicated that the presence of Treponema socranskii and Pseudomonas sp. was a significant predictor of disease (P < 0.05) and that there was no significant difference (P > 0.05) in terms of eubacterial species diversity between health and disease.

Actinobacillus Infections↗

PubMed2000

Evaluation of two PCR-based procedures for typing Clostridium perfringens.

Two polymerase chain reaction (PCR)-based procedures for typing Clostridium, perfringens, which affects most domestic animals, were compared and evaluated for efficiency as substitute to the guinea-pig intradermal test routinely used in our laboratory, namely a multiplex PCR and a protocol based on the individual amplification of gene sequences specific for each toxin. Reference isolates of C. perfringens types A, B, C and D as well as cultures from clinical specimens were tested. The sensitivity and specificity of the PCR was confirmed on reference isolates. There was similarity in results on 43 of the 46 samples typed by all 3 methods. Clear results were obtained by PCR on 5 clinical samples that showed either equivocal or weak skin reactions in guinea-pigs. The multiplex PCR protocol, in combination with the evaluation of bacterial growth, is a better alternative to in vivo toxin typing, since C. perfringens can only be incriminated as cause of a disease when it is present in large numbers in the intestine.

Animals↗

PubMed2003

Molecular detection of anthrax spores on animal fibres.

AIMS: To develop a rapid, specific and sensitive diagnostic test for the detection of the spores of Bacillus anthracis on commercial samples of animal fibres (e.g. wool and cashmere). METHODS AND RESULTS: Extraction of DNA from spores using a mechanical disruption method based on bead beating was evaluated but subsequently abandoned as it compromised the sensitivity of the overall protocol. A multiplex PCR and two nested amplification reactions designed for B. anthracis were developed during this study. CONCLUSIONS: A simple selective incubation step in combination with multiplex PCR was found to be more effective than generic DNA extraction coupled to a sensitive nested amplification reaction. SIGNIFICANCE AND IMPACT OF THE STUDY: The rapid diagnostic test could be applied to the analysis of commercial fibre samples for the detection of anthrax as required by health and safety legislation resulting in considerable savings in time and expense.

Animals↗

PubMed2003

[Establishment and preliminary application of screening methods for Y chromosome microdeletions in male infertility patients].

OBJECTIVE: To develop a multiplex PCR protocol, which could be suitable for routine screening of microdeletions on the Y chromosome in azoospermic and oligozoospermic male infertility patients. METHODS: Five multiplex sets were established. Eighty-seven azoospermic and oligozoospermic patients undergoing intracytoplasmic sperm injection (ICSI) in the in vitro fertilization (IVF) center and 30 azoospermic men undergoing testicular biopsy in the clinic of Urology Surgery were screened for microdeletions of Y chromosome. RESULTS: A total of 19 (16.2%) cases of microdeletions were found in 117 azoospermic and oligozoospermic patients by screening of Y chromosome microdeletions. Of these, 11 cases (18.0%) were found in 61 oligozoospermic patients, and 8 cases (14.3%) were found in 56 azoospermic patients. CONCLUSION: The multiplex PCR protocol presented in this study is an easy-to-do and reliable method for detecting microdeletions on the Y chromosome. Routine screening of microdeletions on the Y chromosome for azoospermic and oligozoospermic patients is essential.

Chromosome Deletion↗

PubMed2004

Simultaneous detection of enteric viruses by multiplex real-time RT-PCR.

A multiplex real-time RT-PCR protocol for the simultaneous detection of noroviruses ("Norwalk-like viruses") of genogroups I and II, human astroviruses and enteroviruses is described. The protocol was developed and evaluated using the LightCycler and corresponding SYBR Green reagents. New primers were designed within conserved genome regions to optimize the detection range of virus subtypes of each genus. To enable the development of a multiplex PCR assay within one tube (capillary), similar mastermix- and cycling-conditions were respected for each individual primer system. Subsequent melting curve analysis allowed the determination of possible dual-contaminations of entero- and noro- or astroviruses by the formation of dual peaks. Special care was taken to minimize the loss of sensitivity, since the detection of small viral contaminations is a crucial parameter especially for food analysis. The multiplex assay was compared successfully to the single SYBR Green assay, and revealed to be at least 10 times more sensitive than the one obtained with an endpoint PCR thermocycler protocol published previously.

Benzothiazoles↗

PubMed1996

Diagnosis and carrier detection in a Duchenne muscular dystrophy family by multiplex polymerase chain reaction and microsatellite analysis.

We used multiplex PCR and a microsatellite or STR analysis for diagnosis and carrier detection in a DMD family. Two affected patients both demonstrated deletion of exon 51 by multiplex PCR. The microsatellite or STR analysis showed that the mother and all sisters except the eldest sister of the patients carried the disease allelle. Therefore, all of them except the eldest sister were carriers. We present the need to introduce the molecular techniques to improve a mode of diagnosis and management of DMD patients in the Thai community.

Adolescent↗

PubMed2003

Use of O-antigen gene cluster-specific PCRs for the identification and O-genotyping of Yersinia pseudotuberculosis and Yersinia pestis.

Yersinia pestis is a very recently evolved clone of Yersinia pseudotuberculosis serotype O:1b. This close relationship causes potential difficulties in DNA-based diagnostic methods. Analysis of the O-antigen gene clusters in these two organisms identified two regions that were used to specifically identify Y. pestis-Y. pseudotuberculosis as a group or Y. pestis alone. Both PCR assays were found to be 100% specific when tested on a large collection of Yersinia species and other Enterobacteriaceae. Furthermore, advantage was taken of the different setups of the O-antigen gene clusters of the 21 known Y. pseudotuberculosis serotypes to develop a multiplex PCR assay to replace the conventional serotyping method of Y. pseudotuberculosis by O-genotyping. The multiplex PCR assay contained nine sets of specific PCRs in a single tube and when used on Y. pseudotuberculosis reference strains allowed the distinction of 14 individual serotypes and two duplex serotypes (O:4a-O:8 and O:12-O:13). Serotype O:7, O:9, and O:10 strains required additional PCRs for O-genotyping. Once applied to Y. pseudotuberculosis strains of various origins, a very good correlation between classical serotypes and O-genotypes was observed, although some discrepancies were found. O-genotyping also proved useful to correct misidentification of some strains and to type Y. pseudotuberculosis isolates that had lost the expression of the O-antigen. The PCR-based O-genotyping can easily be applied in conventional laboratories, without the need for tedious preparation of a large set of specific antisera.

Animals↗

PubMed2001

Identification of equine herpesviruses 1 and 4 by polymerase chain reaction.

OBJECTIVE: To develop and validate specific, sensitive and rapid (< 8 hour) diagnostic tests using polymerase chain reaction (PCR) for the diagnosis of abortion and respiratory disease caused by equine herpesvirus 1 (EHV1; equine abortion virus) and EHV4 (equine rhinopneumonitis virus). DESIGN: Primer sets based on nucleotide sequences encoding glycoprotein H (gH) of EHV1 and gB of EHV4 were designed and used in single round and second round (seminested) PCRs, and in a multiplex PCR for the diagnosis of EHV1 and EHV4 infections. METHODS: Oligonucleotide primers were designed for each virus, PCR conditions were defined and the specificity and sensitivity of the assays were determined. The tests were applied to tissue samples from aborted equine foetuses and to nasopharyngeal swabs from horses with acute febrile respiratory disease. RESULTS: Individual single round and a second round (seminested) EHV1 and EHV4 PCRs were specific in that EHV1 primers amplified all (n = 30) EHV1 isolates and did not amplify EHV4. Similarly EHV4 primers amplified all (n = 6) EHV4 isolates and did not amplify EHV1. Both PCRs were sensitive in that the first round EHV1 PCR detected 1220 molecules of EHV1 plasmid DNA and the first round EHV4 PCR detected 7280 molecules of EHV4 plasmid DNA. The EHV1 second round PCR was 100 times more sensitive in that it detected 12 molecules of EHV1 DNA and the EHV4 second round PCR was 1000 times more sensitive in that it detected 8 molecules of EHV4 DNA. There was a high correlation between detection of EHV1 by virus isolation and PCR when tissue samples from 71 aborted foetuses were examined; all samples positive by virus isolation were positive by PCR. Similarly the EHV4 PCR was at least as sensitive as virus isolation when applied to nasaopharyngeal swabs from horses with respiratory disease in that all samples positive by virus isolation were also positive by PCR. CONCLUSION: Individual single round and second round (seminested) PCRs and a seminested multiplex PCR were developed that enabled reliable, rapid detection of EHV1 and EHV4 in aborted foetal tissues and nasopharyngeal swab samples.

Abortion, Veterinary↗