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 973 records · Page 54Linked to original sources

PubMed2006

[Yersinia pestis pathogenesis and diagnostics].

Plague is an acute bacterial infection caused by Gram negative organism Yersinia pestis. This bacteria is subdivided into three classical biotypes: Orientalis, Medievalis and Antiqua. Plague is transmitted via flea vectors from rodents to humans and by respiratory droplets from animals to humans or humans to humans. This agent is on the top of the CDC list of "Critical Biological Agents"--category A. It appears to be a good candidate agent for a bioterrorist attack. Type III secretion (TTS) is a mechanism by which Y. pestis communicates with eukaryotic cells by injecting bacterial proteins across cellular membranes into the cytosol of these cells. These bacterial proteins take control of the host cells by hijacking their intracellular machinery. A laboratory diagnostics of plague is based on: staining techniques, culture on media, immunochromatography, hemagglutination, immunofluorescence, ELISA, phage tests and genetical techniques including: PCR, multiplex and nested PCR, real time PCR, VNTR, PFGE, ISBF and Microarray.

Animals↗

PubMedDate not supplied

Molecular diagnostic testing for determination of myeloid lineage in acute leukemias.

Determination of myeloid vs. lymphoid cell lineage in acute leukemias is essential for diagnosis, prognosis, and treatment. However, some leukemic cells are too primitive to be identified based on conventional morphology, cytochemistry, and immunophenotype criteria. The gene for myeloperoxidase (MPO) is central to the lineage designation of myeloid cells and their function. Expression of MPO messenger ribonucleic acid (mRNA) may serve to determine the myeloid lineage of primitive leukemic cells that do not express the final product, MPO. Recently, a procedure has been developed for detection of MPO mRNA in leukemic cells, based on message amplification by reverse transcription-polymerase chain reaction (RT-PCR). Three variant polymerase chain reaction (PCR) methods, developed in view of future diagnostic applications of the RT-PCR procedure are reported: (A) nested PCR, using a second pair of internal primers for reamplification of the first PCR product; (B) multiplex PCR for concomitant amplification of up to seven sequences encompassing all regions of MPO mRNA; (C) adaptation of the standard PCR procedure and the two variant methods for use of cellular material from air-dried unstained or Wright stained smears. These variant procedures developed on the HL-60 cell line make the RT-PCR detection of MPO mRNA easily applicable as a new diagnostic test in acute leukemias and confer the versatility needed in the clinical setting. The advantages of our new procedure for MPO mRNA detection are: high sensitivity and specificity, speed, simplicity, and safety. Myeloid lineage of leukemic blasts can be established, confirmed, or ruled out in 6 to 10 hours, making results available at the time of bone marrow sign-out.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗

PubMedDate not supplied

[Molecular biology at the service of the daily medical virology. 1. Methodological principles].

Molecular biology techniques which can rapidly detect a few copy number of viral genome allow to obtain new parameters for the diagnosis, the prognosis, and the monitoring of viral diseases in addition to or in substitution for cell culture and antigen and antibody detection. The methods are based on the virus structural organization and on their replication into cells. They use the tools which have been developed to analyse nucleic acids. Molecular hybridization (dot-blot, southern-blot, liquid phase hybridization, in situ hybridization), enzymatic amplification (PCR, nested-PCR, RT-PCR, NASBA, bDNA, multiplex PCR), DNA and RNA quantification (quantitative PCR or RT-PCR, NASBA, bDNA) and genotyping (virus types, mutations, variant virus) are used. Nucleic acid extracts native or amplified with a reporter molecule (dUTP-biotin or dUTP-digoxigenin) are identified using gel electrophoresis followed by hybridization or using microwell capture hybridization assay and colorimetric immunoenzymatic or bioluminescence assays.

Genetic Engineering↗

PubMed1998

Comprehensive mutational scanning of the p53 coding region by two-dimensional gene scanning.

A comprehensive mutational scanning test for the p53 coding region based on multiplex PCR and two-dimensional DNA electrophoresis was designed and evaluated. In a 2-step multiplex PCR, the p53 coding region (exons 2-11) was amplified as a single 8646-bp fragment by long-distance PCR in step one. This fragment served as a template for the subsequent co-amplification of the individual exons in two multiplex groups in step two. The multiplex products were then separated, first on the basis of size in non-denaturant polyacrylamide gels and then on the basis of sequence by denaturing gradient gel electrophoresis (DGGE). Primers for optimal PCR, melting behavior and 2-D gel distribution were designed using a recently developed computer program. The resulting two-dimensional gene scanning (TDGS) test was evaluated by screening, in a blinded fashion, 29 coded DNA samples from Li-Fraumeni syndrome patients with previously identified germline mutations. All mutations were correctly detected. This assay provides an accurate, cost-effective and non-radioactive method for simultaneous mutational scanning of all p53 coding exons.

Base Sequence↗

PubMed2004

Species-specific PCR method for identification of Streptococcus downei.

AIMS: To establish a rapid method to differentiate Streptococcus downei and S. sobrinus by multiplex PCR. METHODS AND RESULTS: A PCR primer pair specific to S. downei was designed on the basis of the nucleotide sequence of the dextranase gene of S. downei NCTC 11391T. The primer pair specifically detected S. downei, but none of the other mutans streptococci (16 strains of six species). The PCR procedure was capable of detecting 1 pg of genomic DNA purified from S. downei NCTC 11391 and as few as 14 CFU of S. downei cells. The mixture of primer pairs specific to each S. downei (this study) and S. sobrinus (Igarashi et al. 2000) detected only the strains of these two species among all the mutans streptococcal strains, and concomitantly differentiated the two species by species-specific amplicons of different lengths. CONCLUSIONS: The present PCR method is highly specific to S. downei and is useful for detection and identification of S. downei. SIGNIFICANCE AND IMPACT OF THE STUDY: Multiplex PCR using dextranase gene primers is a useful method for simultaneous detection and differentiation of S. downei and S. sobrinus.

DNA, Bacterial↗

PubMed1991

Mutations in p53 as potential molecular markers for human breast cancer.

Based on the high incidence of loss of heterozygosity for loci on chromosome 17p in the vicinity of the p53 locus in human breast tumors, we investigated the frequency and effects of mutations in the p53 tumor suppressor gene in mammary neoplasia. We examined the p53 gene in 20 breast cancer cell lines and 59 primary breast tumors. Northern blot analysis, immunoprecipitation, and nucleotide sequencing analysis revealed aberrant mRNA expression, over-expression of protein, and point mutations in the p53 gene in 50% of the cell lines tested. A multiplex PCR assay was developed to search for deletions in the p53 genomic locus. Multiplex PCR of genomic DNA showed that up to 36% of primary tumors contained aberrations in the p53 locus. Mutations in exons 5-9 of the p53 gene were found in 10 out of 59 (17%) of the primary tumors studies by single-stranded conformation polymorphism analysis. We conclude that, compared to amplification of HER2/NEU, MYC, or INT2 oncogene loci, p53 gene mutations and deletions are the most frequently observed genetic change in breast cancer related to a single gene. Correlated to disease status, p53 gene mutations could prove to be a valuable marker for diagnosis and/or prognosis of breast neoplasia.

Base Sequence↗

PubMed2005

Description of a simple multiplex PCR-SSCP method for AB0 genotyping and its application to the peopling of the Canary Islands.

A simple and affordable multiplex polymerase chain reaction-single-strand conformation polymorphism method is proposed for the molecular study of AB0 polymorphisms. Application of this method to the peopling of the Canary Islands, analyzing a total of 2,200 chromosomes, detected that in addition to Berbers and Basques, the rare alleles 0210 and O303 are also present in the Iberian Peninsula and in the Canary Islands. Allele B101, with the highest frequency in Northwest (NW) Africa, shows a negative correlation (R = -0.822, p = 0.023) between geographic distances from this continent and insular frequencies, congruent with a main aborigine colonization from East to West still detectable today. Similar to previous autosomal studies, admixture estimations point to a major Iberian contribution (82 +/- 0.5%) to the Canary Islands, although, in some islands as La Gomera, the NW African component raised to 62 +/- 4.3%.

ABO Blood-Group System↗

PubMed2003

Characterization of incompletely typed rotavirus strains from Guinea-Bissau: identification of G8 and G9 types and a high frequency of mixed infections.

Among 167 rotavirus specimens collected from young children in a suburban area of Bissau, Guinea-Bissau, from 1996 to 1998, most identifiable strains belonged to the uncommon P[6], G2 type and approximately 50% remained incompletely typed. In the present study, 76 such strains were further characterized. Due to interprimer interaction during the standard multiplex PCR approach, modifications of this procedure were implemented. The modified analyses revealed a high frequency of G2, G8, and G9 genotypes, often combined with P[4] and/or P[6]. The Guinean G8 and G9 strains were 97 and 98%, respectively, identical to other African G8 and G9 strains. Multiple G and/or P types were identified at a high frequency (59%), including two previously undescribed mixed infections, P[4]P[6], G2G8 and P[4]P[6], G2G9. These mixed infections most likely represent naturally occurring reassortance of rotavirus strains. Detection of such strains among the previously incompletely typed strains indicates a potential underestimation of mixed infections, if only a standard multiplex PCR procedure is followed. Furthermore cross-priming of the G3 primer with the G8 primer binding site and silent mutations at the P[4] and P[6] primer binding sites were detected. These findings highlight the need for regular evaluation of the multiplex primer PCR method and typing primers. The high frequency of uncommon as well as reassortant rotavirus strains in countries where rotavirus is an important cause of child mortality underscores the need for extensive strain surveillance as a basis to develop appropriate rotavirus vaccine candidates.

Antigens, Viral↗

PubMed1996

Preparation of mycobacterial DNA from blood culture fluids by simple alkali wash and heat lysis method for PCR detection.

A sodium iodide-isopropanol (NI) method was compared with an alkali wash and heat lysis (AH) procedure for the preparation and extraction of DNA from BACTEC 13A blood culture fluid samples from AIDS patients for use in a PCR for the detection and identification of mycobacteria. The sensitivity and efficiency of the DNA extraction methods were assessed by a multiplex PCR which detected the members of the genus Mycobacterium and differentiated between M. intracellulare, M. tuberculosis, and M. avium isolates with a limit of detection of between 0.28 pg (67 cells) and 120 pg (28,571 cells) of standard mycobacterial DNA. The PCR amplified mycobacterial DNA prepared by the AH procedure from 40 acid-fast bacillus-positive blood cultures with growth index values of > 20 U but not from 48 blood cultures with growth index values of < 21 U. The AH method was about 10 times more sensitive than the NI method for extracting DNA from 13 acid-fast bacillus-positive BACTEC fluid samples for PCR analysis. The study shows that the AH procedure in combination with the multiplex PCR is a simple, specific, and sensitive method which can be used in the routine diagnostic laboratory to detect and identify different members of the genus Mycobacterium in blood culture fluid samples from AIDS patients.

Acquired Immunodeficiency Syndrome↗

PubMedDate not supplied

Screening for microdeletions in human Y chromosome--AZF candidate genes and male infertility.

About 30% of couple infertilities are of male origin, some of them caused by genetic abnormalities of the Y chromosome. Deletions in AZF region can cause severe spermatogenic defects ranging from non-obstructive azoospermia to oligospermia. The intracytoplasmatic sperm injection technique (ICSI) is rapidly becoming a versatile procedure for human assisted reproduction in case of male infertility. The use of ICSI allows Y chromosome defects to be passed from father. The goal of our study is to evaluate the frequency of microdeletions in the long arm of Y chromosome, within the AZF regions, in these cases of infertilities, using molecular genetics techniques. Thirty infertile men with azoospermia or oligozoospermia, determined by spermogram, were studied after exclusion of patients with endocrine or obstructive causes of infertility. Peripheral blood DNA was extracted from each patient, then amplified by multiplex PCR with STS genomic markers from the Y chromosome AZF zones. Each case was checked by multiplex PCR through coamplification with the SRY marker. Three men with microdeletions of the long arm of the Y chromosome were diagnosed among the 30 patients, corresponding to a proportion of 10%. The relatively high proportion of microdeletions found in our population suggest the need for strict patient selection to avoid unnecessary screening for long arm Y chromosome microdeletions. The molecular diagnostics was performed according to the current European Academy of Andrology laboratory guidelines for molecular diagnosis of Y chromosomal microdeletions.

Adult↗

PubMed2004

[Studying dystrophin gene deletion in the northeast of China and applicating].

To detect the distribution characteristics of dystrophin gene deletions of the DMD/BMD patients in the northeast of China and apply for prenatal gene diagnosis, we have analyzed the distribution of the breakpoints of the deleted-patients and the optimized primer-assembly after screening deletions of 120 DMD/BMD patients via multiplex PCR with 12-pair primers and male high-risk fetuses have been detected deletion by multiplex PCR. Results indicated the deletion frequency was 49.2%, about 66.4% deletion breakpoints positioned in introns 44-52, intron 50 was the highest breakpoint (14.8%). The optimized four-primer-assembly was the primers of exon 48, 51, 45 and 8, which could detect 41.7% deletions of all cases; 9 deletions male ones of 29 high-risk fetuses were detected, who had the same deletion-segments as their probands. For the first time screening deletions of DMD/BMD patients in the northeast of China, we have found distribution of the deletions mainly lied in two hot-spots, neighboring regions of exon 8 might be a real deletion 'hot spot' in this area compared with other areas of our country; introns 44-52 of dystrophin gene were highly unstable and prone to break: intron 44 was more stable than the whole molecular region of 'central deletion hot spot' and the unstability of intron 50 changed along with the regional and ethnic difference; the optimized primer-assembly provided the short-cut for detecting patients and making prenatal gene diagnosis, especially it's feasible and advantageous for the isolated pedigrees.

Adolescent↗

PubMed1991

DNA typing and genetic mapping with trimeric and tetrameric tandem repeats.

Tandemly reiterated sequences represent a rich source of highly polymorphic markers for genetic linkage, mapping, and personal identification. Human trimeric and tetrameric short tandem repeats (STRs) were studied for informativeness, frequency, distribution, and suitability for DNA typing and genetic mapping. The STRs were highly polymorphic and inherited stably. A STR-based multiplex PCR for personal identification is described. It features fluorescent detection of amplified products on sequencing gels, specific allele identification, simultaneous detection of independent loci, and internal size standards. Variation in allele frequencies were explored for four U.S. populations. The three STR loci (chromosomes 4, 11, and X) used in the fluorescent multiplex PCR have a combined average individualization potential of 1/500 individuals. STR loci appear common, being found every 300-500 kb on the X chromosome. The combined frequency of polymorphic trimeric and tetrameric STRs could be as high as 1 locus/20 kb. The markers should be useful for genetic mapping, as they are sequence based, and can be multiplexed with the PCR. A method enabling rapid localization of STRs and determination of their flanking DNA sequences was developed, thus simplifying the identification of polymorphic STR loci. The ease by which STRs may be identified, as well as their genetic and physical mapping utility, give them the properties of useful sequence tagged sites (STSs) for the human genome initiative.

Asian People↗

PubMed1999

Use of archival fine-needle aspirates for the allelotyping of tumors.

BACKGROUND: Loss of heterozygosity (LOH) analysis (allelotyping) based on polymorphic microsatellite DNA is one of the most powerful molecular tools currently available for studying carcinogenesis. However, allelotyping studies that require archival paraffin embedded tissues are often hampered by technical difficulties related to microdissection and poor DNA quality. METHODS: The authors compared allelotyping results from 12 paraffin embedded breast carcinoma cases with those from matching alcohol fixed fine-needle aspiration (FNA) cytology slides obtained for routine diagnostic purposes, using 30 polymorphic microsatellite markers at chromosomes 3p, 4p, 4q, 5q, 6p, 8p, 9p, 11q, 17p, and 17q. Cells from the alcohol fixed FNA slides were dissected and processed in three different ways, and DNA dilution experiments were performed to determine the minimum number of cells required for accurate allelotyping. RESULTS: LOH results were identical for paraffin embedded and alcohol fixed tumors for 97% of 114 polymerase chain reactions (PCR) when 1000-2000 cells were dissected from each FNA slide and DNA from 100 cells was used for each multiplex PCR. However, with lower cell numbers, the discordance rate increased and artifactual LOH was observed. Intratumor allelotype heterogeneity could not be documented. CONCLUSIONS: The use of alcohol fixed cytology preparations improves the ease of PCR-based allelotyping and greatly expands the range of archival materials available for study. The allelotyping is accurate and reproducible when DNA from >/=25 cells is used in the initial multiplex PCR. Cancer (Cancer Cytopathol)

Alleles↗

PubMed1998

High level multiplex genotyping by MALDI-TOF mass spectrometry.

A primer extension assay is used to perform highly multiplexed genotyping of single nucleotide polymorphisms (SNPs) present in genomic DNA amplified by a multiplex PCR. The assay uses matrix-assisted laser desorption ionization time-of-flight mass spectrometry to accurately measure the masses of short oligonucleotide primers extended by a single dideoxynucleotide. The multiplexed genotyping assays rely on the natural molecular weight differences of DNA bases. By careful analysis of primer composition complementary to the target, or by judicious addition of one or more noncomplementary 5' bases to the genotyping primers, mass spectra of interleaved genotyping products can be generated with no ambiguity in allele assignment. Using a model multiplex PCR system, we demonstrate the ability to perform 12-fold multiplex SNP analysis.

Base Sequence↗

PubMed2000

Short tandem repeat analysis in Japanese population.

Short tandem repeats (STRs), known as microsatellites, are one of the most informative genetic markers for characterizing biological materials. Because of the relatively small size of STR alleles (generally 100-350 nucleotides), amplification by polymerase chain reaction (PCR) is relatively easy, affording a high sensitivity of detection. In addition, STR loci can be amplified simultaneously in a multiplex PCR. Thus, substantial information can be obtained in a single analysis with the benefits of using less template DNA, reducing labor, and reducing the contamination. We investigated 14 STR loci in a Japanese population living in Sendai by three multiplex PCR kits, GenePrint PowerPlex 1.1 and 2.2. Fluorescent STR System (Promega, Madison, WI, USA) and AmpF/STR Profiler (Perkin-Elmer, Norwalk, CT, USA). Genomic DNA was extracted using sodium dodecyl sulfate (SDS) proteinase K or Chelex 100 treatment followed by the phenol/chloroform extraction. PCR was performed according to the manufacturer's protocols. Electrophoresis was carried out on an ABI 377 sequencer and the alleles were determined by GeneScan 2.0.2 software (Perkin-Elmer). In 14 STRs loci, statistical parameters indicated a relatively high rate, and no significant deviation from Hardy-Weinberg equilibrium was detected. We apply this STR system to paternity testing and forensic casework, e.g., personal identification in rape cases. This system is an effective tool in the forensic sciences to obtain information on individual identification.

Alleles↗

PubMed2004

Multiplex PCR-RFLP assay for detection of factor V Leiden and prothrombin G20210A.

Factor V Leiden and prothrombin G20210A are clinically relevant genetic risk factors for venous thrombosis. Molecular diagnostic testing for factor V Leiden and prothrombin G20210A is widespread, and laboratories use a variety of technical approaches. Here we introduce a multiplex polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) based on single (Mn/l) restriction endonuclease digestion. The assay was shown to simultaneously and accurately detect factor V Leiden and prothrombin G20210A mutations.

Activated Protein C Resistance↗

PubMedDate not supplied

Chromosomal assignment of microsatellite loci in cotton.

Microsatellite markers or simple sequence repeats (SSRs) represent a new class of genetic markers for cotton (Gossypium sp.). Sixty-five SSR primer pairs were used to amplify 71 marker loci and genotype 13 monosomic and 27 monotelodisomic cotton cytogenetic stocks. Forty-two SSR loci were assigned to cotton chromosomes or chromosome arms. Thirty SSRs were not located to specific chromosomes in this study. Nineteen marker loci were shown to occur on the A subgenome and 11 on the D subgenome by screening accessions of G. herbaceum (2n = 2x = 26 = 2A1) and G. raimondii (2n = 2x = 26 = 2D5). The aneuploid stocks proved to be very powerful tools for localizing SSR markers to individual cotton chromosomes. Multiplex PCR bins of the SSR primers and semiautomated detection of the amplified products were optimized in this experiment. Thirteen multiplex PCR bins were optimized to contain an average of 4 SSR primer pairs per bin. This provides a protocol for high-throughput genotyping of cotton SSRs that improves the efficiency of genetic mapping and marker-assisted programs utilizing SSR markers.

Alleles↗

PubMed2003

Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT98-3936.

In a European BIOMED-2 collaborative study, multiplex PCR assays have successfully been developed and standardized for the detection of clonally rearranged immunoglobulin (Ig) and T-cell receptor (TCR) genes and the chromosome aberrations t(11;14) and t(14;18). This has resulted in 107 different primers in only 18 multiplex PCR tubes: three VH-JH, two DH-JH, two Ig kappa (IGK), one Ig lambda (IGL), three TCR beta (TCRB), two TCR gamma (TCRG), one TCR delta (TCRD), three BCL1-Ig heavy chain (IGH), and one BCL2-IGH. The PCR products of Ig/TCR genes can be analyzed for clonality assessment by heteroduplex analysis or GeneScanning. The detection rate of clonal rearrangements using the BIOMED-2 primer sets is unprecedentedly high. This is mainly based on the complementarity of the various BIOMED-2 tubes. In particular, combined application of IGH (VH-JH and DH-JH) and IGK tubes can detect virtually all clonal B-cell proliferations, even in B-cell malignancies with high levels of somatic mutations. The contribution of IGL gene rearrangements seems limited. Combined usage of the TCRB and TCRG tubes detects virtually all clonal T-cell populations, whereas the TCRD tube has added value in case of TCRgammadelta(+) T-cell proliferations. The BIOMED-2 multiplex tubes can now be used for diagnostic clonality studies as well as for the identification of PCR targets suitable for the detection of minimal residual disease.

Chromosome Aberrations↗