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 1,027 records · Page 57Linked to original sources

PubMedDate not supplied

Carrier detection and prenatal molecular diagnosis in a Duchenne muscular dystrophy family without any affected relative available.

In this paper we report a family where the affected DMD patients were not available for study and a molecular strategy was used for female carriers detection and for prenatal diagnosis. Linkage analysis was performed with two markers within the DMD gene, in all family members screened. DMD markers used (pERT87.8/Taq1 and pERT87.15/Xmn1) seemed not to be informative because the propositas mother (II-2) was homozygous for the minor allele at each marker (T2 and X2), however, the proposita and one sister carried only the major allele, which was inherited from the father. These results suggested that a deletion involving both markers could be present, and was inherited from the mother to both daughters. Quantitative multiplex PCR confirmed the deletion in female carriers, involving at least exons 12 to 17. DNA studies of cultured amniotic fluid cells at 14 weeks gestation, by amplification of specific Y-chromosome sequences, followed by multiplex PCR, lead to the diagnosis of a male fetus affected by DMD.

Adult↗

PubMed2002

SNPstream UHT: ultra-high throughput SNP genotyping for pharmacogenomics and drug discovery.

Single nucleotide polymorphism (SNP) genotyping is playing an increasing role in genome mapping, pharmacogenetic studies, and drug discovery. To date, genome-wide scans and studies involving thousands of SNPs and samples have been hampered by the lack of a system that can perform genotyping with cost-effective throughput, accuracy, and reliability. To address this need, Orrhid has developed an automated, ultra-high throughput system, SNPstream UHT, which uses multiplexed PCR in conjunction with our next generation SNP-IT tag array single base extension genotyping technology The system employs oligonucleotide microarrays manufactured in a 384-well format on a novel glass-bottomed plate. Multiplexed PCR and genotyping are performed in homogeneous reactions, and assay results are read by direct two-color fluorescence on the SNPstream UHTArray Imager. The systems flexibility enables large projects involving thousands of SNPs and thousands of samples as well as small projects that have hundreds of SNPs and hundreds of samples to be done cost effectively. We have successfully demonstrated this system in greater than 1,000,000 genotyping assays with >96% of samples giving genotypes with >99% accuracy

Alleles↗

PubMed2006

Genotypes of Listeria monocytogenes strains isolated from 2000 to 2002 in Poland.

Pulsed field gel electrophoresis (PFGE), multiplex PCR and multilocus sequence typing (MLST) methods were used for genotyping study of seventy-three L. monocytogenes isolates collected in Poland between 2000 and 2002 from human, food, environment and a diseased goat. The multiplex PCR, which is an alternative method to classical serotyping, divided the isolates into four PCR groups, IIa (42.5%), IIb (27.4%), IIc (4.1%) and IVb (26%). The isolates displayed 33 distinct PFGE profiles. Twenty eight strains were further characterised by MLST based on sequence analyses of seven housekeeping genes. The combined sequence analyses revealed a total of 10 different allelic profiles from which 3 were not described earlier. It is intended that results obtained in this study will be the first data of a national database of L. monocytogenes genotypes in Poland.

Animals↗

PubMed1999

Development of a quadriplex polymerase chain reaction for human cytomegalovirus detection.

The development of a quadriplex PCR method with amplification of HCMV in a single-step procedure using primers taken from four different regions of the viral genome is described. Different concentrations of dNTPs and MgCl2 were assayed in order to optimize the constitution of the buffer for the multiplex PCR. The specificity of the PCR was tested with 100 ng, 10 ng, and 1 ng of genomic MRC-5 cell DNA infected with CMV in the presence of 10 microg of uninfected MRC-5 cell DNA. The sensitivity of the PCR was evaluated by the amplification of various amounts (100 ng, 10 ng, 1 ng, and 0.1 ng) of genomic MRC-5 cell DNA infected with CMV. The specificity and sensitivity assays were performed for each pair of primers and for the combined four primer pairs in the multiplex PCR. CMV was consistently detected from 10 ng of genomic MRC-5 cell DNA with each primer pair. When all four sets of primers were combined in a single reaction tube, the sensitivity of the assay was equivalent to 10 ng of genomic MRC-5 cell DNA, whereas amplification from 1 ng genomic MRC-5 cell DNA produced only a subset of the amplimers. By amplifying four target-sequences of HCMV simultaneously with minimum incubation time at each temperature, a quadriplex, highly sensitive PCR assay was performed. The use of four primer sets designed in different genomic regions of HCMV allowed the detection of variants and achieved maximal sensitivity and specificity which are essential for a diagnostic utilization.

Cell Line↗

PubMed2000

Preimplantation genetic diagnosis in two families at risk for recurrence of Herlitz junctional epidermolysis bullosa.

The Herlitz type of junctional epidermolysis bullosa (H-JEB) is a severe inherited bullous disease which leads to the early demise of the affected newborn. Mutations in the genes encoding the 3 polypeptides of the anchoring filament protein laminin 5 underlie this condition. We studied 2 families with affected children who previously died from H-JEB. Mutation screening using heteroduplex analysis and direct sequencing of the PCR products revealed a previously described hotspot mutation in LAMB3 (R635X), and a novel delayed termination codon in LAMB3 in the first proband. In the second proband, we found a novel initiation codon mutation in LAMB3, and a novel 2 bp deletion in LAMB3. For preimplantation genetic diagnosis (PGD) in these families, we developed nested multiplex PCR assays, amplifying the mutations and informative intragenic polymorphisms in the probands. Single embryonic cells were biopsied from 8-cell embryos using standard techniques, and subjected to the multiplex PCR assay followed by restriction enzyme digestion. Embryos found not to carry either mutation were transferred to the mothers, and a pregnancy was established in the second family as evidenced by the elevated level of HCG, although the pregnancy did not persist. This study illustrates the feasibility of PGD for an inherited skin disorder for the first time.

Base Sequence↗

PubMed1994

[Molecular biological screening of viruses important to transfusion medicine].

The usefulness of the direct virus detection by polymerase chain reaction (PCR) and reverse transcription/polymerase chain reaction (RT PCR) for blood donor screening was investigated, including the following viruses: cytomegalovirus (CMV), hepatitis B virus (HBV), hepatitis C virus (HCV) and human immunodeficiency type 1 virus (HIV1). Hepatitis C viraemia was detected by RT PCR in 97% of anti-HCV-positive haemophiliacs, in 48% of anti-HCV-positive hepatitis patients, in only 21% of anti-HCV-positive blood donors from North-West Germany, and not at all in 945 blood donors with elevated serum ALT. In order to compare HIV1 detection by PCR and by p24 antigen determination, we tested 34 anti-HIV1-positive AIDS patients for p24 antigen, HIV1 RNA and HIV1 provirus DNA. 97% had HIV1 provirus DNA, 35% had HIV RNA, but only 30% had p24 antigen. A multiplex PCR specific for HBV, HCV and HIV1 (RNA and DNA) was developed for the investigation of a blood donor population from Namibia, where HBV and HIV1 infections occur more frequently than in German blood donors. The prevalence of anti-HIV1 antibodies in this population was 0.6%. HIV1 RNA was never detected in the plasma of 2,569 anti-HIV1-negative donors. HIV1 provirus DNA was present in 75% of the 16 anti-HIV1-positive individuals. None of these anti-HIV1-positive blood donors was also positive for p24 antigen. CMV infections and reactivations in 130 immunocompromised heart transplant patients and in 420 healthy anti-CMV-positive blood donors were monitored using cytochemical detection of CMV early antigen, and PCR. CMV DNA was neither detected in the plasma nor in the leucocytes of any anti-CMV-positive blood donor. During the course of CMV reactivation in immunocompromised heart transplant patients, CMV DNA was always detectable first in granulocytes and afterwards in the plasma. The cytochemical demonstration of CMV early antigen was typically delayed by several days and was observed in only 11% of those blood samples which contained CMV DNA in leucocytes. The determination of CMV DNA in leucocytes proved to be the most sensitive method to detect viraemia. Thus, CMV detection in leucocytes is the method of choice for the monitoring of transplant patients. This method is also promising for blood donor screening. The sensitive routine monitoring of blood donations for virus infections by multiplex PCR is practicable. However, nucleic acid must be extracted both from the plasma and from the cellular compartments of blood in order to detect HIV and CMV provirus DNA. Lysate from EDTA blood is a suitable material for this purpose. The determination of the surrogate marker serum ALT activity is of no use in hepatitis C screening, and determination of p24 antigen is not required in HIV1 screening.

Acquired Immunodeficiency Syndrome↗

PubMed2001

Microarray analysis of microbial virulence factors.

Hybridization with oligonucleotide microchips (microarrays) was used for discrimination among strains of Escherichia coli and other pathogenic enteric bacteria harboring various virulence factors. Oligonucleotide microchips are miniature arrays of gene-specific oligonucleotide probes immobilized on a glass surface. The combination of this technique with the amplification of genetic material by PCR is a powerful tool for the detection of and simultaneous discrimination among food-borne human pathogens. The presence of six genes (eaeA, slt-I, slt-II, fliC, rfbE, and ipaH) encoding bacterial antigenic determinants and virulence factors of bacterial strains was monitored by multiplex PCR followed by hybridization of the denatured PCR product to the gene-specific oligonucleotides on the microchip. The assay was able to detect these virulence factors in 15 Salmonella, Shigella, and E. coli strains. The results of the chip analysis were confirmed by hybridization of radiolabeled gene-specific probes to genomic DNA from bacterial colonies. In contrast, gel electrophoretic analysis of the multiplex PCR products used for the microarray analysis produced ambiguous results due to the presence of unexpected and uncharacterized bands. Our results suggest that microarray analysis of microbial virulence factors might be very useful for automated identification and characterization of bacterial pathogens.

Bacterial Proteins↗

PubMed2003

A simple, low cost and non-invasive method for screening Y-chromosome microdeletions in infertile men.

BACKGROUND: Recent investigations emphasized a high prevalence of Y-chromosome microdeletions in men having severely impaired spermatogenesis. Screening of these men is recommended prior to assisted reproduction techniques. METHODS: The aim of this study was to define a reliable and efficient method to detect Y-chromosome deletions in infertile men. At first the feasibility of using a cytobrush to collect buccal cells as a source of DNA was tested. Then, a multiplex PCR in accordance with European recommendations (European Andrology Academia: EAA) was compared with a commercial kit. The test population consisted of 18 infertile male patients (with a known Y-deletion). Both buccal and blood cells were used for DNA extraction. A specific DNA extraction protocol was carried out on the buccal cells. RESULTS: Between 4-10 micro g of DNA were retrieved per brush, allowing for several PCR attempts. The commercial kit failed to detect an AZFa deletion. Furthermore, markers sY130, sY133 and sY153, included in the kit, are not reliable. Both false negative and false positive results were generated by the commercial kit. CONCLUSION: A multiplex PCR performed pursuant to EAA recommendations is proposed. When the testing is conducted with DNA extracted from buccal cells, this protocol is simple, accurate and affordable.

Blood Cells↗

PubMed1993

A rapid and reliable PCR method for genotyping the ABO blood group.

The ABO blood group has been used extensively as a marker in population studies, epidemiology, and forensic work. However, until the cloning of the gene, it was not possible to determine the genotype of group A and B individuals without recourse to family studies. We have developed a method to determine the ABO genotype directly from human DNA using multiplex PCR and restriction enzyme analysis. Two PCR fragments spanning positions 258 and 700 of the cDNA sequence are amplified. The site at position 258 allows us to differentiate the O allele from the A and B alleles. The site at position 700 allows us to distinguish the B allele from the A and O alleles. Analysis at the two sites thus allows us to distinguish the three alleles. The multiplex PCR product is digested separately with four enzymes, two for each of the sites. The pair of enzymes for each site cut in a reciprocal fashion. Whereas one enzyme for each site is theoretically sufficient for genotyping, the use of complementary pairs of enzymes prevents the assignment of a false genotype as a result of false negative or partial digestion. This method is fast and reliable, does not rely on probing of blots, and should be widely applicable.

ABO Blood-Group System↗

PubMed2005

Use of multiplex polymerase chain reactions to indicate the accuracy of the annealing temperature of thermal cycling.

A condition for multiplex polymerase chain reactions (PCRs) of which outcomes sensitively indicate the actual annealing temperature of thermal cycling is reported. The multiplex reaction was designed to produce four different amplicons of 200, 300, 400, and 480 bp. However, the degree of amplification of each amplicon sensitively responds to a small change in the annealing temperature, by which one can predict the actual annealing temperature of thermal cycling. Deviations between the actual and the designated annealing temperatures as small as 0.5 degrees C were manifested by the banding patterns of the multiplex PCRs in simple agarose gel electrophoresis. For prediction of temperatures in a more objective manner, capillary electrophoresis was also applied to obtain numerical expressions of the relative intensities of the amplicons. By optimizing the multiplex PCR conditions, where concentrations of buffer, dNTPs, and primer pairs were major factors, satisfactory sensitivity and reproducibility of the band patterning were achieved. Blind tests demonstrated the accuracy of the prediction of actual annealing temperatures within +/-0.5 degrees C. The multiplex PCR approach will be further refined and tested for realization of an easily accessible alternative to a physical temperature measurement device in testing the performance of thermal cyclers for PCR.

Buffers↗

PubMed2003

Sensitive and rapid quantitative detection of anthrax spores isolated from soil samples by real-time PCR.

Quantitative analysis of anthrax spores from environmental samples is essential for accurate detection and risk assessment since Bacillus anthracis spores have been shown to be one of the most effective biological weapons. Using TaqMan real-time PCR, specific primers and probes were designed for the identification of pathogenic B. anthracis strains from pag gene and cap gene on two plasmids, pXO1 and pXO2, as well as a sap gene encoded on the S-layer. To select the appropriate lysis method of anthrax spore from environmental samples, several heat treatments and germination methods were evaluated with multiplex-PCR. Among them, heat treatment of samples suspended with sucrose plus non-ionic detergent was considered an effective spore disruption method because it detected up to 10(5) spores/g soil by multiplex-PCR. Serial dilutions of B. anthracis DNA and spore were detected up to a level of 0.1 ng/ microliters and 10 spores/ml, respectively, at the correlation coefficient of 0.99 by real-time PCR. Quantitative analysis of anthrax spore could be obtained from the comparison between C(T) value and serial dilutions of soil sample at the correlation coefficient of 0.99. Additionally, spores added to soil samples were detected up to 10(4) spores/g soil within 3 hr by real-time PCR. As a consequence, we established a rapid and accurate detection system for environmental anthrax spores using real-time PCR, avoiding time and labor-consuming preparation steps such as enrichment culturing and DNA preparation.

Anthrax↗

PubMed2003

Rapid identification of yeasts commonly found in positive blood cultures by amplification of the internal transcribed spacer regions 1 and 2.

A multiplex PCR method using one universal and eight species-specific primers was developed to rapidly identify eight yeast species found in positive blood cultures. The species-specific primers were designed from the internal transcribed spacer regions 1 and 2 of the rRNA gene, whereas the universal primer was located at the 26S rRNA gene. The eight species were Candida albicans, Candida glabrata, Candida guilliermondii, Candida krusei, Candida lusitaniae, Candida parapsilosis, Candida tropicalis, and Cryptococcus neoformans. The PCR products (116 to 630 bp) were different in length and could be effectively separated and recognized by polyacrylamide gel electrophoresis. By testing 234 positive blood cultures (237 isolates), 234 (98.7%) isolates of the above eight species were correctly identified by the multiplex PCR. The present method is simple to perform and can be completed within 6 h.

Base Sequence↗

PubMed1999

A method for the quantitative analysis of human heat shock gene expression using a multiplex RT-PCR assay.

A quantitative multiplex RT-PCR assay is described to measure the levels of messenger RNAs for eight human genes encoding the heat shock proteins (HSP) and molecular chaperones hsp90alpha, hsp90beta, hsp70, hsc70, mtHsp75, Grp78 (BiP), hsp60 and hsp27. The basis of this assay is reverse transcription of total RNA isolated from human cells followed by amplification with PCR. By the careful selection of pairs of oligonucleotide primers corresponding to unique regions of each heat shock gene, selectivity can be attained such that messenger RNAs of multiple heat shock genes can be analyzed simultaneously in a single reaction. This method provides both the absolute and relative levels of each heat shock message by including in the reaction, reference control RNAs corresponding to in vitro transcripts of heat shock gene plasmids carrying small internal deletions.

Cell Line↗

PubMed2005

Development of three multiplex RT-PCR assays for the detection of 12 respiratory RNA viruses.

Three multiplex hemi-nested RT-PCR assays were developed to detect simultaneously 12 RNA respiratory viruses: influenza viruses A, B and C, human respiratory syncytial virus (hRSV), human metapneumovirus (hMPV), parainfluenza virus types 1-4 (PIV-1, -2, -3 and -4), human coronavirus OC43 and 229E (HCoV) and rhinovirus (hRV). An internal amplification control was included in one of the RT-PCR assays. The RT-PCR multiplex 1 and the hemi-nested multiplex 1 detected 1 and 0.1 TCID50 of RSV A, respectively, and 0.01 and 0.001 TCID50 of influenza virus A/H3N2, respectively. Two hundred and three nasal aspirates from hospitalised children were retrospectively tested in comparison with two conventional methods: direct immunofluorescence assay and viral isolation technique. Almost all samples (89/91) that were positive by immunofluorescence assay and/or viral isolation technique were detected by the multiplex assay. This method also detected an additional 85 viruses and 33 co-infections. The overall sensitivity (98%), rapidity and enhanced efficiency of these multiplex hemi-nested RT-PCR assays suggest that they would be a significant improvement over conventional methods for the detection of a broad spectrum of respiratory viruses.

Child↗

PubMed1997

PCR identification of Mycobacterium bovis BCG.

The attenuated bacillus Calmette-Guérin (BCG) vaccine strain is derived from a virulent strain of Mycobacterium bovis. BCG is difficult to differentiate from other strains of M. bovis and other members of the M. tuberculosis complex by conventional methods. Recently, a genomic region designated RD1 was found to be present in all virulent M. bovis and M. tuberculosis strains tested but deleted from all BCG strains tested. With this information, a multiplex PCR method was developed to detect the RD1 deletion. A large collection of BCG and other M. tuberculosis complex strains from diverse host and geographic origins was tested. RD1 was deleted in 23 of 23 BCG strains. RD1 was present in 129 of 129 other M. tuberculosis complex strains. This multiplex PCR method can be used as a tool for the rapid and specific identification of BCG.

Animals↗

PubMed2002

Application of a multiplex polymerase chain reaction assay for the simultaneous confirmation of Listeria monocytogenes and other Listeria species in turkey sample surveillance.

A multiplex polymerase chain reaction was developed to simultaneously identify Listeria monocytogenes and species of the genus Listeria. Two sets of primers were used, with the first amplifying a 938-bp region of the 16S rRNA gene that is highly conserved in all Listeria species and the second amplifying a 174-bp region of the listeriolysin (hlyA) gene of L. monocytogenes. Thus, isolates of Listeria spp. yield a single 938-bp product, whereas L. monocytogenes isolates yield both the 938-bp product and a 174-bp product. The specificity of the assay was verified with all six Listeria species and 11 serotypes of L. monocytogenes, as well as nonrelated bacteria. The multiplex PCR assay was used to determine the incidence of Listeria spp., especially L. monocytogenes, in mechanically separated turkey samples (n = 150 samples). L. monocytogenes strains were selected by using the University of Vermont two-step enrichment protocol and plating to selective Palcam agar. The multiplex PCR assay was used for verification of presumptive Listeria colonies. Approximately 38% of mechanically separated turkey samples (57 of 150) yielded L. monocytogenes; an additional 18% of these samples (27 of 150) harbored other Listeria spp. Fifty-one percent (29 of 57) of the L. monocytogenes isolates were of serogroup 1, 44% (25 of 57) were of serogroup 4, and 2% (1 of 57) were assigned to serogroups other than 1 and 4.

Animals↗

PubMed2005

Molecular strategies for the detection, identification, and differentiation between enteroinvasive Escherichia coli and Shigella spp.

A strategy for the detection, identification, and differentiation of enteroinvasive Escherichia coli (EIEC) and Shigella spp. has been developed. The strategy includes (i) a multiplex PCR for the amplification of two virulence genes, i.e., iuc (222 bp) and ipaH (629 bp); (ii) amplification of the ial gene (a 1,038-bp amplicon) located within a large plasmid; and (iii) restriction fragment length polymorphism (RFLP) of the ial gene amplicon. The multiplex PCR provided three patterns. Pattern 1 (iuc-/ ipaH+) was found in 10 (67%) of 15 EIEC strains tested, pattern 2 (iuc+/ipaH-) in only 2 (4.4%) of 46 non-EIEC isolates, whereas pattern 3 (iuc+/ipaH+) was observed in all Shigella spp. and also in 5 (33%) of 15 EIEC strains tested. The pattern 3 EIEC strains were all positive for the ial gene. The PCR-RFLP of the ial gene amplicon using the endonuclease AclI was used to differentiate Shigella spp. from the EIEC strains that belonged to pattern 3. The ial gene was present in 21 (38%) of 56 and 6 (40%) of 15 Shigella spp. and EIEC strains tested, respectively. The PCR-RFLP of the ial gene amplicon divided the strains in two types. Type 1 did not contain the restriction enzyme site and was found in 6 (100%) of 6 EIEC strains, 4 (80%) of 5 Shigella boydii, and 4 (100%) of 4 Shigella dysenteriae strains tested. Type 2, which gave two fragments of 286 and 752 bp, was observed in 5 (83%) of 6 Shigella fiexneri strains and 6 (100%) of 6 Shigella sonnei strains. Detection, identification, and differentiation of Shigella spp. and EIEC were achieved by analyses of the PCR patterns and RFLP types. To our knowledge, this is the first study to demonstrate a simple and rapid method for detecting, identifying, and differentiating, at the molecular level, Shigella spp. and EIEC strains. This method will have tremendous utility as an epidemiological tool and in helping to develop policies, risk assessments, and national and international methods for Shigella spp.

Bacterial Typing Techniques↗

PubMed2006

Detection, occurrence, and characterization of Escherichia coli O157:H7 from raw ewe's milk in Spain.

A study was carried out in the Castilla y León region of Spain to investigate the presence of Escherichia coli O157:H7 in raw ewe's milk samples collected from several cheese factories during 1 year. All specimens were examined for E. coli O157:H7 by selective enrichment at 41.5 +/- 1.0 degrees C, after both 6 and 22 h of incubation, and then immunomagnetically separated and plated on cefixime-potassium tellurite-sorbitol MacConkey agar. No growth was obtained in the enrichment broth after a 6-h incubation. Presumptive colonies obtained after 22 h of incubation were screened by a multiplex PCR assay for the presence of rfbO157 and fliCH7 genes. Of all the ewe's milk samples studied, three were positive for E. coli O157:H7. The E. coli O157:H7 strains that were positive for the rfbO157 and fliCH7 genes were then analyzed by multiplex PCR for the presence of virulence genes (stx1, stx2, ehxA, and eaeA). All E. coli O157:H7 isolates were Shiga toxigenic and harbored additional genes related to virulence (ehxA and eaeA). The predominant Stx toxin type was stx2. These results demonstrate that raw ewe's milk used in cheesemaking may be sporadically contaminated with E. coli O157:H7 strains that are potentially pathogenic for humans.

Animals↗