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Results for “Multiplex PCR”

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PubMed2004

Molecular detection and characterization of Dichelobacter nodosus in ovine footrot in India.

Dichelobacter nodosus was detected in three clinical cases of ovine footrot in Kashmir, India. The detection was done by PCR in three clinical specimens directly, without isolating the organism, using species-specific 16S rDNA primers. Positive results were indicated by amplification of a 783 bp product. All the three samples were subjected to serogrouping by multiplex PCR using group (A-I) specific primers. All the three samples revealed the presence of serogroup B of D. nodosus by yielding a single band of 283 bps.

Animals↗

PubMed1999

Hprt mutant frequency and molecular analysis of Hprt mutations in Fischer 344 rats treated with thiotepa.

Thiotepa is a bifunctional alkylating anticancer drug that is a rodent carcinogen and a suspected human carcinogen. In order to determine the sensitivity of mutant induction in the Hprt lymphocyte assay for detecting tumorigenic doses of thiotepa, Fischer 344 rats were treated for 4 weeks with thiotepa using a procedure adapted from a carcinogenesis protocol. At various times after beginning the treatment regimen, rats were killed and the lymphocyte Hprt assay was performed on splenic lymphocytes isolated from the animals. The 6-thioguanine-resistant T lymphocyte mutant frequency increased with time during the period of thiotepa exposure and declined slightly thereafter. Significant dose-dependent increases in mutant frequency were found using concentrations of thiotepa that eventually result in lymphoproliferative tumors. Hprt mRNA from mutant lymphocytes was reverse transcribed to cDNA, amplified by PCR and examined for mutations by DNA sequencing. This analysis indicated that the major type of point mutation was G:C-->T:A transversion and that 33% of the mutants contained simple or complex frameshifts. Also, a multiplex PCR performed on DNA from mutant clones that were expanded in vitro indicated that 34% of the clones had deletions in the Hprt gene. These results indicate that the induction of lymphocyte Hprt mutants is a sensitive biomarker for the carcinogenicity of thiotepa and that the types of mutations found in the lymphocyte Hprt gene reflect the kinds of DNA damage produced by thiotepa.

Adenine↗

PubMed1996

Identification of the five most common cystic fibrosis mutations in single cells using a rapid and specific differential amplification system.

We describe a rapid and specific differential amplification system which can detect five of the most common cystic fibrosis mutations from a single cell. In the first round of the polymerase chain reaction (PCR), regions of exons 4, 10 and 11 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene containing the mutations delta F508, G551D, R553X, G542X and 621+1G > T were co-amplified in a single multiplex PCR. To identify potential contamination, we included external amplification primers for the polymorphic human tyrosine hydroxylase (HUMTH01) locus as a fingerprint for the sample. In the second round of PCR, detection of any of the five mutations was achieved using the amplification refractory mutation system (ARMS) in two separate reactions, each containing nested amplification primers for either wild type or mutant sequence. A separate second round PCR for the fingerprinting was performed with nested HUMTH01 primers. Using this procedure we have successfully and accurately detected five cystic fibrosis mutations in 30 single cells with a failed amplification rate of 7% and a contamination rate of 4.6% and that PCR failure or possible contamination will also be easily detected. This procedure allows detection of the five most common point mutations and small deletions responsible for cystic fibrosis from a single cell in < 8 h which could be applicable to preimplantation diagnosis in human embryos.

Cystic Fibrosis↗

PubMed2003

Multiplexed SNP genotyping using the Qbead system: a quantum dot-encoded microsphere-based assay.

We have developed a new method using the Qbead system for high-throughput genotyping of single nucleotide polymorphisms (SNPs). The Qbead system employs fluorescent Qdot semiconductor nanocrystals, also known as quantum dots, to encode microspheres that subsequently can be used as a platform for multiplexed assays. By combining mixtures of quantum dots with distinct emission wavelengths and intensities, unique spectral 'barcodes' are created that enable the high levels of multiplexing required for complex genetic analyses. Here, we applied the Qbead system to SNP genotyping by encoding microspheres conjugated to allele-specific oligonucleotides. After hybridization of oligonucleotides to amplicons produced by multiplexed PCR of genomic DNA, individual microspheres are analyzed by flow cytometry and each SNP is distinguished by its unique spectral barcode. Using 10 model SNPs, we validated the Qbead system as an accurate and reliable technique for multiplexed SNP genotyping. By modifying the types of probes conjugated to microspheres, the Qbead system can easily be adapted to other assay chemistries for SNP genotyping as well as to other applications such as analysis of gene expression and protein-protein interactions. With its capability for high-throughput automation, the Qbead system has the potential to be a robust and cost-effective platform for a number of applications.

DNA↗

PubMed2003

Use of PCR for direct detection of Campylobacter species in bovine feces.

This study reports on the use of PCR to directly detect and distinguish Campylobacter species in bovine feces without enrichment. Inhibitors present in feces are a major obstacle to using PCR to detect microorganisms. The QIAamp DNA stool minikit was found to be an efficacious extraction method, as determined by the positive amplification of internal control DNA added to bovine feces before extraction. With nested or seminested multiplex PCR, Campylobacter coli, C. fetus, C. hyointestinalis, and C. jejuni were detected in all fecal samples inoculated at approximately 10(4) CFU g(-1), and 50 to 83% of the samples inoculated at approximately 10(3) CFU g(-1) were positive. At approximately 10(2) CFU g(-1), C. fetus, C. hyointestinalis, and C. jejuni (17 to 50% of the samples) but not C. coli were detected by PCR. From uninoculated bovine feces, a total of 198 arbitrarily selected isolates of Campylobacter were recovered on four commonly used isolation media incubated at three temperatures. The most frequently isolated taxa were C. jejuni (152 isolates) and C. lanienae (42 isolates), but isolates of C. fetus subsp. fetus, Arcobacter butzleri, and A. skirrowii also were recovered (</=2 isolates per taxon). Considerable variability was observed in the frequency of isolation of campylobacters among the four media and three incubation temperatures tested. With genus-specific primers, Campylobacter DNA was detected in 75% of the fecal samples, representing an 8% increase in sensitivity relative to that obtained with microbiological isolation across the four media and three incubation temperatures tested. With nested primers, C. jejuni and C. lanienae were detected in 25 and 67% of the samples, respectively. In no instance was DNA from either C. coli, C. fetus, or C. hyointestinalis detected in uninoculated bovine feces. PCR was more sensitive than isolation on microbiological media for detecting C. lanienae (17%) but not C. jejuni. Campylobacters are a diverse and fastidious group of bacteria, and the development of direct PCR not only will increase the understanding of Campylobacter species diversity and their frequency of occurrence in feces but also will enhance the knowledge of their role in the gastrointestinal tract of livestock and of the factors that influence shedding.

Animals↗

PubMed1995

The identification of point mutations in Duchenne muscular dystrophy patients by using reverse-transcription PCR and the protein truncation test.

The protein truncation test (PTT) is a mutation-detection method that monitors the integrity of the open reading frame (ORF). More than 60% of cases of Duchenne muscular dystrophy (DMD) result from gross frame-shifting deletions in the dystrophin gene that are detectable by a multiplex PCR system. It has become apparent that virtually all of the remaining DMD mutations also disrupt the translational reading frame, making the PTT a logical next step toward a comprehensive strategy for the identification of all DMD mutations. We report here a pilot study involving 22 patients and describe the mutations characterized. These constitute 12 point mutations or small insertions/deletions and 4 gross rearrangements. We also have a remaining five patients in whom there does not appear to be a mutation in the ORF. We believe that reverse-transcription--PCR/PTT is an efficient method by which to screen for small mutations in DMD patients with no deletion.

Base Sequence↗

PubMed2005

Immunobead multiplex RT-PCR detection of carcinoembryonic genes expressing cells in the blood of colorectal cancer patients.

Circulating cell detection using reverse transcriptase-polymerase chain reaction (RT-PCR) techniques has been studied as a new prognostic factor in colorectal cancer patients. With the view of enhancing detection sensitivity, we developed a new multiplex RT-PCR assay for circulating cell detection based on the expression of carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5; formerly CEA) and CEACAM7 (formerly CGM2). Between November 2002 and December 2003, 45 stage III-IV, 39 stage I-II colorectal cancer patients, 32 non-colorectal cancer patients and 41 healthy individuals were included. Positive selection using HEA-125 immunobeads was applied to blood samples before mRNA extraction, cDNA synthesis and a multiplex CEACAM5/CEACAM7 RT-PCR assay. For both CEACAM5 and CEACAM7, the limit of detection was found to be as low as 1 expressing cell in 10(6) nucleated blood cells. The multiplex RT-PCR assay was negative for the 41 healthy individuals and the 32 non-colorectal cancer patients. The test was positive in 53/84 (63%) of the colorectal cancer patients for CEACAM5 and/or CEACAM7, whereas 32/84 (38%) were positive for both markers. Colorectal cancer patients were positive for one of the two markers in 80% of cases (36/45) for stage III-IV patients (CEACAM5 73%, CEACAM7 51%) and in 44% of cases (17/39) for stage I-II patients. This multiplex RT-PCR assay with two markers proved to be more sensitive than use of a single marker in detecting circulating tumour cells. The discrepant expression of CEACAM5 and CEACAM7 may label circulating tumour cells that have different levels of differentiation and subsequent aggressive behaviour.

Aged↗

PubMed2000

PCR-based identification of MAT-1 and MAT-2 in the Gibberella fujikuroi species complex.

All sexually fertile strains in the Gibberella fujikuroi species complex are heterothallic, with individual mating types conferred by the broadly conserved ascomycete idiomorphs MAT-1 and MAT-2. We sequenced both alleles from all eight mating populations, developed a multiplex PCR technique to distinguish these idiomorphs, and tested it with representative strains from all eight biological species and 22 additional species or phylogenetic lineages from this species complex. In most cases, either an approximately 800-bp fragment from MAT-2 or an approximately 200-bp fragment from MAT-1 is amplified. The amplified fragments cosegregate with mating type, as defined by sexual cross-fertility, in a cross of Fusarium moniliforme (Fusarium verticillioides). Neither of the primer pairs amplify fragments from Fusarium species such as Fusarium graminearum, Fusarium pseudograminearum, and Fusarium culmorum, which have, or are expected to have, Gibberella sexual stages but are thought to be relatively distant from the species in the G. fujikuroi species complex. Our results suggest that MAT allele sequences are useful indicators of phylogenetic relatedness in these and other Fusarium species.

Alleles↗

PubMed2001

IL-18, a product of choriodecidual cells, increases during premature rupture of membranes but fails to turn on the Fas-FasL-mediated apoptosis pathway.

PROBLEM: IL-18 is a novel cytokine, which promotes inflammation and apoptosis. This study examines its expression pattern, site of production, and levels in the amniotic fluid (AF) during pregnancy complications such as preterm labor and preterm premature rupture of membranes (pPROM). The ability of IL-18 to induce the Fas-Fas ligand (FasL)/caspase-mediated apoptotic pathway is also studied. METHODS: Amniochorion collected at term was placed in an organ explant system. IL-18 mRNA expression was studied by RT-PCR. IL-18 mRNA and peptide were localized by in situ hybridization and immunohistochemistry. IL-18 in the AF of women with pPROM, with preterm labor with no rupture of membranes, and at term was measured using ELISA. Multiplex PCR was used to study the expression pattern of proapoptotic genes such as Fas, FasL, caspase 8, and Fas-associated death domain (FADD). ELISA was also used to measure the release of soluble Fas from IL-18-stimulated amniochorion in culture media. RESULTS: IL-18 is a constitutively expressed gene in human chorion and decidua but not in human amnion and increases in the AF of women with pPROM [2.9 +/- 3.3 ng/ml (SD)] compared to women with preterm labor (1.1 +/- 0.67 ng/ml; P < 0.05) and term (0.9 +/- 0.73 ng/ml; P < 0.05). IL-18 induces Fas expression, whereas FADD is a constitutively expressed gene in human fetal membranes. IL-18 failed to induce FasL or caspase 8 expressions. Soluble Fas release from amniochorion was increased after IL-18 stimulation. CONCLUSION: Chorion and decidua are a source of IL-18, whose concentrations are increased in the AF during pPROM. IL-18 induced Fas expression in amniochorion; however, it failed to turn on other genes in the Fas-FasL apoptosis pathway including FasL and caspase 8.

Amniotic Fluid↗

PubMed1999

Differentiation of Asian rice gall midge, Orseolia oryzae (Wood-Mason), biotypes by sequence characterized amplified regions (SCARs).

We developed a polymerase chain reaction (PCR)-based assay that distinguished five different biotypes of the Asian gall midge (Orseolia oryzae), a major insect pest of rice. A total of 400 random primers were screened using random amplified polymorphic DNAs (RAPDs). Five diagnostic PCR products were isolated, cloned, sequenced and converted to sequence characterized amplified regions (SCARs). Primers specific to these SCARs were able to amplify specific DNA fragments from genomic DNAs of five biotypes of gall midge in a multiplexed-PCR-based assay. The amplified DNA fragments were used as diagnostic markers to identify different biotypes of gall midge. The SCAR primers were also capable of differentiating the Asian from the African rice gall midge (Orseolia oryzivora) as well as detecting a variant of biotype 5 which caused an outbreak in Kerala, India. Unlike the use of plant host differentials and midge feeding behaviour for identifying biotypes, this assay is fast, reliable and unaffected by environmental factors.

Animals↗

PubMed2002

The molecular basis of complete complement C4A and C4B deficiencies in a systemic lupus erythematosus patient with homozygous C4A and C4B mutant genes.

The disease course of a complete C4-deficient patient in the U.S. was followed for 18 years. The patient experienced multiple episodes of infection, and he was diagnosed with systemic lupus erythematosus at age 9 years. The disease progressed to WHO class III mild lupus nephritis and to fatal CNS vasculitis at age 23 years. Immunochemical experiments showed that the patient and his sibling had complete absence of C4A and C4B proteins and were negative for the Rodgers and Chido blood group Ags. Segregation and definitive RFLP analyses demonstrated that the patient and his sibling inherited two identical haplotypes, HLA A2 B12 DR6, each of which carries a defective long C4A gene and a defective short C4B gene. PCR and DNA sequencing revealed that the mutant C4A contained a 2-bp insertion in exon 29 at the sequence for codon 1213. The identical mutation was absent in the mutant C4B. The C4B mutant gene was selectively amplified by long range PCR, and its 41 exons were completely sequenced. The C4B mutant had a novel single C nucleotide deletion at the sequence for codon 522 in exon 13, leading to frame-shift mutation and premature termination. Thus, a multiplex PCR is designed by which known mutations in C4A and C4B can be elucidated conveniently. Among the 28 individuals reported with complete C4 deficiency, 75-96% of the subjects (dependent on the inclusion criteria) were afflicted with autoimmune or immune complex disorders. Hence, complete C4 deficiency is one of the most penetrant genetic risk factors for human systemic lupus erythematosus.

Adult↗

PubMed2006

Y-STR analysis of degraded DNA using reduced-size amplicons.

To increase the success rate of Y-STR genotyping for degraded DNA, we have developed two multiplex PCR sets for 21 Y-STR loci. Besides the 17 Y-STR loci of DYS19, DYS385, DYS389-I, DYS389-II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635, and GATA H4.1 contained in a commercial Y-STR kit, AmpFlSTR Yfiler, the other four loci of DYS388, DYS446, DYS447, and DYS449 were also included in the multiplexes to increase the discrimination capacity. Among a total of 21 Y-STR loci, the primers for eight loci (DYS385, DYS390, DYS438, DYS446, DYS448, DYS449, and DYS635) were newly designed in the present study and nine loci (DYS385, DYS390, DYS391, DYS392, DYS438, DYS439, DYS448, and DYS635) have PCR amplicons smaller than those of the AmpFlSTR Yfiler kit. A sensitivity test using serially diluted standard 9948 male DNA showed that all the values of Y-STR loci in the Y-miniplexes are reliable at template concentrations as low as 30 pg. We compared the effectiveness of the two multiplexes with the AmpFlSTR Yfiler kit by using both enzymatically degraded DNA and 30 samples of 50-year-old skeletal remains. This comparison demonstrated that the new Y-miniplex sets can produce a better signal from degraded DNA than the AmpFlSTR Yfiler kit.

Asian People↗

PubMed2006

A novel real-time duplex PCR assay for detecting penA and ponA genotypes in Neisseria gonorrhoeae: Comparison with phenotypes determined by the E-test.

BACKGROUND: For many years, the pathogenic bacterium Neisseria gonorrhoeae, the etiologic agent of gonorrhea, was generally susceptible to penicillin, until the emergence of resistant strains. Well-characterized genetic variations in the penicillin resistance-determining region correlate with decreased susceptibility to penicillin. At least 5 genes (penA, penB, mtrR, ponA, and penC) are involved in the chromosomally mediated resistance to this antibiotic. To date, no development of multiplex PCR assays targeting a range of gonococcal genes and variations as a means of predicting antibiotic resistance has been reported. METHODS: The aim of this study was to develop a duplex assay using DNA from isolated strains. We describe the development and evaluation on the LightCycler platform of a real-time duplex PCR assay (hybridization probe format) for rapid and specific detection of ponA and penA variations, predicting penicillin susceptibilities. RESULTS: The real-time duplex PCR assay successfully detected variations in ponA and penA genes by use of distinct melting temperatures from a total of 120 Neisseria gonorrhoeae isolates. Moreover, the variation profiles obtained with the real-time PCR and the melting analysis showed good correlation with the pattern of penicillin susceptibility generated with classical antibiograms. Nucleotide sequencing data were in complete agreement with multiplex assay results. CONCLUSIONS: The presented assay is suitable for the detection of chromosomally mediated resistant strains of Neisseria gonorrhoeae in genotyping studies and could be valuable in the effective antimicrobial strategy to gonococci.

Anti-Bacterial Agents↗

PubMed2003

Renal C-type natriuretic peptide and natriuretic peptide receptor B mRNA expression are affected by water deprivation in the Spinifex Hopping mouse.

This study investigated the effect of water deprivation on the expression of C-type natriuretic peptide (CNP) and natriuretic peptide receptor B (NPR-B) mRNA, and the ability of NPR-B to generate cGMP in the Spinifex Hopping mouse, Notomys alexis. This rodent is a native of central and western Australia that is well adapted to survive in arid environments. Initially, CNP and NPR-B cDNAs (partial for NPR-B) were cloned and sequenced, and were shown to have high homology with those of rat and mouse. RT-PCR analysis showed CNP mRNA expression in the kidney, proximal and distal colon and small intestine, whilst NPR-B mRNA expression was found in the kidney, proximal and distal colon and the atria. Using a semi-quantitative multiplex PCR technique, the expression of renal CNP and NPR-B mRNA was determined in 7- and 14-day water-deprived hopping mice, in parallel with control hopping mice (access to water). Water deprivation significantly decreased the relative levels of CNP and NPR-B mRNA expression in both the 7- and 14-day water-deprived hopping mice, when compared to control hopping mice. In contrast, the ability of CNP to stimulate cGMP production was significantly increased after 14 days of water deprivation. This study shows that alterations in the renal CNP/NPR-B system may be an important physiological adjustment when water is scarce.

Amino Acid Sequence↗

PubMed1992

Human T lymphotropic virus (HTLV) type I and II DNA amplification in HTLV-I/II-seropositive blood donors of the French West Indies.

To confirm the presence of DNA from human T lymphotropic virus type I (HTLV-I), HTLV-II, or both in individuals found HTLV-I/II-positive through systematic screening of blood donations in Guadeloupe (French West Indies), 42 blood donors repeatedly positive for HTLV-I/II by ELISA were studied by polymerase chain reaction (PCR). Three primer pairs (env, pol, tax) targeted on conserved regions of HTLV-I or -II sequences (or both) and six probes (two generic, two HTLV-I-specific, two HTLV-II-specific) were used in a multiplex PCR. HTLV-I sequences were detected in 31 individuals (74%). All 31 subjects positive by Western blot (WB) harbored HTLV-I sequences. Fifteen individuals (48%) were positive with the three primer pairs used, 10 (32%) with two, and 6 (20%) with one. Subjects indeterminate or negative by WB were all negative by PCR. No HTLV-II sequences were detected with specific probes. The results indicate the absence of HTLV-I and -II infection in individuals with indeterminate WB, the presence of HTLV-I DNA in individuals positive for WB in the French West Indies, and the absence of HTLV-II infection in the cohort.

Adult↗

PubMed2006

In vitro activities of ceftobiprole, tigecycline, daptomycin, and 19 other antimicrobials against methicillin-resistant Staphylococcus aureus strains from a national survey of Belgian hospitals.

The in vitro activities of 22 antimicrobial agents, including ceftobiprole, daptomycin, and tigecycline, against 511 methicillin-resistant Staphylococcus aureus (MRSA) isolates from 112 Belgian hospitals were studied by using the CLSI agar dilution method. Isolates were characterized by pulsed-field gel electrophoresis (PFGE) analysis and by PCR detection of determinants of resistance to aminoglycosides, macrolides-lincosamides-streptogramins, and tetracyclines. A representative set of isolates with different PFGE genotypes was further characterized by multilocus sequence typing, determination of staphylococcal cassette chromosome mec (SCCmec) type, and multiplex PCR for toxic shock syndrome type 1 (TSST-1) and Panton-Valentine leukocidin genes. MRSA isolates belonged to nine epidemic MRSA clones, of which sequence type 45 (ST45)-SCCmec IV and ST8-SCCmec IV were predominant, accounting for 49 and 20% of isolates, respectively. The distribution of antimicrobial resistance and TSST-1 genes was strongly linked to clonal types. Ceftobiprole, daptomycin, and tigecycline showed high activity against all isolates of these sporadic and epidemic MRSA clones, as indicated by MIC(90)s of 2 mg/liter, 0.5 mg/liter, and 0.25 mg/liter, respectively. The MIC distribution of daptomycin and tigecycline was not different in isolates with decreased susceptibility to glycopeptides or tetracyclines, respectively. Ceftobiprole MICs were not correlated with oxacillin and cefoxitin MICs. These data indicate excellent activity of the newly developed agents ceftobiprole, daptomycin, and tigecycline against MRSA isolates recently recovered from hospitalized patients in Belgium, supporting their therapeutic potential for nosocomial MRSA infections.

Anti-Bacterial Agents↗

PubMed2006

Biochemical and molecular characterization of tetracycline-resistant Aeromonas veronii isolates from catfish.

Eighty-one tetracycline-resistant Aeromonas sp. strains were isolated from farm-raised catfish. Morphological and biochemical characteristics indicated that 23 of the 81 aeromonads were Aeromonas hydrophila, 7 isolates were Aeromonas trota, 6 isolates were Aeromonas caviae, 42 isolates were Aeromonas veronii, and 3 isolates were Aeromonas jandaei. However, the AluI and MboI restriction fragment length polymorphism (RFLP) patterns of the PCR-amplified 1.4-kb 16S rRNA gene from all 81 tetracycline-resistant aeromonads from catfish were identical to the RFLP banding patterns of A. veronii ATCC 35626, indicating that all 81 isolates were strains of A. veronii. A multiplex PCR assay successfully amplified the 5 tetracycline-resistant genes (tetA to E) from the genomic DNA of all 81 isolates. The assay determined that tetE was the dominant gene occurring in 73/81 (90.0%) of the aeromonads. Plasmids (2.0 to 20 kb) were isolated from 33 of the 81 isolates. Dendrogram analysis of the SpeI pulsed-field gel electrophoresis identified 15 distinct macrorestriction patterns among the isolates. Our results indicate the need for use of 16S rRNA in the identification of Aeromonas spp. and the prevalence of catfish as a reservoir of tet genes.

Aeromonas↗

PubMed2001

[Molecular identification of sibling Anopheles species: example of the Anopheles minimus and Anopheles dirus complexes, major malarial vectors in Southeast Asia].

Effective control of malaria vectors requires precise identification of species. This is especially important within complexes of species that cannot be distinguished based on morphological features. Two methods based on polymerase chain reaction (PCR) analysis have been developed to identify 2 species in the Anopheles minimus complex and 5 species of the Minimums group as well as 4 species of the Anopheles dirus complex. Association of oligonucleotide couples in the form of multiplex PCR has allowed development of two simple, reliable PCR techniques adapted to each one of these species complexes that comprise the major vectors of malaria in Southeast Asia. Specifically designed to meet the needs of entomologist working in the field for reliable, cost-effective tests, these techniques will facilitate assessment of the geographical distribution of each vector. These data will help to better target vector control measures.

Animals↗