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Results for “Multiplex PCR”

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PubMed1994

Chromosome assignment of human brain expressed sequence tags (ESTs) by analyzing fluorescently labeled PCR products from hybrid cell panels.

Sixty-three human brain cDNA sequences were newly assigned to individual human chromosomes. Ten of these were subregionally localized, and one was also mapped in the mouse genome. Four previously reported assignments were refined. PCR primers were designed from expressed sequence tags (ESTs) and tested for specific amplification from human genomic DNA. DNA was then amplified, often in multiplexed PCR reactions, using DNA from somatic cell hybrid mapping panels as templates. The amplification products were identified using an automated fluorescence detection system. Chromosomal assignments were made by discordancy analysis. Thirteen newly localized cDNAs exhibited homology to previously reported sequences. EST01471 was shown to correspond to human microtubule-associated protein 1B (MAP1B), confirming the previous mapping of this gene to human chromosome 5. Other genes tentatively assigned to chromosomes based on these results were a component of the signal peptide receptor of the endoplasmic reticulum (EST00745) and a cyclic AMP-regulated phosphoprotein (EST01041) on chromosome 1, a protein phosphatase 2A 55-kDa regulatory subunit (EST01650) on chromosome 4, an NAD(P) transhydrogenase (EST01744) on chromosome 5, ribosomal proteins L1a or L1b (EST01627) and L18a (EST01583), a brain transcription factor (BF-1, EST00795) on chromosome 14, a milk fat globule membrane-related protein (EST01678) on chromosome 15, a putative peptide initiation factor (EST00675) on chromosome 17, thiosulfate sulfurtransferase (TST) on chromosome 22, and moesin (MSN) (EST00896) and a human equivalent of rat spot 14 (S14) (EST00887) on Xp11-->cen and Xpter-->p21.3, respectively.

Animals↗

PubMed2004

Molecular typing of enterotoxigenic Staphylococcus aureus isolated from bovine mastitis in Korea.

One hundred and sixty-six Staphylococcus aureus isolates from mastitic milk samples from different cows on 26 farms were investigated for staphylococcal enterotoxins(SEs) and toxic shock syndrome toxin-1(TSST-1) by polymerase chain reaction(PCR) and reverse passive latex agglutination assay(RPLA). SEs and the TSST-1 gene were detected in thirty-seven isolates based on a multiplex PCR; SEA was detected in 32 isolates, SEB in 3 isolates, SEC in 1 isolate, and SEA and the TSST-1 gene in 1 isolate. Of the 37 enterotoxigenic isolates, thirty-three isolates were enterotoxigenic according to RPLA, where 29 isolates produced SEA, 3 isolates produced SEB, and 1 isolate produced SEC. The enterotoxin-producing S. aureus isolates were further characterized by pulsed-field gel electrophoresis(PFGE). A macrorestriction analysis revealed 11 PFGE patterns. Among the 33 enterotoxigenic S. aureus isolates, 45.4% exhibited the same PFGE pattern I. Accordingly, although the enterotoxin-producing S. aureus isolates from bovine mastitis were genetically diverse, 1 common genotype prevailed on the farms, indicating that PFGE pattern I isolates may be the most disseminated in Korea.

Animals↗

PubMed2004

Sex chromosome rearrangements leading to partial aneuploidies and mosaicisms: use of QF-PCR for detection and quantification of the involved cell lines.

Chromosome rearrangements can lead to aneuploidies of specific chromosome regions and could be present in the entire individual or limited to some tissues (mosaicism) depending on the developmental stage of the embryo when the rearrangement occurs. We report 6 cases with sex chromosome rearrangements identified by conventional cytogenetics and tested by quantitative fluorescent polymerase chain reaction (QF-PCR). QF-PCR has been largely employed for rapid detection of common aneuploidies in pre-natal and post-natal diagnosis and consists in DNA amplification by polymerase chain reaction (PCR) using fluorescent labelled primers and the analysis of chromosome specific small tandem repeats (STR). We tested 5 sex chromosome specific STR markers in multiplex PCR amplifications together with other chromosome specific STR markers as control amplifications. The PCR products were analysed by capillary electrophoresis. The results from QF-PCR analysis were obtained within one day and confirmed our cytogenetic observations. This study shows that QF-PCR analysis can detect sex chromosome imbalance and also suspect mosaicism or chromosome rearrangement.

Aneuploidy↗

PubMed2004

Alloiococcus otitidis in acute otitis media.

OBJECTIVE: The bacterium Alloiococcus otitidis has been found to be associated with otitis media with effusion (OME). When the culture method is used, its detection rate is low, whereas applying the polymerase chain reaction (PCR) yields significantly higher frequencies. This study was carried out to investigate the incidence of A. otitidis in children with acute otitis media (AOM). METHODS: Multiplex PCR was used to detect A. otitidis together with Haemophilus influenzae, Moraxella catarrhalis, and Streptococcus pneumoniae in the middle ear effusions (MEEs) of 118 children with AOM. The clinical outcome of AOM and the bacterial findings of MEEs were compared. RESULTS: A. otitidis was detected in 25% (30 of 118) of the tested MEE samples. Children over 2 years of age had significantly more often A. otitidis-positive MEEs (37%; 22 of 59) than younger children (14%; 8 of 59) (chi-square test, P<0.01). There were no significant differences in the duration, clinical failures (after antibiotic treatment), or number of recurrences of AOM between the A. otitidis-positive and A. otitidis-negative children. CONCLUSIONS: A. otitidis is found from the MEEs of AOM. The present data suggest that it has no clinical significance in AOM, and it does not increase the risk of developing OME after AOM.

Acute Disease↗

PubMed2002

Evaluation of selective enrichment PCR procedures for Yersinia enterocolitica.

Four enrichment PCR protocols for detecting unlysed cells of pathogenic Yersinia enterocolitica were studied. First, the probability of detecting Y. enterocolitica cells of known concentrations by a multiplex PCR assay was determined, and it was found to follow a logistic regression model. From this model, the probability of detecting Y enterocolitica at a specific concentration could be estimated; for example, the detection probability of 10(4) CFU/ml was estimated to be 85.4%. The protocols were evaluated on enrichment cultures inoculated with 10(2) CFU/ml Y. enterocolitica and 10(2)-10(6) CFU/ml of a defined background flora. For each protocol, the time for sample withdrawal and the presence of background flora were studied with respect to PCR detection. The optimal point in time of sample withdrawal was found to be different for each protocol employed. Early detection was favoured by concentrating the target cells, and the most rapid PCR detection of Y. enterocolitica was achieved with enrichment in Yersinia-PCR-compatible-enrichment (YPCE) medium for 3 h at 25 degrees C, followed by a centrifugation prior to PCR analysis. For detection of Y. enterocolitica in the presence of high concentrations (10(6) CFU/ml) of background flora, a long incubation time followed by density centrifugation and a dilution step was most successful. The protocol that gave the most reliable PCR detection in the presence of 10(6) CFU/ml background flora included 24 h incubation in Yersinia-selective-enrichment (YSE) broth at 25 degrees C, followed by Percoll density centrifugation, and a 100 times dilution prior to PCR analysis.

Colony Count, Microbial↗

PubMed2005

First report on the occurrence of 'Helicobacter heilmannii' in the stomach of rabbits.

Gastric Helicobacter spp. have been described in a wide range of animal species, including dogs, cats, primates, swine, cattle and rodents. However, in lagomorphs--more specifically rabbits--gastric Helicobacter infections have never been reported. Biopsy specimens were collected from different stomach regions of 23 rabbits, including 10 pet rabbits, 10 industrial animals and 3 research animals. These were subjected to a PCR assay for the detection of Helicobacter DNA. Identification up to the species level was based on 16S rRNA sequence analysis and a recently developed multiplex PCR. Seven rabbits (four pet, one research animal and two industrial animals) tested positive in the Helicobacter genus-specific PCR in the stomach, with the corpus being predominantly positive. H. felis and H. salomonis, hitherto presumed to be naturally hosted by cats and dogs, were detected in three animals and one animal, respectively. One of these animals had been completely devoid of any form of contact with cats or dogs. A H. pullorum/H. rappini-like organism (96% 16S rDNA sequence similarity) was found in an industrially held rabbit. The helicobacters of the two remaining rabbits could not be identified up to the species level. To conclude, this is the first report on the occurrence of Helicobacter spp. in the stomach of rabbits. In view of the fact that H. felis and H. salomonis are put forward as having zoonotic potential, further research is necessary to investigate the implications of these findings not only for the rabbit but also for human health.

Animals↗

PubMed2001

Diagnosing genital ulcer disease in a clinic for sexually transmitted diseases in Amsterdam, The Netherlands.

The most common etiologic agents of genital ulcer disease (GUD) are herpes simplex virus type 1 (HSV-1), HSV-2, Treponema pallidum, and Haemophilus ducreyi. In an outpatient clinic for sexually transmitted diseases in Amsterdam, The Netherlands, specimens from 372 patients with GUD were collected from February to November 1996. Sera were collected at the time of the symptoms and, for most patients, also during follow-up visits. Swabs in viral transport medium were used for HSV culture and for detection of DNA. The most prevalent pathogen found was HSV-2, which was detected by culture in 35% of the patients and by PCR in 48% of the patients. Also, HSV-1 infection was more often detected by PCR (7.8%) than by culture (5.6%). Evidence for an active infection with T. pallidum was found in 1.9% of the patients, using serological tests. A multiplex PCR for simultaneous T. pallidum and H. ducreyi DNA detection was positive for T. pallidum in 3.3% of the samples and for H. ducreyi in only 0.9% (3 out of 368) of the samples. The sensitivity of the PCR was superior to that of culture for HSV detection and to that of serology for T. pallidum detection. Specific H. ducreyi immunoglobulin G antibodies were detected in sera of 5.2% of the patients, with no concordance between serology and PCR. In 37% of the cases, none of the tested microorganisms was detected. Performance of PCR in addition to conventional techniques significantly improved the diagnosis of GUD.

Chancroid↗

PubMed2005

Suspension microarray with dendrimer signal amplification allows direct and high-throughput subtyping of Listeria monocytogenes from genomic DNA.

Listeria monocytogenes is a significant cause of food-borne disease and mortality; therefore, epidemiological investigations of this pathogen require subtyping methods that are rapid, discriminatory, and reproducible. Although conventional microarray subtyping analysis has been shown to be both high resolution and genetically informative, it is still relatively low throughput and technically challenging. Suspension microarray technology eliminates the technical issues associated with planar microarrays and allows high-throughput subtyping of L. monocytogenes strains. In this study, a suspension array assay using dendrimer signal amplification allowed rapid and accurate serovar identification of L. monocytogenes strains using genomic DNA as a target. The ability to subtype genomic DNA without PCR amplification allows probes to be designed for many different regions within the bacterial genome and should allow high-resolution subtyping not possible with multiplex PCR.

Bacterial Typing Techniques↗

PubMed2006

Multiplex detection of Enterocytozoon bieneusi and Encephalitozoon spp. in fecal samples using real-time PCR.

A multiplex real-time polymerase chain reaction (PCR) method was developed for the simultaneous detection of Enterocytozoon bieneusi (n = 30) and Encephalitozoon spp. (n = 3) in stool samples. The multiplex PCR also included an internal control to detect inhibition of the amplification by fecal constituents in the sample. The assay was performed on species-specific DNA controls (n = 22) and a range of well-defined stool samples (n = 140), and it achieved 100% specificity and sensitivity. The use of this assay in a diagnostic laboratory offers the possibility of introducing DNA detection as a feasible technique in the routine diagnosis of intestinal microsporidian infections.

Animals↗

PubMed2005

Simultaneous single-tube PCR assay for the detection of Neisseria meningitidis, Haemophilus influenzae type b and Streptococcus pneumoniae.

Rapid, accurate and inexpensive diagnosis of bacterial meningitis is critical for patient management. This study describes the development and evaluation of a multiplex PCR assay for the detection of Neisseria meningitidis, Streptococcus pneumoniae and Haemophilus influenzae type b, which globally account for 90% of cases of bacterial meningitis. The single-tube assay, based on the ctrA, ply and bex targets, respectively, enabled detection of 5-10 pg DNA. When the assay was tested with clinical samples (n = 425), its sensitivity for the three targets was 93.9%, 92.3% and 88%, respectively, while the overall specificity and positive predictive value of the assay was 100%. The negative predictive value was 99.1-99.5%. The methodology permits rapid and accurate detection of the three main pathogens that cause bacterial meningitis.

Bacterial Proteins↗

PubMed2000

Development and validation of a detection system for wild-type Vibrio cholerae in genetically modified cholera vaccine.

Orochol, a live oral cholera vaccine licensed in Switzerland and in other countries, is based on the genetically modified Vibrio cholerae strain CVD103-HgR. This strain is derived from the wild-type O1 strain Inaba 569B by deletion of a fragment internal to the ctxA gene encoding the A1 subunit of cholera toxin and by replacement of an internal fragment of the hlyA gene with a fragment carrying the mer operon mediating mercury resistance. In this study we describe a polymerase chain reaction (PCR) system for the detection of wild-type Vibrio cholerae and the identification of the vaccine strain for the quality control of production batches. A multiplex PCR system that targets the intact ctxA gene of the wild-type strain and simultaneously the integration site of the mer operon in the hlyA gene (hlyA::mer) of the vaccine strain CVD103-HgR was developed. To evaluate the detection limit of the system, vaccine suspensions were artificially contaminated with wild-type V. cholerae 569B cells and tested by PCR. The detection limit of the system was statistically evaluated and found to be at 11625 wild-type cells per vaccine sachet (95% confidence limit). This number is below the infective dose of wild-type Vibrio cholerae. In Switzerland this test is used in combination with other tests in the official batch-release procedure to assure the safety of each batch of the cholera vaccine Orochol.

Cholera Toxin↗

PubMed2002

Single cell RT-PCR analysis of tyrosine kinase receptor expression in adult rat retinal ganglion cells isolated by retinal sandwiching.

We describe a protocol for analysis of gene expression in single, acutely dissociated adult rat retinal ganglion cells using RT-PCR. Retrograde tracing of retinal ganglion cells from the superior colliculi was conducted using Fluorogold. Retinas were dissected and ganglion cells isolated using retinal layer separation (sandwiching). Single, fluorescently labelled retinal ganglion cells were aspirated using a micropipette and used for PCR. Two PCR protocols are described where single cell cDNA was analysed for TrkB and GAPDH or TrkB, TrkC, Ret, Met, ErbB2 and Beta-actin by multiplex-PCR. All five tyrosine kinase receptors were amplified from single retinal ganglion cells. The method will prove useful for the molecular characterization of adult retinal ganglion cells.

Animals↗

PubMed2005

Identification and quantification of three genetically modified insect resistant cotton lines using conventional and TaqMan real-time polymerase chain reaction methods.

As the genetically modified organisms (GMOs) labeling policies are issued in many countries, qualitative and quantitative polymerase chain reaction (PCR) techniques are increasingly used for the detection of genetically modified (GM) crops in foods. Qualitative PCR and TaqMan real-time quantitative PCR methods to detect and identify three varieties of insect resistant cotton, i.e., Mon531 cotton (Monsanto Co.) and GK19 and SGK321 cottons (Chinese Academy of Agricultural Sciences), which were approved for commercialization in China, were developed in this paper. Primer pairs specific to inserted DNAs, such as Cowpea trypsin inhibitor (CpTI) gene of SGK321 cotton and the specific junction DNA sequences containing partial Cry1A(c) gene and NOS terminator of Mon531, GK19, and SGK321 cotton varieties were designed to conduct the identified PCR assays. In conventional specific identified PCR assays, the limit of detection (LOD) was 0.05% for Mon531, GK19, or SGK321 in 100 ng of cotton genomic DNA for one reaction. Also, the multiplex PCR method for screening the three GM cottons was also established, which could save time and cost in practical detection. Furthermore, a real-time quantitative PCR assay based on TaqMan chemistry for detection of insect resistant gene, Cry1A(c), was developed. This assay also featured the use of a standard plasmid as a reference molecule, which contained both a specific region of the transgene Cry1A(c) and an endogenous stearoyl-acyl carrier protein desaturase (Sad1) gene of the cotton. In quantitative PCR assay, the quantification range was from 0.01 to 100% in 100 ng of the genome DNA template, and in the detection of 1.0, 3.0, and 5.0% levels of three insect resistant cotton lines, respectively, all of the relative standard deviations (RSDs) were less than 8.2% except for the GM cotton samples with 1.0% Mon531 or GK19, which meant that our real-time PCR assays involving the use of reference molecule were reliable and practical for GM insect resistant cottons quantification. All of these results indicated that our established conventional and TaqMan real-time PCR assays were applicable to detect the three insect resistant cottons qualitatively and quantitatively.

Animals↗

PubMedDate not supplied

Genetic deletions of glutathione-S-transferase as a risk factor in squamous cell carcinoma of the larynx: a preliminary report.

PURPOSE: To test whether genetic deletions of glutathione-S-transferase (GST) are associated with squamous cell carcinoma (SCC) of the larynx. GST are a group of detoxifying enzymes that may help reduce the risk of developing cancer in response to environmental carcinogens. Polycyclic aromatic hydrocarbons, found in high concentration in cigarette smoke, are known carcinogens especially for SCC of the larynx. Individuals with absolute or relative deficiency of the GST enzyme system may therefore be at a higher risk of developing laryngeal carcinoma. MATERIALS AND METHODS: Genotyping for GST-M1 and GST-T1 was performed using polymerase chain reaction (PCR) assay on fresh frozen tissue specimens of 20 patients with SCC of the larynx and on 20 control subjects with a similar smoking history. Because this assay results in the absence of a PCR product in individuals expressing the GST-M1/GST-T1 null genotype, oligonucleotide primers that amplify a portion of the albumin gene were included in a multiplex PCR as a positive control for DNA quality and PCR conditions. The chi-square test was used for statistical analysis. RESULTS: GST-M1 gene was deleted in 80% of patients with laryngeal SCC and in 50% of control subjects (P <.05). No statistically significant difference was observed in the incidence of GST-T1 gene deletion in patients with SCC of the larynx and control subjects. CONCLUSION: GST-M1 gene deletion was significantly associated with SCC of the larynx and may produce a risk for this particular disease.

Carcinoma, Squamous Cell↗

PubMed1997

Detection of Neisseria gonorrhoeae and Chlamydia trachomatis in genitourinary specimens from men and women by a coamplification PCR assay.

A coamplification PCR test for the direct detection of Neisseria gonorrhoeae and Chlamydia trachomatis in urethral and endocervical swabs and urine samples from men and women was compared to standard culture techniques. Processed specimens were amplified in single reaction tubes containing primers for both organisms, and PCR products were detected by a colorimetric microwell plate hybridization assay specific for each pathogen. Of 344 specimens from men, 45 (13.1%) urine specimens were PCR positive for C. trachomatis, 51 (14.8%) urethral swab specimens were PCR positive, and 29 urethral swab specimens (8.4%) were culture positive. After analysis of discrepancies, the resolved sensitivity and specificity of PCR for C. trachomatis were 96.2 and 99.3%, respectively, in urethral swab specimens, compared to 88.2 and 98.6% for urine specimens. Of the 192 specimens from women, 28 (14.6%) urine specimens were PCR positive for C. trachomatis, 32 (16.7%) endocervical specimens were PCR positive, and 19 (9.9%) endocervical specimens were culture positive. After analysis of discrepancies, the resolved sensitivity and specificity of PCR for C. trachomatis for endocervical specimens were both 100% compared to 100 and 99.4%, respectively, for urine specimens from women. In men, 68 (19.8%) urine specimens were PCR positive for N. gonorrhoeae, 73 (21.2%) urethral swabs were PCR positive, and 59 (17.2%) urethral swabs were culture positive. After analysis of discrepancies, the resolved sensitivity and specificity of PCR for N. gonorrhoeae were 97.3 and 97.0%, respectively, for urethral specimens compared to 94.4 and 98.5% for urine specimens. In women, 18 (9.4%) urine specimens were PCR positive for N. gonorrhoeae, 23 (12.0%) were endocervical swab PCR positive, and 15 (7.8%) endocervical specimens were culture positive. After analysis of discrepancies, the resolved sensitivity and specificity of PCR for N. gonorrhoeae were 100 and 99.4%, respectively, for endocervical specimens compared to 90.0 and 95.9% for female urine specimens. These results indicate that a multiplex PCR is highly sensitive for detecting both C. trachomatis and N. gonorrhoeae from a single urine or genital swab, providing a more cost-effective way of screening multiple pathogens.

Chlamydia Infections↗

PubMed2006

Correlation between deletion patterns of SMN and NAIP genes and the clinical features of spinal muscular atrophy in Indian patients.

BACKGROUND: Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder involving degeneration of anterior horn cells of spinal cord resulting in progressive muscle weakness and atrophy. AIMS: The molecular analysis of two marker genes for spinal muscular atrophy (SMA) i.e, the survival motor neuron gene (SMN) and the neuronal apoptosis inhibitory protein gene (NAIP) was conducted in 39 Indian patients with clinical symptoms of SMA. Out of these, 28 showed homozygous deletions and the phenotypic features of these SMA patients were compared with the corresponding genotypes. SETTINGS: A tertiary care teaching Hospital. DESIGN: This is a prospective hospital based study. MATERIALS AND METHODS: Polymerase chain reaction (PCR) combined with restriction fragment length polymorphism (RFLP) was used to detect the deletion of exon 7 and exon 8 of SMN1 gene, as well as multiplex PCR for exon 5 and 13 of NAIP gene. RESULTS: Exons 7 and 8 of SMN and NAIP (exon 5) were homozygously deleted in 73% of SMA I and 27% of SMA II patients. SMN exon 7 and 8 deletions without NAIP deletions were seen in 27% of type I SMA and 46% of SMA type II patients. Two patients of type III SMA showed single deletion of SMN exon 7 along with 27% of SMA type II patients. CONCLUSION: With the advent of molecular biology techniques, SMN gene deletion studies have become the first line of investigation for confirmation of a clinical diagnosis of SMA. The findings of homozygous deletions of exons 7 and/or 8 of SMN1 gene confirms the diagnosis of SMA, even in patients with atypical clinical features. Deletions of NAIP gene were mainly seen in severely affected patients, hence is useful for predicting the prognosis.

Adolescent↗

PubMed2004

Development of a PCR test to differentiate between Staphylococcus aureus and Staphylococcus intermedius.

The presence of Staphylococcus intermedius in food remains unclear because routine laboratory analysis does not discriminate between S. intermedius and Staphylococcus aureus, a major cause of food poisoning. Both species share many phenotypic characteristics, including coagulase and thermonuclease production. In both species, some strains can produce enterotoxin and therefore can be the cause of food poisoning outbreaks. Although the ID32 Staph System (bioMérieux, SA, Marcy l'Etoile, France), based on a miniaturized phenotypic characterization, gives satisfactory results for discriminating between these two species, some rapid molecular PCR-based methods have been developed to identify S. aureus specifically, but they do not identify S. intermedius. Here, we developed a rapid, accurate, and discriminative multiplex PCR method that targets species-specific sequences in the nuc gene, which encodes thermonuclease in the two species. The test includes an internal positive control that targets a highly conserved region of 16S ribosomal RNA gene (rDNA). A total of 116 strains were used to validate our test. The test gave no signal on the following Staphylococcus species: S. epidermidis, S. chromogenes, S. hyicus, S. warneri, S. xylosus, S. lentus, and S. sciuri. It allowed a 100% successful discrimination between S. aureus (44 strains tested) and S. intermedius (57 strains) isolated from different origins.

Animals↗

PubMed2003

[Four Y-STR multiplex system by silver staining].

A multiplex PCR system has been developed to amplify 4 Y-chromosome specific short tandem repeats (STR), DYS391, GATA-A4, GATA-A10 and GATA-H4, simultaneously. PCR products were separated by polyacrylamide gels electrophoresis followed by silver stain. When 311 unrelated males from the Han population in Guangdong were detected by the multiplex system, DYS391, GATA-A4, GATA-A10 and GATA-H4 showed 5, 7, 6 and 5 alleles respectively. Total 98 haplotypes could be identified. Gene diversity value for the 4 STR was 0.4623, 0.6972, 0.7173 and 0.6015 respectively. The gene diversity value for the haplotypes of the 4 Y-STR reached 0.9755. The four Y-STR multiplex system will be very powerful for establishing Y-STR database, exploring human origin, paternity testing and personal identification.

English Abstract↗