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PubMedDate not supplied

[Detection of the genes of pyrogenic toxins of superantigens in clinical isolates of methicillin resistant Staphylococcus aureus].

The content of methicillin resistant S. aureus (MRSA) genes, coding the synthesis of staphylococcal enterotoxins A, B, C (sea, seb, sec) and the toxin of the toxic shock syndrome (tst-H) which was classified with pyrogenic toxins of superantigens (PTSAgs), was studied with the use of PCR amplification. The study revealed the specific features of the content of genes sea and sec, detected in epidemic strains, identified earlier and found to circulate in Russian hospitals. Among the isolates, genetically related to international epidemic strain EMRSA-1, isolates containing no gene sea were detected, while among the isolates genetically related to strain EMRSA-2, isolates containing not only gene sea, but also gene sec were detected, which was indicative of the tendence of this epidemic strain in the direction of further acquisition of pathogenicity genes. As revealed in further studies, among the cultures obtained in bacteriemia, 88% contained gene sea. Two out of three isolates obtained from patients with the symptoms of toxic shock also contained this gene. The differences in the content of genes PTSAgs (sea, seb, sec and tst-H) could serve as a genetic criterium for the differention of isolates circulating in a hospital, as well as for a more complete characterization of the epidemic strains MRSA. The determination of the given genetic markers in genetic strains in circulating strains will make it possible to prognosticate the structure, severity and outcomes of hospital infections. The conditions of PCR amplification for the determination of genes sea, seb, sec and tst-H, as well as multiplex PCR for the determination of genes sea and seb, were developed.

Anti-Bacterial Agents↗

PubMed2003

Design of degenerate primers for multiplex nested-PCR detection of human lymphotropic herpesviruses.

To develop the rapid diagnosis and typing of human lymphotropic herpesviruses by using multiplex nested-PCR, the primary PCR (1(o) PCR) primers were redesigned as degenerate primers based on a highly conserved sequences of each DNA polymerase gene of EBV, CMV, HHV-6, HHV-7 and HHV-8. The forward degenerate primer (HHV/1+) contained 12 different sequences, whereas there were 8 different sequences in the reverse degenerate primer (HHV/ 1-). Optimization of multiplex nested-PCR assay conditions were performed to search for the appropriate amount of degenerate primers, dNTP, Taq DNA polymerase, template of secondary PCR (2(o) PCR) and annealing temperature used in 1(o) PCR reaction. Detection sensitivity was the same as described in previous report (approximately 10-100 genome copies). To ensure a true negative result, PCR detection of hepatitis B virus genome was used as internal control. Our presented results, the designed degenerate primers could be used to detect various types of HHV by multiplex nested-PCR.

DNA Primers↗

PubMed2001

Rapid sexing of preimplantation bovine embryo using consecutive and multiplex polymerase chain reaction (PCR) with biopsied single blastomere.

The objective of this study was to establish a rapid and reliable PCR method for the sexing of 8- to 16-cell stage bovine embryos. The BOV97M and bovine 1.715 satellite DNA sequences were selected for amplification of male- and bovine-specific DNA, respectively. But the unequal number of copies of these two repetitive sequences required some modification of the multiplex PCR method. In consecutive and multiplex PCR, the first 10 PCR cycles were done with male-specific primer followed by an additional 23 cycles with bovine-specific primer. In this PCR method, the appearance of male- and bovine-specific bands was independent of the DNA concentration. This PCR method was applied successfully using groups of 8, 4, 2, and 1 blastomeres dissociated from the embryos, and the sexing efficiency was 100.0, 96.3, 94.3 and 92.1%, respectively. The coincident rate of sex determination between biopsied single blastomere and matched blastocyst was 90.0%. Therefore the developmental potential from 8- to 16-cell stage embryos to the blastocyst stage was not significantly different (P>0.2) for intact embryo (42.3%) than for demi-embryos (53.8%), suggesting that trauma to the demi-embryo caused by single-blastomere aspiration using a bevelled micropipette was very small. In conclusion, we developed a rapid (within 2 hours) and effective PCR method for the sexing of 8- to 16-cell stage bovine embryos using a single blastomere.

Animals↗

PubMed1997

The usefulness of single-strand DNA conformation polymorphism analysis to detect mutations in protein C deficiency.

The standard approach for the molecular genetic analysis of protein C deficiency, polymerase chain reaction (PCR) amplification followed by direct sequencing, although very accurate, is time-consuming. The aim of this study is to investigate the usefulness of a simplified, time-saving screening method for the detection of protein C mutations consisting of the combination of multiplex PCR amplifications using the same primers that were designed for sequencing, followed by single-strand DNA conformation polymorphism (SSCP) electrophoresis analysis performed with one set of conditions. The study was designed in two phases. First, we tested six known point mutations located in different exons of the protein C gene by SSCP. Second, we prospectively studied nine patients with protein C deficiency type I using SSCP as the first screening technique. All the exons were amplified with a common PCR protocol, either as single fragments or as multiplex combinations of several of them. In the retrospective study, three out of the six point mutations were visible as a band shift: 40 T-->G (exon 2), 1432 C-->T (exon 3) and 7253 C-->T (exon 8). In the prospective analysis SSCP detected three different mutations. These mutations were: 6128 T-->C (exon 7), 6216 C-->T (exon 7) and in two probands 8631 C-->T (exon 9). In the five remaining patients we identified only two different mutations by direct sequencing: 6246 G-->A (exon 7) in two patients and 8589 G-->A (exon 9) in four patients. In summary, the results from both studies show that only 60% of all mutations can be detected using this simplified method. It also suggests that a multiple set of conditions, smaller PCR fragments, or both, should be used in order to achieve a sensitivity comparable to sequencing.

Genetic Testing↗

PubMed1993

Towards covering immunological genes with highly informative markers: a trans-species approach.

To establish a highly informative screening system for immunologically relevant genes ("immunoprinting") we co-amplified via polymerase chain reaction (PCR) polymorphic exons plus adjacent intronic simple repetitive dinucleotide stretches in the T-cell receptor (Tcr) Vb6 and Major Histocompatibility Complex (MHC)-DRB loci in man and several ungulate species. In both gene families the basic structure of the simple repeat was found to be preserved for more than 70 x 10(6) years in all investigated species. The simple repeats exhibit extensive length variability. Distinct exon sequences are correlated with a defined repeat length and substructure. In addition, PCR and the oligonucleotides for typing were applicable to a broad range of species from different mammalian orders. Multiplex PCR of different members of the Tcr Vb6 family and MHC-DRB resulted in a complex pattern similar to an oligolocus fingerprint. Hence immunoprinting can be employed for searching for associations of immunologically relevant genes with diseases even across species barriers.

Animals↗

PubMed1998

Determination of HPRT mutant frequency and molecular analysis of T-lymphocyte mutants derived from coke-oven workers.

We measured the frequency of mutant (MF) lymphocytes at the hprt locus in a population of 43 coke-oven workers exposed to PAH and in a group of 26 non-exposed workers. A non-significant increase in MF in the exposed group (19.0 +/- 16.3) compared to the non-exposed group (15.8 +/- 14.6) was observed. Moreover, when we considered smoking habits for the overall population, the MF values were higher, although not significantly, in smokers than in non-smokers. For some T-cell mutant clone structural alterations, splicing and coding errors were detected by PCR-based methods. We analysed 161 HPRT- clones, derived from exposed and non-exposed workers by multiplex-PCR and 56 HPRT- clones by reverse transcriptase-PCR. Overall, the percentages of the different types of gene alterations were similar in exposed and non-exposed subjects. Only the frequency of splice mutations in mutant clones derived from coke-oven workers was higher (22%) than in non-exposed donors (11%).

Adult↗

PubMed1998

A multiplex ARMS test for 10 cystic fibrosis (CF) mutations: evaluation in a prenatal CF screening program.

In Maine, prenatal screening for cystic fibrosis (CF) is offered through primary care providers. Cheekbrush (buccal) samples are routinely tested for eight mutations by multiplex PCR amplification of five exons, followed by dot-blot hybridization with pooled allele-specific oligonucleotides (ASO). The ASO methodology is widely used and effective, but somewhat time and labor intensive when applied to CF carrier testing or couple-based prenatal screening in the general pregnant population. Amplification Refractory Mutation System (ARMS) is an improvement of the PCR that allows rapid detection of mutations involving single base changes or small deletions/insertions. In this study, two multiplex ARMS reactions are used to test for 10 common CF mutations. Clinical evaluation of the ARMS test includes a retrospective study of 140 banked samples (54 cell line, proficiency testing, and buccal controls; 86 clinical buccal samples) with known CF genotype (57 with CF mutations, 83 no mutation), followed by a prospective trial in which 309 buccal samples are analyzed con-currently using both methods. The success rate of the ARMS test in buccal lysates is comparable to the ASO method; all CF mutations are successfully identified. For testing nonsterile buccal lysates with low DNA concentrations, optimized performance in the ARMS method is obtained using Amplitaq Gold polymerase. The ARMS method developed is easy, rapid (1 day), and avoids the need for ASO probe labeling, dot-blotting and autoradiography. This study provides further evidence that ARMS methodology is suitable for clinical CF mutation analysis.

Cheek↗

PubMed2006

Comparison of polymerase chain reaction systems for detection of different cdt genes in Escherichia coli strains.

AIMS: Cytolethal distending toxins (CDT) are tripartite toxins encoded by three adjacent or overlapping genes (cdtA, cdtB, cdtC) and found in multiple pathogens. The present knowledge regarding heterogeneity of cdt genes and our previous study revealed that the available polymerase chain reaction (PCR) systems lack adequate specificity. The detection of various cdt genes present in Escherichia coli strains, from different geographical regions demands further assays for wide-range coverage. On the basis of these observations, we were prompted to undertake the present study; hence the specificity of existing PCR systems was addressed using E. coli prototype strains with known cdt gene sequences. METHODS AND RESULTS: A multiplex PCR designed for the detection of E. coli cdt genes was found to be sensitive and specific enough for initial screening. However, for subtyping, the PCR systems yielded nonspecific products upon amplification. These primers are usually designed for sequences of the cdtB locus (the most conserved region of the gene), and since CDT-producing E. coli strains carry different cdt genes, none of the systems are really type specific. Furthermore, PCR systems with type-specific primers for other regions of the gene, i.e. ORF A or ORF C are found to be strain specific and their applications in different geographical regions have limitations. CONCLUSIONS: In conclusion, based on our observations, using these available primers, it seems that the existing PCR systems are not sufficiently accurate to differentiate between different types of cdt genes. SIGNIFICANCE AND IMPACT OF THE STUDY: The results obtained from this study revealed that so-far reported PCR systems are short in specificity. These PCR protocols were not found to be specific enough to detect various cdt genes and have a limited range of application. Moreover, due to similarities in cdt genes the cross-reaction between different sets of primers exists. Hence for epidemiological studies, some additional PCR protocols are required for screening clinical isolates for cdt genes.

Bacterial Toxins↗

PubMed1992

Rapid and sensitive method for detection of Shiga-like toxin-producing Escherichia coli in ground beef using the polymerase chain reaction.

A rapid and sensitive method for detection of Shiga-like toxin (SLT)-producing Escherichia coli (SLT-EC) with the polymerase chain reaction (PCR) is described. Two pairs of oligonucleotide primers homologous to SLTI and SLTII genes, respectively, were used in multiplex PCR assays. The first pair generated a ca. 600-bp PCR product with DNA from all SLTI-producing E. coli tested but not from E. coli strains that produce SLTII or variants of SLTII. The second pair generated a ca. 800-bp PCR product with DNA from E. coli strains that produce SLTII or variants of SLTII but not from SLTI-producing E. coli. When used in combination, the SLTI and SLTII oligonucleotide primers amplified DNA from all of the SLT-EC tested. No PCR products were obtained with SLT primers with DNA from 28 E. coli strains that do not produce SLT or 44 strains of 28 other bacterial species. When ground beef samples were inoculated with SLT-EC strains 319 (O157:H7; SLTI and SLTII), H30 (O26:H11; SLTI), and B2F1/3 (O91:H21; SLTII variants VT2ha and VT2hb) and cultured in modified Trypticase soy broth for 6 h at 42 degrees C, an initial sample inoculum of as few as 1 CFU of these SLT-EC strains per g could be detected in PCR assays with DNA extracted from the broth cultures.

Animals↗

PubMed2002

Genetic diversity of root-knot nematodes from Brazil and development of SCAR markers specific for the coffee-damaging species.

RAPD markers were used to characterize the genetic diversity and relationships of root-knot nematodes (RKN) (Meloidogyne spp.) in Brazil. A high level of infraspecific polymorphism was detected in Meloidogyne arenaria, Meloidogyne exigua, and Meloidogyne hapla compared with the other species tested. Phylogenetic analyses showed that M. hapla and M. exigua are more closely related to one another than they are to the other species, and illustrated the early divergence of these meiotically reproducing species from the mitotic ones. To develop a PCR-based assay to specifically identify RKN associated with coffee, three RAPD markers were further transformed into sequence-characterized amplified region (SCAR) markers specific for M. exigua, Meloigogyne incognita and Meloidogyne paranaensis, respectively. After PCR using the SCAR primers, the initial polymorphism was retained as the presence or absence of amplification. Moreover, multiplex PCR using the three pairs of SCAR primers in a single reaction enabled the unambiguous identification of each species, even in mixtures. Therefore, it is concluded that the method developed here has potential for application in routine diagnostic procedures.

Animals↗

PubMed2000

Allelic loss and microsatellite alterations of chromosome 3p14.2 are more frequent in recurrent cervical dysplasias.

Epidemiological studies have documented the unpredictable clinical progression or recurrence of cervical dysplasia. Recent studies have shown several molecular changes in cervical cancers and their associated dysplasia. We conducted molecular analyses on a retrospectively ascertained cohort of recurrent and nonrecurrent cervical dysplasia cases in an attempt to define molecular biomarkers to predict progressive or recurrent disease. Cases were chosen if long-term follow-up (3-5 years after conization) and biopsy confirmation were available. Paraffin-embedded, postconization cervical tissues from 19 recurrent and 18 nonrecurrent dysplasias were analyzed. Human papillomavirus (HPV) was identified by PCR for general and type-specific (HPV-16 and HPV-18) primers. Allelotyping analysis was performed by multiplex PCR using a panel of 16 microsatellite markers targeting putative tumor suppressor gene regions on chromosomes 3p, 5p, 6p, 9p, 11q, and 17p. The overall rate of HPV infection was similar in both groups. In the allelotyping analysis, loss of heterozygosity at the fragile histidine triad region in 3p14.2 was significantly higher in the recurrent group than in the nonrecurrent group (P = 0.005). Furthermore, microsatellite alterations (MAs) were more frequent in the recurrent group (mean MA index, 0.254) as compared with the nonrecurrent group (mean MA index, 0.085; P = 0.0025). These findings suggest that HPV status alone does not predict recurrence and that loss of heterozygos. ity at the fragile histidine triad region may represent a potential biomarker in predicting recurrence. Frequent MAs in the recurrent group may represent an underlying genomic instability that creates susceptibility for allelic loss, thus increasing the risk for recurrence or progression.

Adult↗

PubMed2000

Paternity testing by PCR-based STR analysis.

We present application of polymerase chain reaction (PCR)-based short tandem repeat (STR) system for use in paternity testing. The process involves a single tube multiplex PCR of 9 STR loci on different chromosomes, in conjunction with Amelogenin sex test and internal size standards, followed by using an automated DNA sequencer to detect amplified products. The results showed that this system provided unambiguously reliable results. In addition, the method is useful for routine use in that it is robust and reproducible and provides a reliable means of paternity testing.

Humans↗

PubMed2005

Detection and identification of Mycobacterium species by polymerase chain reaction (PCR) from paraffin-embedded tissue compare to AFB staining in pathological sections.

BACKGROUND: Polymerase chain reaction (PCR) is a recent, rapid and reliable method in the detection of causative organism. The authors tried to determine the possibility of using PCR technique as an alternative way to detect mycobacterial DNA from paraffin-embedded tissue to avoid repeated biopsy from the patient. MATERIAL AND METHOD: Paraffin-embedded tissue blocks, the corresponding histopathologic slides, and cultural results were retrospectively searched for according to the patient's records, the granuloma clinic, Department of Dermatology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand from 1994-2000. One hundred and thirty-one tissue blocks and slides were found but only 120 cultural results were retrieved Histologic sections were reviewed for AFB findings and PCR was done using 16S rRNA sequences to detect M. tuberculosis by one-tube nested technique and multiplex PCR for M. marinum and M. fortuitum complex. RESULTS: The causative organisms were identified by AFB staining in pathologic sections 31.29%, by PCR 35.87%, and by culture 30.00% of tested samples. The sensitivity of PCR when compared to AFB result was 29.26%, specificity 61.11% but when compared to cultural results, the sensitivity of PCR was 66.67% and AFB sensitivity was 41.66% with specificity 76.19% and 72.61% respectively. CONCLUSION: The low sensitivity of the PCR method may be due to formalin fixation, deparaffinization process, DNA extraction method, the use of 16S rRNA-based primers and the length of the expected product, and the tissue type that may have Taq polymerase inhibitor. Therefore, PCR should be used to augment the information of the conventional method in the diagnosis of mycobacterial infection.

Adolescent↗

PubMed2005

[The discovery of a specific DNA fragment associated with maize cytoplasmic male sterility and its differential display].

Three pairs of PCR primers were designed according to the mitochondrial DNA sequence. PCR amplification was applied to 3 sets of isonuclear alloplasm materials and 3 sets of isoplasm allonuclear materials. Multiplex PCR and general PCR protocol were adopted with total genomic DNA. As for the primers having detected polymorphsim between male sterility and its maintainers, differential display was conducted with mRNA from different development stage of microspore. The results showed as follows: with total genomic DNA template, primer P1-P2 has amplified a specific fragment only in all the male sterile materials, primer P5-P6 has amplified a specific fragment only in maintainer Huangzaosi, primer P3-P4 has no amplification in all the experiment materials. So primer P1-P2 can be used to distinguish male sterile cytoplasm and normal cytoplasm. RT-PCR was conducted with primer P1-P2 in inbred line huangzaosi and 48-2 with male sterile cytoplasm and normal cytoplasm, mRNA was separately isolated from tetrad stage, uninucleate stage and binucleate stage of microspore development, cDNA was obtained with random hexanucleotide primers. With the cDNA template, specific amplified fragments were also detected by primer P1-P2 in the male sterile materials at different development stage of microspore, but there was no amplification by primer P1-P2 in the 2 maintainer lines. This result indicated that primer P1-P2 can be transcripted at 3 development stages of microspore in all male sterile materials, and same transcript was produced by primer P1-P2 among all male sterile materials include 3 sets of isonuclear alloplasm and 3 sets of isoplasm allonuclear. It was suggested from this experiment that the specific DNA sequence detected by primer P1-P2 in all male sterile material total genomic DNA might be related to the cytoplasmic male sterile character.

Cytoplasm↗

PubMed2002

Real-time quantitative polymerase chain reaction diagnosis of infectious posterior uveitis.

OBJECTIVE: To validate a real-time polymerase chain reaction (PCR) assay allowing rapid and sensitive detection and quantitation of 4 common infectious posterior uveitis pathogens. METHODS: A real-time PCR assay using previously validated primer sets for cytomegalovirus, herpes simplex virus, varicella-zoster virus, and Toxoplasma gondii was developed. A standard curve for quantitation of pathogen load was generated for each pathogen using SYBR Green I fluorescence detection. Ocular samples from patients with posterior uveitis and from negative control samples were assayed and compared with standards to identify pathogens and quantify infectious load. RESULTS: Sensitivity for detection of purified pathogen DNA by PCR was not reduced by application of the real-time method. Standard curves for the quantitation of pathogen loads showed sensitivity to fewer than 10 organisms for all pathogens. The technique was applied to 2 clinical problems. First, sensitivities of existing monoplex and multiplex PCR were compared by real-time PCR. No significant difference in sensitivity was observed between multiplex and monoplex techniques. Second, pathogen loads of vitreous specimens from patients previously diagnosed as having infectious posterior uveitis were calculated. Pathogen loads were found to be generally higher for patients with disease caused by varicella-zoster virus than those caused by cytomegalovirus or herpes simplex virus. CONCLUSIONS: Real-time PCR may be applied to infectious agents responsible for posterior uveitis. This technique will likely prove useful for the diagnosis of posterior uveitis as well as the linkage of pathogen to disease. CLINICAL RELEVANCE: Real-time PCR provides a rapid technique for quantitatively evaluating ocular samples for the presence of infectious pathogens.

Animals↗

PubMed1991

Human glandular Kallikrein genes: genetic and physical mapping of the KLK1 locus using a highly polymorphic microsatellite PCR marker.

We describe a highly polymorphic microsatellite repeat sequence, KLK1 AC, which is located 3' to the human glandular kallikrein gene (KLK1) at 19q13.3-13.4. A multiplex PCR was developed to simultaneously genotype the KLK1 AC repeat length polymorphism and a similar repeat at the adjacent APOC2 locus at 19q13.2. Genotypes from these two loci in the 40 large kindred pedigrees from the Centre d'Etude du Polymorphisme Humain were used in conjunction with the background genetic map to establish a multipoint linkage map. The KLK1 locus was also localized physically using somatic cell hybrid DNA templates for polymerase chain reaction analysis. Both genetic and physical mapping studies are consistent with the assignment cen-APOC2-KLK1-D19522-qter. The linkage map places KLK1 approximately 10 cM distal to APOC2. These markers therefore flank the myotonic dystrophy gene and may be useful for diagnosis.

Alleles↗

PubMed2004

Amplification refractory mutation system PCR assays for the detection of variola and Orthopoxvirus.

PCR assays that can identify the presence of variola virus (VARV) sequences in an unknown DNA sample were developed using principles established for the amplification refractory mutation system (ARMS). The assay's specificity utilised unique single nucleotide polymorphisms (SNP) identified among Orthopoxvirus (OPV) orthologs of the vaccinia virus Copenhagen strain A13L and A36R genes. When a variola virus specific primer was used with a consensus primer in an ARMS assay with different Orthopoxvirus genomes, a PCR product was only amplified from variola virus DNA. Incorporating a second consensus primer into the assay produced a multiplex PCR that provided Orthopoxvirus generic and variola-specific products with variola virus DNA. We tested two single nucleotide polymorphisms with a panel of 43 variola virus strains, collected over 40 years from countries across the world, and have shown that they provide reliable markers for variola virus identification. The variola virus specific primers did not produce amplicons with either assay format when tested with 50 other Orthopoxvirus DNA samples. Our analysis shows that these two polymorphisms were conserved in variola virus genomes and provide a reliable signature of Orthopoxvirus species identification.

Animals↗

PubMed2000

Cloning and characterization of two repeated sequences in the symbiotic fungus Tuber melanosporum Vitt.

Two repeated DNA sequences of European strains of the symbiotic fungus Tuber melanosporum were isolated and characterized. One of these, SS14, representing about 0.05% of the fungal genome, was shown to be a T. melanosporum-specific sequence by Southern and dot-blot hybridization. The second one, named SS15, is about 0.0025% of the entire genome, and it is specific not only to T. melanosporum but also to the Asian black truffle Tuber indicum. Neither of these two fragments hybridizes with any of the other European truffle species tested. By sequence analysis of these two fragments, PCR primers were designed and used to selectively amplify DNA from T. melanosporum ascocarps and ectomycorrhizae by simple and multiplex PCR. No amplification products were obtained with DNA from either mycorrhizal roots or fruit bodies of other ectosymbiotic fungi. The two identified genomic traits also provided useful information for a better understanding of the phylogenetic relationships among black truffle species and for testing T. melanosporum intraspecific variability.

Journal Article↗