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Results for “Polymerase Chain Reaction”

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At least 127 records · Page 7Linked to original sources

PubMed1997

Detection of swine vesicular disease virus RNA by reverse transcription-polymerase chain reaction.

Two polymerase chain reaction (PCR) assays are described for the detection of swine vesicular disease virus (SVDV) RNA, a reverse transcription PCR (RT-PCR) and a reverse transcription nested PCR (RT-nPCR). Both the RT-PCR and RT-nPCR were able to detect representative members of each of seven phylogenetically distinct groups of SVDV and gave negative results with a range of porcine enteroviruses and of viruses responsible for vesicular conditions in pigs. When combined with a commercial kit for rapid RNA extraction, the RT-PCR was useful for the detection of SVDV in samples of epithelium and faeces from animals with clinical SVD. The addition of a second amplification step to create a nested PCR (RT-nPCR) increased the sensitivity of the technique for the detection of viral RNA (vRNA) in SVDV infected tissue culture fluid by a factor of approximately 1,000, for 100 TCID50 for the RT-PCR to 0.1 TCID50 for RT-nPCR. When combined with a more elaborate extraction procedure for RNA, the RT-nPCR was considerably more sensitive than virus isolation in tissue culture for detecting SVDV in nasal swabs, tissues, and faeces collected from pigs between 7 days and 176 days after infection with a recent European isolate of SVDV. However, stringent conditions are necessary for carrying out the RT-nPCR to minimise the possibility of contamination.

Animals↗

PubMed1999

Principles and applications of the polymerase chain reaction.

The polymerase chain reaction (PCR), a primer-directed in vitro enzymatic reaction for the production of a specific DNA fragment, has dramatically facilitated molecular biology approaches to fundamental research questions, as well introduced DNA testing into a variety of clinical diagnostic areas. Some of the principles of PCR, several recent advances in PCR technology and selected applications are reviewed. A variety of methods for analyzing allelic sequence diversity in amplified DNA have been developed. Genetic testing of the HLA loci, and other genes encoding cytokines and cytokine receptors, has had a significant impact on disease susceptibility studies and in clinical transplantation.

DNA↗

PubMed1996

Specific detection of Mycoplasma iowae using polymerase chain reaction.

A polymerase chain reaction assay was developed for the specific detection of Mycoplasma iowae, a poultry pathogen. Two primers were chosen in the DNA region located immediately 5' of the ribosomal genes operon. Under our PCR conditions, specific amplification of all M. iowae strains tested was achieved, and none of 17 other Mycoplasma species isolated from birds gave an amplification product. We obtained amplification from culture medium samples of M. iowae by using Tth DNA polymerase instead of Taq DNA polymerase, and we were able to detect as few as 10 organisms. Finally, we described a dot blot hybridization test, using cold labelling and chemiluminescence, which is very convenient for routine detection of M. iowae.

Animals↗

PubMed1992

Multiplex polymerase chain reaction.

The polymerase chain reaction (PCR) is a widely utilized assay for specifically amplifying small fragments of DNA. Multiplex PCR is the amplification of more than one DNA fragment per reaction and has many potential uses. When more than one primer set per reaction tube is utilized, the total number of tubes in any one experiment may be reduced, conserving expensive reagents and decreasing possible contamination. Multiplex PCR allows for an assay of the gene of interest and assures that the amplification process proceeds as expected with the use of a companion control genome primer set. Multiplex PCR is useful in assaying DNA extracted from samples of immunocompromised patients in which more than one infectious agent may be suspected such as simultaneous EBV and CMV detection. Multiplex PCR offers many advantages over single reaction PCR and has been found to be an useful adjunct in our laboratory.

Base Sequence↗

PubMed1994

Capillary electrophoresis: detection of hybridization between synthetic oligonucleotides and HIV-1 genomic DNA amplified by polymerase-chain reaction.

The polymerase chain reaction (PCR) is one of the most efficient techniques for measuring the viral load of HIV-infected samples. Determination of the specificity of PCR products is usually based on Southern blotting and hybridization of the amplified DNA to radioactive oligonucleotide probes specific for sequences comprised between the PCR primers. The recent introduction of capillary electrophoresis (CE) for identification of HIV-1 and HTLV-I PCR products appears interesting in light of its reproducibility, sensitivity and because it is fast and suitable for detection of DNA/DNA and DNA/RNA hybrids. We demonstrate that specific hybridization of a HIV-1 oligonucleotide probe to single-stranded DNA obtained by unbalanced PCR is detectable by capillary electrophoresis. This enabled us the application of a one-step, non-radioactive protocol to demonstrate the specificity of amplification of HIV-1 genomic sequences by PCR. This procedure is simple, reproducible and is suggested as an integral part of automated diagnostic systems based on the use of laboratory work stations for DNA isolation, preparation of PCR reactions and analysis of PCR products.

Base Sequence↗

PubMed1991

[Detection of Chlamydia trachomatis by polymerase chain reaction].

A polymerase chain reaction (PCR) procedure was developed for detection of Chlamydia trachomatis. Two oligonucleotide primers based on sequences within the major outer membrane protein gene from C. trachomatis serovar L2 were used. A single DNA fragment was amplified, when C. trachomatis DNA was template for the PCR. No amplified product was detected in Chlamydia psittaci DNA, Chlamydia pneumoniae DNA or other bacterial DNAs. The amplified DNA fragment was detected, when DNA of greater than or equal to 10(2) C. trachomatis per reaction was used as template for the PCR. Thus, the PCR was shown to be specific for C. trachomatis and more sensitive than the enzyme immunoassays for detection of chlamydial antigen and the chlamydial rRNA:DNA probe hybridization method.

Chlamydia trachomatis↗

PubMed1992

Quantitation of thymidylate synthase, dihydrofolate reductase, and DT-diaphorase gene expression in human tumors using the polymerase chain reaction.

A polymerase chain reaction (PCR)-based method was used to quantitate the expression levels of low abundance genes relevant to cancer drug activity. RNA from tumor samples as small as 20 mg was isolated and converted to cDNA using random hexamers. The 5' primers for the PCR contained a T7 polymerase promoter sequence, allowing the PCR-amplified DNA to be transcribed to RNA fragments. In each sample, the linear ranges of amplification of each cDNA of interest were established. Relative gene expressions were calculated by extrapolating the amounts of PCR products generated within the linear amplification regions of each gene to equal volumes of the cDNA solution. The method was accurate to less than a 2-fold difference in expression levels. Using beta 2-microglobulin and beta-actin gene expressions as internal reference standards and cDNA from HT-29 cells as an external linearity standard, we measured the relative expressions of thymidylate synthase, dihydrofolate reductase, and DT-diaphorase in a number of clinical tumor samples. The expressions of these genes varied from 50- to 100-fold among different tumors, although most of the values were grouped within about a 10-fold range. The amount of thymidylate synthase gene expression in tumor tissues was directly proportional to the content of thymidylate synthase protein. Those tumors with the lowest thymidylate synthase expression had the best response to both the 5-fluorouracil-leucovorin and 5-fluorouracil-cisplatin combinations.

Actins↗

PubMed2001

Detection of rodent parvoviruses by use of fluorogenic nuclease polymerase chain reaction assays.

Polymerase chain reaction (PCR) assays have proven useful for detection of rodent parvoviruses in animals and contaminated biological materials. Fluorogenic nuclease PCR assays combine PCR with an internal fluorogenic hybridization probe, eliminating post-PCR processing and potentially enhancing specificity. Consequently, three fluorogenic nuclease PCR assays were developed, one that detects all rodent parvoviruses, one that specifically detects minute virus of mice (MVM), and one that specifically detects mouse parvovirus 1 (MPV) and hamster parvovirus (HaPV). When rodent parvoviruses and other rodent DNA viruses were evaluated, the rodent parvovirus assay detected only rodent parvovirus isolates, whereas the MVM and MPV/HaPV assays detected only the MVM or MPV/ HaPV isolates, respectively. Each assay detected the equivalent of 10 or fewer copies of target template, and all fluorogenic nuclease PCR assays exceeded the sensitivities associated with previously reported PCR assays and mouse antibody production testing. In addition, each fluorogenic nuclease PCR assay detected the targeted parvovirus DNA in tissues obtained from mice experimentally infected with MVM or MPV. Results of these studies indicate that fluorogenic nuclease PCR assays provide a potentially high-throughput, PCR-based method to detect rodent parvoviruses in infected mice and contaminated biological materials.

Animals↗

PubMed1995

A simple, rapid and sensitive detection of salmonella in food by polymerase chain reaction.

A polymerase chain reaction (PCR) method was developed for detection of salmonella in food. A set of PCR primers was designed to amplify a 199 bp salmonella-specific DNA fragment derived from a repetitive DNA of Salmonella Weltevreden. The assay detected all 52 most prevalent serovars found in contaminated food in Thailand and no cross-reaction was observed with other non-salmonella organisms. The limit of detection was 1 fg of purified target DNA or five bacteria from pure culture. The detection of artificially contaminated food performed following a 6 h enrichment step was three bacteria per gram and the result was obtained within 4 h.

Animals↗

PubMed1992

[Detection of minimal residual disease using reverse transcriptase polymerase chain reaction technique].

Polymerase chain reaction (PCR) is a highly sensitive technique to detect minimal residual disease (MRD) of hematological malignancy by amplifying tumor specific nucleotide sequences. When breakpoint is located over large lesions of genomic DNA, like t(9;22) leukemia, PCR amplifying cDNA of chimeric mRNA, called reverse transcriptase PCR (RT-PCR), can be utilized. We applied this method for the detection of MRD in patients with t(1; 19) acute lymphoblastic leukemia. RT-PCR detection of MRD in patients with leukemia might be useful for estimating of the depth of remission, for disclosing preclinical relapse, and for evaluating the efficacy of in vitro purging in autologous bone marrow transplantation.

DNA, Neoplasm↗

PubMed1989

Human papillomavirus detection in paraffin-embedded cervical carcinomas and metastases of the carcinomas by the polymerase chain reaction.

The polymerase chain reaction (PCR) is used for human papillomavirus (HPV) detection in paraffin-embedded tissue. The specificity of the reaction is unaffected by the method of fixation used before embedding into paraffin. Five HPV 16, 18, 31, and 33 DNA in situ hybridization (DISH)-negative cervical carcinomas were subjected to the PCR. In two patients, HPV 16 DNA could be detected in the cervical squamous cell carcinomas and also in their lymph node metastases. One patient with an adeno-carcinoma of the cervix was found positive for HPV-18. A lymph node of this patient was HPV 18 positive as well. In the tumors of the remaining two patients, no HPV 16, 18, or 33 DNA was detected by the PCR. Both negative patients had cervical squamous cell carcinomas. One had a bladder metastasis, whereas the other had a lymph node metastasis and an additional distant metastasis in the lung. HPV DNA positivity in cervical carcinomas correlated with HPV prevalence in the metastases. This relationship can be of use for diagnostic purposes in the pathologic analysis of metastases.

Blotting, Southern↗

PubMedDate not supplied

Identification of duck plague virus by polymerase chain reaction.

A polymerase chain reaction (PCR) assay was developed for detecting duck plague virus. A 765-bp EcoRI fragment cloned from the genome of the duck plague vaccine (DP-VAC) virus was sequenced for PCR primer development. The fragment sequence was found by GenBank alignment searches to be similar to the 3' ends of an undefined open reading frame and the gene for DNA polymerase protein in other herpesviruses. Three of four primers sets were found to be specific for the DP-VAC virus and 100% (7/7) of field isolates but did not amplify DNA from inclusion body disease of cranes virus. The specificity of one primer set was tested with genome templates from other avian herpesviruses, including those from a golden eagle, bald eagle, great horned owl, snowy owl, peregrine falcon, prairie falcon, pigeon, psittacine, and chicken (infectious laryngotracheitis), but amplicons were not produced. Hence, this PCR test is highly specific for duck plague virus DNA. Two primer sets were able to detect 1 fg of DNA from the duck plague vaccine strain, equivalent to five genome copies. In addition, the ratio of tissue culture infectious doses to genome copies of duck plague vaccine virus from infected duck embryo cells was determined to be 1:100, making the PCR assay 20 times more sensitive than tissue culture for detecting duck plague virus. The speed, sensitivity, and specificity of this PCR provide a greatly improved diagnostic and research tool for studying the epizootiology of duck plague.

Animals↗

PubMed1997

The detection of Escherichia coli DNA in the ancient remains of Lindow Man using the polymerase chain reaction.

The polymerase chain reaction has been applied to the detection of Escherichia coli DNA in the upper gut contents of Lindow Man, an Iron Age bog body dated to ca 300 BC. With sets of primers from the uidA and lacZ genes, E. coli DNA could be detected reproducibly. Initial attempts at detecting DNA from freshly voided faeces from a healthy volunteer were unsuccessful due to inhibition of the reaction. Development of a method, based on guanidine thiocyanate and silica extraction and purification of the DNA fragments, facilitated the detection of the E. coli DNA in both freshly voided faeces and the upper gut contents of Lindow Man. These findings indicate that it may be possible to study the existence of infectious diseases in ancient civilizations and to learn more about the evolution of microbes.

Animals↗

PubMed1990

Identification of three genes coding for aminoglycoside-modifying enzymes by means of the polymerase chain reaction.

A polymerase chain reaction (PCR) was devised for the simultaneous identification of three widespread aminoglycoside-acetylating enzymes (AACs), namely, AAC(3)-I, AAC(3)-II and AAC(3)-IV. Three sets of 20-mer DNA primers were constructed, based on the nucleotide sequences of the genes coding these enzymes. Each of the three gene regions contained a unique restriction site. This property was used to confirm the specificity of the reactions. Evolutionarily-well-conserved sequences of the 16S rRNA bacterial genes were used to construct two 20-mer primers, which served as positive controls for the amplification reactions. Tests of twenty reference strains, containing ten different aminoglycoside-modifying enzymes, confirmed the specificity and the sensitivity of the method. The PCR method confirmed the presence of the aacC2 gene in 63 clinical strains, in which susceptibility data and colony hybridization had previously suggested the presence of the gene.

Acetylation↗

PubMedDate not supplied

Detection of Eimeria acervulina using the polymerase chain reaction.

A polymerase chain reaction (PCR)-based assay was developed for the detection of Eimeria acervulina. Primers were designed to amplify a fragment of the EASZ240/160 sporozoite antigen gene. The PCR assay detected as few as 10 E. acervulina oocysts in a mixed population containing a total of 10(6) oocysts. No nonspecific reaction was observed with any other species of avian Eimeria known to occur in Australia. PCR products from genomic DNA were 237 bp larger than predicted from previously reported cDNA sequences. Sequencing of the product revealed the presence of a probable intron. This work demonstrates the potential of PCR-based assays for identification and detection of avian Eimeria. Potential uses include identification of minor species present in mixed infections and quality control in the production of live vaccines.

Animals↗

PubMed1992

Specific detection of Clostridium botulinum type B by using the polymerase chain reaction.

The polymerase chain reaction (PCR) and a radiolabeled oligonucleotide probe were used to specifically detect proteolytic and nonproteolytic Clostridium botulinum type B. Two synthetic primers deduced from the amino acid sequence data of type B neurotoxin were used to amplify a 1.5-kbp fragment corresponding to the light chain of the toxin. Although, nonspecific priming was observed when the PCR protocol was tested with other clostridial species, only the PCR product from C. botulinum type B isolates reacted with the radiolabeled internal probe. As little as 100 fg of DNA (approximately 35 clostridial cells) could be detected after only 25 amplification cycles.

Base Sequence↗

PubMed1997

Detection of Mycoplasma in avian live virus vaccines by polymerase chain reaction.

The polymerase chain reaction (PCR) was evaluated to detect mycoplasma contamination of avian live virus vaccines. The specificity of the primers showed that 34 strains belonging to nine species of avian mycoplasma DNA could be detected. The sensitivity of PCR to detect mycoplasma DNA was 10(0.2) colony forming units (cfu) of Mycoplasma synoviae and 10(0.7) cfu of Mycoplasma gallisepticum. When M. synoviae and M. gallisepticum were spiked into several avian live virus vaccines, PCR gave a positive reaction except for the avian pox and the avian encephalomyelitis vaccines which were prepared from organ homogenates. Short-term incubation of avian encephalomyelitis vaccines improved the sensitivity of PCR to detect both M. synoviae and M. gallisepticum. Therefore, PCR, combined with the short-term incubation, were shown to be most effective in detecting mycoplasma contamination in all of avian live virus vaccines.

Animals↗

PubMed1994

Detection of mycoplasma contaminations by the polymerase chain reaction.

The polymerase chain reaction (PCR) has been used for the general detection of Mollicutes. 25 Mycoplasma and Acholeplasma species were detected including important contaminants of cell cultures such as M. orale, M. arginini, M. hyorhinis, M. fermentans, A. laidlawii and additional human and animal mycoplasmas. PCR reactions were performed using a set of nested primers defined from conserved regions of the 16S rRNA gene. The detection limit was determined to be 1 fg mycoplasma DNA, which is equivalent to 1-2 genome copies of the 16S rRNA coding region. The identity of the amplification products was confirmed by agarose gel electrophoresis and restriction enzyme analysis. DNA from closely and distantly related micro-organisms did not give rise to specific amplification products. The method presented here offers a much more sensitive, specific and rapid assay for the detection of mycoplasmas than the existing ones.

Base Sequence↗