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Results for “Polymerase Chain Reaction”

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PubMed1991

Successful approach for detection of low numbers of enterotoxigenic Escherichia coli in minced meat by using the polymerase chain reaction.

The polymerase chain reaction (PCR) was used as a tool for the detection of enterotoxigenic Escherichia coli in minced meat. With two synthetic 29-mer oligonucleotides, a 195-bp fragment from the E. coli heat-labile enterotoxin (LT) gene could be amplified specifically. When 6 CFU was added to the reaction mixture as a template, the PCR yielded sufficient amplified product for visualization on an agarose gel. Prior to PCR amplification, the minced meat samples were subjected to enrichment culturing for E. coli. From these cultures, 10 microliters was used in the PCR assay. All 20 25-g samples that were examined in this assay were negative for E. coli LT. However, when 3 CFU of E. coli LT was added to the 25-g samples of minced meat prior to enrichment culturing, the PCR assay yielded positive results.

Bacterial Toxins↗
PubMed1997

Optimisation of the polymerase chain reaction.

The polymerase chain reaction (PCR) is a method by which specific sequences of DNA can be copied many times, allowing detailed molecular studies to be performed on as little as a single cell. Numerous and diverse applications of PCR are being developed across all disciplines of diagnostic pathology and research, and no single protocol is appropriate for all situations. Optimising PCR requires a delicate balance between the amplification of specific products and avoiding the production of non-specific products. Each step, from DNA template extraction to cycling times and temperatures, needs to be considered carefully. The aim of this study is to assess which parameters influence DNA amplification efficiency and specificity. The parameters evaluated are the denaturation, annealing and extension temperatures, the number of cycles performed, and the primer, magnesium chloride, dNTP, Taq DNA polymerase and DNA template concentrations. The important parameters for efficient, specific amplification were denaturation time and temperature, stringent annealing temperatures and magnesium chloride concentration. The importance of DNA concentration was found to depend upon the source from which the DNA was extracted.

DNA Primers↗
PubMed1991

Detection of Mycobacterium tuberculosis in sputum samples using a polymerase chain reaction.

A polymerase chain reaction (PCR) assay for the rapid detection of Mycobacterium tuberculosis in sputum samples is described. The target DNA is a 123-base pair (bp) segment of IS6110, which is repeated in the M. tuberculosis chromosome and is specific for the M. tuberculosis complex. Methodology used to lyse the mycobacteria, extract the DNA, and amplify the 123-bp target DNA is presented. The amplified PCR product is detected by examination of ethidium-bromide-stained acrylamide gels. An internal control using the same primers as the target DNA has been constructed to assess the efficacy of each individual reaction. Of 162 sputum samples tested, 82 were smear-positive for acid-fast bacilli. Of the 94 specimens from patients in whom pulmonary tuberculosis was diagnosed, 51 were culture-positive, smear-positive, or both. Fifty of these were PCR positive. Of the 42 specimens from patients with nontuberculous mycobacterial pulmonary disease, 41 were PCR negative. All 26 specimens from patients without mycobacterial infection were PCR negative. This assay provides a sensitive and specific means for the laboratory diagnosis of tuberculosis within 48 h that is relatively simple to perform.

DNA, Bacterial↗
PubMed1993

Quantitation of enteroviral RNA by competitive polymerase chain reaction.

The polymerase chain reaction (PCR) is a new diagnostic technique for the detection of enteroviral infection; however, it currently provides only qualitative results. The aim of this study was to adapt PCR for the accurate quantitation of enteroviral RNA in clinical specimens. For this purpose, we designed a standard RNA which was homologous to sequences at the 5' end of the coxsackie B3 enterovirus genome but contained a single-base-pair mutation which created a novel internal restriction site. Serial dilutions of this standard template RNA were mixed with a fixed concentration of coxsackie B3 enterovirus RNA. The viral and standard templates were reversed transcribed to cDNA and coamplified by PCR, and a comparison of the radioactive PCR products was made. Since the templates were both present in a single reaction tube and competed for the same primers, the ratio of products remained proportional throughout the amplification process. By this approach, a fourfold-difference in viral titer was clearly distinguishable. Moreover, we were able to accurately quantitate as few as 15 50% tissue culture infectious doses, which reflects common clinical viral titers. This study lays the foundation for quantitation of enteroviral RNA in clinical specimens and establishes a technique that can readily be applied to the diagnosis of enteroviral infection.

Base Sequence↗
PubMed1991

Non-radioactive detection of the rearranged BCR/ABL sequences amplified by polymerase chain reaction.

The polymerase chain reaction (PCR) is a powerful technique for the detection of the bcr/abl rearrangement in chronic myelogenous leukemia (CML). It allows the exponential amplification of the rearranged region, thus facilitating its detection. The specificity of the bcr/abl cDNA sequence amplified by PCR is most commonly verified by hybridization to a 32P-labeled probe. In this paper, an assay for the colorimetric detection of the amplified bcr/abl fragment is described, which offers several advantages over the use of radioactive probes. We adapted the PCR for synthesis and simultaneous labeling of DNA fragments with a non-radioactive steroid compound called digoxigenin. This labeling procedure was used to generate a digoxigenin-labeled internal probe for the chimeric bcr/abl mRNA. The assay described is based on the hybridization of the amplified bcr/abl sequence to the non-radioactively labeled probe and on the subsequent detection by an enzyme-linked immunoassay and enzyme-catalyzed color reaction. Using this protocol, we investigated 20 patients with CML along with six healthy individuals and two cell lines derived from patients with CML for the presence of the bcr/abl rearrangement. It is shown that the assay is both highly sensitive and specific and that it is readily applicable to the routine diagnosis of CML. In addition, the assay could be adapted to a number of clinical diagnostic uses.

Colorimetry↗
PubMed1991

Identification of novel protein tyrosine phosphatases of hematopoietic cells by polymerase chain reaction amplification.

Polymerase chain reaction (PCR) conditions were used to amplify cDNAs that encode putative protein tyrosine phosphatases (PTPs) from a murine interleukin-3-dependent myeloid cell line. Primers for the reactions were based on conserved sequences of the catalytic domain that are shared among all known PTPs. Sequencing of 100 PCR-amplified cDNA clones identified seven different cDNA sequences. Two of these sequences were identical to the murine PTP genes Ly5/CD45/LCA and LRP/R-PTP-alpha. Two of the cDNA sequences were 95% identical to human PTP epsilon (HPTP epsilon) and rat brain PTP (PTP1B), respectively, and are likely to represent their murine homologs. Three of the cDNA sequences encoded novel potential PTPs. One of the putative PTPs was ubiquitously expressed while a second was predominantly expressed in brain, kidney, and liver and at much lower levels in a variety of other cell tkpes and tissues. The third novel putative phosphatase was expressed primarily in hematopoietic cells and tissues in a pattern that was comparable with Ly5/CD45/LCA. Further characterization of these novel PTPs will provide insights into the growth regulation of hematopoietic cells.

Amino Acid Sequence↗
PubMed2006

Introduction to the polymerase chain reaction.

The polymerase chain reaction (PCR) is an in vitro method for the amplification of DNA. Since the introduction of the PCR in 1985, it has become an indispensable technique for many applications in scientific research and clinical and forensic investigations. In this chapter, the principle and setup of PCR, as well as the methods for analyzing PCR products, will be discussed.

DNA↗
PubMed1994

Detection of the Vibrio cholerae heat-stable enterotoxin gene by polymerase chain reaction.

A polymerase chain reaction assay was developed for detection of the Vibrio cholerae heat-stable enterotoxin gene (sto). The assay is based on two oligonucleotide primers suitable for amplification of the entire sto open reading frame. Reaction conditions were defined to obtain optimal results in terms of specificity and sensitivity. Under these conditions the assay was highly sensitive and enabled good results to be obtained using, as a template, crude DNA preparations from single bacterial colonies. A rapid protocol for direct sequence analysis of the amplification product, which may be used in combination with the PCR assay to confirm the product identity and analyse sequence polymorphisms within the sto gene, was also developed. Twenty-two V. cholerae non-O1 isolates from sporadic cases of V. cholerae non-O1-associated gastroenteritis from Cuba were analysed by this PCR assay. Four strains (18.2%) were found to carry the sto gene. The prevalence of V. cholerae non-O1 strains carrying the sto gene among clinical isolates is higher than that reported for other geographical areas, except in the case of epidemics.

Amino Acid Sequence↗
PubMed2000

Rapid and specific identification of medium-chain-length polyhydroxyalkanoate synthase gene by polymerase chain reaction.

A polymerase chain reaction (PCR) protocol was developed for the specific detection of genes coding for type II polyhydroxyalkanoate (PHA) synthases. The primer-pair, I-179L and I-179R, was based on the highly conserved sequences found in the coding regions of Pseudomonas phaC1 and phaC2 genes. Purified genomic DNA or lysate of colony suspension can serve equally well as the target sample for the PCR, thus affording a simple and rapid screening of phaC1/C2-containing microorganisms. Positive samples yield a specific 540-bp PCR product representing partial coding sequences of the phaC1/C2 genes. Using the PCR method, P. corrugata 388 was identified for the first time as a medium-chain-length (mcl)-PHA producer. Electron microscopic study and PHA isolation confirmed the production of mcl-PHA in P. corrugata 388. The mcl-PHA of this organism has a higher molecular weight than that of similar polymers produced by other pseudomonads.

Acyltransferases↗
PubMed1995

[Detection of Toxoplasma gondii DNA in body fluids by polymerase chain reaction].

The polymerase chain reaction (PCR) is used for in vitro amplification of specific DNA fragments. The PCR technique is based on the reiteration of a three-step process: denaturation DNA into single strands, annealing primers (specific synthetic oligonucleotides) to the DNA template and enzymatic extension from the primers along the templates. The usefulness of this new technique to the detection of Toxoplasma gondii DNA has been described.

AIDS-Related Opportunistic Infections↗
PubMed1993

Detection of Pneumocystis carinii DNA in sputum and bronchoalveolar lavage samples by polymerase chain reaction.

A polymerase chain reaction (PCR)-based assay was developed for the detection of Pneumocystis carinii DNA in induced sputum and bronchoscopic alveolar lavage samples. The primer pair was selected from the published sequence of the thymidylate synthase gene of P. carinii derived from infected rats. The amplified DNA fragment of 403 bp was detected by agarose gel electrophoresis and by Southern and slot blot hybridization. No positive reaction was seen with DNA from different microorganisms typically found in the respiratory tract. P. carinii DNA was demonstrated in 30 of 42 sputum samples from immunosuppressed patients, whereas 21 of 42 sputum samples were positive by indirect immunofluorescence (IFL). Among the 42 patients, 14 were receiving prophylactic chemotherapy. In that group, PCR detected P. carinii in nine sputum samples, whereas IFL detected P. carinii in only four sputum samples. A positive PCR result was also seen in 5 of 43 IFL-negative bronchoscopic alveolar lavage samples from patients with respiratory symptoms. The PCR assay detected 10 copies of the target DNA, which corresponds to 10(-18) g of the specific P. carinii sequence. The results indicate that PCR amplification in combination with DNA hybridization is specific and is a more sensitive diagnostic method than IFL for the detection of P. carinii.

Base Sequence↗
PubMedDate not supplied

Quantitative measurement of mRNAs by polymerase chain reaction.

Although polymerase chain reaction (PCR) has been used to detect the presence of specific mRNA species, there are no reports indicating that PCR can be used as a reliable, reproducible assay to quantify the relative level of an mRNA. In this study we examined the enzymatic steps (reverse transcription and PCR) required to analyze RNA by PCR and determined the conditions under which the product obtained reproducibly reflects the relative amounts of amplified species in the starting material. Aliquots of total RNA from rat ovaries and GH3 pituitary cells were used to prepare cDNAs for PCR amplification of beta-actin and prolactin (PRL) sequences, respectively. Assay of equivalent dilutions of ovarian cDNAs made from 10, 2, and 0.4 micrograms of RNA demonstrated that the amount of PCR product obtained was proportional to both the amount of cDNA amplified and the amount of RNA transcribed, with a relatively small variability for both reactions. cDNAs were also made against RNA prepared from GH3 cells cultured in the presence or absence of Ca2+, which induces PRL gene expression. Measurement of PRL mRNA by PCR gave results comparable to those obtained by Northern blot (4.7-fold induction vs. 5.9-fold), and again was highly reproducible. Additionally, PCR analysis of cDNA against GH3 nuclear RNA allowed us to detect an apparent splice variant of the PRL nuclear RNA that is also Ca2+ regulated. These results indicate the sensitivity and reliability of PCR as a quantitative assay for specific mRNAs, and demonstrate the possibilities for obtaining data not readily available by other means.

Actins↗
PubMed2003

Evaluation of methods to improve detection of Escherichia coli O157:H7 in fresh produce by multiplex polymerase chain reaction.

Multiplex polymerase chain reaction (PCR) analysis was used to detect two genes encoding Shiga-like toxins (stx1 and stx2) and a universal Escherichia coli gene (gadA/B) in fresh produce spiked with E. coli O157:H7. Current U.S. Food and Drug Administration procedures for the analysis of fresh produce include the use of the rinsate from an initial rinse for the analysis of several potential pathogens, including E. coli O157:H7. In this study, several procedures were evaluated for their ability to increase the sensitivity of PCR analysis of rinsates from 15 types of produce. The procedures evaluated included the preliminary clarification and concentration of templates by centrifugation and the treatment of templates with compounds reported to facilitate nucleic acid amplification, including polyvinlypolypyrrolidone (PVPP), nonfat dry milk (NFDM), and InstaGene. The preliminary concentration of rinsates resulted in moderate improvements in detection sensitivity. The use of PVPP-treated templates in PCR reaction mixtures did not further improve sensitivity, but the inclusion of NFDM-treated templates increased sensitivity by an order of magnitude for 12 rinsates. The incorporation of InstaGene also improved the detection capability of the analysis; this procedure yielded the strongest gel bands for eight rinsates. However, for four other rinsates, the use of this reagent decreased sensitivity; these four rinsates were those for the produce varieties with the largest surface areas and were the most turbid rinsates. The use of facilitating compounds to block PCR inhibition may enable an analysis for Shiga toxin-producing E. coli in fresh produce to be completed in 1 to 2 days, rather than the 5 days required for current methods.

DNA, Bacterial↗
PubMed1990

Rapid and efficient detection of genetic polymorphism in wheat through amplification by polymerase chain reaction.

The polymerase chain reaction (PCR) was used to amplify genomic DNA of several wheat genotypes. The oligonucleotides used as primers were the terminal sequences of a gamma-gliadin gene. The electrophoretic analysis of the PCR products showed specific bands which revealed both inter- and intra-specific genetic polymorphism among the examined genotypes. The technique is proposed as a very simple and efficient alternative to RFLP markers.

Base Sequence↗
PubMed1994

Sensitive detection of canine parvovirus DNA by the nested polymerase chain reaction.

A polymerase chain reaction (PCR) for the detection of canine parvovirus (CPV) was developed. To increase the sensitivity and specificity of the reaction, the nested PCR with a double-nested primer pair (inner primer pair) was designed. The sequences of the PCR primer pairs were selected from the conserved region in the CPV VP1/VP2 gene. The PCR with the outer or inner primer pair alone (single PCR) could detect 10 fg of viral replicative form (RF) DNA on agarose gel electrophoresis; whereas as little as 100 ag of the RF DNA was detected by the nested PCR, which was shown to be 100 times more sensitive than the single PCR. Samples prepared from feline panleukopenia virus and mink enteritis virus, both having a very close antigenic relationship to CPV, were also amplified by the nested PCR. The specificity of the reaction was confirmed by restriction enzyme analysis and Southern hybridization. Next, fecal samples were examined by the nested PCR. All 10 samples suspected of CPV infection were positive, and two restriction sites (HaeIII and HindIII sites) in the PCR product were conserved among them. On the other hand, specific amplification was not observed in the samples derived from normal dogs. The number of the genome copy in positive samples was estimated about 10(9)-10(11)/g by the single PCR and 10(11)-10(13)/g by the nested PCR. The assay can be completed in 1-1.5 days, and does not need radioisotopes. Thus, the nested PCR seems to be a sensitive, specific and practical method for the detection of CPV in fecal samples.

Animals↗
PubMed1992

Detection of Chlamydia trachomatis by use of polymerase chain reaction.

A polymerase chain reaction (PCR) assay was developed for detection of Chlamydia trachomatis DNA. One primer set was used, from the published sequence of the common C. trachomatis plasmid. Detection of amplified sequences was carried out by agarose gel electrophoresis. Analysis of 106 clinical samples tested by cell culture and PCR showed a sensitivity of 100% when PCR was compared with cell culture.

Base Sequence↗