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Results for “Polymerase Chain Reaction”

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PubMed1992

Detection of Mycobacterium tuberculosis in clinical samples from patients with tuberculosis or other pulmonary diseases by polymerase chain reaction.

Polymerase chain reaction (PCR) using primers targeting the IS6110 repetitive sequence was employed to detect Mycobacterium tuberculosis in 228 samples from patients with tuberculosis or other pulmonary diseases and controls, and the results were compared with culture and clinical findings. None of culture negative samples from 17 healthy controls were PCR positive. Of 109 active tuberculosis patients under chemotherapy, 88 (80.7%) were PCR positive and were significantly higher than 63 (57.8%) positive by culture. Fifty-nine (93.7) of 63 culture positive and 29 (63.0%) of 46 culture negative specimens contained M. tuberculosis detectable by PCR. In 41 specimens from inactive tuberculosis patients who visited to the chest clinic because of chest problems, 16 (39.0%) also gave PCR positive results. In addition, 14 (46.7%) of 30 specimens submitted for M. tuberculosis culture from patients with pulmonary diseases were PCR positive. Presumptive diagnosis of these PCR positive patients was bronchitis, pneumonia, bronchial asthma, etc. Therefore, this study suggests that PCR is sensitive and specific in detecting M. tuberculosis in clinical specimens. However, the interpretation of the PCR results in specimens from patients with pulmonary diseases should be done cautiously in areas with a high prevalence of tuberculosis.

Base Sequence↗

PubMed2004

Pitfalls of quantitative real-time reverse-transcription polymerase chain reaction.

Polymerase chain reaction (PCR)-based assays can target either DNA (the genome) or RNA (the transcriptome). Targeting the genome generates robust data that are informative and, most importantly, generally applicable. This is because the information contained within the genome is context-independent; i.e., generally, every normal cell contains the same DNA sequence--the same mutations and polymorphisms. The transcriptome, on the other hand, is context-dependent; i.e., the mRNA complement and level varies with physiology, pathology, or development. This makes the information contained within the transcriptome intrinsically flexible and variable. If this variability is combined with the technical limitations inherent in any reverse-transcription (RT)-PCR assay, it can be difficult to achieve not just a technically accurate but a biologically relevant result. Template quality, operator variability, the RT step itself, and subjectivity in data analysis and reporting are just a few technical aspects that make real-time RT-PCR appear to be a fragile assay that makes accurate data interpretation difficult. There can be little doubt that in the future, transcriptome-based analysis will become a routine technique. However, for the time being it remains a research tool, and it is important to recognize the considerable pitfalls associated with transcriptome analysis, with the successful application of RTPCR depending on careful experimental design, application, and validation.

Animals↗

PubMed1991

[Genotyping the bovine kappa-casein locus using polymerase chain reaction].

Polymerase chain reaction (PCR) was used for detecting kappa-casein (kappa-casein) genotype in the synthetic breed (Jersey x Black and White x Holstein-Friesian) dairy cattle. The amplified 228 bp fragment includes a region, where relevant mutations lead to both the appearance of different kappa-casein alleles associated with amino acid substitutions and the appearance of new TaqI and HindIII restriction sites in ae-casein B gene. The specificity of the kappa-casein gene fragment amplification was supported by restriction analysis and Southern blot hybridization. Digestion of amplified fragment with endonucleases PstI, HindIII and/or TaqI allows detection of AA-, AB- and BB genotypes of kappa-casein. A total of 32 animals with known (18 samples) and unknown (14 samples) kappa-casein phenotypes were tested using PCR and blot hybridization. In all known cases the detected genotype confirmed the phenotype. Frequencies of the B allele and of the AB genotype in the breeding population are rather high (53.1 +/- 8.8 and 43.7%, respectively). The possibility of effective use of the PCR analysis for genotyping kappa-casein locus in bulls and their offspring has been shown. The advantages of the PCR method in large breeding programs and linkage analysis have been discussed.

Animals↗

PubMed1996

Hepatitis C virus is not recoverable from liver tissue in cryptogenic cirrhosis: failure to identify hepatitis C virus-RNA using reverse transcription-mediated polymerase chain reaction.

Polymerase chain reaction (PCR) has been used to study liver biopsy tissue in patients with known or suspected hepatitis C virus (HCV). Recent studies of cryptogenic cirrhosis using PCR have been based on study of sera, and HCV has not been shown. The failure to show HCV in patients so studied has left unanswered the question of whether or not patients with cryptogenic cirrhosis could still harbor the virus in the liver. The authors studied liver tissue, obtained at the time of orthopic liver transplantation from 10 patients clinically diagnosed as having end-stage liver disease without demonstrable origin, so-called cryptogenic cirrhosis, using reverse transcription (RT)-PCR to try to recover HCV-RNA. Formalin-fixed, paraffin-embedded tissue was used. For comparison, the authors also studied similarly obtained samples from 10 patients with typical hepatitis C-associated cirrhosis and 10 patients with end-stage liver disease resulting from autoimmune hepatitis. The authors recovered HCV-RNA from 9 of 10 livers from patients with cirrhosis resulting from HCV, and 3 of 10 livers from patients with autoimmune hepatitis. HCV-RNA was not recovered from any of the livers of the 10 patients designated as having cryptogenic cirrhosis.

Adolescent↗

PubMed1990

Detection of murine typhus infection in fleas by using the polymerase chain reaction.

Polymerase chain reaction (PCR) amplification of DNA was used to detect the etiologic agent of murine typhus, Rickettsia typhi, in experimentally infected adult fleas. A primer pair derived from the 17-kilodalton antigen sequence of typhus and spotted fever group rickettsiae was used to amplify a 434-base-pair (bp) fragment of the genome of the murine typhus rickettsiae. The amplified 17-kilodalton protein antigen-specific sequence was detected in ethidium bromide-stained agarose gels in individual fleas as early as 2 days after exposure to rickettsemic rats (two of six tested). The 434-bp sequence was not detected in uninfected control fleas. A dot hybridization assay used to detect the 434-bp fragment was also specific and about 100-fold more sensitive than the agarose gel PCR assay. Since the PCR assay employed a boiled extract of triturated fleas, both PCR and an antigen capture enzyme-linked immunosorbent assay (ELISA) could be performed on the same individual flea homogenate. The ELISA identified 12 infected fleas out of 29 randomly selected fleas, compared with 14 specimens which were positive by PCR. The PCR assay detected rickettsiae in samples in which no viable rickettsiae were detected by plaque assay. Like the ELISA, the PCR assay sensitivity was due in part to its suitability for detecting small numbers of both live and dead R. typhi in fleas.

Animals↗

PubMed1992

Detection of the thermostable direct hemolysin gene (tdh) and the thermostable direct hemolysin-related hemolysin gene (trh) of Vibrio parahaemolyticus by polymerase chain reaction.

Polymerase chain reaction (PCR) protocols were established for specific detection of the tdh and trh genes, the virulence marker genes of Vibrio parahaemolyticus encoding two related hemolysins. The tdh and trh genes are known to have sequence divergence of up to 3.3% and 16%, respectively. Attempts were made to find suitable primer pairs and annealing temperatures to detect each gene without fail. DNAs extracted from 36 representative strains of V. parahaemolyticus were used in the initial screening with various combinations of primer pairs and annealing temperatures. The combinations of primer pairs and annealing temperatures selected were then tested with DNAs extracted from 227 more strains of V. parahaemolyticus and from 133 bacterial strains belonging to 40 species other than V. parahaemolyticus. PCR protocols (primer pairs and annealing temperatures) were established that gave identical results to those obtained with the tdh- and trh-specific polynucleotide probes. These protocols established for the tdh and trh genes could detect 400 fg (100 cells) of cellular DNA carrying the respective gene. Spike experiments demonstrated that the sensitivities of the established PCRs were reduced by a factor of 10(4)-10(5) by an inhibitor(s) present in a normal faecal sample, indicating the need for either DNA extraction or enrichment of the faecal sample in alkaline peptone water for 4 h before the PCR of faecal samples.

Base Sequence↗

PubMed1999

Detection of Bordetella bronchiseptica by the polymerase chain reaction.

Polymerase chain reaction (PCR) assays were developed that enabled not only discriminative detection of three Bordetella species, B. pertussis, B. parapertussis, and B. bronchiseptica (Bspp PCR), but also specific detection of B. bronchiseptica (Bb PCR). An upstream sequence of the flagellin gene was used as a target DNA region. This sequence contained differences in B. pertussis, B. parapertussis, and B. bronchiseptica DNA. These species could then be differentiated using two different sets of primers, Bspp and Bb. When oligonucleotide Bspp primers were used, PCR products were obtained from the three species of Bordetella. A fragment of the expected size (164 bp) was amplified using B. bronchiseptica and B. parapertussis DNA, but a fragment with a distinct molecular weight was amplified with B. pertussis DNA (195 bp). This Bspp PCR was specific and sensitive, but it could not differentiate between B. parapertussis and B. bronchiseptica. When Bb primers were used, a 237-bp PCR product was detected only from B. bronchiseptica DNA. No PCR products were identified after Bb PCR amplification of DNAs either from B. parapertussis isolates or B. pertussis isolates, nor from other respiratory pathogen DNAs tested. This second PCR assay had a sensitivity limit of less than 10 organisms of B. bronchiseptica after detection with a specific probe. The specificity and the sensitivity of the fla PCR assay were evaluated with purified DNA, as was its capacity for detecting the bacteria in human clinical samples and in lungs of infected mice.

Animals↗

PubMed1992

Detection of the etiologic agent of human ehrlichiosis by polymerase chain reaction.

Polymerase chain reaction (PCR) primers derived from a variable region of the 16S rRNA gene sequence were used to amplify DNA specifically from Ehrlichia chaffeensis (the recently proposed name for the etiologic agent of human ehrlichiosis). The 389-bp product defined by the specific primers was not detected when DNA samples from any of the other recognized species of Ehrlichia were used as amplification templates. When the PCR was applied to five suitable blood specimens obtained from patients subsequently shown to be serologically positive for E. chaffeensis, all five were positive. The same technique was applied to a total of six control blood specimens, three from febrile patients who had no serologic evidence of infection with Ehrlichia or Rickettsia species and three from patients diagnosed with Rocky Mountain spotted fever, and all six were negative. A chemiluminescent, group-specific oligonucleotide probe was shown to hybridize only with the PCR products obtained upon amplification of the five blood specimens from patients serologically diagnosed as having human ehrlichiosis. The results indicate that PCR, coupled with a nonisotopic method of confirming the identity of the PCR product, is a highly specific and efficient method of detecting the agent of human ehrlichiosis in blood. The results also suggest that E. chaffeensis is the sole etiologic agent of human ehrlichiosis in the United States. The technique was also applied to four ticks that were positive by direct immunofluorescence for Ehrlichia species, and one tick was PCR positive, indicating that E. chaffeensis DNA can be detected in ticks harboring this organism, although the sensitivity may be low.

Animals↗

PubMed1989

Enzymatic amplification of human cytomegalovirus sequences by polymerase chain reaction.

Polymerase chain reaction (PCR) amplification was used to detect human cytomegalovirus (HCMV) sequences. The fragments selected for amplification were fragments of 130 and 152 base pairs (bp) located at two opposite ends of HCMV strain AD169 EcoRI fragment D. Amplification of the 152-bp DNA was consistently greater than that of the 130-bp DNA. At the optimal Mg2+ concentration of 5 mM, specific PCR amplification of 152-bp DNA with Taq polymerase was sensitive; only one AD169-infected fibroblast cell or 0.01 pg of AD169 fragment D DNA was needed for detection. This specific amplification was also found with various clinical HCMV isolates and peripheral blood cells and urine from patients. In 37 urine samples analyzed simultaneously by PCR and by virus cultivation, identical results were found in 35 samples, while 2 scored positive only by PCR. This suggests that specific amplification of 152-bp DNA is sensitive and can be used for rapid detection of HCMV infections.

Base Sequence↗

PubMed2006

Estimation of the reaction efficiency in polymerase chain reaction.

Polymerase chain reaction (PCR) is largely used in molecular biology for increasing the copy number of a specific DNA fragment. The succession of 20 replication cycles makes it possible to multiply the quantity of the fragment of interest by a factor of 1 million. The PCR technique has revolutionized genomics research. Several quantification methodologies are available to determine the DNA replication efficiency of the reaction which is the probability of replication of a DNA molecule at a replication cycle. We elaborate a quantification procedure based on the exponential phase and the early saturation phase of PCR. The reaction efficiency is supposed to be constant in the exponential phase, and decreasing in the saturation phase. We propose to model the PCR amplification process by a branching process which starts as a Galton-Watson branching process followed by a size-dependent process. Using this stochastic modelling and the conditional least-squares estimation method, we infer the reaction efficiency from a single PCR trajectory.

Animals↗

PubMed1995

[Studies on inactivation of hepatitis B virus with polymerase chain reaction].

Polymerase chain reaction-ethidium bromide (PCR-EB) method for detecting hepatitis B virus DNA (HBV-DNA) was established with good specificity and a detection limit of 1 pg HBV-DNA, minimum HBV infection dose in susceptible animal, chimpanzees, could be detected with it. Determination of inactivation of HBV-DNA could be inactivated with active chlorine 1,250 mg/L for 60 minutes, or 2,500 mg/L for 30 minutes, 10 pg HBV-DNA in purified Dane particles could be inactivated by active chlorine 625 mg/L for 10 minutes. Accordingly, use of PCR to evaluate the effects of chlorine disinfectant in inactivating HBV was feasible, and HBV-DNA was a more reliable index for inactivation of HBV than HBsAg.

Base Sequence↗

PubMed2002

Identification of Salmonella enteritidis isolates by polymerase chain reaction and multiplex polymerase chain reaction.

The polymerase chain reaction and the multiplex polymerase chain reaction were developed for detection of Salmonella and for identification of the serotype enteritidis. Three sets of primers were selected from different genomic sequences amplifying a 429 bp fragment specific for the genus Salmonella within a randomly cloned sequence, including a 250 bp fragment within the spv gene, and a 310 bp fragment within the sefA gene specific for Salmonella enteritidis. The polymerase chain reaction and the multiplex polymerase chain reaction were used for detecting S. enteritidis isolated from stool samples during outbreaks of foodborne gastroenteritis between 1992 and 1998 in Taiwan. The sefA gene was detected in all 27 strains of S. enteritidis by this polymerase chain reaction method. Multiplex polymerase chain reaction could detect 3 genes in all strains, but could not detect the spv gene in 2 strains. The sensitivity of the polymerase chain reaction and the multiplex polymerase chain reaction were 10(4) and 10(5) cells/mL, respectively. In double polymerase chain reaction, the sensitivity increased to 100 cells/mL. These data indicate that the specificity and sensitivity of the polymerase chain reaction and the multiplex polymerase chain reaction make them potentially valuable tools for diagnosis of S. enteritidis infection and that they may be used for the identification of S. enteritidis responsible for sporadic enteritis cases.

Animals↗

PubMed1999

Use of ligase chain reaction and polymerase chain reaction on urine specimens to detect Chlamydia trachomatis infections in a sexually transmitted diseases clinic in Singapore.

This study was done to assess the specificity and sensitivity of the DNA amplification assays of ligase chain reaction (LCR) and polymerase chain reaction (PCR) on urine specimens to detect Chlamydia trachomatis infections in both male and female patients seen at a sexually transmitted diseases (STD) clinic in Singapore, compared with other diagnostic methods currently in use. A total of 100 patients were selected; 50 male patients diagnosed with non-gonococcal urethritis based on symptoms and a positive Gram-stained urethral smear and 50 female asymptomatic sex workers were assessed. Automated assays using LCR and PCR were used, and compared to enzyme immunoassays, chlamydial cell cultures and PCR of urethral and endocervical swab specimens. In male patients, LCR and PCR of urine specimens had sensitivities of 100%, compared to 87.0% for PCR of urethral swab specimen, 82.6% for enzyme immunoassay (EIA) and 91.3% for cell cultures. In female patients, LCR and PCR of urine samples achieved sensitivities of 77.8% and 88.9% respectively, compared with 55.6% for PCR of endocervical swab specimens, 22.2% for EIA and 66.7% for cell cultures. LCR and PCR of urine samples provided higher sensitivity compared to cell cultures, EIA and PCR of urethral and endocervical swab specimens. The use of LCR and PCR on urine as a non-invasive means of detecting chlamydial infections is viable, and may have a role to play in population-based screening programmes.

Adult↗

PubMed1998

A comparison of ligase chain reaction to polymerase chain reaction in the detection of Chlamydia trachomatis endocervical infections.

OBJECTIVE: To compare the reliability of ligase chain reaction (LCR) to polymerase chain reaction (PCR) in detecting Chlamydia trachomatis endocervical infections. METHODS: We conducted a prospective study of 486 patients at risk for chlamydial infection of the endocervix. We obtained two endocervical specimens from each patient and used LCR and PCR to detect C. trachomatis. Discrepant results between the two techniques were resolved by repeat testing and by testing for the major outer membrane protein (MOMP) gene, if necessary. We determined the sensitivity, specificity, positive predictive value, and negative predictive value for each test, using concordant results or MOMP gene results as the "gold standard". RESULTS: Of the 486 patients, 42 (8.6%) had evidence of C. trachomatis infection after resolution of discrepant results. Of the 42 true positive specimens, 41 were positive by initial LCR and 38 were positive by initial PCR. Of the 444 true negative specimens, none had a positive initial LCR result, while 2 had a positive initial PCR test. Therefore, compared to the gold standard, LCR had a sensitivity of 97.6% and specificity of 100%, while PCR had a sensitivity of 90% and a specificity of 99.5%. The positive and negative predictive values of LCR were 100% and 99.8%, respectively. PCR had a positive predictive value of 95% and a negative predictive value of 99.1%. The difference in sensitivity of LCR versus PCR was not statistically significant (P = .125). CONCLUSION: LCR and PCR perform equally well in detecting C. trachomatis endocervical infections.

Chlamydia Infections↗

PubMed2004

Quantification of Leishmania infantum parasites in tissue biopsies by real-time polymerase chain reaction and polymerase chain reaction-enzyme-linked immunosorbent assay.

Most of the experimental studies of Leishmania spp. infection require the determination of the parasite load in different tissues. Quantification of parasites by microscopy is not very sensitive and is time consuming, whereas culture microtitrations remain laborious and can be jeopardized by microbial contamination. The aim of this study was to quantify Leishmania infantum parasites by real-time polymerase chain reaction (PCR) using specific DNA TaqMan probes and to compare the efficacy of detection of this technique with a PCR-enzyme-linked immunosorbent assay (ELISA). For this purpose, spleen and liver samples from L. infantum-infected mice were collected during a 3-mo longitudinal study and analyzed by both methods. PCR-ELISA failed to quantify Leishmania spp. DNA in samples with very low or very high numbers of parasites. Real-time PCR was more sensitive than PCR-ELISA, detecting down to a single parasite, and enabled the parasite quantification over a wide, 5-log range. In summary, this study developed a method for absolute quantification of L. infantum parasites in infected organs using real-time TaqMan PCR.

Animals↗

PubMed1998

Comparison of detection techniques for cytokine reverse transcriptase polymerase chain reaction; digoxigenin-labeled polymerase chain reaction permits sensitive detection of cytokine mRNA in rat heart allografts.

The polymerase chain reaction (PCR) is a sensitive method for the analysis of cytokine mRNA expression. The amount of specific mRNA in tissues involved in an inflammatory immune response can be low and therefore requires highly sensitive detection of the PCR products. In our study we have compared different detection techniques in order to replace the commonly used detection by means of radiolabeled probes. Besides the detection of DNA in agarose gels by ethidium bromide (EB), we used detection by digoxigenin (DIG)-labeled probes, as well as the direct incorporation of DIG-labeled nucleotides in the PCR, in comparison to detection by means of 32P-labeled probes. In vitro activated rat lymph node cells, lymph node tissue, and acutely or chronically rejected rat heart allografts were examined for expression of mRNA of the cytokines IL-2 and IFNgamma. The directly DIG-labeled PCR appeared to be the best alternative for detection of PCR products by means of radiolabeled probes. While IL-2 mRNA was not detected by means of EB and IFNgamma mRNA was only detected at the highest PCR cycle numbers in acutely and chronically rejected rat heart allografts, both cytokine mRNA's were readily detected by directly DIG-labeled PCR.

Animals↗

PubMed2002

Simultaneous sequencing of multiple polymerase chain reaction products and combined polymerase chain reaction with cycle sequencing in single reactions.

DNA sequencing is considered the gold standard for nucleic acid identification and mutation detection. However, sequencing is labor intensive because it requires previous amplification and only a single sequence is analyzed at a time. We developed two novel strategies that substantially improve DNA sequencing. The first allows multiple polymerase chain reaction (PCR) products to be sequenced in a single sequencing reaction and analyzed simultaneously in a single lane or capillary. Simultaneous sequencing by this method, designated "SimulSeq," can provide either simultaneous single-direction sequencing of multiple genes or simultaneous forward and reverse sequencing from a single gene. In the second approach, designated "AmpliSeq," we demonstrate a technique combining PCR amplification and sequencing in a single reaction that is analyzed in a single lane or capillary. We demonstrate combined PCR with short bidirectional sequencing, and combined PCR with unidirectional sequencing. We anticipate that these methods will have utility in research and clinical settings where panels of mutations or large numbers of samples are be analyzed and/or when turnaround time is critical.

Base Sequence↗

PubMed1999

Phenotypic characteristics of Enterococcus faecium variants confirmed by intergenic ribosomal polymerase chain reaction and E. faecium polymerase chain reaction.

Enterococcus faecium has recently emerged as a serious nosocomial pathogen. The emergence of multiple antimicrobial agent-resistant E. faecium has been remarkable; with its strains it is one of the most phenotypically heterogeneous of all enterococcal species. About 15% of enterococcal strains isolated from human clinical specimens were found to have atypical biochemical characteristics. In order to determine if these strains were E. faecium variants, intergenic ribosomal polymerase chain reaction (ITS-PCR) and E. faecium PCR (EfPCR) were performed in 45 atypical strains, and the two PCR results were used to analyze phenotypic characteristics of the strains. As many as 60% (27/45) of the atypical strains were identified as E. faecium. Thus, it is concluded that if an enterococcal strain shows positive reaction to arabinose, arginine, and ribose and negative reaction to methyl-alpha-D-glucopyranoside and pigment, it should be identified as E. faecium.

Bacterial Typing Techniques↗