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At least 217 records · Page 12Linked to original sources
PubMed2002

PCR-based assays for the fish pathogen Aeromonas salmonicida. I. Evaluation of three PCR primer sets for detection and identification.

In an effort to develop a rapid diagnostic test for the fish pathogen Aeromonas salmonicida, the performance of 2 polymerase chain reaction (PCR) primer sets (AP and PAAS) targeting the fish pathogen A. salmonicida and 1 PCR primer set (MIY) targeting A. salmonicida subsp. salmonicida were evaluated. Initially, the PCR assays were used to screen purified DNA extracted from 308 A. salmonicida isolates. The AP and PAAS PCR tests were demonstrated to be 100% specific for the species A. salmonicida and did not cross-react with any of the non-target organisms (bacterial species other than A. salmonicida) used in this study. The combined sensitivity of the AP and PAAS tests was 99.4% and offered the best coverage in terms of identifying the target organism. The MIY PCR appeared to be 100% sensitive and specific for A. salmonicida subsp. salmonicida. Studies with tissues, spiked with known quantities of bacteria, were conducted to determine the lower detection limit of the PCR tests, and then the ability of these PCR tests to detect A. salmonicida in experimentally infected salmonids was assessed.

Aeromonas↗
PubMed1998

[Detection of Brugia malayi larva in mosquito vector by PCR and PCR-ELISA].

AIM: To develop sensitive, specific, simple assays for the detection of Brugia malayi larva in mosquito vectors. METHODS: The optimum conditions for PCR and PCR-ELISA were determined. With dissected larvae, detection limits and as well as specificities of the two assays were worked out. The optimized assays were then tested with infected and non-infected mosquito vectors, Anopheles sinensis. RESULTS: Both PCR and PCR-ELISA detected DNA equivalent to a single L1-stage larva(L1). While the actual detecting limits of the two, as estimated and titrated respectively, reached to 1/10 and 1/100 of a L1. When the two assays were used to detect the larvae isolated from 113 infected A. sinensis, all gave specific bands and positive reactions. However, trials on direct amplifications with crude extracts of infected mosquito vectors consistently failed to give specific bands upon electrophoresis and negative results in PCR-ELISA as well. CONCLUSION: Both PCR and PCR-ELISA were preliminarily established for the detection of isolated B. malayi larva in mosquito vector, which proved to be sensitive, specific and easy to manipulate.

Animals↗
PubMed1992

[Application of PCR and RT-PCR method to molecular biology study in nephrology].

Recent advance in molecular biology and genetic engineering has made a profound influence on basic and clinical medicine. Development of PCR (polymerase chain reaction) has transformed our trend of thought in molecular biology. Applications of PCR method has extended to nephrology study. RT (reverse transcription)-PCR method was elaborated to identify mRNA even in a small number of cells. It is also important and advantageous that PCR facilitates the utilization of a non-radioisotopic detection system. Newly developed chemiluminescent procedure yields as high sensitivity just as radioactive probes. Further applications are now being investigated including in situ PCR. PCR and RT-PCR methods are of great value for studying a molecular biological background of intricate kidney functions and diverse renal disorders.

Cloning, Molecular↗
PubMed2004

[Detection of minimal residual disease of chronic myeloid leukemia patients after allogeneic stem cell transplantation by combination of STR-PCR with RT-PCR].

In order to evaluate relapse predication ability of STR-PCR combining with qualitative RT-PCR for the bar/abl transcripts to the patient with chronic myeloid leukemia (CML) fulfilled allogeneic stem cell transplantation (allo-HSCT), 24 patients with CML after allo-HSCT were dynamically investigated for MRD, quantitative analysis of donor chimerism was performed by multiplex PCR amplification of STR markers and capillary electrophoresis with fluorescence detection, qualitative detection of bcr/abl transcripts was detected by nested RT-PCR. The results showed that persistent full donor chimerism (DC >/= 95%) was associated with an absence of MRD. All patients with stable MC (90% </= DC < 95%) and bcr/abl negative had a probability of long-term survival with molecular remission, however the result of bcr/abl positivity was not always associated with leukemia relapse, only the patient with decreasing values of donor chimerism as well as bcr/abl positive proved to be in a higher risk of relapse or graft failure. Decrease of donor chimerism in correlation with MRD positive was detected in 5 patients. Three out of five patients had been proved to have a molecular relapse, one out of five patients had developed to cytogenetic relapse and another patient experienced graft failure. It is concluded that the results of STR-PCR in the range of its sensitivity fully correspond with bcr/abl tests in patients. The combination of STR-PCR with RT-PCR will provide a highly sensitive and valuable tool for MRD detection in CML and can identify these high risk patients with molecular or cytogenetic relapse after allo-HSCT.

Genes, abl↗
PubMed2006

Detection of Mycobacterium avium & M. tuberculosis from human sputum cultures by PCR-RFLP analysis of hsp65 gene & pncA PCR.

BACKGROUND & OBJECTIVE: Identification of mycobacteria by conventional methods is slow, labour intensive and may at times fail to produce precise results. Molecular techniques developed in the recent past, overcome these disadvantages facilitating rapid identification of most species. We undertook this study to characterize mycobacteria isolated from sputa of human patients suspected to have tuberculosis by conventional methods and later, by polymerase chain reaction-restriction fragment length polymorphism analysis (PRA) of hsp65 gene and pncA PCR. METHODS: Twenty two mycobacteria isolated from 30 sputum samples were identified based on growth rate, pigmentation, cultural and biochemical properties and subjected to PRA of hsp65 gene involving amplification of hsp65 gene and digestion of the product with BstEII and HaeIII in separate reactions and analysis of digests by 3 per cent agarose gel electrophoresis. The mycobacteria were simultaneously evaluated by M. tuberculosis-specific and M. bovis-specific pncA PCR assays in separate reactions. RESULTS: With the conventional biochemical tests, the 22 sputum culture isolates were identified as M. tuberculosis (19) and M. avium complex (MAC) (3). PCR of hsp65 gene yielded 439 bp product in all the mycobacteria tested. The RFLP patterns of three MAC isolates with BstEII and HaeIII were identical to reference M. avium strain with two fragments in each of the digest. M. intracellulare reference strain showed a distinct pattern with 3 fragments each in both enzyme digests. The PRA of hsp65 confirmed MAC isolates as M. avium. M. tuberculosis isolates including H37Rv and M. bovis strains could not be discriminated by PRA of hsp65. The two pncA PCR assays (M. bovis-specific and M. tuberculosis-specific) detected specifically the respective organisms with an amplification product of 185 bp. The MAC strains yielded no amplification product in both the pncA PCR assays. INTERPRETATION & CONCLUSION: PRA profiles of hsp65 could differentiate MAC isolates into M. avium and M. intracellulare but could not distinguish between M. tuberculosis and M. bovis. pncA PCR assays were found specific in detecting the respective mycobacterial species. The study confirms utility of pncA PCR assays in differential identification of M. tuberculosis and M. bovis and that of PRA of hsp65 in the identification of M. avium.

Amidohydrolases↗
PubMed1996

Monitoring of CMV infection: a comparison of PCR from whole blood, plasma-PCR, pp65-antigenemia and virus culture in patients after bone marrow transplantation.

Rapid, specific and sensitive methods are essential for early detection of CMV infection in patients after marrow transplantation. Thus, in a prospective study, PCR from whole blood, plasma-PCR, pp65-antigenemia and virus culture were used and compared in 20 consecutive marrow transplant recipients for early diagnosis of CMV infection and monitoring of antiviral therapy. Moreover, semi-quantification of the viral load in blood samples by PCR from whole blood or plasma and pp65-antigenemia was performed. Fifteen out of 20 patients were found to be CMV positive by PCR from whole blood, plasma-PCR and pp65-antigenemia, whereas only 9/20 developed culture-proven viremia and/or viruria. PCR from whole blood, plasma-PCR and pp65-antigenemia revealed identical results in 96 and discordant results in 13 of 109 blood samples (P < 0.01). The efficacy of antiviral therapy was monitored by semi-quantitative scoring of pp65-antigen-positive leukocytes and/or CMV-DNA levels in blood and plasma samples. Twelve of 13 patients were found to be CMV negative by all methods after 14 days of ganciclovir therapy. A good correlation of the semi-quantitative evaluation of the three assays was demonstrated. Thus, all three highly sensitive assays seem to be suitable for screening patients at risk for CMV infection and monitoring the efficacy of antiviral therapy.

Adolescent↗
PubMed1995

Application of fluorescent dUTP on PCR and automated PCR product detection by using internal lane size standard.

A convenient, simple, cost effective and efficient fluorescent PCR labeling for automated detection was developed by using fluorescent deoxyuridine triphosphate-(F)dUTP without changing any PCR reaction parameters. Multiple fluorescent (F)dUTPs in a broad range of concentration were tested on PCR reaction and then titration tests for optimum sample loading amount with automated detection were done by using Genescan 672 software on ABI 373A DNA sequencer. PCR product were automaticaly detected although the sensitivity of each color was different. Colors of (F)dUTP and (F)dUTP/dTTP ratio showed a significant influence on the incorpration of (F)dUTP in PCR reaction, so consequently the ratio gave a great impact on the PCR product detection results. To much incorpration of (F)dUTP changed the PCR fragment mobility and also sizing value. The application of (F)dUTP should have a great potential on linkage studies, mapping with microsatellite and mutation detection.

Color↗
PubMed1996

[Nested polymerase chain reaction (PCR) and multiple cloned antibody capture PCR for the examination of serum hepatitis B virus DNA in negative hepatitis B surface antigen patients suffering from liver diseases].

In order to find out rapidly the causes of the liver diseases suffered by patients with negative hepatitis B surface antigen (HBsAg), nested polymerase chain reaction (PCR) and multiple cloned antibody capture PCR techniques were established to examine serum hepatitis B virus (HBV) DNA. By using both techniques along with the examination of hepatitis C virus (HCV) infection, the causes of chronic liver diseases with negative HBsAg were studied. It is found that nested-PCR can increase the sensitivity of single PCR more than 1,000 fold and multiple cloned antibody capture-PCR can detect concentration of HBV DNA as low as 0.1-0.01 pg/L. HBV DNA positive patients were found in 45.5%, 30.8%, 13.3% and 100% respectively of the patients suffering from liver cirhosis with negative HBsAg (group A, 22 cases), chronic hepatitis with negative HBsAg (group B, 13 cases), normal subjects with negative HBsAg and positive hepatitis B core antibody (HBcAb, group C, 30 cases) and liver cirhosis with positive HBsAg and negative HBeAg (group D, 12 cases). HBV DNA can be also found in the serum of HBsAb positive patients and subjects supposed to be healthy, 81.8% and 53.8% of the patients were infected with HBV and/or HCV in group A and group B respectively. All these results suggest that nested-PCR and multiple cloned antibody capture-PCR are rapid and highly sensitive methods for detection of serum HBV DNA. HBV infection is an important cause of chronic liver diseases in patients with negative HBsAg. The causes of most of the HBsAg-negative chronic liver diseases are related with infection of viruses. The clinical significance of serum HBsAb in naturally infected patients should be reconsidered.

Adult↗
PubMed2004

Comparative evaluation of measles virus specific TaqMan PCR and conventional PCR using synthetic and natural RNA templates.

Comparative evaluation of TaqMan RT-polymerase chain reaction (PCR) methodology developed during this study with the conventional RT-PCR-nested PCR methodology developed earlier, using measles virus RNA templates derived from synthetic and natural sources against a number of primer sets belonging to various regions of the genome, revealed the existence of similar assay thresholds for both methods. An exception to this finding was, however, noted using primer sets of the N and M genes regions with RNA templates extracted from the wild type measles virus strain where the nested PCR method proved to be 10- to 100-fold more sensitive than the end points established with the N gene specific TaqMan RT-PCR method with synthetic RNA templates. These differences were not evident when the same primer sets were evaluated with RNA templates extracted from a brain sample of SSPE patient. These findings indicate that the genetic make up of measles virus strain in any given clinical specimen, in relation to the amplifying primers/probe sequences, can have impact on the overall sensitivity and specificity of the methodology applied. Both methods are equally suitable for the molecular detection of measles virus sequences in clinical specimens, although the TaqMan RT-PCR method may be preferred due to its advantages of contamination control, automation, and real-time product quantitation.

Base Sequence↗
PubMed2025

Performance of Subgenomic RT-PCR for Predicting SARS-CoV-2 Infectivity Compared to Genomic RT-PCR and Culture Isolation.

SARS-CoV-2 clinical samples can be detected as positive for a long period of time using real-time RT-PCR, even when patients are no longer infectious. Viral culture is the gold standard for assessing a patient's infectivity, but it is a time-consuming technique and lacks sensitivity. SARS-CoV-2 subgenomic RNA (sgRNA) detection has been used as a proxy for assessing the infectivity but only a limited number of studies have described its use in vitro and in clinical samples. This study aimed to evaluate the correlation between results from viral culture, genomic RT-PCR (gRT-PCR), and subgenomic RT-PCR (sgRT-PCR) during in vitro infection and in clinical samples. In vitro viral replication kinetics showed that both genomic RNA (gRNA) and subgenomic RNA (sgRNA) levels remained stable up to 21 days in the absence of replication-competent virus. Using clinical samples, sgRNA was detected in 87.5% of culture-positive samples, demonstrating better performances than gRT-PCR (Positive predictive value (PPV) 93.3% and Negative predictive value (NPV) of 87.5%) and an almost perfect agreement with culture results (Cohen &#x3ba;&#x2009;=&#x2009;0.81 [95% CI: 0.66-0.95]). These findings suggest that testing for sgRNA and/or using a gRNA Ct cut-off of 21.2 could be used as a proxy to determine the presence of SARS-CoV-2 replication-competent virus.

Humans↗
PubMed2002

Detection of Epstein-Barr virus genome within thymic epithelial tumours in Taiwanese patients by nested PCR, PCR in situ hybridization, and RNA in situ hybridization.

Epstein-Barr virus (EBV) is known to be associated with a variety of tumours, including Burkitt's lymphoma, nasopharyngeal carcinoma, and some carcinomas of other organs with similar lymphoepithelioma-like features. The association between EBV and thymic epithelial tumours is inconclusive, as reports in this regard are not entirely consistent and the methods employed are of different sensitivity and specificity. This study examined 78 thymomas and 21 thymic carcinomas in Taiwanese patients, to detect the viral genome at both DNA and RNA levels. The tissue blocks were first screened by nested polymerase chain reaction (PCR) targeting on the first tandem internal repeats. The positive cases were further submitted for viral localization by in situ PCR insitu hybridization (ISH) and Epstein-Barr-encoded RNA-1 (EBER-1) ISH. None of the thymomas showed a detectable EBV genome. Eight thymic carcinomas were positive for EBV by nested PCR, of which six displayed nuclear signals within the tumour cells by in situ PCR ISH and/or RNA ISH, one displayed signals within the lymphocytes, and one showed no discernible in situ signals. Most of them exhibited a lymphoepithelioma-like morphology. These results show that nested PCR is a sensitive method for screening the EBV genome in thymic epithelial tumours. In situ PCR ISH is reliable for localization of the virus, in addition to EBER-1 RNA ISH. Thymomas are not related to EBV, even in this endemic area. Thymic carcinomas, especially the lymphoepithelioma-like thymic carcinomas, are more often associated with the virus.

Adult↗
PubMed2002

Quantification of PCR amplification products of Brugia HhaI repeat DNA using a semiautomated Q-PCR system.

The sensitive, rapid and species-specific diagnosis of Brugia infections in humans or animal models is important in determining the level of parasitemia and the efficacy of chemotherapy or vaccinations. The HhaI family of highly repeated DNA sequences from Brugia have been useful in polymerase chain reaction (PCR)-based diagnosis of brugian filarial infections in blood samples and in mosquitoes. A PCR assay was developed using a biotinylated primer, a non-biotinylated primer and a species-specific chemiluminescent probe [tris[2,2'bipyridine] ruthenium (II) chelate, TBR] to detect PCR amplified Hhal family repeats. Individual blood samples from jirds infected with Brugia malayi or B. pahangi and with different levels of microfilaremia were tested in this assay. Known concentrations of Brugia DNA and DNA from the blood of uninfected control jirds were used as positive and negative controls, respectively. The PCR products generated by this method were analyzed using a semi-automated quantitative (Q)-PCR system. The levels of parasite DNA can be calculated from the luminosity units generated. Significant amounts of parasite DNA were detected in blood samples from infected jirds, and these values were correlated with the levels of microfilaremia. In contrast, reductions in circulating microfilaria following treatment with ivermectin correlated with low levels of measurable DNA. Using this system, we were also able to detect HhaI repeat DNA in the spleens of B. pahangi- infected jirds at 56 days post-infection when circulating microfilariae were not readily detectable. The results indicate that the species-specific Hhal Q-PCR detection and quantification method is rapid and sensitive, is useful in the detection of Brugia DNA in blood and other tissues and is suited for use in clinical settings because it does not require radioactive isotopes and gel-based protocols.

Animals↗
PubMed1997

Detection of proviral DNA of bovine immunodeficiency virus in bovine tissues by polymerase chain reaction (PCR) and PCR in situ hybridization.

In this study, experiments were designed to investigate the distribution of bovine immunodeficiency virus (BIV) proviral DNA in the tissues and cells of infected calves by solution-phase polymerase chain reaction (SP-PCR) and PCR in situ hybridization (PCR-ISH). Total DNA samples extracted from tissues of 10 BIV-infected and 5 uninfected calves were amplified by SP-PCR with the primers directed to the BIV conserved pol gene segment. The identity of the SP-PCR product was confirmed by Southern hybridization with a BIV pol gene cDNA probe. SP-PCR results demonstrated that BIV proviral DNA was present predominantly in neural tissues and some lymphoid tissues in BIV-infected calves. It also was detected frequently in other tissues including lung, heart, esophagus, and pancreas. Further investigation on cell location of BIV proviral DNA was performed by in situ amplification of DNA on formalin-fixed tissue sections. The amplified DNA was subjected to in situ hybridization with an internal biotinylated probe and detected with streptavidin-gold followed by silver enhancement. Specific BIV proviral DNA signals were observed in neurons, microglial cells, lymphocytes, septal macrophages, smooth muscle cells, and endothelial cells. On the basis of these results, we conclude that BIV replicates in a variety of bovine tissues in vivo and has a broad cell tropism.

Animals↗
PubMed1998

[In situ PCR and PCR in situ hybridization of paraffin-embedded tissue. New diagnostic possibilities in pathology].

In situ polymerase chain reaction (PCR) and PCR in situ hybridization are new diagnostic tools in dermatopathology. These techniques can combine the sensitivity of PCR with the advantage of in situ hybridization to localize specific cellular structures. With optical control of the PCR reaction product in these techniques, false-positive results due to contamination can be minimized. Several working protocols have been established which allow detection of DNA and RNA sequences in a rapid and reproducible manner. Two different methods have been established to detect the amplification product, the indirect PCR in situ hybridization (PCRisHyb), and the direct in situ PCR (isPCR). An easy and reproducible method for PCRisHyb and isPCR in formalin-fixed and paraffin-embedded tissue is described and the two techniques are compared. Using both isPCR and PCRisHyb, the amplification product can be visualized with an optimal morphological preservation of the tissue. Indirect PCRisHyb showed a slightly higher specificity whereas direct isPCR was the quicker, easier and less expensive method.

Chickenpox↗
PubMed1996

Serotyping, ribotyping, PCR-mediated ribosomal 16S-23S spacer analysis and arbitrarily primed PCR for epidemiological studies on Legionella pneumophila.

Fifty clinical and environmental isolates of Legionella pneumophila were typed serologically and by DNA fingerprinting using arbitrarily primed polymerase chain reaction (AP-PCR). Furthermore, variability in and around ribosomal operons was assessed by conventional ribotyping and PCR-mediated amplification of the spacer region separating the 16S and 23S genes. It appears that serotyping suffers from low resolution capabilities, and ribotyping and spacer PCR display intermediate resolving capabilities, whereas AP-PCR is more discriminating. Results from AP-PCR and both forms of ribotyping analysis correlate with epidemiological and environmental data. It is suggested that AP-PCR typing may be the method of choice for rapidly determining clonality among L. pneumophila isolates.

Bacterial Typing Techniques↗