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PubMed2004

Detection of human papillomavirus DNA in cervical samples: analysis of the new PGMY-PCR compared to the hybrid capture II and MY-PCR assays and a two-step nested PCR assay.

The PGMY-PCR for human papillomavirus (HPV) was evaluated, in parallel with nested PCR (nPCR), in samples with noted Hybrid Capture II (HCII) and MY-PCR results. PGMY-PCR detected HPV DNA in 2.5% of HCII-negative-MY-PCR-negative samples and in 71.7% of HCII-positive-MY-PCR-negative samples; also, it detected the MY-PCR-negative-nPCR-negative types HPV-42, HPV-44, HPV-51, HPV-87, and HPV-89.

Cervix Uteri↗

PubMed2006

Real-time PCR for the detection of Salmonella spp. in food: An alternative approach to a conventional PCR system suggested by the FOOD-PCR project.

A real-time PCR assay using non-patented primers and a TaqMan probe for the detection and quantification of Salmonella spp. is presented. The assay is based on an internationally validated conventional PCR system, which was suggested as a standard method for the detection of Salmonella spp. in the FOOD-PCR project. The assay was sensitive and specific. Consistent detection of 9.5 genome equivalents per PCR reaction was achieved, whereas samples containing an average of 0.95 genome equivalents per reaction were inconsistently positive. The assay performed equally well as a commercially available real-time PCR assay and allowed sensitive detection of Salmonella spp. in artificially contaminated food. After enrichment for 16 h in buffered peptone water (BPW) or universal pre-enrichment broth (UPB) 2.5 CFU/25 g salmon and minced meat, and 5 CFU/25 g chicken meat and 25 ml raw milk were detected. Enrichment in BPW yielded higher numbers of CFU/ml than UPB for all matrices tested. However, the productivity of UPB was sufficient, as all samples were positive with both real-time PCR methods, including those containing less than 300 CFU/ml enrichment broth (enrichment of 5 CFU/25 ml raw milk in UPB).

Animals↗

PubMed2003

Comparison of two DNA extractions and nested PCR, real-time PCR, a new commercial PCR assay, and bacterial culture for detection of Mycobacterium avium subsp. paratuberculosis in bovine feces.

In this study, 5 combinations of 2 DNA extractions and 3 polymerase chain reaction (PCR) techniques were compared with culture for the detection of Mycobacterium paratuberculosis directly from bovine feces. These combinations included a new commercial extraction technique combined with a commercial PCR/Southern blot technique, nested PCR (nPCR), or real-time PCR, and a university-developed extraction combined with nPCR or real-time PCR. Four of the 5 combinations had statistically similar sensitivities between 93% and 100% and specificity between 95% and 100%, when compared with culture results from 63 bovine fecal samples. These results indicated that using a commercial extraction with a commercial PCR/Southern blot, nPCR, or real-time PCR, or a university-developed extraction with real-time PCR would result in similar sensitivities to culture for the identification of M. paratuberculosis from bovine feces and are valid alternatives to culture.

Animals↗

PubMed1992

Recombinant circle PCR and recombination PCR for site-specific mutagenesis without PCR product purification.

Two simple methods for site-specific mutagenesis are described and compared. In each method, the PCR is used in two separate amplifications to mutate the site of interest and to add ends to one PCR product that are homologous to the ends of the other PCR product. In the first method, the two products are combined, denatured and reannealed prior to transformation of E. coli in order to form recombinant circles in vitro, while in the second method, the two linear products are co-transfected directly into E. coli without prior manipulation, resulting in transformation of E. coli with the recombinant of interest by recombination in vivo. Each PCR amplification uses a plasmid template that has been linearized by restriction enzyme digestion outside the region to be amplified. This permits use of unpurified PCR products in these two protocols and generation of the mutant of interest with no other enzymatic manipulation in vitro apart from PCR amplification. In each protocol greater than or equal to 50% of the resulting clones contained the mutation of interest without detected errors.

Base Sequence↗

PubMed2004

Application of 3'-mismatched reverse primer PCR compared with real-time PCR and PCR-RFLP for the rapid detection of 23S rDNA mutations associated with clarithromycin resistance in Helicobacter pylori.

Helicobacter pylori clarithromycin (Cla) resistance dramatically reduces efficacy of eradication therapy. In this study, 3'-mismatched reverse primer PCR (3M-PCR), real-time PCR (LightCycler), and PCR-RFLP assays were investigated to determine their sensitivity for detecting clarithromycin resistance associated with 23S rDNA mutations (A2142G, A2142C, and A2143G). For 84.8% (123/145) of isolates, the same allelic type was detected by each method although methods differed in efficiency of detecting mutations in cultures either containing mixtures of two alleles (24 isolates), or that were dual allelic variants (two isolates). The novel 3M-PCR assay format was the most sensitive, detecting all alleles at > or =0.02 ng/microl in DNA mixtures, and thus provides more precise information to guide clinical management of patients at risk of treatment failure.

Anti-Bacterial Agents↗

PubMed2006

Detection and quantitation of E. faecalis by real-time PCR (qPCR), reverse transcription-PCR (RT-PCR), and cultivation during endodontic treatment.

Enterococcus faecalis is frequently recovered from refractory endodontic infections and has been implicated in endodontic treatment failures. This study compared real-time quantitative PCR (qPCR) assay to cultivation for E. faecalis detection and quantitation during endodontic treatment. A reverse-transcription PCR (RT-PCR) assay was also developed to detect the bacterium clinically in the viable but nonculturable (VBNC) state. Intra-canal samples (n = 87) were collected upon access, post-instrumentation/irrigation and postcalcium hydroxide treatment from 15 primary and 14 refractory infections involving 29 single-rooted teeth, and analyzed by the three methods. The bacterium was up to three times more prevalent in refractory than primary infections at each collection step. Overall, qPCR detected significantly more E. faecalis-positive teeth and samples than cultivation (p < 0.001). VBNC E. faecalis was detected by RT-PCR in four samples that were negative by cultivation. These findings support qPCR and RT-PCR as more sensitive methods than cultivation for detecting E. faecalis in endodontic infections.

DNA, Bacterial↗

PubMed2004

Genetic variability of Brazilian Toxoplasma gondii strains detected by random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) and simple sequence repeat anchored-PCR (SSR-PCR).

The genetic variability of 19 Toxoplasma gondii strains isolated from humans and animals in Brazil was detected by both random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) and simple sequence repeat anchored-PCR (SSR-PCR) for the first time. Two reference strains, RH (highly virulent) and ME49 (avirulent), were submitted to both assays. Besnoitia sp., Plasmodium falciparum and Babesia bigemina were used as outgroups. RAPD-PCR and SSR-PCR profiles were used for building phenetic trees by unweighted pair group method with arithmetic averages (UPGMA). Phenograms built with TREECONW software showed great similarity in the topology of the trees. Both phenograms presented two major clusters that grouped T. gondii strains according to their murine virulence. The strains AS28, BV and N, which are highly virulent for BALB/c mice, were clustered with the reference strain RH, the highly virulent strain of T. gondii that has been most commonly studied. The group formed by the cystogenic strains showed that the strains which presented a level of virulence more similar to that of ME49 strain (avirulent) also presented a closer genetic relationship. Genetic variation within each lineage was significantly lower (P < 0.05) than that between the lineages. Regarding outgroups, Besnoitia sp. presented the closest relationship to T. gondii while P. falciparum was the most distant. The results presented here demonstrate that intraspecific genetic variability separates Brazilian T. gondii strains into two groups which correlate with murine virulence phenotype.

Animals↗

PubMed2002

Characterisation of symbionts of entomopathogenic nematodes by universally primed-PCR (UP-PCR) and UP-PCR product cross-hybridisation.

This work introduces and demonstrates the applicability of universally primed-PCR (UP-PCR) for differentiating bacterial symbionts of entomopathogenic nematodes. Furthermore, typing by UP-PCR product cross-hybridisation was successfully introduced to cluster the bacterial strains. The work was initiated by isolating 10 isolates of Photorhabdus temperata (S172) from the nematode host Heterorhabditis sp. (DK172) and 12 isolates of Xenorhabdus bovienii (S1) from the nematode Steinernema feltiae (DK1). The isolates were compared by UP-PCR using different primers. The two bacterial species (P. temperata and X. bovienii) could be distinguished on the basis of the banding pattern whereas isolates isolated from the same nematode host had identical banding patterns. Three isolates obtained from DK172 and DK1, respectively, were then selected along with a number of reference strains (Hb, HP88, C1, K122, HSH2, HL81, T228, 61, AN6, Q58) and further characterised by UP-PCR product cross-hybridisation. The Xenorhabdus strains (Q58, AN6, 61, T228, S1) represented three species and these species were separated by the hybridisation technique. The Photorhabdus strains (Hb, HP88, C1, K122, HSH2, HL81, S172) represented two species and were also separated according to this in the cross-hybridisation. Within each species of Photorhabdus, two subgroups were formed as a result of intensity of the hybridisation signals. This grouping was in agreement with previous studies in other laboratories.

Animals↗

PubMed2006

Molecular fingerprinting of Vibrio tapetis strains using three PCR-based methods: ERIC-PCR, REP-PCR and RAPD.

Brown Ring Disease (BRD) is a bacterial disease caused by Vibrio tapetis which affects cultured clams and causes heavy economic losses. In this study, 28 V. tapetis strains isolated from 5 different hosts were intraspecifically characterized by 3 different polymerase chain reaction- (PCR-) based typing methods: enterobacteria repetitive intergenic consensus (ERIC)-PCR, repetitive extragenic palindromic (REP)-PCR and randomly amplified polymorphic DNA (RAPD)-PCR. Cluster analysis of genetic profiles obtained from these molecular techniques clearly showed the existence of 3 genetic groups strongly correlated to the host origin. The first group was formed by 23 V. tapetis strains isolated from Manila clam Ruditapes philippinarum, 1 isolated from venus clam Venerupis aurea, and 1 isolated from common cockle Cerastoderma edule, all collected from France and Spain. The second group was formed by 2 strains isolated from carpet-shell clam R. decussatus cultured in the northwest of Spain. The third group was composed of 1 strain isolated from Atlantic halibut Hippoglossus hippoglossus from the UK. We concluded that the 3 typing methods based on PCR were useful for the intraspecific typing of V. tapetis strains, and that they can potentially be used as a fast and reliable tool for epidemiological studies in the future.

Animals↗

PubMed2003

Adapter-tagged competitive PCR (ATAC-PCR)--a high-throughput quantitative PCR method for microarray validation.

Adapter-tagged competitive PCR (ATAC-PCR) is an advanced version of competitive quantitative PCR that is characterized by the addition of unique adapters to cDNA derived from each sample RNA. Using multiple adapters, we can accurately measure the relative expression ratios of many samples, with a calibration curve obtained from internal standards included in the same reaction. ATAC-PCR can identify differences in gene expression as small as twofold, even from very small amounts of sample RNA. This technique is suitable for confirming results obtained with cDNA microarrays or differential display, and it can process more than a thousand of genes per day when used in conjunction with a capillary DNA sequencer.

Animals↗

PubMed1997

French multicentric evaluation of mdr1 gene expression by RT-PCR in leukemia and solid tumours. Standardization of RT-PCR and preliminary comparisons between RT-PCR and immunohistochemistry in solid tumours. French Network of the Drug Resistance Intergroup, and Drug Resistance Network of Assistance Publique-Hôpitaux de Paris.

Since there is no consensus on the techniques for multidrug resistance (MDR) phenotype evaluation, many discrepancies concerning the importance and frequency of mdr1 gene expression in leukemias and solid tumors are observed in the literature. In order to establish an inter-laboratory consensus in France, a multicenter study was carried out to propose further guidelines for MDR phenotype evaluation. The techniques used by the 38 laboratories participating in the trial were: immunodetection (immunohisto and/or cytochemistry, flow cytometry), functional tests, reverse transcription-polymerase chain reaction (RT-PCR) or Northern blot. We present the results obtained by 19 laboratories concerning the measurement of mdr1 gene expression assessed by RT-PCR or Northern blot in: (1)19 samples of tumor cells obtained from leukemic patients; (2) six solid tumor samples obtained at surgery; (3) eight cell lines exhibiting variable levels of resistance, and; (4)10 preparations of RNA and of cDNA obtained from solid tumors. Standardization of the RT-PCR technique and preliminary results comparing RT-PCR with immunohistochemistry in solid tumors are also reported.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2005

Performance of single-step gel-based reverse transcription-PCR (RT-PCR) assays equivalent to that of real-time RT-PCR assays for detection of the severe acute respiratory syndrome-associated coronavirus.

Simple gel-based one-step reverse transcription-PCR (RT-PCR) assays, used to investigate patients during the 2003 severe acute respiratory syndrome (SARS) outbreak in Singapore, were found to be as sensitive as commercial and in-house real-time RT-PCR assays. The detection limit was approximately 1 genome equivalent (GE) per 5 microl PCR mixture. One PFU of SARS coronavirus was estimated to be 258 +/- 46 GE.

Humans↗

PubMed2002

Long PCR-amplified rDNA for PCR-RFLP- and Rep-PCR-based approaches to recognize closely related microbial species.

Long PCR was used to amplify a 5-kb fragment of the bacterial ribosomal operon (16S-intergenic spacer region (ISR)-23S) from several Ralstonia eutropha strains (16S rDNA sequence similarity: 97-99%). Due to the large product size, amplicons from the different strains could be distinguished using restriction enzyme fragment length polymorphisms (RFLP) and repetitive PCR analysis (Rep-PCR) with the primer 1492r. These methods may prove useful in differentiating other bacterial strains with highly similar 16S rDNA sequences.

Cupriavidus necator↗

PubMed2002

Nuclear DNA PCR-RFLPs that distinguish African and European honey bee groups of subspecies. II: Conversion of long PCR markers to standard PCR.

Nuclear DNA PCR-RFLPs previously found in amplifications of three long (> 5 kbp) anonymous regions of DNA were made analyzable using standard PCR procedures. RFLP analyses were simplified by restricting the amplifications to sections, within each locus, that contained most of the informative polymorphic sites. AluI digests of locus L-1 section 2 (L-1S2) revealed three suballeles of which one was African-specific (Apis mellifera scutellata Lepeletier) and one was east European-predominant (A. m. ligustica Spinola, A. m. carnica Pollman, and A. m. caucasica Gorbachev). Alleles found originally at locus L-2 with AvaI were determined in RFLP analysis of two sections, L-2S1int and L-2S2, resulting in two African-specific and two east European-predominant suballeles. Suballele identity was determined by the combination of banding patterns from both fragments. Polymorphisms revealed by HaeIII in locus L-2 were analyzed in amplifications and digests of L-2SM1int. an 830 bpfragment within L-2S1. Seven suballeles were found of which two were African-specific and three were east European-specific or predominant, including one suballele specific to the east European subspecies A. m. caucasica. In locus L-5, RFLPs were detected with HaeIII, DdeI, and SpeI. HaeIII polymorphisms were analyzed by amplification and digestion offragments L-5S1xt and L-5S1ter: Five suballeles were found of which three were African-specific and one east European-predominant. For DdeI, all five alleles originally found with long PCR could be identified in RFLP analyses of three sections. Two African-specific, one east European-specific, and one west European-predominant (A. m. mellifera L. and A. m. iberica Goetze) suballeles were found. A west European-predominant suballele was also found in RFLP analysis of L-5S3 with SpeI. Allele frequency data from Old World and US. populations are presented.

Africa↗

PubMed1998

[Clinical evaluation of RT-PCR method for detection of HCV-RNA: second report availability of RT-PCR system using PCR internal control for detection of HCV-RNA and the influence of interfering substances on the detection].

The COBAS Amplicor system is an automated diagnostic PCR system which contains a PCR internal control (P. I. C.) template to monitor the amplification. The applicability of COBAS Amplicor HCV was examined using sera of patients with hepatitis. Furthermore, the effects of possible interfering substance (total protein, triglyceride, hemoglobin, glucose, total bilirubin, heparin, lysis reagent including guanidium) on HCV-RNA detection were investigated. The sensitivity of COBAS Amplicor HCV was equivalent to the manual method of Amplicor HCV, moreover all of the results in 54 clinical samples analyzed on both COBAS Amplicor HCV and Amplicor HCV were in agreement. Detection sensitivity of HCV-RNA decreased in the presence of total bilirubin and heparin. Ten and 25mg/dl of total bilirubin affected HCV-RNA detection but did not affect P. I. C. This result suggested that total bilirubin interfered with the protein denature caused by the lysis reagent. Fifteen U/ml of heparin in the sample completely inhibited amplification both of the HCV-RNA and P. I. C. One U/ml of heparin did not affect amplification, but heparinized blood samples should not be used for the detection of HCV-RNA. To examine the effect of possible carry over contamination on the lysis reagent which contains guanidium, various concentrations of lysis reagent in P. I. C. were tested. RT-PCR was inhibited by 1/500 volume contamination of lysis reagent in specimen diluent. Other substances did not affect the sensitivity. Our results indicate that the carryover contamination of lysis reagent cause more "false negative" results than interfering substances in sera. In conclusion, HCV-RNA detection system containing P. I. C., such as COBAS Amplicor HCV, will become a very useful to differentiate "false negative" and "true negative" result.

Bilirubin↗

PubMed2002

Comprehensive comparison of FISH, RT-PCR, and RQ-PCR for monitoring the BCR-ABL gene after hematopoietic stem cell transplantation in CML.

The reverse transcriptase-polymerase chain reaction (RT-PCR) was compared with fluorescence in situ hybridization (FISH) and real-time quantitative RT-PCR (RQ-PCR) for minimal residual disease (MRD) monitoring in 266 post-transplant bone marrow samples from 78 patients with chronic myelogenous leukemia (CML). The sensitivities of FISH to BCR-ABL positive samples determined by first-round (1st) RT-PCR, second-round (2nd) RT-PCR, and RQ-PCR were 64.2%, 25.8%, and 20.7%, respectively. The BCR-ABL/ABL ratio by RQ-PCR had a mean of 0.000 13 in the 1st RT-PCR-negative samples and 1.42 in the 1st RT-PCR-positive samples (P<0.001), and means of 0.000 39 and 0.51 in the 2nd RT-PCR-negative and -positive samples (P< 0.001). The mean ratios of BCR-ABL/ABL by RQ-PCR were significantly different in N/N (1st/2nd RT-PCR) or N/P and P/P (P<0.001), but not in N/N and N/P, which showed that the discriminative power of RQ-PCR is confined to the 1st RT-PCR level. In this respect, monitoring of the 1st RT-PCR might be useful for estimating normalized BCR-ABL levels after transplantation. Nested RT-PCR was of limited use, as RQ-PCR quantified the BCR-ABL transcripts in 60 (91%) of 66 samples determined to be negative by 2nd RT-PCR. FISH was significantly correlated with RQ-PCR in FISH-positive samples (n=24, r=0.79, P=0.001). An increase of FISH preceded that of RQ-PCR in a few cases with molecular relapse. By analyzing a large number of samples post-transplant, we found that RQ-PCR might be the most useful assay for MRD monitoring; however, FISH and RT-PCR were found to be useful complementary tools.

Bone Marrow↗

PubMed2000

T-cell clonality determination using polymerase chain reaction (PCR) amplification of the T-cell receptor gamma-chain gene and capillary electrophoresis of fluorescently labeled PCR products.

We compared the effectiveness of polymerase chain reaction (PCR) and DNA blot analysis (DBA) for detecting clonal T-cell populations and investigated whether a nonradioactive PCR method could be used in routine clinical diagnosis. We analyzed DNA from 117 cases for T-cell clonality by PCR amplification. DBA was performed on 77 of these cases. Denaturing polyacrylamide gel electrophoresis (PCR-PAGE) of radiolabeled PCR products and capillary electrophoresis (PCR-CE) of fluorescently labeled PCR products were used for PCR product separation and quantitation. Complete agreement was obtained between PCR-PAGE and DBA in 67 of 77 cases. One case was positive by DBA and negative by PCR-PAGE, and 3 cases were positive by PAGE and negative by DBA. Five cases indeterminate by DBA were positive by PCR-PAGE, and 1 indeterminate case was negative by PCR-PAGE. In the comparison of PCR-PAGE and PCR-CE, of 63 cases with height ratios less than 2.0, all were negative by PCR-PAGE. Of 52 cases with height ratios of 2.0 or more, 50 were positive by PCR-PAGE. We conclude that PCR-CE is analytically equivalent to DBA and PCR-PAGE for detecting clonal T-cell populations. The PCR-CE method is semiquantitative and, therefore, may be more objective than gel-based methods.

Clone Cells↗

PubMed2004

Evaluation of PCR and nested PCR assays currently used for detection of Coxiella burnetii in Japan.

Detection of Coxiella burnetii, the etiologic agent of Q fever, is important for diagnosis of Q fever. PCR-based methods have been widely used for the detection mostly because isolation of C. bumetii is time-consuming. Recent reports showed that PCR-positive rates of Q fever infection widely differed. We have evaluated the PCR and nested PCR assays currently used in Japan. The nested PCR assay detected as few as 6 microorganisms and was 10 times more sensitive than the regular PCR assay. The nested PCR assay did not show any non-specific bands with 12 other bacteria, whereas the PCR assay showed some extra bands for 5 of the 12 bacteria. These results suggest that the nested PCR is more sensitive and specific than the PCR in the detection of C. burnetii. However, nested PCR generally has a risk of cross-contamination during preparation of the 2nd PCR. Using blood specimens serially collected from an acute Q fever patient, the PCR and the nested PCR assays gave very similar results, suggesting that sensitivity of the PCR assay is at an achieved level of the detection for clinical specimens although the nested PCR assay is more sensitive. It is recommended that both the PCR and nested PCR assays should be performed for the detection of C. burnetii to obtain reliable results.

Coxiella burnetii↗