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Detection of hepatitis C viral RNA: comparison of Amplicor single PCR and nested set PCR processes with HCV serology for hemodialyzed patients.

Multicentre quality control studies demonstrated that optimization and standardization of HCV RNA reverse transcription polymerase chain reaction (RT-PCR) amplification techniques were possible: thus, a nested HCV RNA RT-PCR assay was described as a reliable tool for the detection of hepatitis C viral RNA. Besides this, another procedure, the Amplicor HCV RNA qualitative test, a standardized RT-PCR assay, became available. In order to assess the relationship between seropositivity and potential infectivity and to compare both RT-PCR assays, all patients undergoing maintenance hemodialysis were evaluated at hospital de Meaux (Meaux, France) during one year. We conclude that both assays are equally sensitive and that acid guanidinium thiocyanate based methods, which are used to prepare RNA prior to PCR, are more efficient than the usual phenol extraction protocols. Care should be taken with sera from hemodialyzed patients as the presence of inhibitors of the PCR has been demonstrated during the course of HCV RNA testing.

Base Sequence↗

Hybrid PCR sequencing: sequencing of PCR products using a universal primer.

We describe a general method for making template DNA for sequencing of PCR products. The procedure may be particularly useful for PCR products where minimal sequence information is known or as an alternative to primer walking when sequencing long PCR products. A cassette containing the hybridization site for the M13 sequencing primer is ligated to a sample PCR product. Using one phosphorylated primer specific for the cassette together with one primer specific for the sample PCR product, subsequent PCR amplifies one hybrid construct directionally. This allows utilization of the universal M13 primer when sequencing of one strand after the removal of the complementary strand using lambda-exonuclease.

Base Sequence↗

Detection of bcr-abl mRNA in single progenitor colonies from patients with chronic myeloid leukemia by PCR: comparison with cytogenetics and PCR from uncultured cells.

Bone marrow and/or peripheral blood of patients with chronic myeloid leukemia (CML) was investigated by the following three parameters: Ph' chromosome, bcr-abl expression in fresh blood and/or bone marrow, and bcr-abl expression in single hematopoietic progenitor colonies generated from blood and/or bone marrow. Expression of bcr-abl was proven by a reverse "nested primer" polymerase chain reaction (PCR) that is able to detect 1 pg of hybrid mRNA. We performed 108 investigations on 68 patients containing all three parameters: 12 on untreated patients, seven after interferon-alpha (IFN-alpha), seven after low-dose cytosine arabinoside (Ara-C), 22 after cyclic high-dose hydroxyurea (HU), 49 after allogeneic BMT, five before and three after stem cell mobilization, and three after autologous stem cell transplantation (ASCT). In 53 cases (49%), cytogenetics and PCR gave identical results. In 40 cases (37%), PCR from single colonies gave additional information compared to cytogenetics (e.g., mosaic in colonies when all metaphases were positive or negative). Most interesting were the results of one patient after IFN, one patient after ASCT, and 10 patients after BMT (14 investigations = 13%), showing only Ph'-negative mitoses accompanied by a negative nested primer PCR from fresh blood/bone marrow but single bcr-abl-positive progenitor colonies. False-positive results could be widely excluded by repeated insertion of negative controls into the experiments. One explanation for these results could be that CML, progenitors survive in the patient's body by being inactive and not proliferating. These cells express no or very little RNA and bcr-abl is not detectable by reverse PCR. When stimulated ex vivo in a colony assay by external growth factors, cells proliferate and produce detectable amounts of hybrid mRNA. The value of these observations is not clear. A follow-up of the patients will show if such sleeping progenitors can be activated in vivo. Concluding our observations, we can say that in special cases (therapy follow-up, detection of minimal residual disease) it could be useful to perform a PCR analysis of single progenitors in parallel with the routine investigations.

Base Sequence↗

[AP-PCR and AP-PCR-SSCP analyses].

DNA fingerprinting by arbitrarily primed polymerase chain reaction (AP-PCR) is a simple and informative method to investigate genetic changes in cancers. The discovery of a mutator phenotype in hereditary non-polypotic colon cancer by the AP-PCR fingerprinting demonstrated the usefulness of this unbiased method. Due to its semiquantitative amplification of the target DNA, the AP-PCR fingerprinting can be applied to analyze numerical changes of chromosome fragments. A recent breakthrough of the chromosomal assignment of human AP-PCR products enabled us to do PCR-mediated molecular karyotyping of cancers. Application of single-strand conformation polymorphism (SSCP) analysis to AP-PCR products seems to be promising to elucidate somatic point mutations and hidden losses of heterozygosity in the anonymous regions in cancers.

Colonic Neoplasms↗

Increased expression of inducible nitric oxide synthase for human buccal squamous-cell carcinomas: immunohistochemical, reverse transcription-polymerase chain reaction (RT-PCR) and in situ RT-PCR studies.

BACKGROUND: The inducible nitric oxide synthase (iNOS) is involved primarily in inflammatory and carcinogenesis processes. An enhanced expression of iNOS at the protein level has been reported previously for human oral squamous cell carcinoma; however, the expression of iNOS at the mRNA level has not yet been demonstrated. Furthermore, no studies have addressed whether iNOS expression at mRNA level correlates with cervical lymph node metastasis. METHODS: Specimens of the squamous cell carcinoma of the buccal mucosa obtained from tissue samples of surgically resected tumors from 25 male patients were evaluated with immunohistochemical assessment of iNOS protein and IS-RT-PCR, as well as RT-PCR for iNOS mRNA. We also analyzed the iNOS expression status with clinical parameters to determine whether it had any prognostic significance in this homogenous population. RESULTS: Inducible NOS protein (16 of 25, 64%) and mRNA (13 of 25, 53%) activities were detected for the oral carcinoma specimens examined in this study. Cytoplasmic and/or nuclear stainings were observed in the specimens of both well-differentiated SCC (10 of 15, 67%), and moderately to poorly differentiated SCC (6 of 10, 60%). The cellular location of iNOS mRNA was noted to be consistent with the finding using immunohistochemical technique (cytoplasm and/or nuclei stainings of the tumor islands). Using in situ RT-PCR, iNOS mRNA activity was detected in nine specimens of well-differentiated squamous cell carcinoma (9 of 15, 60%) and four specimens of moderately to poorly differentiated squamous cell carcinoma (4 of 10, 40%). With RT-PCR, an electrophoretic band corresponding to a 907-bp PCR product was observed for nine specimens of well-differentiated squamous cell carcinoma (9 of 15, 60%) and four specimens of moderately to poorly differentiated squamous-cell carcinoma (4 of 10, 40%). Neither iNOS protein nor mRNA was noticed in the samples of normal buccal mucosa or in the negative control samples. There was a significant relationship between iNOS expression (at both protein and mRNA levels) and whether patients had nodal disease. CONCLUSIONS: We have demonstrated an enhanced expression of iNOS protein and mRNA in a number of human buccal carcinomas compared with normal buccal mucosa. Such an observation suggests that the iNOS protein and mRNA expression is chiefly derived from a subset of oral cancerous tissues. Our observation also indicates that iNOS expression correlates with cervical lymph node metastasis in oral squamous cell carcinomas. Therefore, it may also indicate that a subset of oral cancers with greater metastatic potential, as evidenced by increased expression iNOS protein and mRNA, is identified.

Adult↗

Comparison of a LightCycler-based real-time PCR for quantitation of Epstein-Barr viral load in different clinical specimens with semiquantitative PCR.

Measurement of viral load is important in predicting and monitoring of Epstein-Barr virus (EBV)-associated diseases especially in immunocompromised patients. The objectives of this study were the development of a LightCycler-based real-time PCR assay using primers and probes which recognize the virus capsid antigen p23-encoding region and its comparison to the semiquantitative PCR. The LightCycler protocol shows a high degree of specificity and inter- and intra-assay reproducibility. Concerning sensitivity, a good correlation between both methods was demonstrated for standard plasmid DNA, reference DNA isolated from the EBV-genome containing Namalwa cell line, and DNA extracted from plasma/cerebrospinal fluid (CSF). The detection limit was determined with 1 copy/microl eluate for the standard plasmid DNA and with 500 copies/ml plasma or CSF. For DNA derived from peripheral blood mononuclear cells (PBMCs), a decrease of sensitivity by factor 10-100 was found when larger amounts of background DNA (500 and 100 ng) were used presuming an inhibitory effect of cellular DNA. This was supported by running dilutions of the plasmid standard carried out with EBV-negative Ramos cell DNA. Thus, the cut-off level was estimated with 100-500 copies/10(5) PBMCs, when 50 or 10 ng total DNA were tested. The results indicate that the real-time PCR described here is a first line tool for the determination of viral load in plasma and CSF. Semiquantitative nested PCR is used for screening of PBMCs viral load. Positive specimens containing more than 500 copies/10(5) cells are measured for exact values by real-time PCR. To circumvent inhibitory effects of cellular DNA, measurements should be carried out generally with 50-10 ng DNA.

Capsid Proteins↗

Fluorometric polymerase chain reaction (PCR) enzyme-linked immunosorbent assay for quantification of immuno-PCR products in microplates.

Immuno-polymerase chain reaction (immuno-PCR) allows the detection of protein amounts as low as a few hundred molecules. This enhanced sensitivity is useful for a variety of applications in analytical and biomedical sciences. Application of this technique as a routine method requires the rapid quantification of the PCR products, preferably as an automated readout by microplate-based assays. Here, three methods are compared for detecting such amplified products, i. e., direct staining with a fluorescent intercalating dye, an enzymatic assay utilizing doubly hapten-labeled products, and gel electrophoresis. The enzymatic assay, carried out with either chromogenic or fluorogenic substrate for enzymatic signal amplification, was found to be the most sensitive method. The optimized assay was tested in direct immuno-PCR assays for detecting immunoglobulins (IgG) from mouse and rabbit as well as in a sandwich immuno-PCR assay for detecting recombinant hepatitis B surface antigen (rec. HBsAg). Sensitivity limits were found to be as low as 15 fg (10(-19) mol) IgG, representing a 1000-fold enhancement compared to enzyme-linked immunosorbent assay detection, and about 70 fg (2 x 10(-18) mol) rec. HBsAg, improving the detection limit of currently available methods by a factor of about 700. The well-reproducible enzymatic amplification signal further enhances the sensitivity of immuno-PCR and should render the method suitable for routine laboratories.

Animals↗

One armed PCR (OA-PCR): amplification of genomic DNA from a single primer domain.

One-armed PCR (OA-PCR) is a novel technique that allows amplification of genomic DNA from a single region of known sequence. Previously described methods are either limited to cDNA or require extensive manipulation of template prior to amplification. In OA-PCR, DNA from a small insert phage Fugu genomic library is amplified using two sequence-specific primers and a tailed vector primer. Single, specific products of between 70 bp and 1.9 kb (mean 600 bp) have been obtained following two rounds of amplification and directly sequenced without cloning. The optimum parameters have been investigated: primer pairs of 18-24 bp, separated by between 0 and 500 bp, have successfully amplified a specific product. OA-PCR represents a rapid, simple method to extend genomic sequence into noncoding and regulatory sequences. In addition, degenerate OA-PCR primer pairs have allowed cross-species amplification of novel Fugu gene homologues.

DNA, Viral↗

Detection of equine arteritis virus (EAV) by polymerase chain reaction (PCR) and differentiation of EAV strains by restriction enzyme analysis of PCR products.

A polymerase chain reaction (PCR) based assay capable of detecting and differentiating seven strains of equine arteritis virus (EAV) from around the world was developed. The primers for the PCR were chosen from the ORF6 gene encoding the unglycosylated membrane protein (M). Viral RNA from cell culture fluids infected with each of the seven EAV strains and RNA from the live vaccine, Arvac, was detected by PCR using four sets of primers. The sensitivity of detection was increased from 100 to 1,000 times by performing nested PCR enabling the detection of RNA at a level of 0.5-5 PFU. Differentiation among the virus strains and the live vaccine was achieved by cutting the PCR-amplified products from three sets of primers with six restriction endonucleases. Using this procedure it was possible to distinguish among the seven EAV strains used.

Base Sequence↗

Application of PCR, rep-PCR and RAPD techniques for typing of Lactococcus lactis strains.

A collection of 34 lactococcal strains were characterized using the polymerase chain reaction (PCR) for the acmA gene, and for the 16S rDNA gene, and DNA fingerprinting methods for randomly amplified polymorphic DNA (RAPD) and repetitive extragenic palindrome-PCR (rep-PCR). PCR experiments corroborated the genotypic identification of Lactococcus lactis strains by RAPD; rep-PCR did not distinguish between L. lactis subspecies. In some cases, phenotypic classification of L. lactis subspecies did not correlate with genotypic characterization.

Bacterial Typing Techniques↗

Comparison of Parental and Transgenic Alfalfa Rhizosphere Bacterial Communities Using Biolog GN Metabolic Fingerprinting and Enterobacterial Repetitive Intergenic Consensus Sequence-PCR (ERIC-PCR).

> Abstract Rhizosphere bacterial communities of parental and two transgenic alfalfa (Medicago sativa L.) of isogenic background were compared based on metabolic fingerprinting using Biolog GN microplates and DNA fingerprinting of bacterial communities present in Biolog GN substrate wells by enterobacterial repetitive intergenic consensus sequence-PCR (ERIC-PCR). The two transgenic alfalfa expressed either bacterial (Bacillus licheniformis) genes for alpha-amylase or fungal (Phanerochaete chrysosporium) genes for Mn-dependent lignin peroxidase (Austin S, Bingham ET, Matthews DE, Shahan MN, Will J, Burgess RR, Euphytica 85:381-393). Cluster analysis and principal components analysis (PCA) of the Biolog GN metabolic fingerprints indicated consistent differences in substrate utilization between the parental and lignin peroxidase transgenic alfalfa rhizosphere bacterial communities. Cluster analysis of ERIC-PCR fingerprints of the bacterial communities in Biolog GN substrate wells revealed consistent differences in the types of bacteria (substrate-specific populations) enriched from the rhizospheres of each alfalfa genotype. Comparison of ERIC-PCR fingerprints of bacterial strains obtained from substrate wells to substrate community ERIC-PCR fingerprints suggested that a limited number of populations were responsible for substrate oxidation in these wells. Results of this study suggest that transgenic plant genotype may affect rhizosphere microorganisms and that the methodology used in this study may prove a useful approach for the comparison of bacterial communities.

Journal Article↗

Directional cloning of native PCR products with preformed sticky ends (autosticky PCR).

A novel method for the directional cloning of native PCR products was developed. Abasic sites in DNA templates make DNA polymerases stall at the site during synthesis of the complementary strand. Since the 5' ends of PCR product strands contain built-in amplification primers, abasic sites within the primers result in the formation of 5' single-stranded overhangs at the ends of the PCR product, enabling its direct ligation to a suitably cleaved cloning vector without any further modification. This "autosticky PCR" (AS-PCR) overcomes the problems caused by end sensitivity of restriction enzymes, or internal restriction sites within the amplified sequences, and enables the generation of essentially any desired 5' overhang.

Cloning, Molecular↗

First detection of Leishmania major in peridomestic Phlebotomus papatasi from Isfahan province, Iran: comparison of nested PCR of nuclear ITS ribosomal DNA and semi-nested PCR of minicircle kinetoplast DNA.

Two PCR methods were compared for their sensitivity in detecting cultured Leishmania major, before being used to estimate infection rates in female sandflies (Phlebotomus papatasi) collected from peridomestic animal shelters and the nearby burrows of the gerbil reservoir hosts, Rhombomys opimus, in Isfahan province, central Iran. A semi-nested PCR was used to amplify a fragment of minicircle kinetoplast (k) DNA with a length and sequence diagnostic for L. major, and a nested PCR was developed to amplify a fragment containing the internal transcribed spacers of the ribosomal RNA genes (ITS-rDNA) with a sequence diagnostic for L. major. The semi-nested PCR was less sensitive than the nested PCR when using DNA extracted from cultured promastigotes of L. major, but it was more sensitive for detecting L. major in wild-caught sandflies. At the edges of two Isfahan villages, infection rates were significantly higher in P.papatasi collected outside gerbil burrows (14/28) compared with those from peridomestic animal shelters (2/21). This is the first record of L. major detected in P.papatasi from peridomestic sites in Isfahan province.

Animals↗

Quantitative real time PCR of deoxycytidine kinase mRNA by Light Cycler PCR in relation to enzyme activity and gemcitabine sensitivity.

Deoxycytidine kinase (dCK) is essential for the phosphorylation of gemcitabine and can predict response to gemcitabine in vivo. Conventional Competitive Template-Reverse Transcriptase-Polymerase Chain Reaction (CT-RT-PCR) was correlated with real time PCR using a Light Cycler (LC) with SYBR-Green detection to enable rapid and sensitive detection of dCK mRNA expression. We used cDNA from human xenografts to establish a relation between dCK activity and gemcitabine sensitivity. A significant correlation of LC-PCR was found with CT-RT-PCR (Pearson: r = 0.956; P < 0.0001), enzyme activity (Pearson: r = 0.972; P = 0.003) and gemcitabine sensitivity (Pearson: r = 0.695; P = 0.048). The LC-PCR was also applied to needle biopsy specimens. In bladder tumors a similar correlation was found, while esophageal tumors with a high dCK expression responded to gemcitabine treatment. The LC is a rapid and reliable method for quantitation of dCK mRNA levels in tumors to predict clinical gemcitabine sensitivity.

Antimetabolites, Antineoplastic↗

Development and applications of a real-time quantitative RT-PCR method (QRT-PCR) for BRCA1 mRNA.

OBJECTIVES: To develop a real-time quantitative RT-PCR method for BRCA1 mRNA and then use it for the study of BRCA1 gene expression in human MCF-7 breast cancer cells after their exposure to antineoplastic agents and gamma irradiation. DESIGN AND METHODS: The developed QRT-PCR method is based on the real-time monitoring of a fluorescein-labeled TaqMan probe, specific for BRCA1 mRNA, during PCR in the LightCycler. A BRCA1 PCR amplicon was purified, quantitated and used as a standard of known concentration for the development and analytical evaluation of the assay. The method was applied to study the alteration of BRCA1 gene expression after exposure to taxol, doxorubicin, 5-fluorouracil, etoposide or gamma irradiation in human MCF-7 breast cancer cells. RESULTS: The developed method is quantitative, highly specific for mRNA and highly sensitive (detection limit of 4 BRCA1 copies per mug of total RNA). We observed a reduction of BRCA1 expression for all antineoplastic agents used, while the gamma irradiated MCF-7 cells had an increase of expression with a peak at the 10 Gy dose. CONCLUSIONS: The developed BRCA1 QRT-PCR method is quantitative, highly sensitive and specific. The proposed method is rapid, automated, and cost effective and can be used to study BRCA1 expression in a variety of clinical samples.

Amino Acid Sequence↗

Refined quantitative fluorescent PCR of Y-chromosome DNA sequences mosaics in Turner's syndrome patients--alternative to real-time PCR.

BACKGROUND: Real-time polymerase chain reactions (PCRs) are the most frequently used techniques for gonosomal mosaics quantification. The primary aim of this work is to assess and optimize the refined technique of quantitative fluorescent polymerase chain reaction (RQF PCR) in the quantification of Y-chromosome sequences in gonosomal mosaics. The method was applied to the analysis of Y-chromosome sequences (amelogenin gene, AMELX/Y-loci) in peripheral lymphocytes and gonadal tissues in Y-positive Turner's syndrome (TS) patients. METHODS: RQF PCR was used for molecular quantification, and fluorescent in situ hybridization (FISH) technique was used for comparison. RESULTS: Based on a formulated calibration curve, DNA mosaics from six Y-positive patients and gonads from one patient were deducted. For calculation of rare mosaics, it is possible to take advantage of a new empirical formula. FISH results were comparable to RQF PCR. CONCLUSION: The sensitivity of RQF PCR brings significant progress in the analysis of gonosomal aberrations. RQF PCR also finds applications in prenatal diagnostics of maternal contaminations of amniotic fluid and foetal DNA in maternal blood and analysis of chimerism in patients after bone marrow transplantation. The method is very convenient for determining the number of testis-specific protein, Y-linked (TSPY) gene repetitions.

Calibration↗

Application of real-time PCR and RT-PCR assays for the detection and quantitation of VT 1 and VT 2 toxin genes in E. coli O157:H7.

Real-time PCR assays, based on hybridisation probes and LightCycler technology, were developed for VT 1 and VT 2 genes and applied to the detection and quantitation of DNA and mRNA of Escherichia coli O157:H7. The qualitative consensus PCR assay for the detection of VT 1 and/or VT 2 genes had a detection limit of 100 fg of E. coli O157:H7 genomic DNA and did not detect DNA from 13 non-VTEC isolates. When E. coli O157:H7 was inoculated into minced beef, enriched and recovered by immunomagnetic separation, the real-time consensus PCR assay had a detection limit of log(10)3.5 ml(-1) E. coli O157:H7 cells. Nineteen E. coli O157:H7 isolates, derived from food, bovine samples and human faeces, were analysed and compared for mRNA expression of three genes, VT 1, VT 2 and gapA (housekeeping gene), using quantitative real-time PCR assays. While there was no statistically significant difference for the expression of the VT 1 (p=0.134) or VT 2 (p=0.52) mRNA in the E. coli O157:H7 isolates from food, bovine and human sources, three clinical isolates did show lower expression of VT 2 compared to other isolates in the study. The study indicates that the consensus qualitative real-time PCR assay for VT 1 and VT 2 is rapid and sensitive and that the quantitative assays reported here have the potential to be used as an alternative method to more conventional methods for studying VT 1 and VT 2 virulence gene expression in E. coli O157:H7 with potential application in other pathogenic E. coli species.

DNA Fingerprinting↗

rpoB gene: a target for identification of LAB cocci by PCR-DGGE and melting curves analyses in real time PCR.

Lactic acid bacteria (LAB) are essential in the quality of many fermented beverages like beer, cider and wine. In the two later cases, they convert malic acid into lactic acid during the malolactic fermentation. After fermentation, microbial stabilization is needed to prevent the development of spoilage bacteria species. Among them, cocci lead to different alterations: Pediococcus sp., and some strains of Leuconostoc mesenteroides and Oenococcus oeni can produce exopolysaccharides which modify wine viscosity and lead to ropiness. They also can produce acetic acid, biogenic amine, ethyl carbamate and volatile phenols. Therefore detection and identification are crucial. Results of phenotypic tests and DNA-DNA probes are not accurate enough. 16S RNA gene which is currently used for bacterial species identification presents intraspecies heterogeneity. The rpoB gene is an alternative to this limitation. However previous PCR targeting partial sequence of rpoB gene could not delimit cocci species. Therefore we compared the rpoB gene sequence of the six main cocci species found in fermented beverages: P. damnosus, P. dextrinicus, P. parvulus, P. pentosaceus, L. mesenteroides and O. oeni. The most discriminating partial sequence of the rpoB gene was chosen for designing primers. By PCR-DGGE the reliability of these primers was verified. It was controlled in a mixture of several cocci and other lactic acid bacteria (Lactobacillus sp.). Then we adapted the primers and the PCR conditions in order to achieve the identification of cocci species by real time PCR program including the fluorescent dye SYBR Green I, which gives faster results. PCR melt curves were established and a specific T(m) was attributed to each species.

Animals↗