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PubMedDate not supplied

Comparison of one-step RT-PCR and a nested PCR for the detection of canine distemper virus in clinical samples.

OBJECTIVE: To develop a rapid and sensitive method for the detection of canine distemper virus (CDV) by nested PCR using clinical specimens. DESIGN: A nested PCR was developed, compared to a one-step RT-PCR and validated. PROCEDURE: Two sets of specific primers for a one-step RT-PCR and a nested PCR, targeting a 640 bp fragment and a 297 bp fragment, respectively, were selected from the highly conserved region of the nucleocapsid protein (NP) gene of CDV. The nested PCR and the one-step RT-PCR were used to amplify a part of the CDV NP gene of a CDV vaccinal strain and samples of urine, blood, nasal discharge and saliva from 29 dogs suspected of suffering CD. RESULTS: Both the one-step RT-PCR and the nested PCR reacted with the CDV vaccinal strain, but not with canine parvovirus. The expected 640 bp fragment of the NP gene was detected in 11/22 (50.0%) blood, 10/20 (50.0%) urine, 5/25 (20.0%) saliva and 6/27 (22.2%) nasal swab samples by one-step RT-PCR, whereas the nested PCR amplified an expected 297 bp fragment of the NP gene in 18/22 (81.8%) blood, 15/20 (75.0%) urine, 14/25 (56%) saliva and 19/27 (70.3%) nasal swab samples. CONCLUSION: The nested PCR detected CDV in blood, urine, nasal swab and saliva more frequently than did the one-step RT-PCR. Therefore, this assay should be a useful aid to antemortem diagnosis of CDV infections in dogs.

Animals↗

PubMed2003

Comparison of a new quantitative ompA-based real-Time PCR TaqMan assay for detection of Chlamydia pneumoniae DNA in respiratory specimens with four conventional PCR assays.

Chlamydia pneumoniae, an important respiratory pathogen, is difficult to culture, and detection rates by conventional PCRs vary considerably. A new quantitative ompA-based real-time PCR assay based on TaqMan technology for detection of C. pneumoniae in respiratory samples is described, and its performance in terms of sensitivity and reproducibility is compared with those of four published conventional PCRs (one single-step PCR targeting a cloned PstI fragment; two nested PCRs, one targeting the 16S rRNA gene followed by hybridization and the other targeting the ompA gene; and a touchdown enzyme time-release [TETR] PCR also targeting the 16S rRNA gene). Both ompA-based PCRs showed the best analytical sensitivity. All five assays could detect even lower target levels from spiked sputum, with the 16S rRNA assays performing better than the ompA-based nested PCR (10(-6) inclusion-forming units [IFU] were detected in four of four and two of four replicates by the 16S rRNA TETR PCR and the 16S rRNA nested PCR, respectively). In general, the ompA-based real-time protocol produced the most consistent positive results for all replicates tested down to 10(-6) IFU. Eight of 45 patient sputum specimens (18%) were C. pneumoniae DNA positive in at least one of four replicates tested by at least one assay. Without taking into consideration the analytical sensitivity or the reproducibility of the test results, the numbers of C. pneumoniae DNA-positive sputum specimens (n = 8) were four, three, two, two, and one for the 16S rRNA TETR assay, the PstI-based single-step PCR, the ompA-based real-time PCR, the ompA-based nested touchdown PCR, and the 16S rRNA-based nested PCR, respectively. However, the overall rate of concordance of positive results was low. Only one cell culture-positive sputum specimen was positive by four of five assays (14 of 16 replicates; mean cycle threshold value, 25; 10(8) particles/ml of sputum). Thirty-seven specimens were C. pneumoniae negative by all five assays for all replicates tested, as were all negative controls (n = 65 to 100 per testing panel). No PCR inhibitors were detected by real-time PCR or by the 16S rRNA-based nested assay. We confirm that the analytical sensitivity of an assay for the detection of C. pneumoniae does not necessarily predict its ability to detect its target in sputum. A quantitative, fast, and easy-to-handle diagnostic approach such as the ompA-based real-time TaqMan PCR described here might improve the detection of C. pneumoniae in respiratory samples.

Adult↗

PubMed2005

Genotypic characterization of Burkholderia cenocepacia strains by rep-PCR and PCR-RFLP of the fliC gene.

Thirty-five strains of Burkholderia cenocepacia from clinical and environmental sources were characterized genotypically by repetitive sequence PCR (ERIC- and BOX-PCR) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the flagellin gene (fliC). In cluster analysis based on the repetitive PCR profiles the strains were composed of five clusters, of which clusters 1, 2 and 3 were more closely related to each other than to clusters 4 and 5. It has been reported that the majority of Burkholderia cepacia complex strains can be separated into two types on the basis of fliC size (types I and II correspond to 1.4 and 1.0 kb, respectively). When the strains were analysed by PCR of fliC, all strains yielded amplified products of 1.0 kb except for three strains. The latter strains gave PCR products of 0.7 kb (atypical type), which belonged to repetitive PCR cluster 5. These results indicated that the majority of B. cenocepacia strains belonged to flagellin type II. In the RFLP analysis of the large fliC amplicons with HaeIII, 10 patterns were observed indicating remarkable variation. Strains grouping in repetitive PCR cluster 4 had a unique fliC RFLP pattern. The results of repetitive PCR typing and PCR-RFLP analysis of fliC showed a strong correlation. Strains belonging to the repetitive PCR clusters 4 or 5 were distinctly different from other B. cenocepacia strains as shown by PCR-RFLP analysis of the fliC gene and phenotypic assays.

Bacterial Typing Techniques↗

PubMed2000

Quantification of VEGF mRNA expression in non-small cell lung cancer using a real-time quantitative reverse transcription-PCR assay and a comparison with quantitative competitive reverse transcription-PCR.

A reliable quantitative method for measuring gene product expression is important in investigating the relationship between growth factors and tumor biological behavior. In this study, we quantified the expression of vascular endothelial growth factor (VEGF) mRNA in 104 paired samples of lung cancer tissue and the surrounding nontumorous lung tissue using a new kinetic quantitative polymerase chain reaction (PCR) method, ie, real-time quantitative reverse transcription-PCR (RTQ RT-PCR). The samples consisted of 46 squamous cell carcinomas, 50 adenocarcinomas, and 8 undifferentiated cell carcinomas. In 17 cases, the results obtained were compared with those obtained using quantitative competitive RT-PCR (QC RT-PCR). Using RTQ RT-PCR, VEGF mRNA expression was detected and quantified in all 104 (100%) lung cancer samples and their normal counterparts. VEGF mRNA expression in the lung cancer tissue was significantly higher than in the normal counterparts (95% CI: 0.575 approximately 1.727, p < 0.001; paired t test). In 68 (65.4%) cases, VEGF mRNA expression was higher in the cancer tissue than normal tissue. VEGF mRNA expression was higher in nonsquamous cell carcinoma (p = 0.02), and higher in tumor with hilar or mediastinal lymph node metastasis (p = 0.024). QC RT-PCR was able to detect and quantify VEGF mRNA expression in 15/17 (88%) lung cancer samples and 12/17 (70.6%) normal tissue samples. The values for VEGF mRNA expression were higher in the tumor in 13 (76%) cases. Comparison of the values of the VEGF mRNA expression quantified using RTQ RT-PCR or QC RT-PCR showed a good correlation between results obtained using these two methods, both in cancer tissue (r = 0.68, p = 0.0025) and normal counterpart (r = 0.53, p = 0.027). Agreement between the results for the relative expression of VEGF mRNA expression in cancer and normal tissue obtained using these two methods was seen in 14/16 cases (88%). We conclude that RTQ RT-PCR is as accurate as QC RT-PCR and is more sensitive than QC RT-PCR in detecting and quantifying VEGF mRNA expression in lung cancer and normal tissues, and both methods reveal that the VEGF mRNA expression is higher in lung cancer tissue than in healthy lung tissue.

Base Sequence↗

PubMed2002

Comparison of competitive-nested PCR and real-time PCR in detecting BCR-ABL fusion transcripts in chronic myeloid leukemia patients.

Real-time RT-PCR has great advantages for estimating transcript levels in a variety of situations. These include relative rapid assay times (hours), reliability and ease of performing replicate analyses. In contrast, competitive PCR is a very labor-intensive procedure requiring a few days to generate useful data. We compared the same samples from CML patients by both methods. Importantly, we used the Bcr-Abl junction plasmid DNA, which is used as a competitor in the manual competitive PCR assay, to generate a standard curve for the real-time assay. This permitted reporting the real-time data as the number of BCR-ABL transcripts per microg of total RNA, which is the same format used for the competitive PCR assay. In this study, a total of 435 peripheral blood and marrow samples from 285 CML patients were analyzed by RT-PCR; these patients were undergoing therapy by STI-571, interferon, and bone marrow transplantation treatment. Most samples also had assay values for the Philadelphia chromosome (Ph), FISH and Western blotting for the Bcr-Abl oncoprotein. Our findings indicated that the real-time assay was less sensitive than the manual competitive RT-PCR assay (t = 5.118; P < 0.001). Of interest, the transcript levels in cell line mixtures with various ratios of K562/KG-1 (BCR-ABL positive/negative) cells were also significantly higher with the competitive RT-PCR assays than real-time RT-PCR, except for levels of BCR-ABL below 200 transcripts per microg of RNA. In both patient and cell line experiments, dividing the BCR-ABL transcripts by the total ABL transcripts virtually eliminated the difference between real-time BCR-ABL transcript values and quantitative competitive BCR-ABL transcript values, indicating that both BCR-ABL and ABL transcripts were underestimated by the real-time assay. In addition, the increased sensitivity of the nested, competitive RT-PCR was readily apparent in patients with minimal residual disease, which by the real-time were negative in the majority of patients but were positive by nested, competitive RT-PCR in 44.6% (n = 29) of samples analyzed (n = 65). These findings indicate that real-time RT-PCR, when normalized for the total ABL transcripts, can be used to monitor CML patients during therapy, but we suggest that nested, competitive RT-PCR be used to determine BCR-ABL/ABL transcript ratios at low transcript values or especially when real-time analyses are negative.

Fusion Proteins, bcr-abl↗

PubMed2006

Improved DOP-PCR-based representational whole-genome amplification using quantitative real-time PCR.

In many cases, only a minute amount of partially degraded genomic DNA can be extracted from archived clinical samples. Diverse whole-genome amplification methods are applied to provide sufficient amount of DNA for comparative genome hybridization, single-nucleotide polymorphism, and microsatellite analyses. In these applications, the reliability of the amplification techniques is particularly important. In PCR-based approaches, the plateau effect can seriously alter the original relative copy number of certain chromosomal regions. To eliminate this distorting effect, we improved the standard degenerate oligonucleotide-primed PCR (DOP-PCR) technique by following the amplification status with quantitative real-time PCR (QRT-PCR). With real-time detection of the products, we could eliminate DNA overamplification. Probes were prepared from 10 different tumor samples: primary and metastatic melanoma tissues, epidermoid and bronchioloalveolar lung carcinomas, 2 renal cell carcinomas, 2 colorectal carcinomas, and a Conn and Cushing adenoma. Probes were generated by using nonamplified and amplified genomic DNA with DOP-PCR and DOP-PCR combined with QRT-PCR. To demonstrate the reliability of the QRT-PCR based amplification protocol, altogether 152 relative copy number changes of 44 regions were determined. There was 85.6% concordance in copy number alterations between the QRT-PCR protocol and the nonamplified samples, whereas this value was only 63.8% for the traditional DOP-PCR. Our results demonstrate that our protocol preserves the original copy number of different chromosomal regions in amplified genomic DNA than standard DOP-PCR techniques more accurately.

DNA Primers↗

PubMed2005

Subtyping of Clostridium difficile PCR ribotype 001 by REP-PCR and PFGE.

The REP-PCR (repetitive sequence-based PCR using repetitive extragenic palindromic primers) typing method and a modified PFGE method were applied to isolates of Clostridium difficile PCR ribotype 001 with the aim of comparing their performance as methods of subtyping this organism. Of 200 isolates from 60 hospitals tested by REP-PCR, eight subtypes were identified and labelled as REP-PCR subtypes 001-008. The predominant subtype, REP-PCR subtype 003, accounted for 47% of the total. Fifty-two of the 200 isolates were analysed by a modified PFGE method and seven subtypes were identified, labelled as PF-A-PF-G. There was excellent correlation between REP-PCR subtypes and PFGE subtypes with both methods displaying broadly similar discriminatory powers. However, REP-PCR subtyping proved to be a much easier, cheaper and more rapid method suitable for application for routine subtyping of C. difficile ribotype 001. Application of REP-PCR subtyping to UK isolates of C. difficile PCR ribotype 001 from 60 different centres revealed a wide distribution of REP-PCR subtype 003 throughout England and Wales, with a regional clustering of REP-PCR subtype 001 around Northwest England and North Wales. Analysis of isolates from a single hospital over a 4-year period revealed a change in predominant subtype over time.

Clostridioides difficile↗

PubMed2003

Real-time PCR assay compared to nested PCR and antigenemia assays for detecting cytomegalovirus reactivation in adult T-cell leukemia-lymphoma patients.

We analyzed the efficiency of the quantitative real-time PCR assay for cytomegalovirus (CMV) reactivation in adult T-cell leukemia-lymphoma (ATL) patients and compared the results with those obtained with qualitative nested PCR and antigenemia assays. The viral load obtained by the real-time PCR assay closely paralleled the number of antigen-positive cells obtained with the antigenemia assay. Real-time PCR revealed that a large number of DNA copies could be present even in samples assessed as negative or low in antigen-positive cells (0 to 10 antigen-positive cells/50,000 cells) by antigenemia assay. CMV copy numbers did not differ between the negative and low-antigen-positive groups. When the input concentration for real-time PCR assay was 2,500 to 5,000 copies/ml, the positivity rate for the nested PCR assay was 47.3%, while the positivity rate was more than 90% at an input concentration of >/=50,000 copies/ml. Real-time PCR is more sensitive than the antigenemia and nested PCR assays. Moreover, real-time PCR was able to detect CMV reactivation earlier than the antigenemia and nested PCR assays through the use of longitudinal analysis in four ATL patients with CMV pneumonia. In longitudinal assessments, analysis of the results suggested that a cutoff level of 5,000 copies/ml might be used to initiate treatment. Real-time PCR is more suitable for monitoring CMV reactivation in ATL patients than the antigenemia and nested PCR assays. CMV viral loads of 5,000 copies/ml are proposed as the cutoff for initiating antiviral therapy in ATL patients.

Adult↗

PubMed2004

Pre-PCR processing: strategies to generate PCR-compatible samples.

Polymerase chain reaction (PCR) is recognized as a rapid, sensitive, and specific molecular diagnostic tool for the analysis of nucleic acids. However, the sensitivity and kinetics of diagnostic PCR may be dramatically reduced when applied directly to biological samples, such as blood and feces, owing to PCR-inhibitory components. As a result, pre-PCR processing procedures have been developed to remove or reduce the effects of PCR inhibitors. Pre-PCR processing comprises all steps prior to the detection of PCR products, that is, sampling, sample preparation, and deoxyribonucleic acid (DNA) amplification. The aim of pre-PCR processing is to convert a complex biological sample with its target nucleic acids/cells into PCR-amplifiable samples by combining sample preparation and amplification conditions. Several different pre-PCR processing strategies are used: (1) optimization of the DNA amplification conditions by the use of alternative DNA polymerases and/or amplification facilitators, (2) optimization of the sample preparation method, (3) optimization of the sampling method, and (4) combinations of the different strategies. This review describes different pre-PCR processing strategies to circumvent PCR inhibition to allow accurate and precise DNA amplification.

Animals↗

PubMed2005

Effectiveness of real-time quantitative PCR compare to repeat PCR for the diagnosis of Charcot-Marie-Tooth Type 1A and hereditary neuropathy with liability to pressure palsies.

The majority of cases of Charcot-Marie-Tooth type 1A (CMT1A) and of hereditary neuropathy with a liability to pressure palsies (HNPP) are the result of heterozygosity for the duplication or deletion of peripheral myelin protein 22 gene (PMP22) on 17p11.2. Southern blots, pulsed-field gel electrophoresis (PFGE), fluorescence in situ hybridization (FISH) and polymorphic marker analysis are currently used diagnostic methods. But they are time-consuming, labor-intensive and have some significant limitations. We describe a rapid real- time quantitative PCR method for determining gene copy number for the identification of DNA duplication or deletion occurring in CMT1A or HNPP and compare the results obtained with REP-PCR. Six patients with CMT1A and 14 patients with HNPP [confirmed by Repeat (REP)-PCR], and 16 patients with suspicious CMT1A and 13 patients with suspicious HNPP [negative REP-PCR], and 15 normal controls were studied. We performed REP-PCR, which amplified a 3.6 Kb region (including a 1.7Kb recombination hotspot), using specific CMT1A-REP and real-time quantitative PCR on the LightCycler system. Using a comparative threshold cycle (Ct) method and beta -globin as a reference gene, the gene copy number of the PMP22 gene was quantified. The PMP22 duplication ratio ranged from 1.35 to 1.74, and the PMP22 deletion ratio from 0.41 to 0.53. The PMP22 ratio in normal controls ranged from 0.81 to 1.12. All 6 patients with CMT1A and 14 patients with HNPP confirmed by REP-PCR were positive by real-time quantitative PCR. Among the 16 suspicious CMT1A and 13 suspicious HNPP with negative REP-PCR, 2 and 4 samples, respectively, were positive by real-time quantitative PCR. Real-time quantitative PCR is a more sensitive and more accurate method than REP-PCR for the detection of PMP22 duplications or deletions, and it is also faster and easier than currently available methods. Therefore, we believe that the real-time quantitative method is useful for diagnosing CMT1A and HNPP.

Charcot-Marie-Tooth Disease↗

PubMed2004

[Prospects and applications of innovated quantitative fluorescent PCR (IQF PCR) in analyses of genetic mosaics using gonosomal sequences].

BACKGROUND: Quantification of fluorescence labelled PCR products on capillary electrophoresis (QF PCR) has limits primarily in the possibility of more sensitive analyses to detect minority cell lines and small time related variations. PCR efficiency and human factor affect measuring error and reproducibility of results in these cases. The aim of this work was to assess and optimise of innovated (I)QF PCR in quantification of Y sequences in gonosomal mosaics. METHODS AND RESULTS: Artificially prepared Y/X mosaics were tested and quantified by IQF PCR, which replaces real-time PCR. Comparison of relative fluorescence to PCR cycles in different Y/X dilutions was plotted on the graphs. Calibration curve for Y sequences quantification was set by the analyses of ratio of Y/X fluorescent signals. An empirical formula was created for the rare mosaic calculation. CONCLUSIONS: QF PCR refined by manual real-time PCR eliminates limits of QF PCR and specifies quantitative analyses based on PCR. The outstanding feature of IQF PCR is its high sensitivity and accuracy in quantification of Y/X gonosomal mosaics.

Cell Line↗

PubMed1994

Detection of cytomegalovirus in liver transplant biopsies. A comparison of light microscopy, immunohistochemistry, duplex PCR and nested PCR.

The polymerase chain reaction was used to detect cytomegalovirus (CMV) in 91 formalin-fixed paraffin-embedded needle biopsies from 38 liver transplant patients with allograft dysfunction. Thirty donor liver biopsies served as negative controls. PCR results were compared with light microscopy (LM), immunohistochemical staining (IH) for CMV early and late antigen, and clinical data. Primers to the major immediate early gene (MIE) and the viral DNA polymerase gene were duplex amplified. PCR product was reamplified with a nested primer set for the MIE and confirmed by electrophoretic mobilities and dot blotting. Primers for human beta-hemoglobin were used as internal controls. Seventeen of 38 patients had clinical evidence of cytomegalovirus disease, 12 of these were IH-positive, 14 were LM-positive, 15 were duplex PCR-positive and 17 were nested PCR-positive. In addition, duplex PCR was positive in one patient without other evidence of CMV disease, while nested PCR was positive in 12 such patients. The sensitivity and negative predictive value of nested PCR was 100%--however, the specificities and positive predictive values were only 42.9 and 58.6%, respectively. The control group was completely negative by LM, IH, and duplex PCR, however, 6 of 30 patients were nested PCR-positive. The number of nested-positive, duplex-negative patients without CMV disease was significantly greater in the transplant group versus the control group (12/21 vs. 6/30, P < 0.009). The incidence of IgG seropositivity was also significantly greater in the transplant group versus the controls (29/32 vs. 15/24, P < 0.02). We conclude that nested PCR may be an overly sensitive technique for the detection of clinically relevant CMV disease. A negative nested PCR assay for CMV may, however, help rule-out symptomatic CMV infection in an individual case. Duplex PCR showed little advantage over LM, while IH was confirmatory but did not add any new information in this study.

Antigens, Viral↗

PubMed2004

Detection of low copy numbers of HIV-1 proviral DNA in patient PBMCs by a high-input, sequence-capture PCR (Mega-PCR).

An internally controlled high-input PCR method, termed HIV-1 Mega-PCR was developed to lower the detection limit of HIV-1 DNA polymerase chain reaction (PCR) and to improve its value as a complementary diagnostic test. It is based on PCR amplification of two target sequences in the gag gene of HIV-1 following the selective capture of the targeted sequence and removal of unselected DNA from up to 500 microg of DNA. Efficient selection and amplification was monitored by inclusion of two mimic plasmids. The method was evaluated with buffy coat cells from healthy blood donors which were spiked with blood from 106 different HIV-1-infected individuals, and with 107 HIV-1 seronegative control buffy coats. All specimens from HIV-infected individuals were positive by a PCR protocol using 1 microg of patient DNA. Amplification of 1 microg DNA of the 106 spiked, diluted samples resulted in 68 double positive, 14 single positive, and 24 double negative reactions. In the Mega-PCR, the average input was 260 +/- 84 microg DNA containing an estimated 1.1 +/- 0.6% of spiked patient DNA. Of the 106 samples tested by Mega-PCR, 102 were positive and three negative. One failed to select the mimic plasmid. Among the 107 negative buffy coat controls, none was false-positive and four exhibited a failure of the internal reaction control. Application of HIV-1 Mega-PCR to clinical specimens from seroreverting newborns of HIV-infected mothers and seroindeterminate, PCR-negative specimens revealed no indication for HIV infection, whereas three samples from confirmed, HIV-1-infected but PCR negative individuals showed evidence of the presence of HIV-1 DNA. Mega-PCR lowers the detection limit of an individual analysis to approximately 0.01 HIV-1 DNA copies/microg of applied DNA and may help to confirm or exclude HIV-1-infection in difficult situations diagnostic.

Adult↗

PubMed2004

One-Step RT-PCR protocols improve the rate of dengue diagnosis compared to Two-Step RT-PCR approaches.

Dengue is the most important arboviral disease transmitted to humans. In our laboratory, we have been working on the standardization of the polymerase chain reaction (PCR) diagnosis of this disease. In this work, we compared five commercial kits regularly used on reverse-transcription polymerase chain reaction (RT-PCR) protocols: two Two-Step kits (SuperScript II RT/Super Mix kit and reverse transcription system/Taq DNA polymerase) and three One-Step kits (ready-to-go RT-PCR Beads kit, QIAGEN One-Step RT-PCR kit, and AcessQuick RT-PCR system). Thirty-one serum samples of patients with clinical diagnosis of dengue fever (DF) were analyzed by RT-PCR and serology. RNA extraction was done with the QIAamp Viral RNA kit, and cDNA synthesis and PCR done according to the manufacturer's protocol for the five kits. Out of the 31 serum samples collected from patients suspected of having dengue, 27 were IgM-positive, confirming the dengue diagnosis. Out of those, 24 were positive by the ready-to-go RT-PCR Beads kit, 25 were positive by AcessQuick RT-PCR system and 27 were positive by QIAGEN One-Step RT-PCR kit. On the other hand, only six samples were positive by the SuperScript II RT/Super Mix kits and 10 were positive by reverse transcription system/Taq DNA polymerase kit. The best performance observed with the One-Step kits was confirmed in spiked samples with known quantities of dengue-1 virus since they detected up 1 x 10(2) PFU/ml, while the most sensitive Two-Step kit detected up 1 x 10(4) PFU/ml. These data show that One-Step RT-PCR kits yielded a higher rate of dengue virus detection than the Two-Step kits and correlated well with the serological diagnosis.

DNA, Complementary↗

PubMed2004

Development of a PCR-and hybridization-based assay (PCR Adenovirus Consensus) for the detection and the species identification of adenoviruses in respiratory specimens.

BACKGROUND: Antigen detection assays and viral isolation techniques are routinely used to detect adenoviruses (Ad) associated with respiratory infections, and the value of the polymerase chain reaction (PCR) has recently been assessed. OBJECTIVES: This paper describes a PCR-hybridization-immunoenzymatic assay (PCR Adenovirus consensus) used to detect Ad and identify Ad species in respiratory specimens. RESULTS: On seven representative serotypes Ad 12, Ad 3, Ad 7, Ad 11, Ad 1, Ad 8, Ad 4, the mean genome equivalents per ml and the mean 50% infectious doses per ml were 10(6.3)and 10(4), respectively. Using 362 nasal aspirates from children, Ad were detected by immunofluorescence (IF) and culture in 97 cases (27%), by the PCR-Ad hexon method in 107 cases (29.5%) and by the PCR Adenovirus Consensus method in 113 cases (31.2%); 13 samples were found positive by both PCR and negative by the IF and culture methods; five samples were only positive according to the PCR Adenovirus Consensus) method. The sensitivity, specificity, predictive positive value and predictive negative value of the PCR Adenovirus Consensus method were 97.9%, 93.2%, 84%, 99.1%, respectively. The method identified the species (sp) from 91 positive amplicons: 1 Ad sp A, 44 Ad sp B, 42 Ad sp C, 3 Ad sp E, and 1 Ad sp F; 85 isolates were identified by IF or the neutralisation in culture, and 86 by a PCR-RE digestion method. The PCR Adenovirus Consensus detected six positive samples that were negative according to the IF and culture methods, and it identified the precise species of nine IF-positive and culture-negative nasal aspirates. CONCLUSION: The PCR Adenovirus Consensus technique is more efficient than the classical IF or culture techniques for the detection of Ad in respiratory samples. An internal control is included to validate the screening results, and specific probes are used to identify the Ad species.

Adenovirus Infections, Human↗

PubMed2004

PCR and PCR-RFLP techniques targeting the DNA topoisomerase II gene for rapid clinical diagnosis of the etiologic agent of dermatophytosis.

BACKGROUND: We have focused on the DNA topoisomerase II genes of several pathogenic fungi, and developed polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP) methods targeting this gene for identification of dermatophytes. OBJECTIVE: To assess the availability of the PCR-based identification for an etiologic study of dermatophytosis, by testing these PCR and PCR-RFLP methods for stability and reproducibility. METHODS: Three hundred and fifty-six dermatophyte strains were isolated from 305 patients with tinea, and their genomic DNAs were used as templates for the PCR using primer mixes (PsT, PsME, dPsD1 or dPsD2) composed of gene-specific primers for identification of dermatophytes to the species level. The genomic DNAs of Trichophyton rubrum were further subjected to subrepeat element analysis of the nontranscribed spacer (NTS) of ribosomal DNA (rDNA). RESULTS: In this study, six dermatophyte species (T. rubrum, Trichophyton mentagrophytes, Trichophyton tonsurans, Microsporum canis, Microsporum gypseum, and Epidermophyton floccosum) were obtained. In all cases, the identifications obtained from the PCR and PCR-RFLP targeting the DNA topoisomerase II gene coincided with those from the conventional morphological features-based identification technique. The sensitivity of the PCR-based identification was found to be a colony of approximately 3mm in diameter. Furthermore, T. rubrum was divided into three groups (17 types) on the basis of the sizes and numbers of the products generated from the TRS-1 region, and three types from the TRS-2 region. CONCLUSION: The PCR and PCR-RFLP targeting the DNA topoisomerase II gene were rapid, stable, and reproducible for species identification of dermatophytes, and thus are convenient tools for an etiologic study of dermatophytosis.

Adolescent↗

PubMed2004

Detection and quantitation of bovine respiratory syncytial virus using real-time quantitative RT-PCR and quantitative competitive RT-PCR assays.

A single tube, fluorogenic probe-based, real-time quantitative reverse transcription-polymerase chain reaction (Q-RT-PCR) assay was developed for detection and quantitation of bovine respiratory syncytial virus (BRSV) using BioRad's iCycler iQ. Real-time Q-RT-PCR was compared with quantitative competitive RT-PCR (QC-RT-PCR) and viral titers. Viral mRNA levels were measured in BRSV-infected bovine turbinate cell lysate harvested at eight time points (1.5, 6, 12, 24, 36, 48, 60, 72 h) post-infection. A homologous BRSV cRNA standard was used for quantitation of the mRNA by plotting a standard curve of cycle threshold (Ct) values versus standard 10-fold dilutions of cRNA of known concentrations. Detection as low as 171 copies/microl of standard BRSV cRNA was possible. For QC-RT-PCR, a competitor RNA molecule having a deletion was designed and used for quantitation of the BRSV viral mRNA. The results of real-time Q-RT-PCR and QC-RT-PCR assays showed a positive correlation. Real-time Q-RT-PCR was a sensitive, specific, rapid, and efficient method that eliminates the post-PCR processing steps when compared to QC-RT-PCR. Quantitation of BRSV using real-time Q-RT-PCR will have application in studies aimed at understanding the pathogenesis of BRSV.

Animals↗

PubMed2003

A comparison of in situ hybridisation, reverse transcriptase-polymerase chain reaction (RT-PCR) and in situ-RT-PCR for the detection of canine distemper virus RNA in Paget's disease.

Previous evidence implicating Paramyxoviruses in the aetiopathology of Paget's disease of bone has proved controversial. Whilst several groups have demonstrated Paramyxoviruses using techniques such as in situ hybridisation (ISH), reverse transcriptase-polymerase chain reaction (RT-PCR), and in situ-RT-PCR (IS-RT-PCR), others have found no evidence of viruses using only RT-PCR. To investigate this latter finding, we have now compared detection of canine distemper virus by ISH, RT-PCR (three different methods) and IS-RT-PCR, in 10 patients with Paget's disease, and samples of non-diseased bone from four patients. Canine distemper virus was detectable in six of the samples by ISH, but only in five of the samples by RT-PCR, using one of the methods. Neither of the other RT-PCR methods detected canine distemper virus. IS-RT-PCR demonstrated canine distemper virus in all 10 samples. There was no evidence of virus in the control samples. We have shown that the ability to detect canine distemper virus in bone is dependent on the technique used. IS-RT-PCR clearly showed that canine distemper virus was present in 100% of Pagetic samples, whereas canine distemper virus was only found in 60% by ISH and in 50% using one particular RT-PCR method. These results provide conclusive evidence that canine distemper virus is present within Pagetic bone, and provide a possible explanation for the failure of some groups to detect Paramyxovirus sequences. These findings also have wider implications for other studies investigating viral expression.

Distemper Virus, Canine↗