Rapid quantification of hepatitis B virus DNA by automated sample preparation and real-time PCR

Monitoring of hepatitis B virus (HBV) DNA in serum by molecular methods has become the standard for assessment of the replicative activity of HBV. Several molecular assays for the detection and quantification of HBV DNA have been described. However, they usually lack automated sample preparation. Moreover, those assays, which are based on PCR, are limited by a short dynamic range (2 to 3 log units). In the present study, the use of RealArt HBV LC PCR Reagents in conjunction with automated extraction on the COBAS AMPLIPREP analyzer was evaluated. Members of an HBV proficiency program panel were tested; linearity, interassay, and intra-assay variations were determined. The performance of the assay in a routine clinical laboratory was evaluated with a total of 117 clinical specimens. When members of the HBV proficiency program panel were tested by the new molecular assay, the results were found to be within +/-0.5 log unit of the results obtained by reference laboratories. Determination of linearity resulted in a quasilinear curve over more than 6 log units. The interassay variation of the RealArt HBV LC PCR Reagents by use of the automated sample preparation protocol ranged from 16 to 73%, and the intra-assay variation ranged from 9 to 40%. When clinical samples were tested by the new assay with the automated sample preparation protocol and the results were compared with those obtained by the COBAS AMPLICOR HBV MONITOR Test with manual sample preparation, the results for 76% of all samples with positive results by both tests were found to be within +/-0.5 log unit and the results for another 18% were found to be within between 0.5 and 1.0 log unit. In conclusion, the real-time PCR assay with automated sample preparation proved to be suitable for the routine molecular laboratory and required less hands-on time.     

Normalized quantification of human cytomegalovirus DNA by competitive real-time PCR on the LightCycler instrument

The development of a novel normalized quantitative competitive real-time PCR on the LightCycler instrument (NQC-LC-PCR) and its application to the quantification of cytomegalovirus (CMV) DNA in clinical samples are described. A heterologous competitor DNA was spiked into test samples and served as an internal amplification control. The internal control (IC) DNA in the test samples was coamplified with the CMV DNA and was tested against a calibrator sample that contained equal amounts of IC DNA and CMV reference standard DNA. An algorithm was developed to normalize possible varying amplification efficiencies between the standard and the samples. After normalization, CMV DNA copy numbers were determined in absolute terms. In a routine clinical setting, normalized quantification by NQC-LC-PCR using a single IC concentration led to results ranging from 500 to 50,000 CMV DNA copies/ml. The results obtained with conventional real-time quantification on the LightCycler instrument were almost identical to those obtained with the NQC-LC-PCR-based quantification. This was the case only for samples in which the PCR was not inhibited. With partially inhibited samples, NQC-LC-PCR was still able to correctly quantify CMV DNA copy numbers even when the PCR was inhibited by about 70%. By analyzing 80 undefined clinical samples, we found that NQC-LC-PCR was suitable for the routine assessment of CMV DNA in clinical plasma samples. Since the ICs were already added to the samples during the DNA purification, almost the entire assay was controlled for sample adequacy. Thus, false negative results were precluded. The NQC-LC-PCR approach developed should be adaptable for additional microbiological applications.     

A quantitative, internally controlled real-time PCR Assay for the detection of parvovirus B19 DNA

Parvovirus B19 is an erythrovirus causing diverse clinical manifestations ranging from asymptomatic or mild, to more severe outcomes in, for example, immune-compromised patients. B19 is spread primarily via the respiratory route, but it can also be transmitted via blood and blood products. Viral loads in blood or plasma donations amount up to 10(11) genome equivalents/ml. Therefore, screening of plasma for fractionation for the presence of B19 and removal of highly loaded donations is a way to limit considerably the input of B19 into production pools and to improve further the safety of plasma products.An assay for the quantitative detection of B19 DNA, based on real-time PCR using ABI Prism SDS7700 (TaqMan) is described here. This assay allows precise quantitation of viral loads over 7 orders of magnitude. An exogenous internal control (internal quality marker) is included in each individual sample to prevent false negative results. A linearized plasmid is used as an internal quality marker that contains the identical sequence of the B19 target sequence but with an altered probe hybridization site. This allows co-amplification of B19 and internal quality marker and co-detection of FAM (6-carboxyfluorescein) or VIC labeled probes respectively. The assay is validated according to current guidelines (of the International Conference on Harmonization, Paul Ehrlich Institute, and the Council of Europe) and is optimized for high throughput screening.     

Multilaboratory evaluation of real-time PCR tests for hepatitis B virus DNA quantification

The performance characteristics of four different assays for hepatitis B virus (HBV) quantification were assessed: the Abbott RealTime HBV IUO, the Roche Cobas AmpliPrep/Cobas TaqMan HBV test, the Roche Cobas TaqMan HBV test with HighPure system, and the Qiagen artus HBV TM ASR. Limit of detection (LOD), linear range, reproducibility, and agreement were determined using a serially diluted plasma sample from a single chronically infected subject. Each assay was tested by at least three laboratories. The LOD of the RealTime and two TaqMan assays was approximately 1.0 log(10) IU/ml; for artus HBV (which used the lowest volume of extracted DNA), it was approximately 1.5 log(10) IU/ml. The linear range spanned 1.0 to at least 7.0 log(10) IU/ml for all assays. Median values were consistently lowest for artus HBV and highest for Cobas AmpliPrep/Cobas TaqMan HBV. Assays incorporating automated nucleic acid extraction were the most reproducible; however, the overall variability was minor since the standard deviations for the means of all tested concentrations were ≤0.32 log(10) IU/ml for all assays. False-positive results were observed with all assays; the highest rates occurred with tests using manual nucleic acid extraction. The performance characteristics of these assays suggest that they are useful for management and therapeutic monitoring of chronic HBV infection.     

A genotype-independent real-time PCR assay for quantification of hepatitis B virus DNA

Accurate quantification of hepatitis B virus (HBV) DNA levels is important for monitoring patients with chronic HBV infection and for assessing their responses to antiviral therapy. This study aimed to develop a real-time PCR assay that is sensitive and can accurately quantify a wide range of HBV DNA levels across the known HBV genotypes. An "in-house" real-time PCR assay using primers and a TaqMan probe in a highly conserved region of the HBV surface gene was designed. The assay was standardized against a WHO standard and validated against plasmids of HBV genotypes A through H. The linear quantification range was approximately 5 x 10(0) to 2.0 x 10(9) IU/ml. Results of samples from patients infected with HBV genotypes A through H tested using our real-time "in-house" PCR assay showed an excellent correlation with those of the Cobas Amplicor HBV Monitor (R2=0.9435) and the Cobas TaqMan HBV (R2=0.9873) tests. We have established a real-time PCR assay that is genotype independent and can accurately quantify a wide range of HBV DNA levels. Further studies of additional samples are ongoing to validate the genotype independence of our assay.     

Detection of herpes simplex virus DNA in genital and dermal specimens by LightCycler PCR after extraction using the IsoQuick, MagNA Pure, and BioRobot 9604 methods

We evaluated two automated systems, MagNA Pure (Roche Molecular Biochemicals, Indianapolis, Ind.) and BioRobot 9604 (Qiagen, Inc., Chatsworth, Calif.) as effective replacements for the manual IsoQuick method (Orca Research, Inc., Bothell, Wash.) for extraction of herpes simplex virus (HSV) DNA from dermal and genital tract specimens prior to analysis by LightCycler PCR. Of 198 specimens (152 genital, 46 dermal), 92 (46.2%) were positive for HSV DNA by LightCycler PCR after automated extraction of specimens with either the MagNA Pure or BioRobot 9604 instrument. The manual IsoQuick method yielded HSV DNA (total n = 95) from three additional specimens that were negative by the automated method (P = 0.25, sign test). Although the mean numbers of LightCycler PCR cycles required to reach positivity differed statistically significantly among all three of the methods of extraction, the estimated means differed by no more than 1.5 cycles (P < 0.05). Seventy (76%) of the 92 specimens that were LightCycler PCR positive by all three extraction methods were also positive by shell vial cell culture assay. HSV DNA was detected by a lower LightCycler PCR cycle number (26.1 cycles) in specimens culture positive for the virus than in culture-negative samples (33.3 cycles) (P < 0.0001). The manual IsoQuick and automated MagNA Pure and BioRobot 9604 methods provide standardized, reproducible extraction of HSV DNA for LightCycler PCR. The decision to implement a manual versus an automated procedure depends on factors such as costs related to the number of specimens processed rather than on the minimal differences in the technical efficiency of extraction of nucleic acids among these methods.     

Development of an internally controlled real-time PCR assay for detection of Chlamydophila psittaci in the LightCycler 2.0 system

A real-time PCR assay with a DNA purification and inhibition control (internal control; IC) was developed to detect Chlamydophila psittaci DNA in human clinical samples. Novel C. psittaci-specific primers targeting the ompA gene were developed. The IC DNA contained the same primer-binding sites and had the same length and nucleotide content as the C. psittaci DNA amplicon, but had a shuffled probe-binding region. The lower limit of detection was 80 target copies/PCR, corresponding to 6,250 copies/mL in a clinical sample. Specificity was tested using reference strains of 30 bacterial species. No amplification was observed from any of these samples. Respiratory samples from eight patients were positive with this PCR. Six of these patients were confirmed as positive for C. psittaci with serological testing. Two patients had increasing antibody titres, but did not fulfil criteria proposed previously for serologically proven Chlamydia spp. infection. The real-time PCR described in this paper is a sensitive, specific and rapid method to detect C. psittaci DNA in human clinical respiratory samples.     

Reduced PCR sensitivity due to impaired DNA recovery with the MagNA Pure LC total nucleic acid isolation kit

The increasing demand for molecular diagnostics in clinical microbiology laboratories necessitates automated sample processing. In the present study, we evaluated the performance of the MagNA Pure LC total nucleic acid isolation kit (M extraction) in comparison with the manual method (Si extraction) according to Boom et al. (R. Boom, C. J. A. Sol, M. M. M. Salimans, C. L. Jansen, P. M. Wertheim-van Dillen, and J. van der Noordaa, J. Clin. Microbiol. 28:495-503, 1990) for the detection of viral DNA by competitive quantitative PCR. Reconstruction experiments with HindIII-digested phage lambda DNA and HaeIII-digested phiX174 DNA showed that the recovery of DNA from phosphate-buffered saline, cerebrospinal fluid, EDTA-anticoagulated plasma, and EDTA-anticoagulated whole blood by M extraction is, on average, 6.6-fold lower compared to Si extraction. PCR signals of spiked PCR control DNAs for Epstein-Barr virus and varicella-zoster virus were also between 1.9- and 14.2-fold lower after M extraction compared to Si extraction, also suggesting impaired DNA recovery. M extraction of spiked cytomegalovirus strain AD 169 in whole blood showed a 5- to 10-fold reduction in PCR sensitivity compared to Si extraction. This reduction of PCR sensitivity was also observed when clinical whole blood samples were processed by M extraction. Before implementing M extraction, the clinical consequences of the reduced recovery should first be considered, especially when maximal sensitivity is required.     

Comparison of specimen processing and nucleic acid extraction by the swab extraction tube system versus the MagNA Pure LC system for laboratory diagnosis of herpes simplex virus infections by LightCycler PCR

A total of 563 specimens (234 dermal and 329 genital swabs) from patients suspected of having herpes simplex virus (HSV) infections were processed using two different extraction methods (the MagNA Pure LC system and the swab extraction tube system [SETS]); HSV DNA was amplified by LightCycler PCR. HSV DNA was detected in 157 of 563 specimens (27.9%) processed by the MagNA Pure LC system and in 179 of 563 specimens (31.8%) processed by SETS (P < 0.0001). There was no specimen processed by the MagNA Pure LC extraction method that was positive only for HSV DNA. Of 157 specimens positive by both methods, HSV DNA copy levels were higher (using cycle crossover points [cycle threshold {C(T)}]) with SETS (mean C(T), 25.9 cycles) than with the MagNA Pure LC system (mean C(T), 32.0 cycles) (P < 0.0001). The time to process 32 samples was longer with the MagNA Pure LC extraction system (90 min) than with SETS (35 min). HSV DNA extraction using SETS is faster, less expensive, and more sensitive than the MagNA Pure LC system and could replace the latter for the laboratory diagnosis of HSV infections using LightCycler PCR.     

Routine use of a highly automated and internally controlled real-time PCR assay for the diagnosis of herpes simplex and varicella-zoster virus infections.

BACKGROUND: Detection of herpes viruses can be significantly improved by PCR. The development of real-time PCR, which has overcome several limitations of conventional PCR, improved the prospects for implementation of PCR-based assays in diagnostic laboratory. OBJECTIVES: To compare the diagnostic performance of an automated sample extraction procedure in combination with an internally controlled real-time PCR assay for detection of herpes simplex virus (HSV) and varicella-zoster virus (VZV) to conventional shell vial culture. STUDY DESIGN: One hundred eighty-two consecutive specimens from patients suspected of HSV or VZV infection were examined by internally controlled PCR and shell vial culture. An internal control consisting of phocine herpes virus was processed along with the specimens during the entire procedure and permitted to monitor extraction and amplification efficiency, including inhibition. RESULTS: A total of 48 (26.4%) specimens were positive for HSV or VZV by culture, and 77 (42.3%) by real-time PCR. Thus, overall sensitivity increased by 60.4%. All culture-positive specimens were detected and typed correctly by PCR, except for a single specimen that contained PCR inhibitors. Specifically, the real-time PCR assay increased the detection rate for HSV-1 and HSV-2 by 43.9% and 62.5%, respectively. In PCR-positive specimens, lower levels of viral DNA were found in culture-negative than in culture-positive specimens. The increase of HSV detection rates by PCR varied with the origin of specimen and was particularly significant for skin specimens (7/14 versus 3/14 detected by culture) and bronchoalveolar lavages (8/8 versus 1/8). In addition, real-time PCR significantly increased the detection rate for VZV. CONCLUSIONS: Compared to shell vial culture, our real-time PCR assay demonstrated a superior sensitivity and an added value of using internal control for checking the quality of examination of each specimen. These results provide a solid basis for implementation of real-time PCR in the routine diagnosis of HSV and VZV infections in various clinical specimens.     

Detection of Tropheryma whipplei DNA in clinical specimens by LightCycler real-time PCR

A real-time PCR method using the LightCycler (Roche Applied Science, Indianapolis, IN) was compared to a conventional PCR assay for the detection of Tropheryma whipplei in 321 clinical specimens. The LightCycler method had sensitivity and specificity comparable to the conventional method but required considerably less labor and time (3 h versus 3 to 5 days).     

Performance of the Cobas AmpliPrep/Cobas TaqMan real-time PCR assay for hepatitis B virus DNA quantification

Hepatitis B virus (HBV) DNA quantification is used to establish the prognosis of chronic HBV-related liver disease, to identify those patients who need to be treated, and to monitor the virologic response and resistance to antiviral therapies. Real-time PCR-based assays are gradually replacing other technologies for routine quantification of HBV DNA in clinical practice. The goal of this study was to evaluate the intrinsic characteristics and clinical performance of the real-time PCR Cobas AmpliPrep/Cobas TaqMan (CAP/CTM) platform for HBV DNA quantification. Specificity was satisfactory (95% confidence interval, 98.1 to 100%). Intra-assay coefficients of variation ranged from 0.22% to 2.68%, and interassay coefficients of variation ranged from 1.31% to 4.13%. Quantification was linear over the full dynamic range of quantification of the assay (1.7 to 8.0 log(10) IU/ml) and was not affected by dilution. The assay was accurate regardless of the HBV genotype. Samples containing HBV DNA levels above 4.5 log(10) IU/ml were slightly underestimated relative to another accurate assay based on branched-DNA technology, but this is unlikely to have noteworthy clinical implications. Thus, the CAP/CTM HBV DNA assay is sensitive, specific, and reproducible, and it accurately quantifies HBV DNA levels in patients chronically infected by HBV genotypes A to F. Samples with HBV DNA concentrations above the upper limit of quantification need to be diluted and then retested. Broad use of fully automated real-time PCR assays should improve the management of patients with chronic HBV infection.     

Use of the real-time PCR assay in conjunction with MagNA Pure for the detection of mycobacterial DNA from fixed specimens.

Tuberculosis in immunocompromised patients is often caused by Mycobacterial species other than Mycobacterium tuberculosis. Thus, detection of and differentiation between M. tuberculosis and nontuberculosis species is necessary for diagnosis of disease in these patients. Furthermore, when tissue changes show granulomatous inflammation, quick confirmation testing for mycobacterial infection is needed for conclusive diagnosis. The aim of this study was to validate the utility of a real-time polymerase chain reaction (PCR) assay in conjunction with the MagNA Pure LC automated extraction system for the detection of mycobacterial DNA from formalin-fixed, paraffin-embedded specimens. A total of 46 archived, paraffin-embedded, fixed specimens showing granulomatous inflammation were studied for mycobacterial infection by real-time PCR. Bacterial DNA was extracted and isolated using the MagNA Pure extraction system. Real-time PCR was performed on the LightCycler using the Artus Real Art Mycob Diff ASR kit from Qiagen. Thirteen of the 46 patient specimens were positive for mycobacterial infection by acid-fast bacilli (AFB) stain. Of the13 reported positive by AFB stain, 12 where positive by real-time PCR. All 13 specimens reported positive by AFB were sent for culture confirmation. Eleven of 13 were returned positive by culture. Specimens reported as negative by culture and positive by real-time PCR were confirmed positive by a second PCR method from another reference laboratory. We believe that these studies are beneficial in the differential diagnosis of mycobacterial infection from fixed tissue specimens where tuberculosis might not have been clinically initially suspected and when specimens are not suitable for microbiologic examination.     

COBAS AmpliPrep-COBAS TaqMan hepatitis B virus (HBV) test: a novel automated real-time PCR assay for quantification of HBV DNA in plasma

Success in antiviral therapy for chronic hepatitis B is supported by highly sensitive PCR-based assays for hepatitis B virus (HBV) DNA. Nucleic acid extraction from biologic specimens is technically demanding, and reliable PCR results depend on it. The performances of the fully automatic system COBAS AmpliPrep-COBAS TaqMan 48 (CAP-CTM; Roche, Branchburg, NJ) for HBV DNA extraction and real-time PCR quantification were assessed and compared to the endpoint PCR COBAS AMPLICOR HBV monitor (CAHBM; Roche). Analytical evaluation with a proficiency panel showed that CAP-CTM quantitated HBV DNA levels in one single run over a wide dynamic range (7 logs) with a close correlation between expected and observed values (r = 0.976, interassay variability below 5%). Clinical evaluation, as tested with samples from 92 HBsAg-positive patients, demonstrated excellent correlation with CAHBM (r = 0.966, mean difference in quantitation = 0.36 log(10) IU/ml). CAP-CTM detected 10% more viremic patients and longer periods of residual viremia in those on therapy. In lamivudine (LAM)-resistant patients, the reduction of HBV DNA after 12 months of Adefovir (ADF) was higher in the combination (LAM+ADF) schedule than in ADF monotherapy (5.1 logs versus 3.5 logs), suggesting a benefit in continuing LAM. CAP-CTM detected HBV DNA in liver biopsy samples from 15% of HBsAg-negative, anti-HBcAg-positive graft donors with no HBV DNA in plasma. The amount of intrahepatic HBV DNA was significantly lower in occult HBV infection than in overt disease. CAP-CTM can improve the management of HBV infection and the assessment of antiviral therapy and drug resistance, supporting further insights in the emerging area of occult HBV infection.     

Evaluation of a real-time PCR assay using the LightCycler system for detection of parvovirus B19 DNA

We evaluated the artus RealArt Parvovirus B19 LC PCR reagent (artus biotech USA, San Francisco, Calif.) for real-time PCR detection of parvovirus B19 DNA by retesting 71 specimens previously submitted to our laboratory. The artus assay, which produces a quantitative result and provides an internal PCR control, appeared to be slightly more sensitive than our conventional qualitative PCR assay.     

Detection of porcine teschoviruses and enteroviruses by LightCycler real-time PCR

Porcine picornaviruses comprising at least 23 serotypes grouped into six species were described as causative agents of neurological disorders, reproductive failure, and aphthae-like dermal lesions of swine. Other viruses such as classical swine fever virus (CSFV), African swine fever virus, pseudorabies virus (PRV), vesicular stomatitis virus, vesicular exanthema virus, porcine respiratory and reproductive syndrome virus, and porcine parvovirus (PPV) may cause diseases with similar clinical symptoms. Therefore, rapid and reliable PCR detection of the most frequent porcine picornaviruses is of interest. A real-time RT-PCR protocol employing LightCycler technology to detect all known serotypes of the three porcine enterovirus (PEV) cytopathic effect (CPE) groups was established. It uses three sets of primer pairs and group-specific hybridisation probes. The primer pairs were designed to amplify highly conserved sequences of the 5'-non-translated region (5'-NTR) of the relevant virus species. The one-step real-time PCR based on the LightCycler technology is more rapid and less contamination-prone than the nested RT-PCR and allows the precise quantitation of the virus load in the tested specimens. All acknowledged serotypes of the three PEV CPE groups and all tested field strains isolated from clinical specimens were detectable. Viruses of the PEV CPE group III can be distinguished from the closely related swine vesicular disease virus (SVDV).     

Detection and quantification of hepatitis B virus DNA by SYBR green real-time polymerase chain reaction

A single-round real-time polymerase chain reaction (PCR) assay based on SYBR green dye technology for the detection and quantification of hepatitis B virus (HBV) DNA in serum was evaluated and compared with a qualitative nested PCR and the Cobas Amplicor HBV Monitor assay (Roche Molecular Diagnostics, Milan, Italy). The performance of the real-time PCR assay was evaluated in a routine clinical laboratory setting with a total of 212 clinical specimens. The sensitivity of the real-time PCR corresponded to 31 IU/ml (70 copies/ml), and comparison with the qualitative nested PCR showed significant concordance for 94% of samples. The linear curve over 7 log units, spanning 10³–10⁹ IU/ml (2.28 × 10³ to 2.28 × 10⁹ copies/ml), was observed in the quantitative determination. The interexperimental variability coefficient of the assay ranged from 0.22 to 0.39 and the intraexperimental variability coefficient from 0.24 to 0.41. By excluding values outside of the dynamic ranges of both tests, the HBV Monitor and the real-time PCR gave an agreement within ±1 log unit for 90% of samples, while those for the remaining 10% were found to be above 1 log unit but less than 1.5 log units. When the results inside and outside the dynamic range of the HBV Monitor were examined, 90% of the results were in agreement. In conclusion, the real-time PCR based on SYBR green technology proved suitable for routine diagnostic purposes, showing good sensitivity, high specificity, high reproducibility, and good linearity over a broad dynamic range of quantification.     

Automated extraction of genomic DNA from medically important yeast species and filamentous fungi by using the MagNA Pure LC system

A fully automated assay was established for the extraction of DNA from clinically important fungi by using the MagNA Pure LC instrument. The test was evaluated by DNA isolation from 23 species of yeast and filamentous fungi and by extractions (n = 28) of serially diluted Aspergillus fumigatus conidia (10(5) to 0 CFU/ml). Additionally, DNA from 67 clinical specimens was extracted and compared to the manual protocol. The detection limit of the MagNA Pure LC assay of 10 CFU corresponded to the sensitivity when DNA was extracted manually; in 9 of 28 runs, we could achieve a higher sensitivity of 1 CFU/ml blood, which was found to be significant (p 相似文献