Parvovirus B19 infection in pregnancy: quantitative viral DNA analysis using a kinetic fluorescence detection system (TaqMan PCR)

Human parvovirus B19 infections are common in the general population, and infection during pregnancy may cause hydrops fetalis and fetal death. To initiate adequate treatment, accurate laboratory diagnosis is essential. The most sensitive tests are nested PCR systems, but these assays provide semiquantitative results at best. A parvovirus B19 DNA assay was developed based on the real time TaqMan PCR. This method was calibrated on the basis of serial plasmid dilutions and tested with an international parvovirus B19 standard. The assay was capable of quantifying parvovirus B19 DNA from one to about 5 x 10(7) genome equivalents per reaction (corresponding to 100 to 5 x 10(9) genome equivalents per ml serum). Samples from 51 pregnant women with suspected acute parvovirus B19 infection were tested, and positive PCR results were obtained in at least one of the materials investigated in 41 cases. The median viral DNA load in maternal blood samples was 1.3 x 10(4) copies/ml (range 7.2 x 10(2)-2.6 x 10(7)). Maternal virus DNA concentration was not associated with the presence of maternal symptoms and/or fetal complications. As the stage of infection was not known in the majority of cases, our data do not exclude an association between peak levels of parvovirus B19 DNA and the development of complications. Maternal sera and corresponding fetal material were available for concurrent testing from 15 DNA-positive cases: in most fetal samples, viral DNA concentrations were several orders of magnitude higher (up to 2.1 x 10(12) copies/ml) compared to the corresponding maternal blood samples.     

Rapid detection and quantitation of hepatitis B virus DNA by real-time PCR using a new fluorescent (FRET) detection system.

BACKGROUND: The diagnosis of hepatitis B virus (HBV) has until recently been based on traditional serologic methods targeting viral antigens and antibodies to viral proteins. The development of molecular methods allowing for the quantitation of HBV DNA is proving clinically valuable for monitoring therapy and detecting early treatment failures. OBJECTIVES: Here we report a new real-time (LightCycler) quantitative PCR for the detection of HBV DNA based on sequence specific hybridisation probes (designed in-house), targeting the HBV surface antigen. STUDY DESIGN: The assay was evaluated using a 10-fold dilution series of standard HBV DNA [Eurohep standard reference 1, genotype A, HBsAg subtype adw with a unitage of 10(6) WHO. i.u./ml] and 89 clinical serum samples. The performance was measured against a quantified standard HBV DNA working reagent (NIBSC code 98/780) and the sensitivity compared with our conventional thermal-block PCR. RESULTS AND CONCLUSION: Real-time PCR detected HBV DNA in 45% (40/89) and thermal-block PCR in 16% (14/75) of clinical samples. Results for 26 samples were below the detection limit of the thermal-block PCR but could be quantified by real-time (LightCycler) PCR. The LightCycler assay was at least 5 logs more sensitive than thermal-block PCR and could detect HBV in a linear range between 5 and 10(7) i.u. per reaction. The broad generic nature of the PCR primers coupled with the enhanced sensitivity and specificity of the fluorescent hybridisation probes makes this assay potentially valuable for both routine diagnostic and epidemiological work.     

Evaluation of the COBAS TaqMan 48 real-time PCR system for quantitation of hepatitis B virus DNA

The purpose of this study is to evaluate the usefulness of the new real-time PCR COBAS TaqMan 48 analyzer, comparing it to the existing COBAS AMPLICOR HBV MONITOR based on conventional PCR technology. The study used 104 samples from different patients. No differences were found in the sensitivity of the tests. There was an excellent correlation between the sample with a viral load within the dynamic range of the two tests (r = 0.938). The COBAS TaqMan test has a wider linear range, and this fact enables quantifying of the viral load without diluting the sample.     

Real-time PCR for detection and quantitation of hepatitis B virus DNA

A sensitive and reproducible real-time PCR assay based on TaqMan technology was developed for the detection and quantitation of hepatitis B virus (HBV) DNA in serum, and compared with an "in-house" qualitative PCR assay. HBV DNA was measured in 125 serum samples from 76 hepatitis B patients, consisting of 22 patients with an acute infection, 20 patients with a previous history of hepatitis B infection, and 34 patients with a chronic hepatitis B. Four patients with a chronic infection were treated with either an IFN-alpha monotherapy or a combination of IFN-alpha and lamivudine. Twenty-nine sera from healthy individuals and non-hepatitis B patients served as negative controls. The assay was validated by using a 10-fold dilution series of the World Virological Quality Control (VQC) sample containing 3.73 x 10(7) genome equivalents per ml. The detection limit for the real-time PCR was 3.73 x 10(2) genome equivalents per ml (geq/ml), while it was 3.73 x 10(3) geq/ml for the in-house PCR. The real-time PCR assay had an 8-logarithm dynamic range spanning from 10(2) to 10(10) geq/ml. In clinical serum samples, the real-time PCR and the in-house PCR detected HBV DNA in 81% (101/125) and 66% (83/125) of samples, respectively. HBV DNA was not detected among the negative controls by either of these assays. In conclusion, real-time PCR is a sensitive, specific, and a reproducible approach for the detection and quantitation of HBV DNA in clinical serum samples, useful also for monitoring the efficacy of antiviral treatment.     

Sensitive and specific detection of Salmonid alphavirus using real-time PCR (TaqMan)

Pancreas disease is responsible for major economic losses in the European salmonid farming industry. It was previously believed that a single subtype (salmon pancreas disease virus) of the virus species Salmonid alphavirus (SAV) was responsible for all outbreaks of pancreas disease in the UK and Norway. However, the recent discovery that pancreas disease in Norway is caused by a new and distinct subtype of salmonid alphavirus, exclusively found in Norway, has advocated the need for better diagnostic tools. In the present paper, three real-time PCR assays for all known subtypes of salmonid alphavirus have been developed; the Q_nsP1 assay is a broad-spectrum one that detects RNA from all subtypes, the Q_SPDV assay specifically detects the salmon pancreas disease virus subtype, and the Q_NSAV assay only detects the new Norwegian salmonid alphavirus subtype. The results demonstrated the assays to be highly sensitive and specific, detecting <0.1TCID50 of virus stocks. Regression analysis and standard curves calculated from the Ct-values from 10-fold serial dilutions of virus stocks showed that the assays were highly reproducible over a wide range of RNA input. Thirty-nine field samples were tested in triplicate and compared with traditional RT-PCR. Overall, the real-time assays detected 35 positive compared to 29 positives in standard RT-PCR, and were thus shown to be more sensitive for detecting salmonid alphaviruses in field samples.     

Real-time PCR assay using molecular beacon for quantitation of hepatitis B virus DNA

Levels of hepatitis B virus (HBV) DNA in the blood serve as an important marker in monitoring the disease progression and treatment efficacy of chronic HBV infection. Several commercial assays are available for accurate measurement of HBV genomic DNA, but many of them are hampered by relatively low sensitivity and limited dynamic range. The aim of this study was to develop a sensitive and accurate assay for measuring HBV genomic DNA using real-time PCR with a molecular beacon (HBV beacon assay). The performance of this assay was validated by testing serial dilutions of the two EUROHEP HBV DNA standards (ad and ay subtypes) of known concentrations. The assay showed low intra-assay (<7%) and interassay (<5%) variations for both subtypes. Its dynamic range was found to be 10(1) to 10(7) copies per reaction (1.0 x 10(2) to 1.0 x 10(9) copies ml(-1)). The assay was further evaluated clinically using serum samples from 175 individuals with chronic hepatitis B. The HBV DNA level measured by this assay showed good correlation with that measured by the commercially available COBAS AMPLICOR HBV Monitor test (r = 0.901; P < 0.001). The higher sensitivity and broader dynamic range of this assay compared to the existing commercial assays will provide an ideal tool for monitoring disease progression and treatment efficacy in HBV-infected patients, in particular for those with low levels of HBV viremia.     

Fully automated quantitation of hepatitis B virus (HBV) DNA in human plasma by the COBAS AmpliPrep/COBAS TaqMan system.

BACKGROUND: The measurement of HBV DNA levels has become the most direct and reliable method used for accurate diagnosis and prognosis of acute and chronic HBV infection []. Nucleic acid amplification testing (NAT detection) also reduces the pre-seroconversion window period. The method can be used to aid in the management of HBV infection, by the identification of individuals with high levels of viral replication who might benefit from antiviral therapy, monitoring patients on therapy, and identification of the development of resistance. OBJECTIVES: Evaluation of the novel COBAS AmpliPrep/COBAS TaqMan HBV Test, which combines automated extraction of DNA on the COBAS AmpliPrep Instrument (CAP), coupled with real-time PCR on the COBAS TaqMan Analyzer (CTM), thus greatly reducing hands-on time during sample preparation and amplification/detection. The assay fulfils the current requirements: a highly sensitive HBV DNA detection reagent which is calibrated to the International WHO Standard to reliably quantify HBV genotypes A-G in plasma with a very broad measuring range, thereby minimizing laborious repeat testing. STUDY DESIGN: The test was evaluated for sensitivity, dynamic range, precision, clinical and analytical specificity, genotype inclusivity, interfering substances, and correlation with other tests for the detection of HBV DNA (COBAS Amplicor HBV Monitor Test, COBAS TaqMan HBV Test for Use with the High Pure System and VERSANT HBV DNA 3.0 Assay). RESULTS AND CONCLUSION: A fully automated system for sample preparation, amplification and quantitation of HBV DNA was developed that demonstrates a nearly 7-log dynamic range up to 1.1E+08IU/mL, an assay sensitivity (95% hit-rate) of 4-12IU/mL and an equivalent detection of genotypes A-G plus a prevalent pre-core mutant. The results obtained with the new test show a good correlation to titers obtained with other platforms.     

Quantitation of hepatitis B virus DNA by real-time PCR using internal amplification control and dual TaqMan MGB probes

Hepatitis B virus (HBV) DNA quantitation is used extensively for monitoring of antiviral treatment of HBV infection. A real-time PCR assay was developed using a TaqMan minor groove binder probe and primers corresponding to HBV pre-core region for HBV DNA quantitation. A 228 bp fragment from this genomic region of HBV was cloned and serial dilutions of plasmid DNA were used as an external DNA standard. Comparison of the real-time PCR quantitation results from 35 clinical serum samples with those obtained by COBAS Amplicor HBV DNA monitor kit (Roche Diagnostics) revealed a significant correlation (r = 0.92) for all the samples. The assay showed wide dynamic linear range between 2.5 x 10(2) and 2.5 x 10(10) copies/ml serum. Sera from 25 healthy individuals tested negative indicating the high specificity of the assay. The median coefficients of variation for both intra- and inter-experimental variability were 4.9% and 10.6%, respectively, which indicated remarkable reproducibility. An internal amplification control (IC) was developed to detect the presence of PCR inhibitors in the samples to avoid false negative results. The IC had the same primer binding sites but different internal sequence and it competed with the virus-derived target. The optimum concentration of IC was found to be 100 copies/reaction. The assay was validated by testing serial dilutions of the WHO international HBV DNA standard. Since conserved regions were considered during primer and probe design, the assay should be applicable to all HBV genotypes. The real-time assay will be useful for monitoring HBV-infected patients in routine diagnostic laboratories and in clinical practice enabling analysis of a wide dynamic range of HBV DNA in a single, undiluted sample.     

Real-time PCR quantitation of hepatitis B virus DNA using automated sample preparation and murine cytomegalovirus internal control

Quantitation of circulating hepatitis B virus (HBV) DNA is important for monitoring disease progression and for assessing the response to antiviral therapy. Several commercial and 'in house' assays for HBV DNA quantitation have been described but many of these have limitations of relatively low sensitivity and limited dynamic range. This study describes the development and evaluation of a FRET-based real-time PCR assay designed to overcome these limitations and to provide accurate quantitation of DNA from all eight genotypes of HBV (A-H). The assay employs a fully automated nucleic acid extraction system permitting high-sample throughput with minimal 'hands-on' time and incorporates a murine cytomegalovirus (mCMV) internal control to prevent false negative results and under-reporting due to unrecognised problems with viral lysis, DNA purification or PCR amplification. Sensitivity, assessed by Probit analysis at the 95% detection level, was 24.4 IU/ml, associated with an extremely wide dynamic range (approximately 9 log10). Coefficients of variation were low for both intra-assay and inter-assay variability (CV%, 7-11%) and quantitative data correlated well (R2 = 0.97) with the Digene hybrid capture assay. This assay provides an ideal system for therapeutic monitoring and for studying the relationship between HBV viral load and stage of disease.     

Development of TaqMan real-time PCR assay for detection and quantitation of reticuloendotheliosis virus

A highly sensitive real-time PCR method was developed in this study for reticuloendotheliosis virus (REV) detection and quantitation. The real-time PCR method, with a minimum detection limit of 10 proviral DNA copies, was 100 times more sensitive than the conventional PCR. It was also shown to be highly specific, as no positive signals were detected for other common avian DNA viruses. The coefficients of variation of intra- and inter-assay reproducibility were both less than 2%. The chicken β-actin gene was co-amplified and used as the internal control to monitor the efficiency of DNA extraction and PCR amplification. Specific pathogen free chickens were infected with REV at different ages and the blood was detected with the real-time PCR method. High levels of proviral DNA were detected in the blood of REV-infected chickens during the experiment and chickens infected early had higher proviral loads from 2 weeks post-infection compared with late infected chickens. This study provides an excellent research and diagnostic tool that can be used for REV detection and quantitation.     

Liquid-phase hybridization and capture of hepatitis B virus DNA with magnetic beads and fluorescence detection of PCR product

The polymerase chain reaction (PCR) exceeds all hitherto known detection limits. This sensitivity could lead to false positive results. Every manipulation increases the risk of contamination via, for example, aerosols. Most protocols for the extraction of template nucleic acids are complicated and possible centrifugation steps do not reduce the risk of aerosols. In addition, most of the methods for analysis are time-consuming and cannot be applied to different template materials. An alternative extraction method has been developed. The fast chemical denaturation of template by guanidine thiocyanate was followed by liquid hybridization to biotinylated oligonucleotides. The template nucleic acid could be washed after binding to streptavidin-coated paramagnetic beads to reduce influence on the enzymatic amplification steps. PCR of hepatitis B virus deoxyribonucleic acid was used to demonstrate how easy, versatile, and time-saving this method is without centrifugation. The level of extracted nucleic acids was quantitated and the properties for sensitive extraction were evaluated. After PCR an additional step was developed which used fluorescent staining to detect positive amplifications. This is useful to identify positive results in predominantly negative samples.     

TaqMan real-time PCR for detection and quantitation of squash leaf curl virus in cucurbits

A real-time PCR assay based on the TaqMan chemistry was developed for reliable detection and quantitation of the squash leaf curl virus (SLCV) in melon and squash plants. This method was highly specific to SLCV and it was about one thousand times more sensitive than the conventional PCR method. The protocol of the real-time PCR established in this study enabled detection of as little as 10² copies of SLCV DNA with CP gene as the target. This TaqMan real-time PCR assay for detection and quantitation of SLCV would be a useful tool for application in quarantine and certification of SLCV in cucurbits as well as in the research of disease resistance and epidemiology.     

锁核酸捕获TaqMan探针实时聚合酶链反应构建及检测乙型肝炎病毒DNA

建立和检测新型乙型肝炎病毒(HBV)DNA检测技术锁核酸(LNA)捕获TaqMan探针实时聚合酶链反应(PCR)。方法 2009年8月至2010年3月选取本院20例健康献血者和肝病专科住院的75例经COBAS Amplicor检测HBV DNA阳性慢性乙型肝炎(CHB)患者,分别采集血液样本进行LNA捕获TaqMan探针实时PCR特异性试验。同时对LNA-实时PCR方法的重复性、特异性和线性范围等进行分析。结果 其线性范围为10～2.3×109 IU/ml。将10 IU/ml作为检测下限,具有95％可信区间。线性回归分析显示其斜率接近1.0 (R2=0.999)。批内变异值为3.88％～4.36％,批间变异值为8.5％～11.7％。20例健康献血者的阴性对照血清HBV-DNA检测均为阴性,而75例经COBAS Amplicor检测HBV DNA阳性患者的血清样本除1例阴性外,其余都为阳性。结论 LNA捕获TaqMan探针实时PCR方法为HBV-DNA检测的一种快捷灵敏、准确度高的新方法,值得临床实验室推广应用。     

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.     

荧光定量聚合酶链反应检测乙型肝炎病毒DNA

目的 建立检测HBV病毒DNA的荧光定量PCR法（FQ-PCR）,并与运用常规凝胶电泳技术观察特异扩增带检测的结果加以比较。方法 合成扩增HBV DNA314bp特异保守序列的1对引物及1条带2个荧光基团的寡核苷酸探针。用PE-5700型定量PCR仪完成PCR反应及产物的荧光定量检测。同是PCR产物经琼脂糖凝胶电泳,EB染色,UVP（凝胶成像仪）检出有314bp带者为阳性或弱阳性。结果 建立了检测HBVDNA的荧光定量PCR技术,用已知HBV阳性模板不同拷贝数的标准溶液测得标准曲线Ct,原始拷贝数在10^5/ml以上者为阳性,用定量及定性PCR2种方法检测698例血清标本的结果表明：用FQ-PCR技术共检测出204例为阳性,阳性率29.2%;用定量及定性PCR2种方法检测698例血清标本的结果表明：用FQ-PCR技术共检测出204例为阳性,阳性率29.2%;用定性PCR观察到193例有阳性特异带,阳性检出率为27.65%。没有发现用定性PCR检测为阳性而用FQ-PCR检测为阴性者。结论 FQ-PCR检测HBVDNA较普通定性PCR技术具有操作简便,灵敏度更高、减少发生污染可导致假阳性结果的可能性及自动化程度高等优点,值得在临床检验中推广应用。     

Mutation analysis of lamivudine resistant hepatitis B virus strains by TaqMan PCR

Hepatitis B virus infected patients on long-term lamivudine treatment are exposed to a 15-32% risk compounded annually of developing resistance mutations. Such resistance results in a progression of the liver damage caused by chronic hepatitis B, and may also impair the effect of other antivirals through cross-resistance. At present lamivudine is used frequently as monotherapy because of its relatively low price and negligible side effects. Thus, simple methods for identifying resistance mutations are required. A method based on real-time polymerase chain reaction with TaqMan chemistry is described. The method combines both primer specificity, in order to target wild type and mutant viral strains at codon 180, and a mixture of three minor groove binding probes distinguishing the YMDD wild type and the YVDD and YIDD variants at codon 204. The accuracy of the method was verified by concordance with results of direct sequencing and restriction fragment length polymorphism when examining 27 samples from five patients, in whom lamivudine resistance was known to have developed. This method is rapid, cost effective, and should prove useful for monitoring patients treated with lamivudine.     

Performance evaluation of ExiStation HBV diagnostic system for hepatitis B virus DNA quantitation

The performance of a recently developed real-time PCR system, the ExiStation HBV diagnostic system, for quantitation of hepatitis B virus (HBV) in human blood was evaluated. The detection limit, reproducibility, cross-reactivity, and interference were evaluated as measures of analytical performance. For the comparison study, 100 HBV-positive blood samples and 100 HBV-negative samples from Korean Blood Bank Serum were used, and the results of the ExiStation HBV system showed good correlation with those obtained using the Cobas TaqMan (r² = 0.9931) and Abbott real-time PCR systems (r² = 0.9894). The lower limit of detection was measured as 9.55 IU/mL using WHO standards and the dynamic range was linear from 6.68 to 6.68 × 10⁹ IU/mL using cloned plasmids. The within-run coefficient of variation (CV) was 9.4%, 2.1%, and 1.1%, and the total CV was 11.8%, 3.6%, and 1.7% at a concentration of 1.92 log₁₀ IU/mL, 3.88 log₁₀ IU/mL, and 6.84 log₁₀ IU/mL, respectively. No cross-reactivity or interference was detected. The ExiStation HBV diagnostic system showed satisfactory analytical sensitivity, excellent reproducibility, no cross-reactivity, no interference, and high agreement with the Cobas TaqMan and Abbott real-time PCR systems, and is therefore a useful tool for the detection and monitoring of HBV infection.     

Development of a TT virus DNA quantification system using real-time detection PCR

Although TT virus (TTV) was isolated from a cryptogenic posttransfusion hepatitis patient, its pathogenic role remains unclear. It has been reported that the majority of the healthy population is infected with TTV. To elucidate the differences between TTV infection in patients with liver diseases and TTV infection in the healthy population, a quantification system was developed. TTV DNA was quantified by a real-time detection PCR (RTD-PCR) assay on an ABI Prism 7700 sequence detector. With this system, TTV DNA was quantified in 78 hepatitis C virus (HCV)-infected patients (63 with elevated serum alanine aminotransferase [ALT] levels and 15 with normal ALT levels) and in 70 voluntary blood donors (BDs). The quantification range was 2.08 to 7.35 log copies/ml. The intra-assay and interassay coefficients of variation were 0.37 to 6.33% and 0.60 to 7.07%, respectively. The mean serum TTV DNA levels in the HCV-infected patients with both elevated and normal ALT levels and BDs were 3.69 +/- 0.89, 3.45 +/- 0.76, and 3.45 +/- 0.67 log copies/ml, respectively. Comparison of the serum TTV DNA levels among the HCV-infected patients revealed that they were not related to the serum ALT and HCV core protein levels or to the histopathological score on liver biopsy. This study showed that (i) the RTD-PCR assay for the detection of TTV was accurate and had a high degree of sensitivity, (ii) the mean serum TTV DNA level was similar among HCV-infected patients, irrespective of their ALT level, and also among BDs, and (iii) a high serum TTV DNA level does not affect the serum ALT and HCV levels or liver damage in HCV-infected patients.     

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.     

Real-time PCR quantitation of hepatitis B virus total DNA and covalently closed circular DNA in peripheral blood mononuclear cells from hepatitis B virus-infected patients

It remains unclear whether hepatitis B virus (HBV) replicates in extrahepatic tissues, and particularly in peripheral blood mononuclear cells (PBMCs), which may serve as a reservoir for the maintenance of infection. A real-time PCR assay for the quantitation of total and covalently closed circular (ccc) HBV DNA in serum and in PBMCs was developed. This assay was highly sensitive (detection limit: 27 IU/mL), linear over a wide range (9 log10), and was displayed high inter- and intra-assay reproducibility for the quantitation of total DNA. Genotypes A to E were detected and the results were consistent with those obtained with the COBAS Amplicor HBV Monitor Test. The specificity of the methodology was increased by prior treatment with an enzyme that digests relaxed circular DNA, and the elimination of background signals from virus adsorbed to the surface of PBMCs. HBV DNA was detected in the serum and PBMCs of 12 HBsAg-positive patients, with less than 1% in the cccDNA form. In conclusion, the quantitation of total and ccc HBV DNA in PBMCs is potentially useful as a non-invasive marker, and may help to increase our knowledge of the natural history of hepatitis B.