Fast and reliable titration of recombinant adeno-associated virus type-2 using quantitative real-time PCR

In this study, a quantitative real-time PCR (qPCR) was developed to determine genomic rAAV-2 titers using the Light-Cycler technology. Since the CMV promoter is the most commonly used promoter in gene therapeutic approaches, primers were designed which hybridize with the human CMV promoter sequence. PCR products were detected by the addition of SYBR green. qPCR of a 5 log spanning serial dilution of the vector plasmid containing one CMV promoter per plasmid molecule yielded a high amplification efficiency of 1.99 per cycle. To quantify the copy number of viral genomes, the qPCR curves of adeno-associated virus type 2 (AAV-2) samples were related to a standard curve assessed by the 5 log spanning serial vector plasmid dilution (0.01-100 pg DNA). For validation of the method, rAAV-2 preparations were analyzed by a standard method and qPCR in parallel. As standard method, flow cytometry was used for titration of infectious viral particles on HeLa cells using the Enhanced Green Fluorescent Protein as a marker. A significant correlation was found between the results obtained by flow cytometry and the results from the qPCR over a 5 log range (r=0.85, P<0.0001). The mean ratio between infectious rAAV-2 particles titrated via flow cytometry and genomic copies of rAAV-2 measured by qPCR of the same sample was 1:253. The higher titers found by qPCR might be due to multiple transduction of a single cell or to non-infectious particles generated during rAAV-2 preparation. In conclusion, qPCR is a fast and reliable method for determination of rAAV-2 titers and might be a powerful tool for standardization of rAAV-2 preparations particularly in the context of clinical studies.     

Cellular chromosome DNA interferes with fluorescence quantitative real-time PCR detection of HBV DNA in culture medium

Fluorescence quantitative real-time PCR (FQ-PCR) is a recently developed technique increasingly used for clinical diagnosis by detection of hepatitis B virus (HBV) DNA in serum. FQ-PCR is also used in scientific research for detection of HBV DNA in cell culture. Understanding potential FQ-PCR interference factors can improve the accuracy of HBV DNA quantification in cell culture medium. HBV positive serum was diluted with culture medium to produce three test groups with HBV DNA levels of 5 x 10(7) copies/ml (high), 5 x 10(5) copies/ml (medium), and 5 x 10(3) copies/ml (low). Chromosome DNA was extracted from HepG2 cells and then added to high, medium, and low group samples at final concentrations of 0, 12.5, 25, 50, and 100 microg/ml. The samples were quantified by FQ-PCR and data were evaluated using statistical software. No marked changes were seen in the quantitative curves for high level HBV DNA samples when the samples were supplemented with 0-100 microg/ml of chromosome DNA. Interference was observed in medium level samples when 50 and 100 microg/ml of chromosome DNA was added. Interference was also observed in low level HBV DNA samples when the concentration of added chromosome DNA was greater than 25 microg/ml. The interference was eliminated when samples were digested by DNase I prior to PCR detection. In Conclusions, the presence of cellular chromosome DNA can interfere with the detection of HBV DNA by FQ-PCR. Removal of cellular chromosome DNA from culture media prior to FQ-PCR is necessary for reliable HBV DNA quantitative detection.     

细胞人基因组DNA对荧光实时定量PCR检测细胞培养液中HBV DNA的影响

 目的 研究细胞人基因组 DNA 对荧光实时定量 PCR（FQ-PCR）检测细胞培养上清液中 HBV DNA 含量的可能干扰影响，提高细胞培养上清液中 HBV DNA 定量检测的准确性。方法 取 HBV DNA 阳性血清，以 DMEM 培养基分别配制出 HBV DNA 拷贝数为 5 × 107/ml（高拷贝组）、5 × 105/ml（中拷贝组）、5 × 103/ml（低拷贝组）的不同样本组；各组内再分 5 个亚组，分别加入终浓度为 0（对照组）、12.5、25、50、100 μg/ml 的细胞人基因组 DNA（提取自人肝癌细胞系 HepG2）。以不含 HBV DNA 阳性血清的 DMEM培养基加入上述浓度细胞人基因组 DNA 为空白组。采用基于 TaqMan 技术的 FQ-PCR 检测各组样本的 HBV DNA 拷贝数并绘制 HBV DNA 定量曲线。每组均重复检测 10 次。根据 HBV DNA 定量检测结果确定受干扰样本和未受干扰样本，分别计算其定量曲线线性期斜率和平均扩增效率。 结果 高拷贝组中加入不同浓度细胞人基因组 DNA 的各个亚组与相应对照组比较，HBV DNA 拷贝数差异均无统计学意义。中拷贝组中加入细胞人基因组 DNA 浓度为 50 和 100 μg/ml 的 2 个亚组，其 HBV DNA 拷贝数结果明显高于相应对照组，增高可达 50 ~ 100 倍，且重复性差。低拷贝组中只有加入细胞人基因组 DNA 浓度为 12.5 μg/ml的亚组可通过提高基线值取得与相应对照组接近的定量检测结果，而其他亚组 HBV DNA 定量曲线指数扩增期斜率明显异常，无法确定其定量检测结果。空白组各亚组均未观察到明显的 HBV DNA 指数扩增期。受干扰样本线性期斜率（-1.01 ± 0.06）和平均扩增效率（90.0% ± 2.1%）与对照组样本（分别为 -1.52 ± 0.06，97.0% ± 0.4%）比较，差异均有统计学意义（P < 0.01）。 结论 细胞人基因组 DNA 对应用 FQ-PCR 技术进行的HBV DNA 定量检测可产生非特异性干扰，尤其在 HBV DNA 拷贝水平较低时。在细胞培养上清液作 HBV DNA 定量检测时应尽量去除细胞人基因组 DNA。     

A real-time TaqMan PCR for routine quantitation of cytomegalovirus DNA in crude leukocyte lysates from stem cell transplant patients

A real-time TaqMan PCR based on the cytomegalovirus (CMV) polymerase (pol) gene was developed for quantitation of CMV DNA in crude peripheral blood leukocyte (PBL) lysate from stem cell transplantation (SCT) patients. The dynamic range of the assay was between 10 and 4x10(6) copies. Both intra- and inter-assay variability were well within +/-0.25 log10 S.D. Thus, a pooled PBL sample that was used as positive control in 57 consecutive TaqMan PCR runs over 7 months showed a stable CMV quantity (4.12+/-0.13, log10 mean+/-S.D.). The sensitivity of the pol TaqMan PCR was validated by parallel analysis of 177 PBL samples with a nested PCR. The use of crude PBL lysate as PCR input did not cause PCR inhibition. We demonstrated further the clinical utility of the newly developed TaqMan PCR by monitoring changes in CMV levels in eight patients receiving antiviral therapy. This TaqMan PCR was highly sensitive, reproducible, and stable and has served a useful tool for monitoring CMV DNA levels in large number of clinical samples in a routine diagnostic setting for over 1 year.     

Quantitation of cell-free and cell-associated Kaposi's sarcoma-associated herpesvirus DNA by real-time PCR

A real-time PCR assay for quantitation of Kaposi's sarcoma-associated herpes virus (KSHV or human herpesvirus 8) DNA was evaluated. The linear dynamic range was 10 to 10(5) copies of KSHV DNA (r(2) > 0.99). The accuracy of DNA purification and quantitation was less than +/-0.4 log(10) copies for samples that contained from 10 to 10(5) copies of KSHV DNA. Cell-associated KSHV DNA was quantitated over a range of infected cell frequencies from 0. 1 to 10(-5), and cell-free KSHV DNA in plasma was quantitated over a range of 100 to 10(6) copies/ml. Real-time PCR provides a convenient method for quantitation of cell-free and cell-associated KSHV DNA in laboratory and clinical specimens.     

Detection of human cytomegalovirus DNA by real-time quantitative PCR

A real-time PCR assay was developed to quantify human cytomegalovirus (CMV) DNA. This assay was used to demonstrate a higher CMV DNA load in plasma of bone marrow transplant patients than in that of blood donors. The CMV load was higher in CMV antigen-positive patients than in antigen-negative patients.     

实时定量PCR检测HBV核酸载量测量不确定度的构成分析

 目的 研究实时荧光定量PCR用于乙型肝炎病毒（HBV）核酸载量检测时的测量不确定度构成。 方法 采用实时荧光定量PCR检测乙型肝炎患者血清标本中HBV核酸载量，收集检测过程中的各类数据，计算以下6种来源的测量不确定度，对该方法的测量不确定度构成进行评估。⑴标本浓缩的不确定度（uenrich）；⑵核酸提取的不确定度（uex）；⑶扩增反应体系的不确定度（upcr）；⑷热循环仪的不确定度（uins）；⑸数据处理的不确定度（uana）；⑹加样枪的不确定度（upip）。其中热循环仪的不确定度检测样本数为7份，其他各种不确定度检测的样本数为10份。 结果 核酸浓度为5.610E+07拷贝/ml的标本，其浓缩过程来源的相对不确定度达0.437；核酸提取来源的浓度真值无偏估计在6.585E+03、9.067E+04、7.223E+06拷贝/ml样本的相对不确定度分别为0.249、0.173、0.140；热循环仪和数据分析来源的相对不确定度分别为0.020和不大于0.050。 结论 标本制备过程中的浓缩和DNA提取步骤所带来的不确定度是实时荧光定量PCR检测HBV核酸载量测量不确定度的主要分量，因此标本制备处理的方法与标准操作程序能否有效地提取核酸并去除PCR反应的抑制物对于该项检测十分关键；起始模板浓度与测量不确定度相关，低浓度的样本显示出更大的相对不确定度。     

实时荧光定量PCR检测乙型肝炎病毒核酸载量的测量不确定度构成分析

目的 研究实时荧光定量PCR用于HBV核酸载量检测时的测量不确定度构成.方法 采用实时荧光定量PCR检测乙型肝炎患者血清标本中HBV核酸载量,收集检测过程中的各类数据,计算以下6种来源的测量不确定度,对该方法的测量不确定度构成进行评估.(1)标本浓缩的不确定度(uenrich);(2)核酸提取的不确定度(uex);(3)扩增反应体系的不确定度(upcr);(4)热循环仪的不确定度(uins);(5)数据处理的不确定度(uana);(6)加样枪的不确定度(upip).其中热循环仪的不确定度检测样本数为7份,其他各种不确定度检测的样本数为10份.结果 核酸浓度为5.610E+07拷贝/ml的标本,其浓缩过程来源的相对不确定度达0.437核酸提取来源的浓度真值无偏估计在6.585E+03、9.067E+04、7.223E+06拷贝/ml样本的相对不确定度分别为0.249、0.173、0.140;热循环仪和数据分析来源的相对不确定度分别为0.020和不大于0.050.结论 标本制备过程中的浓缩和DNA提取步骤所带来的不确定度是实时荧光定量PCR检测HBV核酸载量测量不确定度的主要分量,因此标本制备处理的方法与标准操作程序能否有效地提取核酸并去除PCR反应的抑制物对于该项检测十分关键;起始模板浓度与测量不确定度相关,低浓度的样本显示出更大的相对不确定度.     

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.     

Rapid and quantitative detection of human adenovirus DNA by real-time PCR

Rapid diagnosis of human adenovirus (HAdV) infections was achieved by PCR in the recent years. However, conventional PCR has the risk of carry-over contamination due to open handling with its products, and results are only qualitative. Therefore, a quantitative "real-time" PCR with consensus primer and probe (dual fluorescence labelled, "TaqMan") sequences for a conserved region of the hexon gene was designed and evaluated. Real-time PCR detected all 51 HAdV prototypes. Sensitivity of the assay was 相似文献   

Detection of Herpes simplex virus DNA by real-time PCR

Molecular detection of herpes simplex virus (HSV) DNA is recognized as the reference standard assay method for the sensitive and specific diagnosis of central nervous system infections caused by HSV. In this study, a molecular assay based on real-time PCR on the LightCycler (LC) instrument was evaluated and compared with a home-brew molecular assay. The detection limit of the LC assay was determined with 10-fold dilutions of plasmid pS4 with the SalI restriction fragment of the DNA polymerase gene and with the First European Union Concerted Action HSV Proficiency Panel. A total of 59 cerebrospinal fluid (CSF) specimens were investigated for the comparative study. With plasmid pS4, the detection limit of the LC assay was found to be 10(4) copies per ml, i.e., 12.5 copies per run. When samples of the First European Union Concerted Action HSV Proficiency Panel were tested, 2x10(3) to 5x10(3) HSV type 1 genome equivalents (GE) per ml, i.e., 2.5 to 6.3 GE per run, could consistently be detected. There was a correlation between the LC assay and the home-brew assay in 55 of 59 specimens. In conclusion, the LC assay allows very rapid detection of HSV DNA in CSF. It was found to be laborsaving and showed sufficient sensitivity.     

Quantitation of varicella-zoster virus DNA in whole blood, plasma, and serum by PCR and electrochemiluminescence

We describe a highly sensitive assay for quantitation of varicella-zoster virus (VZV) DNA in blood, involving PCR amplification, solution hybridization with Tris-(2, 2'-bipyridine)-ruthenium(II) chelate-labeled probes, and measurement by electrochemiluminescence (ECL). Extraction and amplification efficiencies were monitored by the inclusion of internal control (IC) DNA, mimicking the VZV target, in the DNA extraction. Viral DNA load was calculated from the ratio of VZV and IC ECL signals. The lower limit of sensitivity was 20 VZV DNA copies/ml of plasma or serum and 80 copies/ml of whole blood. In reconstruction experiments, expected and calculated VZV DNA loads were in excellent accordance. Blood specimens from 42 VZV-infected patients were tested for the presence of VZV DNA and showed detection rates of 86% in patients with varicella and 81% in patients with herpes zoster. In specimens obtained during the first week after onset of the rash, detection rates were 100 and 89%, respectively. Viral DNA was detected in all immunocompromised patients with herpes zoster, emphasizing the risk of disseminated disease in this patient group. VZV DNA load was similar in patients with varicella and multidermatomal herpes zoster and lower in patients with unidermatomal zoster. Despite the cell-associated nature of the virus, VZV DNA was detected in serum and plasma at high copy numbers, and at similar frequencies compared to whole-blood specimens. Quantitation of VZV DNA in blood is of potential importance for diagnosis and clinical management of VZV-infected patients. Plasma and serum provide convenient matrices for this purpose.     

Quantitation of ranaviruses in cell culture and tissue samples

A quantitative real-time PCR (qPCR) based on a standard curve was developed for detection and quantitation of ranaviruses. The target gene for the qPCR was viral DNA polymerase (DNApol). All ten ranavirus isolates studied (Epizootic haematopoietic necrosis virus, EHNV; European catfish virus, ECV; European sheatfish virus, ESV; Frog virus 3, FV3; Bohle iridovirus, BIV; Doctor fish virus, DFV; Guppy virus 6, GV6; Pike-perch iridovirus, PPIV; Rana esculenta virus Italy 282/I02, REV282/I02 and Short-finned eel ranavirus, SERV) were detected with the qPCR assay. In addition, two fish cell lines - epithelioma papulosum cyprini (EPC) and bluegill fry (BF-2) - were infected with four of the isolates (EHNV, ECV, FV3 and DFV), and the viral quantity was determined from seven time points during the first three days after infection. The qPCR was also used to determine the viral load in tissue samples from pike (Esox lucius) fry challenged experimentally with EHNV.     

Multiplex,quantitative, real-time PCR assay for cytomegalovirus and human DNA

We created a multiplex, quantitative, real-time PCR assay that amplifies cytomegalovirus (CMV) and human DNA in the same reaction tube, allowing for a viral load determination that is normalized to measured human DNA. The assay targets a conserved region of the CMV DNA polymerase gene that is not affected by known drug resistance mutations. All 36 strains of CMV detected by culture or qualitative PCR in a population of lung transplant recipients were detected. The assay detected 1 to 10 copies of CMV plasmid DNA. The analytic sensitivity was not affected by the presence of DNA from 10(6) human cells but was reduced approximately 10-fold by alkaline lysates of leukocyte preparations. CMV quantitation was linear over a range of 10(1) to 10(6) copies. The intraassay and interassay coefficients of variation were 29 and 40%. Human DNA was regularly detected in patient plasma samples, and the amount was increased by storage of blood at room temperature before plasma separation and by plasma separation techniques that allowed leukocyte contamination. Applied to whole blood, the assay provides a measurement of CMV DNA in relation to cellular content without a need for cell counting procedures. Applied to plasma, the assay can reveal artifactual increases in plasma CMV levels resulting from leukocyte contamination. Further study of the utility of this assay to monitor patient populations at risk for CMV disease is warranted.     

Optimization of quantitative detection of cytomegalovirus DNA in plasma by real-time PCR

Previous studies have shown that detection of cytomegalovirus (CMV) DNA in plasma is less sensitive than the antigenemia assay for CMV surveillance in blood. In 1,983 blood samples, plasma PCR assays with three different primer sets (UL125 alone, UL126 alone, and UL55/UL123-exon 4) were compared to the pp65 antigenemia assay and blood cultures. Plasma PCR detected CMV more frequently in blood specimens than either the antigenemia assay or cultures, but of the three PCR assays, the double-primer assay (UL55/UL123-exon 4) performed best with regard to sensitivity, specificity, and predictive values compared to antigenemia: 122 of 151 antigenemia-positive samples were detected (sensitivity, 80.1%), and there were 122 samples that were PCR positive-antigenemia negative (specificity, 93%). Samples with discrepant results had a low viral load (median, 0.5 cells per slide; 1,150 copies per ml) and were often obtained from patients receiving antiviral therapy. CMV could be detected by other methods in 15 of 29 antigenemia positive-PCR negative samples compared to 121 of 122 PCR positive-antigenemia negative samples (P < 0.001). On a per-subject basis, 21 of 25 patients (antigenemia positive-PCR negative) and all 57 (PCR positive-antigenemia negative) could be confirmed at different time points during follow-up. The higher sensitivity of the double-primer assay resulted in earlier detection compared to antigenemia in a time-to-event analysis of 42 CMV-seropositive stem cell transplant recipients, and two of three patients with CMV disease who were antigenemia negative were detected by plasma PCR prior to the onset of disease. Interassay variability was low, and the dynamic range was >5 log(10). Automated DNA extraction resulted in high reproducibility, accurate CMV quantitation (R = 0.87, P < 0.001), improved sensitivity, and increased speed of sample processing. Thus, primer optimization and improved DNA extraction techniques resulted in a plasma-based PCR assay that is significantly more sensitive than pp65 antigenemia and blood cultures for detection of CMV in blood specimens.     

Detection of transfusion transmitted virus DNA by real-time PCR.

BACKGROUND: Little is known about the pathogenic role and the endemic situation of transfusion transmitted virus (TTV). OBJECTIVES: In this study, a molecular assay for detection of TTV based on automated nucleic acid extraction and real-time PCR was developed and evaluated. The new assay includes an internal control. STUDY DESIGN: After optimization of the molecular assay, 103 clinical samples were studied retrospectively. All sera had been tested for anti-HCV and anti-HIV-1 antibodies earlier. RESULTS: The amplification efficiency was found to be 102%. When clinical specimens were tested, 79 of 103 serum samples were found to be positive for TTV. There was no significant difference between various groups of patients. The internal control was detected in all negative and weak positive samples. CONCLUSIONS: This molecular assay proved to be suitable for routine detection of TTV in clinical samples. Moreover, a relative statement on the TTV serum load can be done.     

Quantitation of putative glycoprotein X in bioengineered pseudorabies vaccine virus culture medium by ELISA

An enzyme-linked immunosorbent assay has been developed for the detection and quantitation of putative pseudorabies glycoprotein X (gX) in bulk bioengineered PRV delta gX delta tk-1 pseudorabies vaccine virus culture medium supernatants. The assay has a dynamic range of 0.2-25 ng, with a best linear region of 0.4-12.5 ng (correlation coefficient = 0.99) which permits 1 ppm discrimination for gX.     

Quantitation of human cytomegalovirus in recipients of solid organ transplants by real-time quantitative PCR and pp65 antigenemia

Human cytomegalovirus (HCMV) infections and anti-HCMV treatment are usually monitored by measuring pp65 antigenemia. This method is time-consuming, labour-intensive and requires skilled operators. We have compared results obtained using real-time Light Cycler quantitative PCR (QPCR) and the pp65 antigen assay on serial samples collected from recipients of solid organ transplants. We collected 198 blood samples from 14 solid organ transplant recipients and assayed them for pp65 antigen and with Light Cycler PCR. HCMV DNA was extracted from leukocytes and measured using primers and probe located in the UL83 region. The quantity of HCMV DNA was calculated using a standard curve prepared from a plasmid containing the target sequence. There was a good correlation between the number of pp65-positive cells and the DNA copy number (r = 0.57, P < 0.0001). A clinical threshold of 50 positive polymorphonuclear leukocytes/200,000 cells was equivalent to two log10 genome copies per capillary by Light Cycler PCR. HCMV DNA was detected before pp65 antigen in three patients at a mean time of 10 days, whereas the two tests were positive simultaneously for eight patients. Both the pp65 antigen data and DNA copy number decreased over time during antiviral treatment, although the QPCR was positive 28.2 days after the pp65 antigen assay had become negative. The real-time Light Cycler quantitative PCR assay is a rapid and labour-saving technique. This molecular method could be useful for monitoring infections and antiviral treatment in recipients of solid organ transplants.     

Detection and quantitation of hepatitis E virus in human faeces by real-time quantitative PCR

Hepatitis E Virus (HEV) is the causative agent of an acute and self-limited form of hepatitis. The virus is transmitted by the faecal-oral route and is a major cause of viral hepatitis in much of the developing world where it causes sporadic infections and large-scale epidemics. A simple and rapid protocol for the measurement of HEV faecal shedding by a real-time polymerase chain reaction (PCR) with the SYBR Green method on a LightCycler instrument, is described. After only 3h the real-time quantitative PCR method detected 10 molecules of HEV cDNA fragment per reaction tube and showed a high linear dynamic range of quantitation (10-10(6) molecules of cDNA/reaction) with a good correlation (r = -1.00). Its specificity was confirmed by assay in human faecal samples.