New design and optimization of an in-house quantitative TaqMan Real-Time PCR-based assay for the detection and monitoring of occult hepatitis B virus (genotype A-J) infection |
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| Affiliation: | 1. Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran;2. Department of Pathology, School of Medicine, Fasa University of Medical Sciences, Fasa, Iran;3. Department of Research and Development, Production and Research Complex, Pasteur Institute of Iran, Tehran, Iran;4. Department of Medical Biotechnology, School of Medicine, Shahroud University of Medical Sciences, Shahroud, Iran;1. Max Hospital, New Delhi, India;2. PGIMER, Chandigarh, India;1. Department of Laboratory Medicine, Nanjing Drum Tower Hospital, the Affiliated Hospital of Nanjing University Medical School, Nanjing, Jiangsu Province, 210008, China;2. Department of Rheumatology and Immunology, Nanjing Drum Tower Hospital, the Affiliated Hospital of Nanjing University Medical School, Nanjing, Jiangsu Province, 210008, China;3. Department of Infectious Diseases, Nanjing Drum Tower Hospital, Clinical College of Nanjing Medical University, Nanjing, Jiangsu Province, 210008, China;1. Department of Emergency Medicine, Christian Medical College and Hospital, Vellore, 632004, India;2. Department of Clinical Virology, Christian Medical College and Hospital, Vellore, 632004, India;1. Department of Experimental Animal Facility, National JALMA Institute for Leprosy and Other Mycobacterial Diseases, Agra, Uttar Pradesh, 282004, India;2. Clinical Division, National JALMA Institute for Leprosy and Other Mycobacterial Diseases, Agra, Uttar Pradesh, 282004, India;3. Department of Biochemistry, National JALMA Institute for Leprosy and Other Mycobacterial Diseases, Agra, Uttar Pradesh, 282004, India;4. National JALMA Institute for Leprosy and Other Mycobacterial Diseases, Agra, Uttar Pradesh, 282004, India |
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| Abstract: | PurposeHBV DNA quantification is used for individuals with uninterpretable serological tests, occult HBV infections, decreasing the window period of the disease, and treatment follow-up. Although there are commercial qPCR assays, they are expensive. In this study, we developed a highly sensitive quantitative TaqMan Real-Time PCR with an exogenous internal control to quantify HBV DNA in serum/plasma.MethodsA specific primer/probe set was designed for the S conserved region of various HBV genotypes. The primer/probe set was evaluated experimentally and in-silico. An exogenous internal control was included to monitor the effects of inhibitors. The standard plasmid was titrated using three different methods to prepare the seven standards for the assay. The functional characteristics of the in-house assay were evaluated using the standards. Two hundred clinical specimens were also tested.ResultsThe LOD of the in-house assay was 40 IU/mL, and the assay was linear from 3.26Log10 to 9.26Log10 IU/mL. The analytical and clinical sensitivity of the assay was 100% and 92.15%, respectively. The analytical and clinical specificity of the assay was 100% and 98.97%, respectively. The positive and negative predictive values of the assay were determined to be 98.94% and 92.38%, respectively. The highest coefficient of variation of the inter/intra-assay was 5.1%. The accuracy was close to 100% for all standards, and the correlation between the in-house assay and commercial kit AltoStar® PCR Kits 1.5 was remarkable. The results of the clinical samples using the standards titrated using AcroMetrix? HBV Panel, Artus® HBV RG PCR Kit, and AltoStar® PCR Kits 1.5 were comparable (r ?= ?0.942, 0.951, 0.951).ConclusionsThe results indicate that the in-house assay is highly sensitive and specific, reproducible, and cost-benefit. Thus, it can be used to detect and quantify HBV DNA in research and clinical settings. |
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| Keywords: | Occult hepatitis B virus Quantitative TaqMan Real-time PCR Novel primer-probe set |
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