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

Mohammad Ghorbani; Rahman Shokri; Vahid Kia; Fatemeh Yari; Zohreh Sharifi; Mahdi Paryan

doi:10.1016/j.ijmmb.2022.07.003

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

Indian J Med Microbiol. 2022 Oct-Dec;40(4):560-566. doi: 10.1016/j.ijmmb.2022.07.003. Epub 2022 Jul 30.

Authors

Mohammad Ghorbani¹, Rahman Shokri², Vahid Kia³, Fatemeh Yari⁴, Zohreh Sharifi⁵, Mahdi Paryan⁶

Affiliations

¹ Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran; Department of Pathology, School of Medicine, Fasa University of Medical Sciences, Fasa, Iran.
² Department of Research and Development, Production and Research Complex, Pasteur Institute of Iran, Tehran, Iran.
³ Department of Medical Biotechnology, School of Medicine, Shahroud University of Medical Sciences, Shahroud, Iran.
⁴ Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran.
⁵ Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran. Electronic address: z.sharifi@ibto.ir.
⁶ Department of Research and Development, Production and Research Complex, Pasteur Institute of Iran, Tehran, Iran. Electronic address: mparyan@gmail.com.

PMID: 35914958
DOI: 10.1016/j.ijmmb.2022.07.003

Abstract

Purpose: HBV 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.

Methods: A 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.

Results: The 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).

Conclusions: The 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.

Keywords: Novel primer-probe set; Occult hepatitis B virus; Quantitative TaqMan Real-time PCR.

MeSH terms

DNA, Viral / analysis
DNA, Viral / genetics
Genotype
Hepatitis B virus* / genetics
Hepatitis B* / diagnosis
Humans
Real-Time Polymerase Chain Reaction / methods
Sensitivity and Specificity
Viral Load / methods

Substances

DNA, Viral