Development of highly sensitive Real-time COLD-PCR taqman lna probe assay for detection of tenofovir resistant RT-A194T mutation in chronic hepatitis B
Main Article Content
Abstract
The rtA194T mutation on the gene coding for RTase of Hepatitis B Virus (HBV) has been reported to be associated with tenofovir disoproxil fumarate (TDF)-resistance in Chronic Hepatitis B (CHB). In this study, we successfully developed a novel real time COLD-PCR TaqMan LNA probe assay with high sensitivity of 0.1% (mutant/wild-type, mt/wt) for early detection rtA194T mutation in TDF-experienced patients with CHB. This assay was then applied in 75 serum samples, and as results no rtA194T mutation was found in any samples. In conclusion, the real time COLD-PCR TaqMan LNA probe assay with high sensitivity (0,1% mt/wt) and time-saving of 3 h performance is potential for early detection of rtA194T mutations, thereby providing a tool for consulting and efficient follow-up the efficacy of TDF treatment regimens for chronic hepatitis B patients in future.
Article Details
Keywords
HBV, rtA194T, real-time COLD-PCR, TaqMan LNA probe, Tenofovir disoproxil fumarate (TDF)
References
2. Sharma V, Sharma R, Gaurav G. First Report of Primary Tenofovir Resistance in a Hepatitis B Viral Hepatitis Patient from India without Human Immunodeficiency Virus Co-infection. J Liver Res Disord Ther. 2016;2(5):37. doi:10.15406/jlrdt.2016.02.00037
3. Cho WH, Lee HJ, Bang KB, Kim SB, Song IH. Development of tenofovir disoproxil fumarate resistance after complete viral suppression in a patient with treatment-naïve chronic hepatitis B: A case report and review of the literature. World J Gastroenterol. 2018;24(17):1919-1924. doi:10.3748/wjg.v24.i17.1919
4. Park ES, Lee AR, Kim DH, et al. Identification of a quadruple mutation that confers tenofovir resistance in chronic hepatitis B patients. J Hepatol. 2019;70(6):1093-1102. doi:10.1016/j.jhep.2019.02.006
5. Rawal R, Konreddy A, Chu C. Mechanism of Adefovir, Tenofovir and Entecavir Resistance: Molecular Modeling Studies of How A Novel Anti-HBV Agent (FMCA) Can Overcome the Drug Resistance. Curr Med Chem. 2015;22(34):3922-3932. doi:10.2174/0929867322666150904144802
6. Liu C, Lin J, Chen H, et al. Detection of Hepatitis B Virus Genotypic Resistance Mutations by Coamplification at Lower Denaturation Temperature-PCR Coupled with Sanger Sequencing. J Clin Microbiol. 2014;52(8):2933-2939. doi:10.1128/JCM.01127-14
7. Wong DKH, Tsoi O, Huang FY, et al. Application of Coamplification at Lower Denaturation Temperature-PCR Sequencing for Early Detection of Antiviral Drug Resistance Mutations of Hepatitis B Virus. McAdam AJ, ed. J Clin Microbiol. 2014;52(9):3209-3215. doi:10.1128/JCM.00343-14
8. Phung TTB, Chu SV, Vu ST, et al. COLD-PCR Method for Early Detection of Antiviral Drug-Resistance Mutations in Treatment-Naive Children with Chronic Hepatitis B. Diagnostics. 2020;10(7):491. doi:10.3390/diagnostics10070491
9. Sun Z, Zhou L, Zeng H, Chen Z, Zhu H. Multiplex locked nucleic acid probes for analysis of hepatitis B virus mutants using real-time PCR. Genomics. 2007;89(1):151-159. doi:10.1016/j.ygeno.2006.07.011
10. Truong H, Nguyen VA, Nguyen HL, Pham VA, Phan TN. Sensitive quantification of mitochondrial mutation using new Taqman probes. Cent Eur J Med. 2014;9(6):839-848. doi:10.2478/s11536-013-0325-8
11. Motahar M, Arabzadeh SA, Mollaei H, Iranmanesh Z, Nikpour N, Soleimani F. Evaluation of HBV resistance to tenofovir in patients with chronic hepatitis B using ZNA probe assay in Kerman, southeast of Iran. Asian Pacific J Trop Dis. 2016;6(7):513-516. doi:10.1016/S2222-1808(16)61079-4
12. Alacam S, Karabulut N, Yolcu A, et al. Evaluation of drug resistance mutations in patients with chronic hepatitis B. Folia Microbiol. 2019;64(2):237-243. doi:10.1007/s12223-018-0650-z