Multiplex Detection of 60 Hepatitis B Virus Variants by MALDI-TOF Mass Spectrometry

Abstract

Background: Variations in the hepatitis B virus (HBV) genome may develop spontaneously or under selective pressure from antiviral therapy. Such variations may confer drug resistance or affect virus replication capacity, resulting in failure of antiviral therapy. Methods: A duplex PCR was used to amplify the region of the reverse transcriptase gene, the precore promoter, and the basal core promoter of the HBV genome. Four multiplex primer-extension reactions were used to interrogate 60 frequently observed HBV variants during antiviral therapy. Automated MALDI-TOF mass spectrometry (MS) was used for mutation detection. Capillary sequencing was used to confirm the MS results. Results: The limit of quantification was 1000 HBV copies/mL for multiplex detection of HBV variants. Fifty-three variants (88.3%) were analyzed successfully in at least 90% of the sera from 88 treatment-naive patients and 80 patients with virologic breakthrough. MS was able to detect twice as many minor variants as direct sequencing while achieving close to full automation. MS and direct sequencing showed only 0.1% discordance in variant calls. Conclusions: This platform based on multiplex primer extension and MALDI-TOF MS was able to detect 60 HBV variants in 4 multiplex reactions with accuracy and low detection limits.

Rethinking therapeutic decisions for hepatitis B infection in Syria: insights into molecular monitoring

“…HBV demonstrates a high mutation rate due to its errorprone reverse transcriptase that lacks exonuclease proofreading activity [4]. Hence drug-resistant variants may emerge throughout nucleos(t)ide analogues-based therapy leading to relapse and hepatic decompensation [5,6]. Moreover, the HBV genotype plays an essential role in driving viral evolution [5] as well as response to interferon-based therapy [7,8].…”

Replacing Reverse Line Blot Hybridization Spoligotyping of the Mycobacterium tuberculosis Complex

“…Methods similar to spoligotyping for the typing of other bacterial isolates with CRISPRs are being developed and can utilize the MassARRAY system. In general, the technology and the automated assay design tool are flexible and able to replace dot blot detection assays and membrane-based approaches, surpassing previous MALDI-TOF MS applications with respect to high-throughput single nucleotide polymorphism (SNP) genotyping (19) and variant detection (16,27). Likewise, the MassARRAY platform has already been shown to have the ability to be used for bacterial pathogen identification through comparative sequencing (9)(10)(11).…”

High-Throughput Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry as an Alternative Approach to Monitoring Drug Resistance of Hepatitis B Virus

“…The first step of the MALDI-TOF MS-based HBV genotyping assay was amplification of a 754-bp HBV DNA fragment containing the polymorphic site of our interest (codons 80, 169, 173, 180, 181, 184,194, 202, 204, 233, 236, and 250). PCR conditions were the same as those described previously (26). In order to remove the remaining, nonincorporated dNTPs from the amplification products, 5 l of each PCR product was treated with 2 l of shrimp alkaline phosphatase (SAP) solution.…”