Rapid Detection of Hepatitis B Virus Variants Associated with Lamivudine and Adefovir Resistance by Multiplex Ligation-Dependent Probe Amplification Combined with Real-Time PCR

Abstract

C hronic hepatitis B virus (HBV) infection remains a major public health problem worldwide. Two drugs, lamivudine (LAM) and adefovir (ADV), are presently used as first-line therapies against HBV in China. Antiviral drug resistance is the major obstacle to successful long-term therapy for chronic hepatitis B infection (1, 2). The detection of mutations associated with resistance to LAM and ADV is very important for the clinical selection of drugs used against HBV. At present, there are many methods to detect drug-resistant mutations of HBV, including direct sequencing from PCR products, reverse hybridization INNO-LiPA, microarray, and real-time PCR. Direct sequencing and INNOLiPA are well-known methods used in clinical practice. Although direct sequencing is the gold standard for the detection of mutations, it cannot detect mutations that are present at a low frequency (Ͻ20%) in the viral population (3). The INNO-LiPA (4, 5) is more sensitive, but the reagents and equipment are costly. Microarray is high-throughput but is also costly; therefore, a microarray cannot be widely used in clinical practice (6). With the development of new technologies in molecular diagnostics, realtime PCR was applied to detect HBV mutations. For many realtime PCRs, the mutants and wild-type strains were distinguished by changes in the melting temperatures (T m ) of the fluorescence probes (7), which result in a low rate of identification of mutations with similar T m values, adjacent mutations, and silent mutations. Moreover, the mutation sites need to be located in the middle of the fluorescence probes, such as in a type-specific minor groove binder probe (8-10) or a locked nucleic acid probe (11), thus restricting the design of probes and the detection flux. In most cases, only single-codon mutations can be detected by traditional real-time PCR. We have developed a new detection method, multiplex ligation-dependent real-time PCR (MLP-RT-PCR), which combines multiplex ligation-dependent probe amplification (MLPA) with real-time PCR. The method can be applied to quickly detect five HBV mutations associated with resistance to LAM and ADV in one tube, including the mutations rtM204V/I, rtA181V/T, and rtN236T in the reverse transcriptase domain of the HBV P gene (12)(13)(14). Schouten et al. (15) reported on the first MLPA assay in 2002. Forty pairs of MLPA probes were designed to detect changes in gene copies. The assay can also be used to detect single nucleotide polymorphism (SNPs) and point mutations (16), and it has been applied for detecting multiple-drug-resistant mutations and genotyping in Mycobacterium tuberculosis (17,18). In the traditional MLPA, pathogens are quantified and genotyped by capillary electrophoresis (18) or microarray (17). The synthesis of many different lengths of MLPA probes or complex operations, such as the fixation of probes, hybridization, and elution, are needed. However, using a TaqMan probe inserted into the MLPA probe as a tag, the MLPA probe can be directly amplified by realtime PCR and the results ...

A systematic review of hepatitis B virus (HBV) drug and vaccine escape mutations in Africa: A call for urgent action

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Enzymological description of multitemplate PCR—Shrinking amplification bias by optimizing the polymerase–template ratio

“…Classical PCR is a method of choice to obtain a sufficient amount of DNA for subsequent analyses, including drug-resistance evaluation (Jia et al, 2014) or construction of libraries (e.g., for next-generation sequencing van Dijk et al, 2014), and it is involved in many applications including species diversity studies (Shokralla et al, 2012).…”

Multiple Cross Displacement Amplification Linked with Nanoparticles-Based Lateral Flow Biosensor in Screening of Hepatitis B Virus in Clinical Application

“…Currently, enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) related technologies are the most commonly used methods for HBV identification in clinical diagnostic application. 7 , 8 Immunoassay is based on a serological response targeting the HBV antigens or antibodies, which can present better accuracy. 9 However, the sensitivity of the ELISA method is low at the early stages of HBV infection.…”

“…In the present study, MCDA amplification combined with LFB detector (MCDA-LFB) was developed for cost-effective, sensitive, specific, rapid, simple, and visual identification of HBV carrying the S gene, 7 which appeared to be uniquely present in HBV as it showed no homology with other microbial genomes at GenBank by BLAST searches. The detection performance was analyzed with HBV nucleic acid (HBV DNA) standard substance and clinical serum samples.…”