Rapid detection of hepatitis B virus mutations using real-time PCR and melting curve analysis

Abstract

Current methods of detecting hepatitis B virus (HBV) mutations are time consuming, labor intensive, and not suitable for screening large numbers of samples. In the present study, we documented the advantages of a system that exploits differences in thermal stability between perfect match and mismatch hybrids, and thereby distinguishes between wild-type and mutants. Hybridization probes were designed complementary to specific wild-type HBV sequences in surface (S), precore, and basal core promoter (BCP) regions of the HBV genome (nt 587, 1896, and 1762/1764, respectively). Two probes were designed for each mutation: anchor probes were 3 labeled with fluorescein and sensor probes, 5 labeled with LC-Red 640, and 3 phosphorylated. Temperatures for each probe melted from amplification products were then determined in a melting program. Sera from 12 patients, each containing identified HBV mutants (6 S-escape, 1 precore, 1 BCP, and 4 mixed precore and BCP), and 5 control sera from patients with wild-type virus were analyzed. Genomic sequences of mutant and wild-type viruses were confirmed by direct sequencing. Real-time polymerase chain reaction (PCR) with fluorescent hybridization probes accurately identified each mutant and wild-type genome. Melting temperatures obtained from probe-product duplexes for the 3 mutants were distinguished from wild-type (>4.0°C, minimal) within 45 minutes. The sensitivity of the system was 100 copies/mL and as few as 5% of mutant among wild-type virus were detected. In conclusion, real-time PCR with fluorescent hybridization probes is a specific, sensitive, quantitative, and rapid means of detecting clinically relevant HBV mutants. (HEPATOLOGY 2002;36:723-728.) T he hepatitis B virus (HBV) is one of the most common infectious agents in humans and a major cause of chronic hepatitis, cirrhosis, and hepatocellular carcinoma. [1][2][3] It is a partially double-stranded DNA molecule virus that replicates through an RNA intermediate by reverse transcription. 4,5 The reverse transcriptase enzyme responsible for this aspect of the replication cycle lacks proofreading function and, therefore, mutations to the genome are common. Indeed, it is now recognized that the majority of chronic HBV infections are associated with emergence of mutations throughout the viral genome. Some of these mutations result in the generation of diverse viral populations and different clinical outcomes. Those in the surface, core, precore, and basal core promoter (BCP) regions appear to have the greatest clinical relevance. 6,7 Additional mutations have also been described after therapeutic interventions such as antiviral therapy and vaccination. 8,9 Thus, the ability to detect these mutants is essential to further our understanding of the natural history of HBV and its response to antiviral therapy and immunoprophylaxis.Presently, the detection of HBV mutants is largely performed by restriction fragment length polymorphism analysis, 10,11 gap ligase chain reaction assay and/or direct sequencing. 12,13 These methods ...

Occult hepatitis B virus infection in a North American adult hemodialysis patient population

“…13 Briefly, HBV DNA was extracted from sera using a High Pure Viral Nuclei Acid Isolation Kit (Roche Diagnostics, Laval, Quebec, Canada). The sequences of the primers, anchor and sensor probes used in this study are summarized in Table 1.…”

Comparison of an in-House Qualitative PCR Assay for Detection of Hepatitis B Virus DNA with Real Time PCR using Sybr® Green I

“…It is important to monitor this process because of the emergence of drug resistant HBV strains carrying mutations associated with the treatment (12,13).…”

Ultrasensitive Quantification of Hepatitis B Virus A1762T/G1764A Mutant by a SimpleProbe PCR Using a Wild-Type-Selective PCR Blocker and a Primer-Blocker-Probe Partial-Overlap Approach

“…Thus, the SimpleProbe PCR has a built-in "self-check" function. Detection of HBV mutations by melting curve analysis using hybridization probes (also named Hyb probes or fluorescence resonance energy transfer [FRET] probes) was reported (22,28). However, the hybridization probes consist of an anchor probe and a sensor probe, and the total probe length is more than 50 nucleotides (the sensors were 27 to 29 nucleotides in length).…”