Quantification of the relative levels of wild‐type and lamivudine‐resistant mutant virus in serum of HBV‐infected patients using microarray

Abstract

During the course of lamivudine administration in hepatitis B virus (HBV)-infected patients, a dynamic development of the viral population in serum is observed. Total HBV level is initially reduced, then lamivudine-resistant mutants appear, and finally, the viral level is increased. All methods of mutant detection so far described can only identify mutants in the serum, and cannot determine the proportion of those mutants. In this paper, we report the development of a novel technique that can quantify the relative proportion of mutants in serum utilizing gene microarray technology. Based on the nucleotide sequence at the loci of the mutations in lamivudine-resistant HBV mutants, 28 probes were designed and dotted on glass film to prepare the oligonucleotide microarray. Ten standard curves were established by employing 15 reference plasmids as templates. Ten standard math functions were simulated, which allowed quantification of the proportion of mutants in the sample by measuring the value of fluorescent intensity on the microarray. By utilizing the standard math function, the relative proportion of two different mutation sequences in the mixed template could be detected with an error <10%. The HBV-lamivudine oligonucleotide microarray is reliable to quantify the relative proportion of wild-type HBV vs HBV mutants in patient's sera.

HBV drug resistance: Mechanisms, detection and interpretation

“…Other genotyping methods that are still being developed rely on hybridization coupled to a variety of detection techniques including mass spectrometry [63,64], computer assisted microarray analysis [65][66][67], luciferase assays [68], or electrochemical methods based on oxidation of guanine [69]. All are rapid and accurate but require expensive equipment and highly trained personnel.…”

Use of Massively Parallel Ultradeep Pyrosequencing To Characterize the Genetic Diversity of Hepatitis B Virus in Drug-Resistant and Drug-Naive Patients and To Detect Minor Variants in Reverse Transcriptase and Hepatitis B S Antigen

“…In addition, this technique does not allow a mutation frequency estimate, so it is not suitable for dynamic studies. Other sequence-specific genotypic resistance tests are available or are being developed, such as restriction fragment length/mass polymorphism (11,33,41), oligonucleotide microarray and gene chip technology (19,36), mutation-specific real-time PCR (39), matrix-assisted laser desorption ionization-time of flight mass spectrometry (17), and pyrosequencing (14,20). On the whole, all of the sequence-specific methods present the same drawbacks of reverse hybridization.…”

Clinical significance of periodic detection of hepatitis B virus YVDD mutation by ultrasensitive real-time amplification refractory mutation system quantitative PCR during lamivudine treatment in patients with chronic hepatitis B

“…Previous reports indicated that during the course of LMV therapy, HBV DNA in patient serum often reduces to an undetectable level -a situation which lasted for a long time and then reversed to the pre-treatment level (Li et al, 2005;Pallier et al, 2006). During this process, resistant variants were generated which finally replaced all WT YMDD strains and then the pathological effects appeared.…”