Detection of lamivudine resistance-associated hepatitis B virus mutations by multi-analyte suspension array

Abstract

Objective To develop and establish a rapid, convenient method with high-throughput, high sensitivity and specificity for detection of lamivudine (LAM) resistance-associated mutations in the hepatitis B virus. Methods HBV DNA was extracted as PCR templates. The target sequence at liT region was amplified using primers with 5'-biotin label. The biotin-labelled target PCR products interacted with those bead-bound probes in aqueous suspension, and then streptomycin and avidin-modified straptavidin phycoerythrin (SAPE) were added to the aqueous suspension after the free HBV DNA was washed out, which had not hybridized with probes on the beads. Finally, mutations were tested in luminex analyzer using the bicolor laser (red and green laser). The mutated type and relative quantity were identified according to the different ratios of red fluorophores incorporated into the beads (for qualitative analysis) and green fluorophores imparted by a specific reporter molecule SAPE (for quantitative analysis). Subsequently, the multi-analyte suspension array (MASA) was applied to detect plasmids containing fnll-length HBV genome with LAM-resistance associated HBV mutations and 25 serum specimens from patients with chronic hepatitis B, who were treated with LAM and experienced virological breakthrough due to LAM-resistanee. The specificity of this method was compared with direct DNA sequencing. Meanwhile, we also determined the sensitivity of this method by mixing the wild and mutant type plasmids in different proportions. Results MASA can rapidly detect mutations at rt180 and rt204 positions in RT region simultaneously. 25 samples with LAM-resistant mutations confirmed by DNA sequencing were found to contain consistent mutations by this method, with 15 cases hybridizing to a particular 20411 probe type( negative and positive median signal values were 5 and 106 respectively), 11 cases hybridizing to 204V probe( negative and positive median signal values were 9 and 114 respectively) (Z value =-4.588 and -4.520, P<0.05), and only 1 specimen hybridizing to 20412 probe. However, two samples were found harboring mixed mutated types (rtM204V and rtM204I). More importantly,MASA was able to detect 5% mutation frequency with up to 100% of specificity. Conclusions MASA could be used for early detection of LAM resistance-associated mutations in hepatitis B virus. This method has high sensitivity, specificity and high-throughput in detection of LAM resistance-associated mutations compared with direct DNA sequencing. This method may also significantly save the time of diagnosis and facilitate the clinical application of large samples. Key words: Hepatitis B virus; Lamivudine; Drug resistance; viral; Microarray analysis