Rapid Detection Of Hepatitis B Virus Variants Associated With Lamivudine And Adefovir Resistance By MLPA Combined With Real-time PCR

Drug-resistant mutations of hepatitis B virus (HBV) are the major obstacles tosuccessful therapy for chronic hepatitis B. Although there are many methods to detect theantiviral drug-resistant mutations of HBV, their applications are restricted because of theshortcomings such as low sensitivity, time required and high cost. Here a multiplexligation-dependent probe real-time PCR (MLP-RT-PCR) method was developed to detectlamivudine (LAM) and adefovir (ADV) resistant HBV mutants (rtM204V/I, rtA181V/T andrtN236T) simultaneously. The new method combined the high-throughout of multiplexligation-dependent probe amplification (MLPA) with the rapid and sensitive detection ofreal-time PCR. In this report, the MLP-RT-PCR was evaluated by detecting drug-resistantmutants in116patients with chronic hepatitis B. By MLP-RT-PCR analysis, LAM-resistantmutations were detected in41patients (35.3%), ADV-resistant mutations were detected in17patients (14.7%), LAM and ADV-resistant mutations were detected in5patients (4.3%).Based on the results of MLP-RT-PCR, the mutations rtM204V, rtM204I, rtA181T, rtA181Vand rtN236T were95.7%(111/116),98.3%(114/116),99.1%(115/116),98.3%(114/116),and99.1%(115/116), respectively, in concordance with those of direct sequencing. TheMLP-RT-PCR assay was more sensitive than direct sequencing when detecting mutationswith low percentage.The channels for rtM204V, rtM204I, rtA181T and rtN236T detectedmutants whose percentage were over0.1%, while the channels for rtA181V detectedmutants whose percentage was over1%. Four samples containing the low percentage(<10%) mutants were identified by MLP-RT-PCR and further confirmed by clonalsequencing. MLP-RT-PCR is a rapid and sensitive method that enables the detection ofmulti-drug-resistant HBV mutations in clinical practices.