Rapid detection of hepatitis B virus variants associated with lamivudine and adefovir resistance by multiplex ligation-dependent probe amplification combined with real-time PCR.
Drug-resistant mutations of hepatitis B virus (HBV) are the major obstacles to successful therapy for chronic hepatitis B infection. Although there are many methods for detecting the antiviral drug-resistant mutations of HBV, their applications are restricted because of their shortcomings, such as low sensitivity, the time required, and the high cost. For this study, a multiplex ligation-dependent probe real-time PCR (MLP-RT-PCR) method was developed to simultaneously detect lamivudine (LAM)- and adefovir (ADV)-resistant HBV mutants (those with the mutations rtM204V/I, rtA181V/T, and rtN236T). The new method combined the high-throughput nature of multiplex ligation-dependent probe amplification (MLPA) with the rapid and sensitive detection of real-time PCR. In this report, MLP-RT-PCR was evaluated by detecting drug-resistant mutants in 116 patients with chronic hepatitis B infection. By MLP-RT-PCR analysis, LAM-resistant mutations were detected in 41 patients (35.3%), ADV-resistant mutations were detected in 17 patients (14.7%), and LAM- and-ADV-resistant mutations were detected in 5 patients (4.3%). Based on the results of MLP-RT-PCR, the mutations rtM204V, rtM204I, rtA181T, rtA181V, and rtN236T were 95.7% (111/116 patients), 98.3% (114/116 patients), 99.1% (115/116 patients), 98.3% (114/116 patients), and 99.1% (115/116 patients) concordant, respectively, with those of direct sequencing. The MLP-RT-PCR assay was more sensitive than direct sequencing for detecting mutations with low frequencies. Four samples containing the low-frequency (<10%) mutants were identified by MLP-RT-PCR and further confirmed by clonal sequencing. MLP-RT-PCR is a rapid and sensitive method that enables the detection of multidrug-resistant HBV mutations in clinical practice.
Department of Clinical Laboratory, Institute of Surgery Research, Daping Hospital, Third Military Medical University, Chongqing, China.
Shuangrong
S
Jia
Feng
F
Wang
Fake
F
Li
Kai
K
Chang
Shaojun
S
Yang
Kejun
K
Zhang
Wenbin
W
Jiang
Ya
Y
Shang
Shaoli
S
Deng
Ming
M
Chen
D000225:Adenine / Q000031:analogs & derivatives* / Q000627:therapeutic use; D000998:Antiviral Agents / Q000627:therapeutic use; D024882:Drug Resistance, Viral; D006515:Hepatitis B virus / Q000235:genetics* / Q000302:isolation & purification; D019694:Hepatitis B, Chronic / Q000188:drug therapy* / Q000821:virology; D006801:Humans; D019259:Lamivudine / Q000627:therapeutic use*; D008826:Microbial Sensitivity Tests / Q000379:methods; D025202:Molecular Diagnostic Techniques / Q000379:methods; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D009154:Mutation; D063065:Organophosphonates / Q000627:therapeutic use*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013995:Time
D000998:Antiviral Agents; D063065:Organophosphonates; D019259:Lamivudine; C053001:adefovir; D000225:Adenine