Application of coamplification at lower denaturation temperature-PCR sequencing for early detection of antiviral drug resistance mutations of hepatitis B virus

Abstract

Nucleoside/nucleotide analogue for the treatment of chronic hepatitis B virus (HBV) infection is hampered by the emergence of drug resistance mutations. Conventional PCR sequencing cannot detect minor variants of <20%. We developed a modified co-amplification at lower denaturation temperature-PCR (COLD-PCR) method for the detection of HBV minority drug resistance mutations. The critical denaturation temperature for COLD-PCR was determined to be 78°C. Sensitivity of COLD-PCR sequencing was determined using serially diluted plasmids containing mixed proportions of HBV reverse transcriptase (rt) wild-type and mutant sequences. Conventional PCR sequencing detected mutations only if they existed in ≥25%, whereas COLD-PCR sequencing detected mutations when they existed in 5 to 10% of the viral population. The performance of COLD-PCR was compared to conventional PCR sequencing and a line probe assay (LiPA) using 215 samples obtained from 136 lamivudine- or telbivudine-treated patients with virological breakthrough. Among these 215 samples, drug resistance mutations were detected in 155 (72%), 148 (69%), and 113 samples (53%) by LiPA, COLD-PCR, and conventional PCR sequencing, respectively. Nineteen (9%) samples had mutations detectable by COLD-PCR but not LiPA, while 26 (12%) samples had mutations detectable by LiPA but not COLD-PCR, indicating both methods were comparable (P = 0.371). COLD-PCR was more sensitive than conventional PCR sequencing. Thirty-five (16%) samples had mutations detectable by COLD-PCR but not conventional PCR sequencing, while none had mutations detected by conventional PCR sequencing but not COLD-PCR (P < 0.0001). COLD-PCR sequencing is a simple method which is comparable to LiPA and superior to conventional PCR sequencing in detecting minor lamivudine/telbivudine resistance mutations.

FIG 1

Principle of COLD-PCR. Mutation sites in the minority mutant template are shown as open circles. A, B, C, and D on top denote the four thermal-cycle steps. (Adapted from reference by permission from Macmillan Publishers Ltd., copyright 2008.)

FIG 2

Real-time PCR result for the determination of T_c. (a) Melting curve analysis. (b to f) Real-time PCR amplification with denaturation temperature at (b) 94°C, (c) 82°C, (d) 80°C, (e) 79°C, (f) 78°C, (g) 77.5°C, and (h) 77°C. Green color indicates wild-type HBV; magenta, HBV with lamivudine resistance mutations (rtV173L, rtL180M, and rtM204V); blue, HBV with telbivudine resistance mutations (rtM204I); turquoise, HBV with adefovir resistance mutations (rtA181T and rtN236T); black, HBV with entecavir resistance mutations (rtL180M, rtT184S, rtS202I, rtM204V, and rtM250V), red, negative control.

FIG 3

Representative conventional PCR sequencing and COLD-PCR sequencing results with different ratios of wild-type (ATG) and rtM204V (GTG) sequences. The position of the A-to-G mutation is shown on top.