COLD-PCR for early detection of hepatitis B virus antiviral drug resistance mutations.

Nucleos(t)ide analogues (NAs) are effective therapeutic agents for the treatment of chronic hepatitis B virus (HBV) infection. However, longterm use of NAs is often hampered by the emergence of drug resistance mutations, causing potentially serious consequences such as liver decompensation and mortality. A more sensitive method for early detection of drug resistance mutations is needed. Conventional PCR amplification of HBV DNA followed by direct sequencing of the purified amplicons for detection of drug resistance mutations has two advantages. First, it can detect any novel mutation within the amplicons. Second, it is relatively inexpensive. However, it cannot detect a low level of mutations that comprise <20% of the total viral population. Co-amplification at lower denaturation temperature PCR (COLD-PCR) has been used for enrichment of a low level of variants within a mixed pool of sequences.1 COLD-PCR relies on slight changes to the melting temperature (Tm) in the DNA sequence caused by mutations within the sequence. For each DNA sequence, there is a critical denaturation temperature (Tc) below which PCR efficiency decreases abruptly.1 Tc is lower than Tm and is dependent on the DNA sequence itself. When the denaturation temperature of PCR is set to Tc (instead of the usual 94oC), DNA amplicons with different mutations will have different amplification efficiencies. This property enables selectively enrich low-level mutations in a mixed pool. Hong Kong Med J 2015;21(Suppl 7):S8-10 RFCID project number: 09080682