Mutation-sensitive On/off Switch Based Assays For The Detection Of HBV Lamivudine-resistant Mutations

Objective The purpose of this study was to develop methods for detecting themutations of YMDD, rtL180M, and rtV173L by nanoscale mutation-sensitive “on/offswitch” consisting of high fidelity polymerase and phosphorothioate-modified allelespecific primers.Methods Sequencing analysis information show the most common drug resistanceof HBV occurs in the reverse transcriptase domain of the HBV polymerase. A438bptemplate for assay development covered all the hotspot mutations was synthesized invitro and named mut1（the mutation of YMDD was YVDD）； The mutant template2（the mutation of YMDD was YIDD） was obtained using the overlap extension PCRtechnology. The final PCR product was gel purified and then subcloned into thePMD19-T vector. Cloning products were confirmed by direct sequencing；Wild typetemplate was obtained by regular PCR amplification and subcloning. The extracted wildtype HBV DNA was used as the template. The PCR product was then subcloned intothe PMD19-T vector and confirmed by sequencing. Five primers used for the detectionof the four mutations were designed according to the reference sequences ofNC₀03977.1in genbank. All primers were designed to match the mutant type and thenucleotides in3’ end were phosphorothioate modified. A nanometer-scaled proofreadingpolymerase mediated on/off switch was employed in these SNP （single nucleotidepolymorphism） assays for the detection of YMDD, rtL180M and rtV173L mutationsconferring resistance to lamivudine. Different results were shown on mutant plasmidsand wild plasmids. And the PCR reaction conditions for normal PCR and realtime PCRwere optimized with which the sensitivities and specificities of the assays weredetermined.Results In the present study, quick assays were developed for the detection ofM204V,M204I,V173L,L180M lamivudine-resistant mutations. Specific amplificationproducts were obtained on mutant type templates and no amplification product was yielded on wild type templates. The in vitro prepared plasmid DNA templates rangedfrom101-107copies were used to test the sensitivity and specificity of the four pairs ofthe phosphorothioate-modified allele-specific primers. The allele specific primer forM204V was able to detect the perfectly matched template at a sensitivity of about100copies. The same assay conditions do not yield primer extension PCR products until thetemplate was at or more than105copies from the mismatched amplicon. The specificityof M204V assay was about1000copies. Similar results were obtained for the otherthree hotspot mutations. The sensitivities for M204I, L180M and V173L wererespectively100copies,10copies, and10copies. The specificities were respectively105copies,103copies, and104copies for M204I, L180M and V173L. Multiplex PCRfor detection of M204V, V173L and L180M mutation or M204I, V173L, and L180Mwere established. The sensitivity for multiplex PCR was around100-1000copies andthe specificity was1000copies.Conclusion This work successfully established PCR based assays with specificprimers for detection of the HBV lamivudine-resistant mutations. The mutationsensitive “molecular switch”, consisting of high fidelity DNA polymerase andphosphorothioate modified primers can detect the micro-mutations. Thus, thistechnology has a greater potential in monitoring lamivudine-resistant mutations at theearly stage before and during the lamivudine therapy.