Optimization and assessment of a reverse hybridization system for the detection of HBV drug-resistant mutations

• Published in: Zhonghua gan zang bing za zhi Vol. 19; no. 12; p. 884
• Main Authors: Liu, Yan-chen, Huang, Ai-long, Hu, Yuan, Hu, Jie-li, Lai, Guo-qi, Zhang, Wen-lu
• Format: Journal Article
• Language: Chinese
• Published: China 01.12.2011
• Subjects: DNA, Viral - genetics; Drug Resistance, Viral - genetics; Hepatitis B virus - drug effects; Hepatitis B virus - genetics; Humans; Hybridization, Genetic; Mutation; Nucleic Acid Hybridization - methods; Oligonucleotide Array Sequence Analysis; Sensitivity and Specificity
• ISSN: 1007-3418
• DOI: 10.3760/cma.j.issn.1007-3418.2011.12.002

To establish a detection method for HBV drug-resistant mutations related to lamivudine, adefovir and entecavir by optimization and assessment of reverse hybridization system. 26 degenerated probes covering 10 drug-resistant hotspots of 3 drugs were synthesized and immobilized on the same positively charged nylon membrane. PCR products labeled with digoxigenin were hybridized with corresponding probes. To improve the sensitivity and specificity, 4 reaction steps of reverse hybridization were optimized including the number of labeled digoxigenin, the energy intensity of UV cross-linking, hybridization and stringency wash conditions. To prove the feasibility, the specificity, sensitivity and accuracy of this system were assessed respectively. Sensitive and specific results are obtained by the optimization of the following 4 reaction steps: the primers labeled with 3 digoxigenin, energy intensity of UV cross-linking for 1500 x 0.1 mJ/cm², hybridization at 42 degrees C and stringency wash with 0.5 x SSC and 0.1% S