| Objective: YMDD variant HBV is a discovered drug-resistance mutation after 1996. The mutations particularly emerge in some patients during long-term Lamivudine (3TC) treatment. 3TC has been receiving attention as an effective antiviral agent for treatment of patients with chronic Hepatitis B. As 3TC has been recently approved worldwide, the problem of resistance is becoming a significant clinical issue that need to be addressed for each patient in terms of identification and monitoring of prolong 3TC treatment.Methods: SS-SPPCR was used to identify genetic variant worldwide. This method is not only as sensitive as Southern Blot hybridization, but also simple and rapid and most importantly, require only the standard facilities of our clinical laboratory. The principle of this method was one of the primers used for HBV RT DNA amplification labeled with digoxigenin to allow incorporation into the amplicon. Biotin labeled probes specific for the desired sequence were used to identify specific amplicons during fluid-phase hybridization and extension, hybridized products were boundto anti-digoxigenin on microplate wells, and sequence-specific products were detected with ELISA. YMDD motif mutations were detected by three procedures that can be classified as follows : Site-Directed Mutagenesis; Solid-phase detection of PCR products; Evaluation of sensitivity and specificity of the assay.In order to determine the arising of YMDD variant HBV, each 3TC resistance mutation (nt 171-985) was engineered into the HBV geneome by two rounds amplification respectively. Then it was cloned into pGEM-T vector. Four recombinant plasmids (p-Vgtg, p-Iatt, p-Iatc, p-Iata) were identified by restriction enzyme and sequenced. During the solid-phase detection of PCR products,, two groups of probes for use in the SS-SPPCR were designed to hybridize with a DNA sequence specifmg the wt strains and YMDD motif mutations. One of them was labeled with biotine, the other was sythesized without labeled. The primers for standard PCR was labeled with digoxigenin. The probes were bound to the PCR product containing the digoxigenin -labeled primer by fluid-hybridization. Hybrid reaction parameters in solution were estimated with changing level of reanneal temperature of hybrid process in this assay. The reanneal temperature of pMS was developed with risen per 5℃ from 35℃ to 45℃. Similarly, The reanneal temperature of platt was choiced. Finally, ELISA was used to determine hybrized products. A series fold dilution of anti-digoxigenin andavidine-HRP was made to obtain the 'working dilution' of ELISA. We examined sensitivity of SS-SPPCR method to detect wt when it was diluted at different concentration. Moreover, specificity of the assay was evaluated with detecting YIDD ( att) when wt and YIDD (att) were occurred in mixed.Results: As described above, we got four recombinant plasmids: p-Vgtg, p-Iatt, p-latc, p-Iata. The parameters of SS-SPPCR were shown as follows: pMS and p-Iatt were given a reanneal temperature of 40℃ and 65℃ respectively. In terms of Working dilution concentration of coating-antidody concentration and avidine-HRP, the former is 2μ g/ml, the latter is 1: 400.The cut-off of positive OD volume was over 0.15. Sensitivity of SS-SPPCR to detect pMS : when purified pMS was mixed with different concentration, the method could detect when mixed 1/106. Specificity of SS-SPPCR to detect p-Iatt: pMS was mixed with different concentration of the p-Iatt, SS-SPPCR could detect p-Iatt when it represented 1/105 of the virus mixture.Conclusions: These results suggested that SS-SPPCR is nucleic acid-based ELISA-linked highly sensitive and specific diagnosis method, and may be generally applicable for detection of large kinds of virus. |
PDF Full Text Request