Study On A Novel Technology For The High-Sensitive Detection Of The Hepatitis B Virus Mutations Related To Drug Resistance And Carcinogenesis

We established a novel detection technique which is rapid, multiplex, array-based, and super sensitive for the detection of point mutations relevant to drug resistance and carcinogenesis of HBV. This technique is based on fluorescent quantitative PCR and modules divided on a 96-well plate. Mutation sites relevant to drug resistance to LAM (rtL180M,rtM204I/V) and ADV (rtA181V/T,rtN236T) as well as carcinogenesis (A1762T/G1764A) are detected and identified simultaneously and quantitatively using AllgloTM probe and the specific fluorescent groups (MAR, JUP, SAT, PLU, NEP, and URA). This technique can effectively differentiate wild type, mutation, and the mixture of wild type and mutation with high sensitivity, high specificity, easy operation, and high throughput. This technical platform is also applicable to the detection of the mutations relevant to drug resistance and carcinogenesis of other virus to achieve highly efficient, precise, and sensitive detection and to facilitate clinical diagnosis and customerized treatment.We constructed the engineered plasmids of HBV LAM rt180L (wild type), rt180M (mutation), rt204M (wild type), rt204I/V (mutation); ADV rt181A (wild type), rt181V/T (mutation), rt236N (wild type), rt236T (mutation); HBV BCP 1762A/1764G (wild type), and 1762T/1764A (mutation). For each array module we established the multiplex fluorescent quantitative method to detect the mutaion sites relevant to the drug resistance to LAM and ADV and in the BCP region using the above plasmids as standards. We evaluated the sensitivity, specificity, precision, and stability of the method. Results showed that the detection limits for the mutations of HBV LAM, ADV, and BCP are all below 1×102 copies/ml using the established array module type multiple fluorescence quantitative method in this study. Experiments for the mixtures of wild type and mutation showed high specificity and the precision ranged from 1.1% to 2.2%. Accelerated destruction test at 37℃for 2h (which is equivalent to storage at -20℃for 6 months) showed good results. In a word, the established methods in this study can detect single-base mutation effectively and differentiate wild type, mutation and mixture of wild type and mutation. Meanwhile, the mutation rate can be roughly quantitated using the standard curve, providing references for clinical diagnosis and treatment.We detected 245 samples (115+130+60) using this method.115 samples (among them 20 samples showed drug resistance phenotype during LAM treatment) were detected for drug resistance to LAM. Results revealed 11 samples with rtL180M mutation,14 samples with rtM204I (YIDD) mutation,7 samples with rtM204V (YVDD) mutation,4 samples with rtL180M/rtM204I (YIDD) mutation,3 samples with rtL180M/rtM204V (YVDD) mutation, and 2 samples with rtL180M/rtM204I (YIDD)/rtM204V (YVDD) mutation. The mutation rates are 9.57%,12.17%,6.09%, 3.47%,2.61%, and 0.86%, respectively.130 samples (10 of them are suspected samples of drug resistance to ADV) were detected for drug resistance to ADV. Results revealed 5 samples with rt A181V mutation,6 samples with rtA181T mutation,3 samples with rtN236T mutation, and 1 sample with rtA181V/T and rtN236T mutation. The mutation rates are 3.85%,4.62%,2.31%, and 1.74%, respectively.60 samples (15 of them are hepatocellular carcinoma samples) were detected for 1762T/1764A mutations in the BCP region. Results revealed 9 samples with 1762T/1764A double mutations. Meanwile, the above 245 samples were detected in parallel using DNA sequencing method. The results perfectly coincide with multiplex fluorescent quantitative method.In a word, the established array module type multiple fluorescence quantitative method for the detection of point mutation has high sensitivity and specificity and can be used to detect the mixed infection of wild type and mutation. Problems of low differentiation and sensitivity of traditional methods were resolved. The new method can better meet the clinical requirements of the early diagnosis of the drug resistance of HBV and hepatic cancer. It facilitates the early diagnosis, early finding, and early treatment and provides sufficient time for the appropriate medication and treatment.