Establish A Branched DNA Technology Used For HBV DNA Quantitation And Evaluate The Performance Of A DNA Microarray Method For Detecting HBV Mutations

Objective1. To establish a method based on branched DNA signal amplification technology forHBV DNA quantitation, to evaluate it’ s main characteristic and clinical applicationvalue, and to apply this approach to clinical laboratory diagnosis and therapeuticeffect judgment.2. To evaluate the performance of a DNA microarray method for detecting HBVmutations conferring antiviral drug resistance to lamivudine and adefovir.Methods1. Design a set of probes labeled with alkaline phosphatase according to full genomesof the two most common HBV genotypes in China(genotype B and genotype C),which contains: capture probes, capture extenders, label extenders, preamplifierprobes, amplifier probes, and label probes. All capture probes were attached to96-well plate, and then hybridized to HBV DNA, preamplifier probes, amplifierprobes and label probes respectively. The hybridization signal was cascade amplified.The chemiluminescence signal value was detected by luminometer(Beijing yuandeBiological company,JETLIA-962).2. Prepare standard substance:5-ml-serum samples were collected from5HBV-infected patients with high HBV load, mixed each adequately, and quantitatedHBV DNA20times for4-day by RT-qPCR, respectively. The average test resultssaying1.87E+08copies/ml were carried out as the standards.3. Improved reaction conditions referred to lysates content, reaction time as well asthe linear range, specificity, reproducibility of the assay and interference test were performed.4. HBV DNA level of84serum samples collected from HBV-infected patients wererespectively quantified by b-DNA and RT-qPCR, and then evaluate the clinicalapplication value of b-DNA. All analyses were performed with SPSS statisticalsoftware version11.5.5.48serum samples were obtained from patients with CHB who confirmed resistanceto lamivudine and adefovir, The mutations within the HBV reverse transcriptaseregion, which included rtL180、rtM204、rtA181、rtN236were analyzed by DNAmicroarray as well as direct sequencing. Compared the consistency of two methods’results.Results1. The linear range of b-DNA was104copies/ml~107copies/ml. This method has anexcellent reproducibility, the CV of within group was2.60%,1.49%and1.35%forHBV DNA concentration of107,105and104copies/ml, respectively. While the CV ofbetween groups were11.6%,9.82%and5.61%, respectively. This technology alsopossesses a good specificity, and the signal value was confirmed not to rise whensamples coexisted with HCV, CT, HSV, HPV.2. Of all the84serum samples detected, the positive coincident rate between theresult of b-DNA and that of RT-qPCR was85.7%(72/84, Kappa=0.694, P＜0.05).Theaverage HBV DNA content was4.24log10copies/ml (0～8.36log10,4.24±3.08log10) when detected by RT-qPCR, while3.85log10copies/ml (0～8.27log10,3.85±3.42log10) by b-DNA. The average HBV DNA level determined by RT-qPCR wassignificant higher than that by b-DNA. Differences between the two methods showeda statistical significance(P<0.05) and they were closely correlated(r=0.904,p<0.01).3. Both the DNA microarray and direct sequencing method successfully detected192resistance genotypes of the48samples, with40drug-resistant mutation casesand8wild type cases. The two methods showed a consistency of91.67%.44casesshowed the same results. However,4samples did not (1was found mutationsincluded rtA181T, rtL180M and rtM204V by DNA microarray method, but only rtA181T by direct sequencing. Another was appeared mutations included rtA181T andrtN236T by DNA microarray method, but rtA181T alone by direct sequencing. Theother2was revealed mutations included rtL180M, rtM204V and rtM204I by DNAmicroarray method, but only rtM204I by direct sequencing.).Conclusion1. A method based on branched DNA signal amplification technology for HBV DNAquantitation was established successfully. Its results were stable, accurate and reliable,and it can be applied to the diagnosis and curative effect monitoring ofHBV-infected patients.2. DNA microarray method is highly consistent with direct sequencing in detectingHBV drug-resistant mutations, and has the characteristics of high throughput. DNAmicroarray method can detect the trace mutations which cannot be detected by directsequencing method in the several known mutations covered by the detection ofmicroarray method. Consequently, it is useful for early detection of drug resistance.