Two Methods For The Quantification Of Hepatitis B Virus (hbv) Covalently Closed Circular Dna (cccdna) By Cross-single-gap And Cross-double-gap Real-time Pcr

Hepatitis B virus infection affects more than 400 million people worldwide, chronic Hepatitis B virus infection is one of the leading causes of death. Now, most antiviral agents used to date successfully lower viral load to undetectable levels, but easy to reactive after cessation of antiviral therapy,it is believed that HBV covalently closed circular DNA(cccDNA) contributes to the relapse of discontinuation of antiviral therapy. So, to better treate this disease, it's necessary to monitor the change of cccDNA level. But, there isn't have mature quantitative PCR detection methods of HBVcccDNA, expecifily, lack of a rapid,accurate,specific,sensitive,economical and easy assay. The reason is that there are different levels of false positive problems between every detection methods, mainly the interference from RcDNA. At present, there are three main detection methods: Invader assay, chimeric primers fluorescence quantitative PCR, selective fluorescence quantitative PCR, but no matter which kind of the detection methods, its strategies are used to different of structure continuous of cccDNA and RcDNA to achieve specific differences of detection. The former two methods can not be excluded that the impact of the context of high RcDNA,but can't remove the interference caused by illegitimate copy. The latter includes two types: cross-single-gap specific primers and fluorescent labeled with the TaqMan specific probe,cross-two-gap specific primers and fluorescent labeled with specific Hybridization probe. Because can't exclude illegitimate copy (generated double-stranded linear DNA) and interference of high RcDNA background, so simple cross-single-gap fluorescence quantitative PCR detection method is considered relatively low specificity essay, but many HBVcccDNA quantitative PCR detection kit that is with cross-single- gap specific primers and TaqMan probe technology, and at the same time ,many articles which have been reported used this HBVcccDNA detection kit to detect HBVcccDNA , then research correlation of cccDNA with HBV-DNA, etc. or used it to evaluate the clearance effect on HBVcccDNA of some antiviral drugs. Cross-double-gap fluorescence quantitative PCR can exclude disruption of illegitimate copy, but can not completely rule out interference of high RcDNA background. Many scholars use Plasmid-safe ATP-dependent DNase to digest extracted total DNA, which can effectively degrade RcDNA and linear DNA. At present, HBVcccDNA detection method that has been many foreign scholars recepted is a fluorescent quantitative PCR which use Plasmid-safe ATP-dependent DNase to digest extracted total DNA and with cross-double-gap specific primers. However, this method also has its shortcomings, for example, large PCR product, limited choice in the probes, hybridization probe of highly technical, complicated operation, high cost, limited application was choice, but can not choose the TaqMan probes which is the most mature,stability,easy to operate and low cost.Objective To detect the effect of Plasmid-safe ATP-dependent DNase to remove RcDNA and linear DNA; to establish cross-single-gap fluorescence quantitative PCR detection method (TaqMan probe)of HBVcccDNA which is rapid,accurate,high sensitivity,low cost,easy to operation. To confirm the specificity of this method, the results of the test was compared with the results of cross-double-gap fluorescence quantitative PCR detection method (hybridization probes) of HBVcccDNA.Methods According to the differences of RcDNA and cccDNA in structure and Physicochemical nature, To establish detection methods of HBVcccDNA that are cross-single-gap (TaqMan probe) and cross-double-gap (hybridization probes) fluorescence quantitative PCR. Total DNA was extracted from serum and liver tissue of the same patient, part of the product was digested with DNase, the sample which was digested with or without DNase were detected using cross-single-gap and cross-double-gap fluorescence quantitative PCR of HBVcccDNA. The products of the two PCR detection methods were recovred, then, the products were cloned, screened, sequenced and analyzed, and use SPSS13.0 statistical software to count the disparity among the three kinds of fluorescence quantitative PCR detection method which are cross-single-gap fluorescence quantitative PCR with or without plasmid-safe ATP-dependent DNase and cross-double-gap fluorescence quantitative PCR with plasmid-safe ATP-dependent DNase.Results Successfully established two detection methods of HBVcccDNA which are cross-single-gap fluorescence quantitative PCR (TaqMan probe) and cross-double-gap fluorescence quantitative PCR (Hybridization Probes); there are obviously difference among the three kinds of fluorescence quantitative PCR detection methods which are cross-single-gap fluorescence quantitative PCR with or without plasmid-safe ATP-dependent DNase and cross-double-gap fluorescence quantitative PCR with plasmid-safe ATP-dependent DNase, it showed: the serum and liver tissue were digested with DNase whose HBVcccDNA copy number significantly lower than the undigested, serum Z =- 3.920, P <0.000, liver Z =- 3.920, P <0.000; serum and liver tissue were digested with DNase, then were detected using cross-double-gap and cross-single-gap fluorescence quantitative PCR, copy number of HBVcccDNA of the former significantly lower than the latter, serum Z =- 3.296, P = 0.001, liver tissue Z =- 3.808, P <0.000; The HBVcccDNA copy number come from serum that was detected using the three kind fluorescence quantitative PCR detection methods of HBVcccDNA which are cross-single-gap fluorescence quantitative PCR with or without plasmid-safe ATP-dependent DNase and cross-double-gap fluorescence quantitative PCR with plasmid-safe ATP-dependent DNase are significantly lower than those in the experimental results of liver tissue, P <0.001.Conclusion Plasmid-safe ATP-dependent DNase can effectively reduce the false positive problem of HBVcccDNA detection methods, but can not completely eliminate interference originate from RcDNA,linear DNA and others; Successfully established HBVccDNA cross-single-gap fluorescence quantitative PCR detection method (TaqMan probe) and cross-double-gap fluorescence quantitative PCR detection method (Hybridization Probes); The specificity of cross-single-gap fluorescence quantitative PCR combined DNase to digest samples is lower than cross-double-gap fluorescence quantitative PCR combined DNase to digest samples. That to say, The specificity of HBVcccDNA cross-double-gap fluorescence quantitative PCR higher than HBVcccDNA cross-single-gap fluorescence quantitative PCR.