Establishment And Application Of A Highly Sensitive Method For The Detection Of HBV PgRNA In Microvolume Samples

Hepatitis B virus(HBV)is the leading pathogen of Chronic hepatitis B,which belongs to the Orthohepadnavirus genus of the hepadnavividae family.Viral hepatitis belongs to a common infectious disease caused by a variety of virus,including Hepatitis A virus,Hepatitis B virus,Hepatitis C virus,Hepatitis D virus,Hepatitis E virus,Hepatitis G virus,EB virus,Cytomegalovirus,etc.Chronic hepatitis B is a global contagious disease caused by HBV,which can lead to hepatic fibrosis,cirrhosis and liver cancer.China is a country with a high incidence of Hepatitis B.Nearly a half of global HBV surface antigen(HBs Ag)carriers come from China.Sixty percent of Chinese have been infected by HBV and about 8-10% are HBs Ag positive carriers.The incidence of primary liver cancer(HCC)increases at least 300 times in patients infected with HBV,and 80% of what leads to HCC are related to HBV infection.As a region with a high incidence of liver cancer,how to effectively treat patients with Chronic hepatitis B to avoid liver cancer has become the primary problem to be addressed for liver health in China.The detection of HBV markers has a great relationship with the treatment and prognosis of Chronic hepatitis B.The following five main markers have been found in traditional HBV serological testing: HBs Ag,anti-HBs,HBe Ag,anti-HBe,and antiHBc.HBs Ag,the hepatitis B surface antigen,can reflect the stage and treatment progress of Hepatitis B disease and can also be used to guide the treatment of recombinant human interferon and pegylated interferon-α(Peg-IFN-α).Nucleic acid testing for HBV includes quantitative HBV DNA testing and quantitative HBV RNA testing.Quantitative HBV DNA testing mainly uses fluorescent quantitative PCR technology,currently an important method for evaluating the efficacy of antiviral therapy,to identify the level of HBV,which can be further implemented to assess the level of viral replication in HBV-infected patients.During antiviral therapy,if the viral content decreases,the risk of liver cirrhosis and HCC can be significantly reduced.HBV RNA assay is a quantitative assay for HBV pgRNA,which is a novel marker of HBV load,as well as a vital marker to evaluate the risk of relapse after nucleoside/nucleotide analogs(NAs)discontinuation,since the level of HBV pgRNA has a strong correlation with ccc DNA transcriptional activity in hepatocytes.However,current quantitative HBV RNA assays have some limitations,i.e.,different research teams do not take exactly the same detection approach and perform different standards.Since the presence or absence of HBV pgRNA in blood can indirectly reflect the transcriptional activity level of HBV ccc DNA in hepatocytes,this thesis studies the novel marker HBV pgRNA.With the technology of single-cell analysis,this thesis creates a new assay method,which can precisely detect HBV pgRNA in microsamples with high discrimination.The research result can throw new light on effective clinical treatment of Chronic hepatitis B and the subsequent safe discontinuation of the drug.