A Novel Duplex Quantitative Reverse Transcription PCR Assay For Simultaneously Determining Hepatitis B Virus Pregenomic RNA And DNA

The existing antiviral drugs such nucleotide analogues(NAs),PEGylated interferon alpha(PEG-IFNα)are effective to suppress duplication of hepatitis B virus(HBV).However,they are not easily able to eliminate the intrahepatic HBV covalently closed circular DNA(cccDNA).The remaining cccDNA tend to lead to a relapse or even exacerbation of chronic hepatitis B(CHB).Hence intrahepatic cccDNA is a key biomarker to estimate if CHB has been cured or not.But liver biopsy is a kind of invasive operation which makes it cannot be a routine diagnosis.So non-invasive substitution,for example the serologic examination,is needed to reflect the level of intrahepatic cccDNA.HBV cccDNA can transcribe transcriptions of four different length: 0.7kb,2.1kb,2.4kb,3.5kb.Among these transcriptions,the3.5 kb long prgenomic RNA(pgRNA)is the only one that can be reverse transcribed into the minus strand of relaxed circular DNA(rc DNA).rc DNA is an indispensable part of the intact HBV particle.So pgRNA plays a key role of HBV’s life cycle.It has been proved that serum pgRNA correlates well with intrahepatic cccDNA,especially in patients who is undergoing NAs treatment,because serum pgRNA can better reflect transcription activities of intrahepatic cccDNA than serum HBV DNA.For these reasons,serum pgRNA has the potential to become a new biomarker which has a key referential significance to judge the drug withdraw timing during antiviral treatment.Although serum HBV RNA has unique clinical significance,it still cannot replace HBV DNA as a better clinical marker: HBV DNA level is not only an important basis for deciding whether patients should receive antiviral treatment,but also an important indicator for evaluating patients’ virological response.In addition,studies have shown that the ratio of HBV RNA to DNA is significantly different among CHB patients with different natural infection histories,so the comprehensive levels of HBV DNA and RNA are of great significance in determining the HBV infection stage and disease prognosis.However,currently there is only a standardized HBV DNA detection kit in clinical practice,and there is no standard detection method for HBV RNA.If HBV RNA becomes a new clinical marker in the future,both HBV DNA and RNA level will be needed to determine.But if they are determined separately,more manpower,time,reagent and samples will be consumed.On the other hand,currently there is no widely accepted method for the detection of HBV RNA,and the detection methods in previous articles are limited.For example,our study in this subject proved that quantitative PCR with direct RNA extractionafter reverse transcription with oligo d T or gene specific primer(GSP)may have quantitative inaccuracy due to HBV DNA contamination which is difficult to eliminate,and the DNA in RNA extract can not be completely eliminated even by DNase digestion.Other articles have reported usage of Quantigene nucleic acid quantification,droplet digital PCR(dd PCRds)and other new technologies for the detection of HBV RNA.Although it has the advantages of high sensitivity and specificity,it is currently only applicable to laboratory research due to the need for advanced and expensive supporting equipment,so it is difficult to be popularized in clinical practice by now.Our research developed a new method of duplex fluorescence quantitative PCR to detect pgRNA and DNA simultaneously in nucleic acid extract.This method overcomes a shortage in some other articles,that the RNA extract may be contaminated by HBV DNA which causes biased outcome.On the other hand,our method can save time,reduce sample requirements,and keep specificity and accuracy.We designed two detection strategies in our research.The first quantitative strategy is to subtract copy numbers after reverse transcription,which is suitable for the detection of cell culture supernatant specimens and also theoretically suitable for the detection of serum.The second strategy is an improvement on the first one.The method is to specifically amplify c DNA with anchored sequence and then perform quantification PCR.This improved detection strategy is capable of detecting both cell culture supernatant and cell specimen.In conclusion,we designed a new method that can simultaneously determine HBV DNA and HBV RNA in one quantitative PCR.The method is proved to have accuracy and specificity.Also,it can save time,manpower and sample costs.The method can fulfil clinical needs,with a good clinical practicability.