Establishment Of Real-time Fluorescence Quantitative PCR For Detection Of Serum PgRNA Level In HBV Infections And Evaluation Of Its Clinical Implication

?Objectives?1 To establish real-time fluorescence quantitative PCR?Real-time qPCR?for detection of serum pregenomic RNA?pgRNA?level in HBV infections and to evaluation its performance and clinical implication.2 To detect the level of serum HBV pgRNA in different clinical outcomes of patients with hepatitis B virus?HBV?infection and to analyse the correlation of serum pgRNA and traditional HBV serological markers levels.?Methods?1 Establishment of Real-time qPCR for detection of serum HBV pgRNA1.1 Successfully established Real-time qPCRThe present study was comprised of PCR reaction system,amplification conditions and preparation of standard plasmids for calibration curves,etc.1.2 Methodological evaluation of Real-time qPCR1×10¹⁰ copies/ml¹×10¹ copies/ml recombinant plasmids were tested by Real-time qPCR.The methodological evaluation of Real-time qPCR included sensitivity test,linear range test,specificity test,accuracy test,reproducibility test,and etc.1.3 The evaluation of pgRNA for clinical implicationThe effect of antiviral drugs on pgRNA was monitored by using self-built method to detect the levels of serum pgRNA in HBV patients,thus providing reference for selecting suitable timing for drug withdrawal and judging the curative effect.2 Exploring the correlation of serum pgRNA and traditional HBV serological markers levels in different clinical outcomes of patients with hepatitis B virus?HBV?infection2.1 Patients and testsThe study cohort included 400 different clinical outcomes with hepatitis B virus infection who received NAs therapy and 15 HBsAg-negative controls and other people infected with viruses in The First Affiliated Hospital of Fujian Medical University.The400 different clinical outcomes with hepatitis B virus infection were comprise of 100asymptomatic hepatitis b virus carriers,100 chronic hepatitis B,100 liver cirrhosis and100 hepatocellular carcinoma.The HBsAg-negative controls were comprise of hepatitis c patient,EB virus infection,herpes simplex infection.The HBV DNA and HBV RNA were extracted by magnetic beads method and spin column method,respectively.HBsAg and HBeAg concentrations were quantified using Architect i4000commercially available chemiluminescence immunoassay.Detection of HBV DNA load using the LightCycler 480 fluorescent quantitative PCR.The quantitative real-time PCR?qPCR?analysis of HBV pgRNA levels were performed using the StepOne plus Real-time PCR Detection System with TaqMan probe and according to the instructions provided by the manufacturer.2.2 Correlation analysisAnalyzing the correlation between HBV pgRNA and traditional HBV serological markers and comparing the differences between HBV pgRNA and HBV serological markers in different groups?ASC group,CHB group,LC group and HCC group?of patients with different clinical outcomes to provide reference for clinical antiviral therapy.?Results?1 Establishment of Real-time qPCR for detection of serum HBV pgRNA1.1 Establishment of Real-time qPCRThe plasmid standard and standard curves were successfully constructed,and the composition and amplification conditions of the reaction system were determined.1.2 Methodological evaluation of Real-time qPCRThe linear range of Real-time qPCR was between 1×10¹⁰ copies/ml and 1×10³copies/ml.The low detection limit was 1×10³ copies/ml.The intra-run coefficient of variation?CV?was between 0.13%⁰.73%.The inter-run coefficient of variation?CV?was between 0.16%⁰.63%.The serum HBV pgRNA levels of other virus-infected patients were negative,indicating that the method had better specificity.The recovery rate of the mixture sample of accuracy test was 93.785%and the result was in line with the standard requirements of the pharmaceutical industry of the People's Republic of China.1.3 Evaluation of pgRNA for clinical implicationThe levels of HBV pgRNA,HBV DNA,and HBsAg in different clinical outcomes of patients with hepatitis B virus?HBV?infection were measured by Real-time qPCR,the results showed that the level of HBV pgRNA in ASC group and LC group,ASC group and HCC group,CHB group and LC group,CHB group and HCC group had significant difference?4.32±2.36 copies/ml vs.3.22±2.13 copies/ml,4.32±2.36copies/ml vs.3.14±2.12 copies/ml,4.41±2.51 copies/ml vs.3.22±2.13 copies/ml,4.41±2.51 copies/ml vs.3.14±2.12 copies/ml,respectively.all P<0.05?.The level of HBV DNA in ASC group and LC group,ASC group and HCC group,CHB group and LC group,CHB group and HCC group had significant difference?4.42±1.72 copies/ml vs.3.68±0.89 copies/ml,4.42±1.72 copies/ml vs.3.64±0.65 copies/ml,4.7±1.77copies/ml vs.3.68±0.89 copies/ml,4.7±1.77 copies/ml vs.3.64±0.65 copies/ml,respectively.all P<0.05?.The level of HBsAg in ASC group and LC group,ASC group and HCC group,CHB group and LC group,CHB group and HCC group had significant difference?7393.32±16302.77 IU/ml vs.1831.56±6005.84 IU/ml,7393.32±16302.77 IU/ml vs.1276.72±1697.07 IU/ml,6157.9±11583.95 IU/ml vs.1831.56±6005.84 IU/ml,6157.9±11583.95 IU/ml vs.1276.72±1697.07 IU/ml,respectively.all P<0.05?,suggesting that the HBV pgRNA analogy to traditional HBV markers had similar clinical significance in different clinical outcomes of HBV infection.2 Exploring the relevance of serum pgRNA and HBV serological markers levels in different clinical outcomes of patients with hepatitis B virus?HBV?infectionWe successfully tested 400 HBV infected patients,including 100 in each of the ASC group,the CHB group,the LC group,and the HCC group.Furthermore,we found positive correlation between HBV DNA,HBV pgRNA and HBsAg?HBV pgRNA g levels vs.HBV DNA levels,r=0.58,P<0.001,HBV pgRNA levels vs.HBsAg levels,r=0.47,P<0.001,HBsAg levels vs.HBV DNA levels,r=0.45,P<0.001?in HBV infected patients.In addition,the levels of HBV pgRNA and HBV DNA were higher in HBeAg positive group?5.04±2.49 copies/ml and 4.85±1.97 copies/ml,respectively?than HBeAg negative group?3.11±1.99 copies/ml and 3.72±0.91 copies/ml,respectively?,and the differences of HBV pgRNA and HBV DNA between HBeAg positive group and HBeAg negative group were statistically significant?t=9.987,P<0.001 and t=7.545,P<0.001,respectively?,and we found positive correlation between HBV pgRNA and HBV DNA in HBV infected patients?HBeAg positive group,r=0.57,P<0.001,HBeAg negative group,r=0.32,P<0.001?.In addition,we analyzed the different serological markers of 400 HBV-infected patients and found that the detection rate of HBV pgRNA in the samples with HBV DNA<500 IU/ml was 78.9%,suggesting that the level of HBV DNA and the detection rate of HBV pgRNA were there some kind of connection?This aroused our great interest.We selected 108 patients with hepatitis B from them,then according to HBV DNA levels,which were divided into HBV DNA<20 IU/ml group?74 cases?and 20 IU/ml<HBV DNA<500 IU/ml group?34 cases?,and the HBV pgRNA detection rate was statistically analyzed.The detection rates of HBV pgRNA were 17.57%?13/74?and 41.18%?14/34?,respectively,and the undetected rates were 82.43%?61/74?and 58.82%?20/34?,respectively.In other words,in HBV DNA<20 IU/ml group?74 cases?,the HBV DNA level and HBV pgRNA level of 82.43%of patients infected with HBV were lower than the detection limit.?Conclusions?1 The real-time fluorescence quantitative PCR assay which can quantify the serum HBV pgRNA with highly accuracy,sensibility,specificity and wide linear range was successfully developed,which could be used widely in clinical laboratories.2 HBV pgRNA levels had good correlation with HBV DNA and HBsAg levels.When the level of HBV DNA was below the detection limit,part of HBV-infected individuals could still detect HBV pgRNA levels,only when the two indexes were below detection limit,which could be used as a criterion to achieve pera-functional cure.