Establish The Real-time PCR Method For The Mutations Detection Of Hepatitis B Virus X Gene Rapidly And Its Application Research

Objective Establish the real-time quantitative PCR method for the detection of mutant HBX gene,and to explore its clinical application value.Methods Collect whole blood samples of 30 from the patients with chronic HBV infection which HBsAg positive is more than half a year,serum HBV-DNA positive or negative confirmed.And 27 samples of HBV-related Hepatic Cirrhosis,25 samples of HBV related HCC.The whole patients come from Hang zhou Normal University Affiliated Hospital and outpatient,in February 2015-2015 August.Extracting the DNA in each sample by PBMC.The HBx gene in the 382-401 base which searched in genebank was designed to detect mutant probe,and design primer by the the two sides which is relatively conserved region sequence of the 382-401 base.So we can Establish a reliable response system.The SYBR Green real time PCR was used to amplify all the DNA,Screening samples which contain HBX gene by analysing the melting curve and agarosegel electrophoresis,recovery of the amplified products,through cloning,plasmid transformation and extraction,to sequence HBX gene by related company.At the same time,The samples with HBX gene are detected by the TaqMan real time Pcr to screen the mutation of HBX gene.To obtain the HBx gene sequencing results for sequence comparison,the sensitivity,specificity and accuracy of the established TaqMan probe real-time fluorescence quantitative PCR analysis,its application value in clinical research,and to explore the clinical significance of detection of mutant HBx gene.Comparing the HBX gene sequencing.To analysis the sensitivity,specificity and accuracy of Taqman probe method,and to study its application value in clinic,exploring the clinical significance of the detection of mutant HBx gene.Results The results of the mutation in PBMC of the HBx gene in 382-401 base detected by the real time PCR,10 cases of chronic hepatitis B patients,15 cases of hepatitis B cirrhosis,18 cases of patients with HBV related HCC,and no mutation samples 13 cases;56 samples were sequenced,except 3 cases of sequencing without results,the rest of the samples are completely consistent with the direct sequencing results,coincidence rate was 94.64%(53/56).Three groups of mutation detection rate were 71.43%、75.00%and 81.82%,there was no significant difference because of mutation rate by statistical analysis(P>0.05).After sequencing,the amplified products of the mutant samples showed that there were different point mutation at different sites between the 382-401 bases,but it did not cause the generation of the stop codon.At the same time,in the 2 cases of HBV related HCC samples,we found two samples which one lack of 380-399,another lack of 375-396 base fragment,The former leading to the termination codon TAA produced in advance,thus causing the C terminal truncated HBX mutant due to the 131 amino acid premature termination;The latter did not cause termination of code generation.We found two hot spot mutations by sequencing,The A389T/G391A double mutation rate was 66.67%,77.78%in hepatitis B cirrhosis and HCC patients,and T380C mutation incidence was 22.67%,27.78%respectively,significantly higher than in patients with CHB(20%,10%)(P>0.05);In the 43 mutation patients,contain 26 cases of HBeAg(+),and 17 cases of HBeAg(-),the A389T/G391A double mutations in 14 cases of HBeAg(-),mutation rate was 82.35%(14/17),significantly higher than the HBeAg(+)patients(46.15%)(12/26);and 3 patients were HBeAg(+)of cases T380C mutation,the mutation rate was 11.54%(3/26),7 cases of HBeAg(-),mutation rate 41.18%(7/17),HBeAg(-)were obviously mutation rate is higher than that of HBeAg(+);The total of the amplified fragment are 148bp,outside the 382-401 base fragment still found other mutations,in particular,there was 2 cases of chronic hepatitis whose the 360th g->A hypermutation(TGG->TGA),resulting the performance of 120 tryptophan early termination of truncated HBx mutants,and anther lack of the 369 base g,causing termination codon TAA produced in advance,that lead to the performance of 129 tryptophan early termination of truncated HBx mutants.Conclusion TaqMan-MGB probe real-time PCR method sensitivity high specificity,can be primary for mutations,suitable for large-scale detection of clinical samples;Small sample studies show that chronic hepatitis B to liver cirrhosis and hepatocellular carcinoma progression,and the PBMC HBx gene mutation rate was gradually increasing trend;We found the HBX-Mut 120、HBX-Mut 129、HBX-Mut 131 by sequencing;The G391A/T380C double mutation and A389T/mutation rate of HBeAg(-)and liver cancer patients were significantly higher than those of HBeAg(+)and chronic hepatitis patients.