| About 350 million people worldwide are infected with chronic hepatitis B.chronic hepatitis B can lead to cirrhosis and liver cancer,and it is a serious threat to human health.The treatment of chronic hepatitis B is of great significance to the prevention and treatment of liver cirrhosis and liver cancer.Previously,diagnosis and disease of hepatitis B detection always using biochemical index of immunology,clinical diagnosis and treatment of molecular biology in recent years has also been widely used in hepatitis,such as hepatitis B virus nucleic acid quantitative detection and genotype of hepatitis B virus detection.The quantitative detection of hepatitis B virus using real-time fluorescent PCR method for diagnosis of chronic hepatitis B patients,evaluation and prognosis after treatment in patients with the timing of the decision and the treatment of judgment is important.This paper aims at the significance of according to different HBV types,comparing the domestic real time fluorescence quantitative PCR reagent(FQ-PCR)and Roche COBAS AmpliPrep/COBAS TaqMan(CAP/CTM)method for detection of hepatitis B virus(HBV)DNA results,find out the gap of domestic and COBAS reagents in sensitivity and specificity,lay the theoretical foundation for the improvement of domestic reagent performance.This study also used direct sequencing(gold standard of HBV genotyping)to verify reverse dot blot hybridization HBV genotyping method,to study the method of reliability,and whether it can be used for clinical HBV virus genotyping.The contents of this paper are as follows:(1)Select a total of 72 cases of chronic hepatitis B serum samples,using FQ-PCR reagent COBAS Amplicor(A)and FQ-PCR(B)real-time domestic reagent HBV-DNA to test serum samples,analysis of different types of HBV samples,using the COBAS Amplicor test results to evaluate the sensitivity,specificity and the correlation of domestic reagent between COBAS reagent.Results:The A&B two reagents test results overall good correlation(r=0.94),there is no significant difference of quantitative detection results(P>0.05).A B two reagents for low load samples,the correlation coefficient is low(r = 0.31),the reliability of B reagent in the detection of low load sample is poor.A&B two detection kit of different genotype samples results good correlation(r>0.85),and the difference is statistically significant and significant.(2)Do the HBV virus genotyping of the 72 HBV virus serum samples by reverse dot hybridization Chip method,then select parts of the 72 samples randomly(20 samples)and do the HBV,virus genotyping using Sanger dideoxy sequencing method.Study on the difference of two kinds of virus genotyping methods.Results:PCR reverse dot blot hybridization method and Sanger double sequencing method have high coincidence rate,PCR reverse dot hybridization chip method can be used for clinical HBV virus genotyping.(3)According to the experimental results of the analysis are discussed and summarized that two kinds of domestic import reagents for the detection of the main difference is that the detection of low load samples of domestic reagent(reagent B)reliability is lower than that of imported reagent(A reagent),domestic reagent(reagent B)occurred in two cases of missed detection of low viral load(mL<104 IU/samples genotypes is type B and mixed type C/E).(4)In order to further study the HBV virus typing method and accumulate scientific research material,this paper designs to test the HBV DNA virus classification.The result of the experiment is consistent with the reverse hybridization,the commercial kit and the direct sequencing reagent kit,the result is high and the cost is low.It can be used in clinical examination and research.Conclusion:From the results of this study,suggests that domestic reagent modify nucleic acid extraction method,use a high yield extraction method(such as magnetic bead method),improve technical condition to develop mechanical automatic extraction equipment,and increase the overall PCR reaction system,add the internal standard quality control,use multiple pairs of primers in the conserved region,improve sensitivity,reliability and specificity of domestic reagent B.Finished above all,the domestic reagent B is expected to replace the COBAS reagent,reduce the treatment cost and economic burden of patients.This study using Sanger dideoxy sequencing method commercial kit and self-design experiment system based on Sanger dideoxy sequencing method to verify the reverse dot blot hybridization method,the overall compliance rate is high,simple operation,strong specificity,so the reverse dot blot hybridization method can be used for clinical detection of HBV genotype. |
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