To establish a new and simple method of genotyping of hepatitis B virus by PCR-RFLP to analysis on S region

Abstract

Objective A method was established for genotyping of hepatitis B virus (HBV A-D genotype),based on the PCR-restriction fragment length polymorphism (RFLP) created by HinfⅠ,EarⅠ,ApoⅠ action on an amplified segment of the S region. Methods Clinical diagnosis research.One hundred and twenty-eight HBV S sequences obtained from GeneBank were analyzed for restriction enzyme sites that would be genotype-specific.Restriction patterns following digestion with restriction enzymes HifⅠ,EarⅠ,ApoⅠ were determined to identify A-D HBV genotypes.The method was used to detect the HBV genotype of fifty severe hepatitis patients due to chronic hepatitis B in China. Then the detection results were confirmed by direct sequencing. Results The new genotyping method was established,named simple PCR-RFLP,which could identify HBV genotypes A to D.Genotypes B,C,B/C and A or D could be determined by a single step digestion with HifⅠ. Eight patients of genotype A/B/C classified by single step digestion with HifⅠ were conformed as genotype B variant by further digestion and direct sequencing.Extracted randomly and diluted into different concentration,three specimens were tested for genotype of HBV repeatedly and respectively.The results were all in accord with the originals,and the lowest detection limit of HBV DNA was 7~9 IU/ml.This was particularly useful in China where genotypes B and C were predominant.Twenty-three of genotype B and ten genotype C patients were classified from these fifty severe hepatitis B patients by a single step digestion with HifⅠ through the simple PCR-RFLP method.The same results were also obtained by direct sequencing of PCR products (Kappa=1.00,P=0.001). The simple PCR-RFLP method was superior to direct sequencing in detecting HBV B/C polyinfection (9 cases and 0 case;χ2=18.00,P=0.001). Conclusions Both the sensitivity and repetitiveness of Simple PCR-RFLP method are satisfactory.It is superior to direct sequencing in detecting HBV B/C polyinfection,and simple,convenient.(Chin J Lab Med,2013,36:420-424) Key words: Hepatitis B virus; Genotype; Polymerase chain reaction; Polymorphism; restriction fragment length