Detection of hepatitis B virus nucleic acid by PCR method.

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Hepatitis B virus is a major etiologic agent of various liver diseases. It is estimated that there are about 2 million virus carriers in Japan. So far, detection of hepatitis B virus DNA (HBV DNA) is performed using Southern blot or spot test method, but the limit of sensitivity by these methods is about 0.1-1.0 picogram (equivalent to 10^4-10^5 viral particles). We established a method to detect HBV DNA at the level of one virion using polymerase chain reaction (PCR) method which can exponentially amplify nucleic acids. Using this method, we detected HBV DNA in sera from HBsAg positive patients with chronic liver diseases. We found HBV DNA in all the sera from HBeAg positive patients. We also found it in the majority of patients with HBeAb positive patients who were supposed to have no HBV DNA in their serum so far. We also revealed that HBeAb positive patients who have normal liver function tests for long period (up to 7 years) have no HBV DNA in serum. Thus these patients were supposed to have cleared off hepatitis B virus from their serum. In addition,we analyzed the PCR product by direct sequencing method and showed analysis of HBV DNA sequence could be easily performed by these methods. With this technique we confirmed the report that the HBsAg subtype d/y and r/w was defined by the amino acid of the 122nd and 160th codon of HBsAg.