Detection of hepatitis B virus nucleic acid by PCR method.

乙型肝炎病毒是多种肝脏疾病的主要病原。据估计，日本约有200万名病毒携带者。迄今为止，乙型肝炎病毒DNA （HBV DNA）的检测主要采用Southern blot或斑点试验方法，但这些方法的灵敏度极限约为0.1-1.0 picogram（相当于10^4-10^5个病毒颗粒）。建立了一种以指数扩增核酸的聚合酶链式反应（PCR）检测单个病毒粒子水平HBV DNA的方法。我们用这种方法检测了慢性肝病患者血清中HBsAg阳性的HBV DNA。我们在所有HBeAg阳性患者的血清中发现HBV DNA。我们还在大多数HBeAb阳性患者中发现了它，这些患者到目前为止应该在血清中没有HBV DNA。我们还发现，长期（长达7年）肝功能检测正常的HBeAb阳性患者血清中没有HBV DNA。因此，这些患者应该已经从他们的血清中清除了乙肝病毒。此外，我们用直接测序法对PCR产物进行了分析，结果表明该方法可以方便地进行HBV DNA序列分析。通过该技术，我们证实了HBsAg亚型d/y和r/w由HBsAg第122和160密码子的氨基酸定义的报道。

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.