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Detection of hepatitis B virus nucleic acid by PCR method.

PCR法检测乙型肝炎病毒核酸。

基本信息

批准号：
63570310
负责人：
YOKOSUKA Osamu
金额：
$ 1.54万
依托单位：
Chiba University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1988
资助国家：
日本
起止时间：
1988 至 1989
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-63570310/
关键词：
hepatitis B virus HBV DNA polymerase chain reaction HBeAg Ab direct sequencing chronic liver disease HBs抗原 HBs抗原サブタイプ慢性肝炎ポリメラーゼ・チェイン・リアクション法 B型肝炎ウイルス合成DNA HBVDNA 超高感度検出法 HBe抗体肝疾患

项目摘要

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.

乙型肝炎病毒是多种肝脏疾病的主要病原体。据估计，日本约有200万病毒携带者。迄今为止，乙型肝炎病毒DNA（HBV DNA）的检测采用Southern blot或斑点测试方法进行，但这些方法的灵敏度极限约为0.1-1.0皮克（相当于10^4-10^5病毒颗粒）。我们建立了一种使用聚合酶链式反应 (PCR) 方法在一个病毒颗粒水平上检测 HBV DNA 的方法，该方法可以指数扩增核酸。利用这种方法，我们检测了 HBsAg 阳性慢性肝病患者血清中的 HBV DNA。我们在 HBeAg 阳性患者的所有血清中都发现了 HBV DNA。我们还在大多数 HBeAb 阳性患者中发现了这一点，而到目前为止，这些患者的血清中应该没有 HBV DNA。我们还发现，长期（长达7年）肝功能检查正常的HBeAb阳性患者的血清中没有HBV DNA。因此，这些患者应该已经从血清中清除了乙型肝炎病毒。此外，我们通过直接测序方法对PCR产物进行了分析，结果表明通过这些方法可以轻松地进行HBV DNA序列分析。通过这项技术，我们证实了 HBsAg 亚型 d/y 和 r/w 是由 HBsAg 第 122 和 160 密码子的氨基酸定义的报告。