SPECIFIC DETECTION OF POSITIVE AND NEGATIVE STRANDED HEPATITIS-C VIRAL-RNA USING CHEMICAL RNA MODIFICATION
SPECIFIC DETECTION OF POSITIVE AND NEGATIVE STRANDED HEPATITIS-C VIRAL-RNA USING CHEMICAL RNA MODIFICATION
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DOI：
10.1007/bf01310568
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发表时间：
1994-01-01
影响因子：
2.7
通讯作者：
SHIMOTOHNO, K
中科院分区：
文献类型：
--
作者：
GUNJI, T;KATO, N;SHIMOTOHNO, K
由于丙型肝炎病毒（HCV）是输血后感染的主要病原体。非甲非乙型肝炎病毒是一种正链RNA病毒，它应该通过负链RNA复制。虽然链特异性逆转录-聚合酶链反应（RT-PCR）方法最近被开发用于检测HCV RNA的每条链，但该策略的特异性仍有待确定。在这项研究中，使用体外转录的正链和负链HCV RNA与来自正常肝脏的肝细胞RNA混合，我们发现这种策略不能区分两条RNA链，但在3'端对RNA样品进行化学修饰，然后进行链特异性RT-PCR，使特异性检测成为可能。采用RT-PCR结合RNA修饰的新方法，对10例慢性HCV感染者的肝组织、血清和外周血单个核细胞（PBMC）进行了检测。10例（100%）肝组织HCVRNA阳性，9例（90%）肝组织HCVRNA阴性。5例（50%）患者血清中检测到负链RNA，9例（90%）患者血清中检测到正链RNA。在PBMC中，8例（80%）检测到HCVRNA的正链，而仅1例（10%）检测到HCVRNA的负链，表明HCV对PBMC的细胞嗜性远低于对肝细胞的嗜性。
Since hepatitis C virus (HCV), a major causative agent of posttranfusional. non-A, non-B hepatitis, is a positive stranded RNA virus, it is supposed to replicate via a negative RNA strand. Although strand specific reverse transcription-polymerase chain reaction (RT-PCR) method was recently developed to detect each strand of HCV RNA, the specificity of the strategy has remained to be determined. In this study, using in vitro transcribed positive and negative stranded HCV RNAs mixed with hepatic cellular RNA from normal liver, we found that this strategy did not distinguish between the two RNA strands, but that chemical modification of RNA samples at the 3' end followed by strand specific RT-PCR made specific detection possible. Liver tissues, sera and peripheral blood mononuclear cells (PBMC) from ten patients with chronic HCV infection were analyzed with the novel strategy of RT-PCR combined with RNA modification. Positive and negative strands of HCV RNA were detected in liver tissues of ten (100%) and nine (90%) cases, respectively. Negative RNA strand was detected also in sera of five cases (50%), positive strand being detected in nine cases (90%). In PBMC, positive strand of HCV RNA was detected in eight cases (80%), whereas negative strand in only one case (10%), suggesting that HCV has much less cellular tropism to PBMC than to hepatocytes.