TTV在非甲-庚型肝炎患者中的检测、克隆及测序

目的分析ＴＴＶ（Ｔｒａｎｓｆｕｓｉｏｎｔｒａｎｓｍｉｔｅｄｖｉｒｕｓ）深圳分离株ＯＲＦ１部分基因序列。方法在ＴＴＶＯＲＦ１设计引物，建立巢式聚合酶链反应（Ｎｅｓｔｅｄ－ＰＣＲ），检测４０例广东地区非甲－庚型肝炎患者血清中ＴＴＶＤＮＡ。对ＰＣＲ产物进行分子克隆，以荧光法（ＡｐｐｌｉｅｄＢｉｏｓｙｓｔｅｍｓ，３７３Ａ）测序。结果４０例非甲－庚型肝炎中２１例ＴＴＶＤＮＡ阳性（５２．５％）。对其中一株ＴＴＶ（ＳＺ１）ＯＲＦ１部分基因克隆、测序，并与Ｏｋａｍｏｔｏ等报道的２株（Ｎ２２、Ｇ１ａ）相比较，其核苷酸序列的同源性分别为９６．７％与９７．４％。结论本研究证实我国华南地区存在ＴＴＶ感染。ＴＴＶ的分子克隆与测序及ＰＣＲ诊断技术的建立对国内进一步开展ＴＴＶ感染的诊断与流行病学调查均具有重要意义

Molecular cloning and sequencing of TT virus and its detection in patients with non A G hepatitis

Objective To analyse partial nucleotide sequence of ORF 1 of TTV (transfusion transmitted virus) isolated from a patient with non A G hepatitis in Shenzhen. Methods A nested polymerase chain reaction (PCR) assay with primers from ORF 1 of TTV genome was established to detect TTV DNA in 40 patients with non A G hepatitis in Guangdong.PCR product was cloned and sequenced with the Applied Biosystems (373A). Results TTV DNA was positive in 21 of 40 patients (52.5%) with non A G hepatitis.Partial gene of ORF 1 of a TTV isolate (SZ1) was cloned,sequenced and compared with known sequences of TTV isolates (N22,G1a) reported by Okamoto.The nucleotide homology were 96.7% between SZ1 and N22,97.4% between SZ1 and G1a.Conclusion The results of this study confirmed the epidemic of TTV infection in Southern China.The cloning and sequencing of the TTV isolate (SZ1) and the development of a PCR assay for detecting TTV DNA has important implication for the diagnosis and epidemiological investigation on TTV infection.