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目的探讨聚合酶链反应（ＰＣＲ）扩增ＴＴＶ核酸的优化条件，初步测定慢性丙型肝炎及慢性非甲－戊，非庚肝炎患者中ＴＴ病毒的检出率．方法将标本分别予以４℃保存１ｗｋ、室温保存１ｗｋ及１ｗｋ内将标本于－３５℃与室温间反复冻融６次３种储存方法．分别以ＳＤＳ－蛋白酶Ｋ方法（方法１），ＮＰ－４０法（方法２）及聚乙二醇法提取ＴＴＶＤＮＡ，分别以套式ＰＣＲ与单管套式ＰＣＲ检测ＴＴＶ．以ＰＣＲ直接序列分析方法测定扩增产物以验证扩增产物的特异性．结果在１０份已知病毒标志均为阴性的血清中，方法１与方法２提取核酸后可在４份血清中检出ＴＴＶ，方法３提取核酸后的检出率为０．１次ＰＣＲ、单管ＰＣＲ与单管套式ＰＣＲ对１０份已知病毒标志均为阴性血清的ＴＴＶ检出率分别为１０％，４０％和４０％．将２份血清按原血清、１０－１～１０－１０系列稀释后检测，发现，１次ＰＣＲ、套式ＰＣＲ和单管套式ＰＣＲ的检出水平分别为原倍、１０－５与１０－４及阴性、１０－４与１０－４．４℃保存１ｗｋ、室温保存１ｗｋ及反复冻融标本的最低检测稀释度为１０－５，１０－４，１０－３与１０－４，１０－４，１０－３．对一份ＰＣＲ产物进行序列分析证实为ＴＴＶ特异性基因．检测３１份慢性丙型?
Objective To investigate the optimal conditions for the amplification of TTV nucleic acid by polymerase chain reaction (PCR) and preliminary detection of the detection rate of TT virus in patients with chronic hepatitis C and chronic non-A-hepatitis and non-hepatitis C virus. Methods The specimens were stored at 4 ℃ for 1wk, stored at room temperature for 1wk and 1wk, respectively. The samples were frozen and thawed repeatedly at -35 ℃ and room temperature for 6 times. TTVDNA was extracted by SDS-proteinase K method (method 1), NP-40 method (method 2) and polyethylene glycol method respectively, and TTV was detected by nested PCR and single-tube nested PCR respectively. Amplification products were determined by PCR direct sequence analysis to verify the specificity of the amplified product. Results In 10 serums with known virus sign negative, TTV was detected in 4 serums after extracting nucleic acids by method 1 and method 2, and the detection rate after method 3 extraction was 0.1 PCR Tuberculosis PCR and single-tube nested PCR for 10 samples of known viruses were negative serum TTV detection rates were 10%, 40% and 40%. The two serum samples were diluted according to the original serum and 10-1-10-10. The results showed that the detection rates of primary PCR, nested PCR and single-pipe PCR were 10 times, 10-5 and 10- 4 and negative, 10-4 and 10-4.4 ℃ preservation 1wk, 1wk at room temperature and repeated freeze-thaw specimens of the lowest detection dilution of 10-5,10-4,10-3 and 10-4,10-4 , 10-3. Sequence analysis of a PCR product confirmed TTV-specific genes. Detect 31 chronic C?