荧光定量PCR检测TTV方法学建立及临床应用

目的　建立一种灵敏度高、特异性好、操作简便的荧光实时定量PCR技术。方法　设计针对TT型肝炎病毒(TTV)基因保守序列非转录区的一对引物和一条荧光基团双标记探针。将PCR扩增所得的产物片段克隆 ,作为定量检测的标准品。对 36例丙肝患者及 12 3例不同血清标志物阳性的乙肝患者、16 6例健康体检者血清中TTVDNA定量检测 ,评估TTVDNA在不同人群中检出阳性率的差异显著性。结果　该方法检测TTVDNA的灵敏度高于普通套式PCR ELISA方法 ,线性范围为 10 2～ 7拷贝 /ml;批间CV为 13.2 %～ 16 .0 % ,批内CV为 6 .5 %～ 11.3%。健康体检者、丙肝患者、乙肝患者三组人群阳性检出率无显著性差异 ;且乙肝患者 (TTVDNA )血清ALT水平与TTVDNA载量无显著相关性 (P >0 .0 5 ) ;乙肝患者血清ALT异常与正常两组TTVDNA阳性检出率无显著性差异 (P >0 .0 5 )。结论　该方法操作简单、灵敏度高、特异性好、结果数据化 ,适合于临床实验室进行临床标本的TTVDNA定量检测。

Quantification of TT Virus DNA by fluorescence real time PCR and Its clinical significance

Objective To establish a simple, sensitive, specific and quantitative method for the TT virus (TTV) DNA in clinical samples.Methods TTV DNA was extracted with proteinase K-UNIQ Kit. The designed primers and probe located at UTR and ORF2. TTV DNA was quantified by a real-time detection PCR assay on ROCH lithtcycler. The PCR products were cloned into the PUCm-T cloning vector and transfected into DH5a,the cloned plasmid was used as standard after quantification by terminal dilution method. With this system.TTV DNA was quantified in 166 healthy persons and 123 hepatitis B virus infected patients and 36 hepatitis C virus infected patients.Results The same sensitive and specific results were obtained with two TTV DNA extraction methods in 22 serum samples. The linear range of the system was from 10 2 to 10 7 copies/ml in all kinds of clinical samples. The CV value was 6.5% to 11.3% in batch assay and 13.2% to 16.0% in day to day assay.There was no relationship between serum ALT levels and TTV DNA load in CHB patients. The positive rate of serum TTV DNA was same between high ALT level group and normal ALT level group.Conclusion For its simplicity, sensitivity, specificity and digitized results, the real time PCR for quantification of TTV DNA in clinical samples was available.