特异核酸捕获-PCR技术在从高背景标本中检测TT病毒DNA的应用

摘要

Objective To improve the amplification of non-specificity of TTV DNA detection using PCR from high background nuclear acids because of its low level in samples. Methods Specific nuclear acid captured PCR(SNAC-PCR) was established through capturing TTV DNA onto microplate with specific nuclear acid by means of avidin-biotin system, then amplifying captured TTV DNA as usual. Its specificity was evaluated by comparing with the detection of routine extraction samples using nested-PCR with or without subsequent DNA sequencing in 10 samples of sera and liver tissues in pairs. Results Positive results were obtained using routine protocol in 4 and 9 out of 10 samples of the sera and the liver tissues respectivly. The PCR products were proved to be specific using RFLP analysis with Kpn I . However,positive results were found in only 2/10 and 3/10 of samples of sera and liver tissue using DNA sequencing. The results of SNAC-PCR were the same as those of routine protocol with subsequent DNA sequencing. Furthermore, sharper electrophoreais bands were obtained. Conclusion SNAC-PCR could dramatically increase the specificity of the detection of TTV DNA,especially in high background nuclear acid samples. It can be widely used in the detection of other pathogens with low seral level or in high background nuclear acid samples. The level of TTV infection might be over-evaluated by currently used TTV DNA detection when the PCR products were not confirmed by DNA sequencing.%目的解决因TTV在样本中的水平极低而导致从高背景核酸样本中检测TTV DNA存在非特异性扩增的问题。方法采用亲和素生物素系统将TTV特异的核酸片段包被在微孔板上，利用特异核酸捕获样本中的TTV DNA，从而建立特异核酸捕获-PCR检测TTV DNA方法，并以10份血清(4份TTV DNA阳性)和肝组织配对标本为对象与抽提法进行比较，阳性产物进行DNA序列分析，最后评价特异核酸捕获-PCR的特异性。结果抽提法在血清和肝组织中的检出率分别为4/10和9/10。RFLP初步分析产物具有特异性，但经DNA序列分析证实的检出率仅分别为2/10和3/10。采用特异核酸捕获-PCR检测的结果与抽提法经DNA序列分析后的结果一致，获得的电泳条带也更为清晰。结论特异核酸捕获-PCR方法能显著提高TTV DNA检测的特异性，特别是针对高背景核酸标本，表明该方法在其他低水平病原体检测及高背景核酸样本检测中也有广泛的应用前景。目前广泛采用的TTV DNA检测方法如不进行DNA序列分析则可导致过高地估计了TTV的感染率。