聚合酶链反应-微孔板杂交法检测输血传播病毒DNA

目的 建立聚合酶链反应（ＰＣＲ）－微孔板杂交检测ＴＴ病毒（ＴＴＶ）ＤＮＡ的方法，探讨不国因素对方法敏感度和特异性的影响。方法 硫氰酸胍裂介异丙醇沉淀提取ＴＴＶ ＤＮＡ，以ＰＣＲ扩增，扩增产物上标记的生物素与包被在微孔板上的链亲和素结合标记有荧光素的探针与产物杂交，再与酶标抗荧光素抗体结合，经底物显果在各种影响因素中，当ＮａＯＨ浓度为０．３ｍｏｌ／Ｌ，杂交时间６０ｍｉｎ，酶反应时间为４５ｍｉｎ时，结

Detectionof TT virus DNA by polymerase chain reaction-microplatehybridization.

Objective To establish a method for the detection of TT virus (TTV) DNA by polymerase chain reaction (PCR) microplate hybridization and to find out various influential factors of the assay.Methods TTV DNA extracted by guanidinium thiocyanate was amplified by PCR, the PCR primers were pre labelled with biotin. By conjugation of biotin with streptavidin, the amplified products were coated to the microplate well, and hybridized with FITC probe simultaneously. After being reacted with Anti Fluorescein POD, the products which contained FITC probe were coloured by TMB H 2O 2, and then measured the optical density using a microplate reader at 450 nm wave length.Results The optimal concentration of NaOH was 0 3 mol/L, and the optimal time of hybridization and enzymatic reaction was 60 minutes and 45 minutes, respectively. The results also showed that the positive rate of TTV infection in normal population, hepatitis A G and hepatitis non A G was 13 6%,20 3% and 28 1%, respectively.Conclusion Comparing the PCR microplate hybridization with the electrophoresis assay, the former is simple,rapid, sensitive and specific and is expected to be a new technique instead of electrophoresis assay as a routine method for TTV DNA detection.