Strappe P M, Wang T H, McKenzie C A, Lowrie S, Simmonds P, Bell J E
Department of Pathology, University of Edinburgh, Western General Hospital, UK.
J Virol Methods. 1998 Feb;70(2):119-27. doi: 10.1016/s0166-0934(97)00156-0.
A direct in situ polymerase chain reaction (IS-PCR) assay is described for the detection of HIV-1 proviral DNA in formalin fixed paraffin embedded brain tissue. Biotin-16-dUTP is incorporated during the PCR process and microwave pretreatment of tissue sections ensures that no non-specific incorporation into damaged or nicked genomic DNA occurs. Two methods are compared to detect the biotinylated amplified product, the use of an avidin-biotin-alkaline phosphatase complex (ABC) and the application of tyramide signal amplification (TSA) which allows both chromogenic and fluorescence detection. TSA detection enhances the sensitivity of IS-PCR, permitting fewer PCR cycles and preserving tissue morphology.
本文描述了一种直接原位聚合酶链反应（IS-PCR）检测方法，用于检测福尔马林固定石蜡包埋脑组织中的HIV-1前病毒DNA。在PCR过程中掺入生物素-16-dUTP，组织切片的微波预处理可确保不会非特异性掺入受损或有切口的基因组DNA中。比较了两种检测生物素化扩增产物的方法，即使用抗生物素蛋白-生物素-碱性磷酸酶复合物（ABC）和应用酪胺信号放大（TSA），后者可同时进行显色检测和荧光检测。TSA检测提高了IS-PCR的灵敏度，减少了所需的PCR循环次数，并保留了组织形态。
