通过巢式聚合酶链反应检测福尔马林固定、石蜡包埋脑组织中的牛病毒性腹泻病毒RNA

Detection of bovine viral diarrhea virus RNA in formalin-fixed, paraffin-embedded brain tissue by nested polymerase chain reaction.

作者信息

Gruber A D, Greiser-Wilke I M, Haas L, Hewicker-Trautwein M, Moennig V

机构信息

Institute of Pathology, School of Veterinary Medicine, Hannover, Germany.

出版信息

J Virol Methods. 1993 Aug;43(3):309-19. doi: 10.1016/0166-0934(93)90149-l.

DOI:10.1016/0166-0934(93)90149-l

PMID:8408445

Abstract

Isolation and amplification of RNA from formalin-fixed, paraffin-embedded tissues is delicate due to its fragility and ubiquitous ribonucleases. For retrospective studies, however, a convenient procedure for the detection of RNA in archived material is of great value. Bovine viral diarrhea (BVD) virus is a member of the pestivirus genus in the family Flaviviridae. Different protocols for the isolation of BVD virus RNA from fresh and autolytic as well as from routinely formalin-fixed and paraffin-embedded brain tissue of BVDV-infected calves were compared. The polymerase chain reaction (PCR) after reverse transcription (RT-PCR) was carried out subsequently for the detection of viral RNA. Using proteinase K digestion of deparaffinized tissue sections without additional ribonuclease inhibitors and subsequent nested PCR, a 803 bp fragment of the gene coding for the nonstructural protein p125 of BVD virus could be consistently detected. In addition, BVD virus RNA was detected by RT-PCR from non-fixed brain tissue after 10 days of autolysis.

摘要

从福尔马林固定、石蜡包埋的组织中分离和扩增RNA是一项精细的工作，因为RNA很脆弱且存在普遍的核糖核酸酶。然而，对于回顾性研究而言，一种用于检测存档材料中RNA的便捷方法具有重要价值。牛病毒性腹泻（BVD）病毒是黄病毒科瘟病毒属的成员。比较了从感染BVDV的犊牛的新鲜和自溶以及常规福尔马林固定和石蜡包埋的脑组织中分离BVD病毒RNA的不同方案。随后进行逆转录后的聚合酶链反应（RT-PCR）以检测病毒RNA。使用蛋白酶K消化脱石蜡组织切片而不添加额外的核糖核酸酶抑制剂，随后进行巢式PCR，可以持续检测到BVD病毒编码非结构蛋白p125的基因的803bp片段。此外，在自溶10天后，通过RT-PCR从未固定的脑组织中检测到了BVD病毒RNA。