用于检测福尔马林固定、石蜡包埋组织中猪戊型肝炎病毒的巢式逆转录聚合酶链反应的开发及其与原位杂交的比较

Development of nested RT-PCR for the detection of swine hepatitis E virus in formalin-fixed, paraffin-embedded tissues and comparison with in situ hybridization.

作者信息

Choi Changsun, Ha Seung-Kwon, Chae Chanhee

机构信息

Department of Veterinary Pathology, College of Veterinary Medicine and School of Agricultural Biotechnology, Seoul National University, San 56-1, Shillim-Dong, Seoul, Kwanak-Gu 151-742, South Korea.

出版信息

J Virol Methods. 2004 Jan;115(1):67-71. doi: 10.1016/j.jviromet.2003.09.007.

DOI:10.1016/j.jviromet.2003.09.007

PMID:14656462

Abstract

Swine hepatitis E virus (HEV) was detected in formalin-fixed, paraffin-embedded tissues from naturally infected pigs by nested reverse transcription-polymerase chain reaction (RT-PCR). The results for seminested RT-PCR were compared with those determined by in situ hybridization. The results obtained show that use of xylene deparaffinization, digestion with proteinase K followed by nested RT-PCR is a reliable detection method. Swine HEV nucleic acid was detected in formalin-fixed, paraffin-embedded hepatic tissues from 40 pigs. Distinct positive signals for swine HEV were obtained in the same hepatic tissues by in situ hybridization. Swine HEV nucleic acid was localized to the cytoplasm of hepatocytes and had a granular staining pattern. The rate of agreement between nested RT-PCR and in situ hybridization for the detection of swine HEV in formalin-fixed, paraffin-embedded hepatic tissues was 100%.

摘要

通过巢式逆转录聚合酶链反应（RT-PCR）在自然感染猪的福尔马林固定、石蜡包埋组织中检测到猪戊型肝炎病毒（HEV）。将半巢式RT-PCR的结果与原位杂交的结果进行了比较。所得结果表明，采用二甲苯脱蜡、蛋白酶K消化后进行巢式RT-PCR是一种可靠的检测方法。在40头猪的福尔马林固定、石蜡包埋肝组织中检测到猪HEV核酸。通过原位杂交在相同肝组织中获得了猪HEV明显的阳性信号。猪HEV核酸定位于肝细胞的细胞质中，呈颗粒状染色模式。在福尔马林固定、石蜡包埋肝组织中检测猪HEV时，巢式RT-PCR与原位杂交的符合率为100%。