寡核苷酸探针原位检测石蜡切片中PRRSV核酸

根据GenBank收录的美洲型猪繁殖与呼吸综合征病毒（PRRSV）ATCCVR-2332株ORF6和ORF7基因序列，用O1igo软件设计并合成大小为37bp的寡核苷酸探针，经生物素标记后，成功建立了原住检测石蜡组织切片中PRRSV核酸的方法。该探针能检测到56PgPRRSV核酸的RT—PCR产物DNA，能特异检测出PRRSV核酸及其PCR产物，而对猪瘟病毒（HCV）、猪细小病毒（PPV）、猪伪狂犬病病毒（PRV）、猪乙脑病毒（JEV）的核酸呈阴性反应。应用该方法检测PRRSVSC-1株人工感染的28日龄仔猪，在感染后7d即可在肺脏、肾脏、扁桃体、胸腺、肺门淋巴结、十二指肠和大脑检测到PRRSV核酸。该法可用于仔猪PRRSV感染的诊断和组织中核酸的定位及分布研究，也可用于甲醛固定组织的回顾性诊断。

Detection of Porcine Reproductive and Respiratory Syndrome Virus(PRRSV) RNA in Paraffin-embedded Tissues by in situ Hybridization with Biotin-labelled Oligonucleotide Probe

According to ORF6 and ORF7 gene sequence of ATCC VR-2332 strain in GenBank,A 37 bp oligonucleotide probe was synthesized and then labelled with biotin,and the method for detecting the RNA of porcine reproductive and respiratory syndrome virus(PRRSV) in paraffin-embedded tissues by in situ hybridization was established successfully.The probe could sensitively detect 56 pg RT-PCR product of PRRSV,specifically the RNA and RT-PCR product of PRRSV,but did not to the viral nucleic acid of HCV,PPV,PRV and JEV.28-day-old piglets infected with PRRSV SC-1 were diagnosed by the method,demonstrating that the positive signals were found at 7 days post infection in these tissues,including the lung,kidney,tonsil,thymus,hilar node,duodenum and cerebrum.The result shows this method can be used in the diagnosis of PRRSV infection and the research for PRRSV distribution in the infected tissues,and the retrospective diagnosis in formalin-fixed tissues.