猪圆环病毒2型原位杂交检测技术的建立与应用

参照GenBank发表的猪圆环病毒2型（PCV2）ORF2基因序列设计引物，利用PCR扩增得到PCV2BF株341bp的核酸片段，用随机引物法制备出地高辛标记的核酸探针。制备的探针与PCV1、PRRSV、PPV、PRV等不发生反应，可检测的最低PCV2DNA含量为1．78Pg。对30份临床组织样本进行了检测，并与PCR比较，结果表明，阴性符合率为100％，阳性符合率为88．9％。应用原位杂交技术分析了PCV2在人工感染仔猪主要组织中的分布，结果表明，感染后3d，从仔猪的淋巴结、胸腺、肺脏、脾脏、鼻黏膜可检测到阳性信号，感染后21d，肝脏、肾脏、胰腺和回肠可检出阳性信号，至感染后42d，可从心脏、胃、脑检出阳性信号。在整个试验过程中会厌软骨、膀胱、皮肤、肌肉等组织均为阴性。本研究结果表明，建立的PCV2原位杂交技术具有良好的敏感性和特异性，可用于PCV2的实验室诊断和感染靶细胞的定位分析。

Development and Application of an in situ Hybridization for Detection of Porcine Circovirus Type 2

A fragment of PCV2 BF strain with a size of 341 bp was amplified by PCR with a set of primer designed according to the genomic sequence of porcine circovirus type 2 available on GenBank.A nucleic acid probe labeled with digoxigenin-dUTP was prepared by random priming.Further research indicated that the probe was not reactive to PCV1,porcine reproductive and respiratory syndrome virus(PRRSV),porcine parvovirus(PPV) and pseudorabies virus(PRV),and could detect 1.78 pg DNA of PCV2.Thirty clinical samples were detected with the developed in situ Hybridization(ISH) and polymerase chain reaction(PCR).The results indicated that the ISH had a specificity of 100% and a sensitivity of 88.9%.Distribution of PCV2 in tissues of piglets inoculated with PCV2 was analyzed with ISH.The lymph nodes,thymus gland,tonsil,lung,spleen and nasal mucosa from the inoculated piglets exhibited positive signal at day 3 post-inoculation.The liver,kidney, pancreas and ileum were detected to be positive at day 21 post-inoculation.The heart,stomach and brain were positive at day 42 post-infection.The epiglottis cartilage,urinary bladder,skin,muscle and the tissues from un-inoculated piglets were negative during the experiment.The results suggested the in situ hybridization were sensitive and specific,and could be applied for clinical and laboratory detection of PCV2,as well as location of infected target cells of PCV2.