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猪脑心肌炎病毒一步法RT-PCR检测方法的建立及应用
作者: 施开创  莫胜兰  胡丽萍  杨 荣  郑 敏  李 军*  
单位: (广西动物疫病预防控制中心  南宁 530001)  
关键词:   脑心肌炎病毒(EMCV)  3D基因  一步法RT-PCR  
分类号:S858.28
出版年,卷(期):页码:2011 ,42 ( 3 ): 页码:324-327
摘要:

【Objective】The present experiment was aimed to develop a rapid and accurate method for the detection of porcine encephalomyocarditis (EMCV) pathogen and its clinical diagnosis. 【Method】A one-step reverse transcription-polymerase chain reaction (RT-PCR) for detection of porcine encephalomyocarditis virus (EMCV) was developed using a pair of specific primers based on the 3D gene sequences of EMCV strains obtained from GenBank. The specificity, sensitivity and repetition of the developed method were also conducted. 【Result】The developed one-step RT-PCR could specifically amplify a 286 bp fragment from EMCV BJC3 strain. The test gave negative results for the strains of foot-and-mouse disease virus (FMDV), classical swine fever virus(CSFV), porcine reproductive and respiratory syndrome virus(PRRSV), porcine pseudorabies virus(PRV), porcine parvovirus (PPV) and normal BHK-21 cells. The detection limit of the developed assay was determined to be as little as 2 tissue culture infectious dose (TCID50) of EMCV. Repeated detection from 10 EMCV-infected cell cultures by the one-step RT-PCR resulted in the same results. The assay was used for 207 clinical samples from suspected piglets, of which 19 gave the positive results, with 9.18% of positive rate. 【Conclusion】 The established one-step RT-PCR assay was specific, sensitive, efficient and rapid for the clinical detection and epidemiological investigation of EMCV infection.

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