摘要
【目的】实现快速、准确地对猪脑心肌炎进行临床诊断和病原学检测。【方法】根据GenBank中已发表的猪脑心肌炎病毒(EMCV)3D基因序列设计合成1对特异性引物,优化反应条件,建立了EMCV一步法RT-PCR检测方法,并对该方法的特异性、敏感性和重复性进行验证。【结果】一步法RT-PCR可以从EMCVB JC3株中扩增出与试验设计相符的286bp特异性片段,而对猪口蹄疫病毒(FMDV)、猪瘟病毒(CSFV)、猪繁殖与呼吸综合征病毒(PRRSV)、猪伪狂犬病病毒(PRV)、猪细小病毒(PPV)、BHK-21正常细胞的扩增结果均为阴性;对EMCVBJC3株细胞毒的敏感度达到2TCID50;对10份EMCVB JC3株细胞毒进行重复性检测,结果均一致;对207份临床疑似发病猪的病料进行检测,其阳性检出率为9.18%。【结论】所建立的EMCV一步法RT-PCR检测方法具有特异、灵敏、高效、快速等特点,可用于EMCV的临床检测及流行病学调查。
【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-poly- merase 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-in-fected 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.
出处
《南方农业学报》
CAS
CSCD
2011年第3期324-327,共4页
Journal of Southern Agriculture
基金
广西青年科学基金项目(桂科青0728047)
广西科技创新能力与条件建设基金项目(08-05-01D)
