猪瘟疫苗毒株和流行毒株RT-nPCR检测方法的建立
Establishment of a RT-nPCR Method for Detection and Differentiation of Wild-type and Vaccine Viruses of Classical Swine Fever Virus

为给临床快速鉴别诊断猪瘟提供技术依据，根据GenBank公布的CSFV全基因序列，结合猪瘟序列测定结果，针对 NS5B基因区域设计2条通用外扩引物、2条通用内扩引物和1条疫苗毒株特异性内扩引物，建立一种可鉴别猪瘟疫苗毒株和流行毒株的RT-nPCR诊断方法。结果显示，该方法可从HCLV株扩增出 2条 大小分别为261 bp和157 bp的片段，可从Shimen、SXYL2006、GX54和SD2002等流行毒株扩增出1条261 bp的片段，其他几种常见猪病毒检测均呈阴性；灵敏度试验表明，检测的cDNA质量浓度极限为3.4×107 pg/L；116份临床样品中有37份样品检测呈阳性，阳性率为32%；分析部分阳性病料E2基因序列也证实检测结果的准确性。说明：建立的RT-nPCR鉴别诊断方法灵敏度高、特异性好，能直观区分猪瘟疫苗毒株和流行毒株。
To establish a method of a reverse transcription-multiplex nested polymerase chain reaction for easy differentiation of classical swine fever vaccine C-strain and wild-type strains, according to the CSFV complete genome sequences in GenBank and some of the wild-type CSFV sequences in Shaanxi province, we designed two universal external expansion primers for the NS5B gene region and two universal internal primers and one special primer of expansion of C-strain, the RT-nPCR diagnostic methods were established for the identification of wild-strain CSFV and C-strain. We acquired 261 bp and 157 bp two bands from C-strain, but only acquired 261 bp band from Shimen, SXYL2006, GX54 and SD2002 other wild-strains by using the RT-nPCR method, and several other common pig virus were negative. The sensitivity test showed that the method detection limit of cDNA content was 3.4×107 pg/L. 116 clinical samples test results showed that 37 samples were positive, the positive rate was 32%. We analyses some of E2 gene sequence of positive tissues confirmed the accuracy of the test results. The establishment of a reverse transcription-multiplex nested polymerase chain reaction method has high sensitivity, specificity, and can intuitively differentiate between C-strain and wild-strains of CSFV.