猪瘟病毒和猪繁殖与呼吸综合征病毒双重RT-LAMP检测方法的建立与应用  

Development and preliminary application of duplex RT-LAMP for simultaneous detection of classical swine fever virus and porcine reproductive and respiratory syndrome virus

作　　者：袁向芬[1] 吴绍强[1] 吕继洲[1] 张永宁[1] 林祥梅[1] 

机构地区：[1]中国检验检疫科学研究院动物检疫研究所,北京100029

出　　处：《中国兽医科学》2015年第7期721-728,共8页Chinese Veterinary Science

基　　金：质检公益性行业专项(201210018);国家科技支撑计划项目(2012BAK11B00;2013BAD12B00)

摘　　要：根据GenBank中猪瘟病毒(CSFV)和猪繁殖与呼吸综合征病毒(PRRSV)的保守序列各设计一套LAMP引物,在同一体系中进行RT-LAMP扩增,通过对体系内引物浓度及反应温度等进行优化,建立了可在63℃恒温下对CSFV和PRRSV进行同步检测的LAMP方法,并进行了敏感性和特异性试验。结果显示,该方法的灵敏度与荧光定量RT-PCR相当,与常规RT-PCR相比高10倍;用该方法对猪传染性胃肠炎病毒、猪流行性腹泻病毒、猪轮状病毒、伪狂犬病病毒、猪圆环病毒2型进行RT-LAMP扩增,均未发生交叉反应。用该法对采自天津市4个猪场的52份临床样品进行检测,与荧光定量RT-PCR检测结果的符合率为100%。结果表明,该方法具有良好的敏感性和特异性,可实现两病原的同步快速检测,是临床疫病初筛技术的强有力候选。Two sets of loop-mediated isothermal amplification（LAMP） primers were designed accor- ding to the conserved nucleic acid sequences of CSFV and PRRSV deposited in GenBank, respectively. After optimizing the contents of inner primers and choosing the proper reaction temperature in the same reaction solution, the developed LAMP detection assay can be completed within 60 minutes in the constant tempera- ture of 63℃ ,and the sensitivity and specificity were test. In result,the sensitivity of the developed RT- LAMP is same as qRT-PCR and 10 times higher than that of conventional RT-PCR. For the specificity test,all of the other porcine pathogens, including transmissible gastroenteritis virus of swine, porcine epi- demic dia-rrhea virus, porcine rotavirus, pseudorabies virus, and porcine circovirus type 2, showed negative results. A total of 52 field samples collected from 4 Tianjin pig farms were tested by duplex RT-LAMP and qRT-PCR. The coincidence rate of duplex RT-LAMP with qRT-PCR was 100% ,indicating that the method has excellent reliability. In conclusion,the developed duplex RT-LAMP may be a useful tool for rapid and sensitive etiological diagnosis for CSFV and PRRSV in piglets simultaneously and provide an effective tech- nical support for the screening of clinical disease.

关 键 词：猪瘟病毒 猪繁殖与呼吸综合征病毒 双重RT-LAMP 检测 

分 类 号：S852.659.6[农业科学—基础兽医学]