猪Linda病毒巢式RT-PCR检测方法的建立

Establishment of a Nested RT-PCR Assay for Porcine Linda Virus

猪Linda病毒能引起仔猪先天性震颤,严重危害养猪业的发展,目前在我国暂未发现感染病例。本研究根据猪Linda病毒Core-E~(ms)基因序列,设计并合成巢式RT-PCR引物,通过对退火温度、引物浓度等进行优化,建立了猪Linda病毒巢式RT-PCR检测方法。结果显示,该方法具有良好的特异性,与非洲猪瘟病毒、猪瘟病毒、猪繁殖与呼吸综合征病毒、猪伪狂犬病病毒、猪圆环病毒2型均无交叉反应;敏感性好,对慢病毒阳性对照品的最低检出限为10~1 copies/μL,比普通RT-PCR灵敏10倍。使用该方法检测模拟病毒样品,发现最低检出限为10~1 TU/mL,与荧光定量RT-PCR方法一致。本研究首次建立了可检测猪Linda病毒的巢式RT-PCR方法,其特异性强、灵敏度高,为在口岸以及条件有限的场地对猪Linda病毒进行精准且快速的检测提供了有效技术手段,也为防范Linda病毒传入提供了有力技术支撑。

Porcine Linda virus could lead to piglet congenital tremors,posing a serious danger to pig industry.Fortunately,no case had been found in China.In the paper,nested RT-PCR primers were designed and synthesized based on the Core-Es gene sequence of porcine Linda virus,a nested RT-PCR assay was established through optimizing annealing temperature and concentration of the primers.The results revealed that the established method was with good specificity,without cross reactivity with African swine fever virus(ASFV),classical swine fever virus(CSFV),porcine reproductive and respiratory syndrome virus(PRRSV),porcine pseudorabies virus(PRV)and porcine circovirus type 2(PCV-2);with good sensitivity,and a minimum detection limit of 10'copies/μL for lentivirus positive control samples,which was 10 times more sensitive than general RT-PCR assay.When simulated virus samples were detected by the method,the detection limit was 1o'TU/mL,which was consistent with the results by fluorescence quantitative RT-PCR assay.In conclusion,a nested RT-PCR assay was firstly established for porcine Linda virus,which has strong specificity and high sensitivity,and could be used as an effective technical tool for accurate and rapid detection of the virus at ports and in the premises with limited conditions.Also,a technical support for prevention of any introduction of the virus was provided.