猪伪狂犬病病毒PCR-LFD检测方法的建立及应用

Development and Application of PCR-LFD Method for Detection of Pseudorabies Virus

旨在将PCR技术与可视化的横向流动试纸条(lateral flow dipstick,LFD)相结合,建立猪伪狂犬病病毒野毒株的PCR-LFD快速检测技术。依据猪伪狂犬病病毒gE基因保守区域设计2条特异性引物和对应的特异性探针,其中下游引物和探针分别标记了荧光素(FITC)和生物素(Biotin)。通过对PCR退火温度、引物浓度、探针杂交温度的筛选,确定PCR-LFD方法的最佳反应条件,并对其敏感性、特异性和重复性进行检测。结果显示,所建立的检测方法能够特异性的检测出猪伪狂犬病病毒野毒株,与猪伪狂犬病病毒Bartha-K61株、6种对照病毒和3种对照细菌均无交叉反应,其最低检测限为100TCID50/100μL。该方法对同批次和不同批次猪伪狂犬病病毒DNA分别进行5次重复检测,结果完全一致。该方法对50份临床样品进行检测,PCR-LFD方法和病毒分离培养法检测出16份阳性样品,常规PCR检测出14份阳性样品,PCR-LFD方法的敏感性高于PCR,但两者间差异不显著P>0.05。以上结果表明,所建立的方法具有良好的特异性、敏感性和重复性,且不需要琼脂糖凝胶电泳可直接判读结果,适合养殖场、基层实验室的现场检测。

The experiment was aimed to establish a rapid PCR-LFD method for the detection of pseudorabies virus wild-type strains based on the PCR method and visualization by a lateral flow dipstick(LFD).In this study,two spectific PCR primers and one nucleic acid probe were designed based on the conservative regions of gE gene of pseudorabies virus,downstream primer and nucleic acid probe were respectively labled by fluorescein isothiocyanate(FITC)and biotin.The optimum reaction conditions of PCR-LFD method were determined by screening the annealing temperature of PCR,the concentration of primers and the hybridization temperature of the probe,while its specificity,sensitivity and reproducilbility were tested.The results showed only pseudorabies virus wild-type strains were positive,and no cross-reaction were observed with the PRV Bartha-K61 strain and other six control virus strains and three control bacterial strains.The sensitivity of the assay was 100TCID 50/100μL.This method had five repeated tests for the same batch and different batch of pseudorabies virus DNA,and the results were identical.For the 50 suspected samples,16 positive samples were identified by PRV isolation and the PCR-LFD strip method,while 14 positve samples were identified by regular PCR.The PCR-LFD method had higher sensitivity than the PCR,but P>0.05,that was not in significant level.The above results indicated that the method of PCR-LFD for detecting pseudorabies has good specificity,sensitivity and repeatability,and it does not require agarose gel electrophoresis to read the results,which is suitable for field testing of farm and basic-level laboratory.