猪繁殖与呼吸综合征病毒LAMP检测方法的建立与应用

Development and Application of a Loop-mediated Isothermal Amplification Method for Detection of Porcine Reproductive and Respiratory Syndrome Virus

为建立一种具有敏感性高、特异性强、方便、快速等特点的检测猪繁殖与呼吸综合征病毒(PRRSV)的环介导等温扩增技术(LAMP),针对美洲型PRRSV的ORF5基因设计了一套LAMP引物,建立了LAMP反应体系,并对其敏感性、特异性等进行了评估。结果表明,该方法可在65℃、1h内完成最低限度5.49pg/μL含量的PRRSV核酸扩增,结果仅肉眼就能判定。该检测方法特异性强,与猪瘟病毒(CSFV)、猪圆环病毒2型(PCV-2)和猪伪狂犬病病毒(PRV)等病原体无交叉反应;对25份临床样品检测表明,该方法的检出率高于常规RT-PCR法,且操作更为方便、快速。该检测方法特异、敏感、准确,可应用于临床检测PRRSV。

To develop a loop-mediated isothermal amplification method（LAMP）with high sensitivity,good specificity,convenience and fastness for detection of porcine reproductive and respiratory syndrome virus（PRRSV）,a set of LAMP primers were designed according to the ORF5 gene of PRRSV American type and a LAMP reaction system was established.The sensitivity and specificity of the system were evaluated.The results showed that the assay could carry out nucleic acid amplification reaction of PRRSV within 1hunder 65℃ with the detection limit of 5.49pg/μL and the results could be read by naked eye without gel electrophoresis.This detection method was highly specific,and had no cross reaction with pathogens such as classical swine fever virus（CSFV）,porcine circovirus type 2（PCV-2）and porcine pseudorabies virus（PRV）;detection results of 25 clinical samples showed that the detection rate of this assay was higher than that with conventional RT-PCR and this assay operated quickly and conveniently.This detection method was specific,sensitive and accurate,and could be used for clinical detection of PRRSV.