基于猪繁殖与呼吸综合征病毒ORF2a基因的RT-RAA-LF检测方法的建立

Development of RT-RAA-LF assay to detect PRRSV based on ORF2a gene

本研究针对猪繁殖与呼吸综合征病毒ORF2a基因序列,设计特异性RAA引物和标记探针,通过反转录酶与重组酶扩增结合,旨在建立一种高效检测PRRSV的基于侧流层析试纸条的可视化RT-RAA方法。结果,建立的RT-RAA-LF方法的最佳反应条件是39℃恒温扩增10 min,可在2 min内实现对检测结果的可视化判定;该方法特异性强,仅对PRRSV出现阳性扩增,对本研究中其他病原均无交叉反应;以含PRRSV ORF2a基因全段序列的重组质粒作为模板,RT-RAA-LF的检出限为160 copies/μL。应用建立的RT-RAA-LF方法对临床采集的160份样本进行检测,与PCR方法进行比较,两种方法的符合率为96.8%(65/68),一致性检验为χ2<3.84,在95%的置信区间内没有显著性差异,当中3份临床样品在PCR检测中为阴性。综上,基于PRRSV ORF2a建立的RT-RAA-LF方法操作简单,对仪器设备要求不高,能够高效且直观的实现临床检测,满足现场检测的要求,对猪养殖场快速诊断和防控PRRSV具有重要意义。

A rapid visual assay of RT-RAA based on PRRSV ORF2a gene with lateral flow was developed,which designed specific primers and probe of RAA combined with Reverse Transcriptase M-MLV and recombinase.The optimal reaction condition of RT-RAA-LF was ampilfication for 10 min at 39℃and within 2min can detect 160 copies of PRRSV c DNA molecules by lateral flow strips.The assay has high specificity and does not cross-react with other swine pathogens.The RT-RAA-LF was used to detect 160 clinic samples compared with PCR assay,the results showed that the specificity of two assays was 96.8%(65/68),the chi-square test isχ~23.84 and was no significant difference within the 95%confidence interval.There were 3 clinic samples were negative in the results of PCR assay.In conclusion,the development of the RT-RAA-LF assay based on PRRSV ORF2a gene has simple instructions,which has low requirements for equipmen and can achieve rapid visualized clinic detection.It has significance for rapid diagnosis and control of PRRSV in pig farms.