绵羊肺炎支原体降落PCR-侧向层析快速检测方法的建立

Visualized detection of Mycoplasma ovipneumoniae by real-time touchdown PCR-lateral flow assay

为建立一种简易、快速、特异性强的绵羊肺炎支原体(Mo)检测方法,本研究结合降落PCR(TD PCR)和侧向层析(LFA)技术,采用40nm的胶体金标记抗地高辛抗体,检测线和质控线分别包被链霉亲和素和羊抗鼠IgG,组装成LFA方法通用胶体金层析试纸条。两条特异性引物5'端分别标记地高辛和生物素,以Mo的全基因组为模板进行降落PCR,产物滴加于试纸条上样垫,检测结果可在15min内读取。通过对各环节的调整优化,建立了检测Mo病原的TD PCR-LFA快速检测方法。结果显示:该方法仅对MO检测呈阳性,对其它病原菌检测均为阴性,表明该方法特异性良好。其检测下限为3×102ccu/mL,敏感性较强。不同批次的PCR产物、不同批次的试纸条读取结果均一致,表明重复性和稳定性较好。本研究建立的Mo检测方法整合了TD PCR的特异性、敏感性和金标条快速、简单的特性,在2h内可完成检测,为建立适用于基层的临床检测方法奠定了基础。

The study combined touchdown PCR（TD PCR） with gold nanoparticle（GN） based lateral flow assay by conjugating 40nm GNs with anti-Digoxin antibody, pre-immobilizing streptavidin and goat anti-mouse IgG on the test line and control line, respectively, which was conducted to establish a simple, rapid and effective assay of the TD PCR-LFA for Mycoplasma ovipneu- moniae detection. The 5＇ termina of specific primers labled Digoxin, biotin respectively, and TD PCR was carried out with templates of M.ovipneumoniae whole genome. The result was easily achieved by visual observation within 15 minutes after loading of the PCR products onto the LFA device. This assay was highly specific to M.ovipneumoniae without cross reactions with other related bacteria, and showed good repeatability and stability both in inter and intra assay. In addition, the detection limit of the assay was 3 ×10^2 ccu/mL, suggesting that the assay had a high sensibility to M.ovipneumoniae. The detection for M.ovip- neumoniae was completed within 2 hours and this assay provides the foundation for clinical examination used for the grassroots.