绵羊肺炎支原体降落PCR-侧向层析快速检测方法的建立
发布时间：2018-11-14 07:55
【摘要】：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。
[Abstract]:In order to establish a simple, rapid and specific (Mo) method for detection of mycoplasma pneumoniae in sheep, the method of landing PCR (TD PCR) and lateral chromatography (LFA) was used to label anti-digoxin antibody with colloidal gold of 40nm. The detection line and the quality control line were coated with streptavidin and sheep anti-mouse IgG, respectively, to form a general colloidal gold chromatographic test strip for LFA. Digoxin and biotin were labeled with two specific primers at the 5 'end. The whole genome of Mo was used as template to drop the PCR, product onto the sample pad. The results could be read in 15min. By adjusting and optimizing each link, a rapid detection method of TD PCR-LFA for detecting Mo pathogen was established. The results showed that the method was only positive for MO and negative for other pathogens, which indicated that the method had good specificity. The detection limit is 3 脳 10 ~ 2 ccur 路mL ~ (-1), and the sensitivity is strong. The results of different batches of PCR products and different batches of test strips are consistent, indicating that the repeatability and stability are good. The Mo detection method established in this study integrates the specificity, sensitivity and rapid and simple characteristics of TD PCR, and can be completed within 2 hours, which lays a foundation for the establishment of clinical detection method suitable for basic level.
【作者单位】： 石河子大学生命科学学院;石河子大学动物科技学院;石河子大学西部地区高发人兽共患传染性疾病防治协同创新中心;
【基金】：国家自然科学基金项目(31360226) 高层次人才科研启动资金专项(CZX201138)
【分类号】：S852.62
[Abstract]:In order to establish a simple, rapid and specific (Mo) method for detection of mycoplasma pneumoniae in sheep, the method of landing PCR (TD PCR) and lateral chromatography (LFA) was used to label anti-digoxin antibody with colloidal gold of 40nm. The detection line and the quality control line were coated with streptavidin and sheep anti-mouse IgG, respectively, to form a general colloidal gold chromatographic test strip for LFA. Digoxin and biotin were labeled with two specific primers at the 5 'end. The whole genome of Mo was used as template to drop the PCR, product onto the sample pad. The results could be read in 15min. By adjusting and optimizing each link, a rapid detection method of TD PCR-LFA for detecting Mo pathogen was established. The results showed that the method was only positive for MO and negative for other pathogens, which indicated that the method had good specificity. The detection limit is 3 脳 10 ~ 2 ccur 路mL ~ (-1), and the sensitivity is strong. The results of different batches of PCR products and different batches of test strips are consistent, indicating that the repeatability and stability are good. The Mo detection method established in this study integrates the specificity, sensitivity and rapid and simple characteristics of TD PCR, and can be completed within 2 hours, which lays a foundation for the establishment of clinical detection method suitable for basic level.
【作者单位】： 石河子大学生命科学学院;石河子大学动物科技学院;石河子大学西部地区高发人兽共患传染性疾病防治协同创新中心;
【基金】：国家自然科学基金项目(31360226) 高层次人才科研启动资金专项(CZX201138)
【分类号】：S852.62
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