【摘要】：為建立一種簡(jiǎn)易、快速、特異性強(qiáng)的綿羊肺炎支原體(Mo)檢測(cè)方法,本研究結(jié)合降落PCR(TD PCR)和側(cè)向?qū)游?LFA)技術(shù),采用40nm的膠體金標(biāo)記抗地高辛抗體,檢測(cè)線和質(zhì)控線分別包被鏈霉親和素和羊抗鼠IgG,組裝成LFA方法通用膠體金層析試紙條。兩條特異性引物5'端分別標(biāo)記地高辛和生物素,以Mo的全基因組為模板進(jìn)行降落PCR,產(chǎn)物滴加于試紙條上樣墊,檢測(cè)結(jié)果可在15min內(nèi)讀取。通過對(duì)各環(huán)節(jié)的調(diào)整優(yōu)化,建立了檢測(cè)Mo病原的TD PCR-LFA快速檢測(cè)方法。結(jié)果顯示:該方法僅對(duì)MO檢測(cè)呈陽(yáng)性,對(duì)其它病原菌檢測(cè)均為陰性,表明該方法特異性良好。其檢測(cè)下限為3×102ccu/mL,敏感性較強(qiáng)。不同批次的PCR產(chǎn)物、不同批次的試紙條讀取結(jié)果均一致,表明重復(fù)性和穩(wěn)定性較好。本研究建立的Mo檢測(cè)方法整合了TD PCR的特異性、敏感性和金標(biāo)條快速、簡(jiǎn)單的特性,在2h內(nèi)可完成檢測(cè),為建立適用于基層的臨床檢測(cè)方法奠定了基礎(chǔ)。
[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.
【作者單位】： 石河子大學(xué)生命科學(xué)學(xué)院;石河子大學(xué)動(dòng)物科技學(xué)院;石河子大學(xué)西部地區(qū)高發(fā)人獸共患傳染性疾病防治協(xié)同創(chuàng)新中心;
【基金】：國(guó)家自然科學(xué)基金項(xiàng)目(31360226) 高層次人才科研啟動(dòng)資金專項(xiàng)(CZX201138)
【分類號(hào)】：S852.62

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本文編號(hào)：2330541