【摘要】：目的建立基于DNA環(huán)介導(dǎo)恒溫核酸擴(kuò)增法(LAMP)快速檢測空腸彎曲桿菌。方法以空腸彎曲桿菌馬尿酸酶基因(hip O基因)為靶序列,設(shè)計(jì)6套特異性引物進(jìn)行LAMP反應(yīng),通過比較擴(kuò)增效率篩選出最佳引物用以快速檢測空腸彎曲桿菌。對(duì)檢測方法的特異性進(jìn)行驗(yàn)證,敏感性與PCR法比較。結(jié)果實(shí)時(shí)濁度儀監(jiān)測反應(yīng)結(jié)果表明,LAMP反應(yīng)在62℃恒溫條件60 min內(nèi)完成;如果在反應(yīng)前添加鈣黃綠素與氯化錳混合液(FD),黃綠色的陽性結(jié)果明顯區(qū)別于橙色陰性結(jié)果。LAMP法最低檢出限為6.97×10~2拷貝/μl,敏感性為PCR法(6.97×10~3拷貝/μl)的10倍。結(jié)論 LAMP法用于快速檢測空腸彎曲桿菌具有檢測過程簡單、實(shí)驗(yàn)裝置簡便、反應(yīng)結(jié)果判讀方便、敏感性高、特異性強(qiáng)等特點(diǎn),特別適于現(xiàn)場和基層檢疫及醫(yī)療機(jī)構(gòu)快速診斷。
[Abstract]:Objective To establish a DNA loop-mediated isothermal nucleic acid amplification (LAMP) method for the rapid detection of Campylobacter jejuni. Methods Six sets of specific primers were designed for LAMP reaction with the hippocampal uridase gene (hip O gene) of Campylobacter jejuni. The best primers were screened for rapid detection of Campylobacter jejuni by comparing amplification efficiency. Results The results of real-time turbidimetry showed that the LAMP reaction was completed within 60 minutes under the constant temperature of 62 C. If the mixture of calcein and manganese chloride (FD) was added before the reaction, the positive result of yellow-green was obviously different from the negative result of orange. ConclusionLAMP is a simple and convenient method for rapid detection of Campylobacter jejuni, which is suitable for on-site and grass-roots quarantine and rapid diagnosis of medical institutions.
【作者單位】： 解放軍總醫(yī)院;解放軍疾病預(yù)防控制所;解放軍總醫(yī)院海南分院;
【基金】：國家科技支撐計(jì)劃資助項(xiàng)目(2015BAI13B07) 解放軍總醫(yī)院“百病妙訣”扶植項(xiàng)目(MJ201403)
【分類號(hào)】：R440

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