【摘要】：目的研究水中產(chǎn)氣莢膜梭菌的熒光定量PCR檢測方法,縮短水中產(chǎn)氣莢膜梭菌的檢測時間。方法根據(jù)產(chǎn)氣莢膜梭菌的α-toxin基因設(shè)計引物并擴增,構(gòu)建α-toxin-T重組質(zhì)粒,對重組質(zhì)粒進行梯度稀釋后進行熒光定量PCR檢測;用該方法檢測水中常見的15種細(xì)菌,確定方法的特異性;檢測產(chǎn)氣莢膜梭菌加標(biāo)水樣,驗證方法的可行性。結(jié)果成功擴增出產(chǎn)氣莢膜梭菌的α-toxin基因并連接T載體(417 bp)。α-toxin-T重組質(zhì)粒的稀釋度在2.17×10~0~2.17×10~9 copies/μl的范圍內(nèi),PCR反應(yīng)的特異性良好,且重組質(zhì)粒各濃度梯度間間隔的Ct值相近;所得回歸方程為y=31.69-3.11x,r=0.997 73;該方法的檢出限可達到為10 copies/μl。15株受試細(xì)菌DNA的檢測結(jié)果均為陰性,產(chǎn)氣莢膜梭菌加標(biāo)水樣檢測結(jié)果陽性。結(jié)論該方法操作快速,具有較好的特異性和靈敏性,適用于水中產(chǎn)氣莢膜梭菌的快速檢測。
[Abstract]:Objective to study the fluorescence quantitative PCR method for the detection of Clostridium perfringens in water, and to shorten the detection time of Clostridium perfringens in water. Methods according to the 偽 -toxin gene of Clostridium perfringens, primers were designed and amplified to construct 偽 -toxin-T recombinant plasmid. The recombinant plasmid was detected by fluorescence quantitative PCR after gradient dilution, and 15 common bacteria in water were detected by this method, and the specificity of the method was determined. Detection of Clostridium perfringens plus standard water samples to verify the feasibility of the method. Results the 偽 -toxin gene of Clostridium perfringens was successfully amplified and ligated into T vector. The dilution of the recombinant plasmid was 2.17 脳 10 ~ (10) ~ 0 ~ 2.17 脳 10 ~ (9) copies/ 渭 l. The regression equation was obtained as YYI 31.69-3.11xrnr 0.99773.The detection limit of this method was 10 copies/ 渭 l.15 strains of bacteria DNA were negative, and the results of Clostridium perfringens plus standard water samples were positive. Conclusion the method is rapid, specific and sensitive, and can be used for rapid detection of Clostridium perfringens in water.
【作者單位】： 中國疾病預(yù)防控制中心環(huán)境與健康相關(guān)產(chǎn)品安全所;
【基金】：國家衛(wèi)生和計劃生育委員會公益性衛(wèi)生行業(yè)科研專項(201302004)
【分類號】：R123

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