發(fā)布時(shí)間：2018-03-26 20:34

  本文選題：海洋　切入點(diǎn)：創(chuàng)傷弧菌　出處：《國際檢驗(yàn)醫(yī)學(xué)雜志》2016年05期

【摘要】：目的利用環(huán)介導(dǎo)等溫?cái)U(kuò)增(LAMP)技術(shù),建立海洋創(chuàng)傷弧菌快速、簡便、特異且敏感的檢測方法,并對該方法的特異性和靈敏度進(jìn)行評價(jià)。方法選取海洋創(chuàng)傷弧菌溶細(xì)胞素(vvhA)基因作為靶基因,根據(jù)GenBank公布的序列設(shè)計(jì)4條LAMP引物;對47株細(xì)菌(包括20株弧菌屬細(xì)菌)進(jìn)行LAMP和聚合酶鏈?zhǔn)椒磻?yīng)(PCR)擴(kuò)增,并做特異性比較;對創(chuàng)傷弧菌M06株增菌液10倍倍比稀釋,提取DNA后進(jìn)行靈敏度的檢測,并與常規(guī)PCR作比較;構(gòu)建含vvhA基因片段的重組質(zhì)粒,作為LAMP反應(yīng)體系的標(biāo)準(zhǔn)陽性對照。結(jié)果常規(guī)PCR實(shí)驗(yàn)出現(xiàn)假陽性結(jié)果,而LAMP實(shí)驗(yàn)只有海洋創(chuàng)傷弧菌出現(xiàn)陽性擴(kuò)增,其他樣品均為陰性,無假陽性和假陰性結(jié)果,表明引物的特異性較好,加入鈣黃綠素和電泳結(jié)果相一致;針對vvhA基因建立的LAMP技術(shù)其最低檢測下限為每個(gè)反應(yīng)4×10CFU,是常規(guī)PCR(每個(gè)反應(yīng)4×10~2 CFU)的10倍,呈現(xiàn)較好的靈敏度。重復(fù)實(shí)驗(yàn)過程兩遍,PCR和LAMP技術(shù)檢測結(jié)果穩(wěn)定。結(jié)論建立了一種用于檢測海洋創(chuàng)傷弧菌的LAMP檢測方法,該方法特異性強(qiáng),靈敏度高,方便快捷,特別適合用于現(xiàn)場和床旁的快速檢測。
[Abstract]:Objective to establish a rapid, simple, specific and sensitive method for the detection of marine vibrio vulnificus by using loop mediated isothermal amplification (Lampp) technique. The specificity and sensitivity of the method were evaluated. Methods four LAMP primers were designed according to the sequence published by GenBank. 47 strains of bacteria (including 20 strains of Vibrio) were amplified by LAMP and polymerase chain reaction (PCR) and their specificity was compared. The recombinant plasmid containing vvhA gene fragment was constructed as the standard positive control of LAMP reaction system. Results there were false positive results in routine PCR test and positive amplification in LAMP assay only in Vibrio vulnificus. All the other samples were negative, no false positive and false negative results, which showed that the specificity of primers was better, and the results of adding calcium yellowish were consistent with those of electrophoretic electrophoresis. The minimum detection limit of LAMP technique for vvhA gene was 4 脳 10 CFU per reaction, which was 10 times as high as that of conventional PCR (4 脳 10 ~ 2 CFU per reaction). The results of repeated experiments were stable. Conclusion A method for detection of marine Vibrio vulnificus by LAMP was established, which was specific, sensitive, convenient and rapid. It is especially suitable for quick detection on the spot and beside the bed.
【作者單位】： 遼寧醫(yī)學(xué)院;威海市立醫(yī)院;濟(jì)南博科生物科技有限公司;濟(jì)南軍區(qū)總醫(yī)院;
【基金】：全軍“十二五”科研重點(diǎn)項(xiàng)目(BWS12J014)
【分類號】：R440;R378

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