【摘要】：本研究針對(duì)大腸桿菌rfbE基因和副溶血弧菌pR72H基因分別設(shè)計(jì)了LAMP特異性引物,利用LAMP技術(shù)擴(kuò)增基因組DNA,建立了大腸桿菌和副溶血弧菌的快速LAMP檢測(cè)技術(shù),并初步應(yīng)用于食物樣品的檢測(cè),主要研究結(jié)果如下。1.建立并優(yōu)化了大腸桿菌LAMP反應(yīng)條件,并對(duì)引物特異性進(jìn)行了研究。結(jié)果表明大腸桿菌最佳反應(yīng)條件為：25μL反應(yīng)體系中包含10μmol/L內(nèi)引物FIP-E和BIP-E各3.0μL,缺省外引物F3-E和B3-E,10μmol/L dNTP 2.0μL,10×buffer 2.5μL, 100mmol/LMg2+0.5μL,DNA模版2.0μL,BstDNA聚合酶16U,雙蒸水10μL補(bǔ)足25μL,反應(yīng)溫度65℃,反應(yīng)時(shí)間60min,75℃保溫10min終止反應(yīng)。引物特異性實(shí)驗(yàn)表明,該引物能擴(kuò)增5株供試大腸桿菌,其他供試菌為陰性。在此條件下LAMP反應(yīng)對(duì)大腸桿菌的檢出限為100fg/反應(yīng)。2建立并優(yōu)化了副溶血弧菌LAMP反應(yīng)條件,并對(duì)引物特異性進(jìn)行了研究。結(jié)果表明副溶血弧菌最佳反應(yīng)條件為：25μL反應(yīng)體系中包含10μmol/L內(nèi)引物FIP-V和BIP-V各3.0μL,缺省外引物F3-V和B3-V,10μmol/L dNTP 2.0μL,10×buffer 2.5μL, 100mmol/L Mg2+ 0.5μL, DNA模版2.0gL,BstDNA聚合酶16U,雙蒸水10μL補(bǔ)足25gL,反應(yīng)溫度65℃,反應(yīng)時(shí)間60min,75℃保溫10min終止反應(yīng)。引物特異性實(shí)驗(yàn)表明,該引物能擴(kuò)增3株供試副溶血弧菌,其他供試菌為陰性。在此條件下LAMP反應(yīng)對(duì)副溶血弧菌的檢出限為100fg/反應(yīng)。3.大腸桿菌和副溶血弧菌的雙重LAMP檢測(cè)方法的建立經(jīng)過優(yōu)化實(shí)驗(yàn)確定了大腸桿菌和副溶血弧菌的雙重LAMP反應(yīng)的最佳條件為：25μL反應(yīng)體系中包含大腸桿菌內(nèi)引物FIP-E和BIP-E各3.0μL,副溶血弧菌內(nèi)引物FIP-V和BIP-V各3.0μL,缺省外引物F3-E和B3-E、F3-V和B3-V,10mmol/L dNTP 2.0μL, 10×buffer 2.5μL, 100mmol/L Mg2+0.5μL,大腸桿菌DNA模板2.0μL,副溶血弧菌DNA模板2.0μL,BstDNA聚合酶16U,雙蒸水2.0pL補(bǔ)足25μL,反應(yīng)溫度65℃,反應(yīng)時(shí)間60min,75℃保溫10min結(jié)束反應(yīng)。此條件下雙重LAMP反應(yīng)檢測(cè)大腸桿菌和副溶血弧菌的靈敏度均達(dá)到100fg/反應(yīng),是傳統(tǒng)PCR的靈敏度的100倍。在交互實(shí)驗(yàn)中,對(duì)人工污染大腸桿菌和副溶血弧菌的食物樣品進(jìn)行檢測(cè),結(jié)果表明雙重LAMP反應(yīng)可以特異性檢測(cè)出供試樣品中含有大腸桿菌或者副溶血弧菌的樣品。本研究建立了大腸桿菌和副溶血弧菌的雙重LAMP檢測(cè)方法,并應(yīng)用于人為污染病原菌的食品樣品的檢測(cè),能達(dá)到同時(shí)快速檢測(cè)兩種病原菌的目的。
[Abstract]:In this study, specific LAMP primers were designed for the rfbE gene of Escherichia coli and pR72H gene of Vibrio parahaemolyticus, and a rapid LAMP technique for the detection of E. coli and Vibrio parahaemolyticus was established by using LAMP technique to amplify genomic DNA,. It has been applied to the detection of food samples. The main results are as follows. The LAMP reaction conditions of Escherichia coli were established and optimized, and the specificity of primers was studied. The results showed that the optimal reaction conditions of Escherichia coli were as follows: 10 渭 mol / L internal primer FIP-E and 3.0 渭 L BIP-E, default external primer F 3 渭 E and B 3 渭 E, 10 渭 mol / L dNTP 2.0 渭 L, 10 脳 buffer 2.5 渭 L, 100mmol/LMg2 0.5 渭 L, respectively. DNA template 2.0 渭 L, BstDNA polymerase 16U, disteaming water 10 渭 L supplemented with 25 渭 L, reaction temperature 65 鈩，

本文編號(hào)：2453488