CAJ | 학술논문

建立食品中全血清型霍乱弧菌的交叉引物等温扩增检测方法.针对霍乱弧菌设计特异性引物及探针,建立交叉引物等温扩增法,利用免疫金标试纸条对结果进行检测.用57株霍乱弧菌及相近株细菌进行特异性试验;通过定量DNA、纯菌液计数、样品中添菌检测及与环介导等温扩增方法进行灵敏度验证及比较工作.结果表明,建立方法具有较好特异性;DNA检测灵敏度为79.28 fg/test,增菌液检测灵敏度为4.2×102 CFU/mL,当每25克样品中有5.6 CFU菌时经增菌步骤后即可检出.建立的交叉引物恒温检测方法可作为食品中霍乱弧菌快速初筛检测方法使用,较环介导等温扩增方法灵敏且易于操作.
건립식품중전혈청형곽란호균적교차인물등온확증검측방법.침대곽란호균설계특이성인물급탐침,건입교차인물등온확증법,이용면역금표시지조대결과진행검측.용57주곽란호균급상근주세균진행특이성시험;통과정량DNA、순균액계수、양품중첨균검측급여배개도등온확증방법진행령민도험증급비교공작.결과표명,건립방법구유교호특이성;DNA검측령민도위79.28 fg/test,증균액검측령민도위4.2×102 CFU/mL,당매25극양품중유5.6 CFU균시경증균보취후즉가검출.건립적교차인물항온검측방법가작위식품중곽란호균쾌속초사검측방법사용,교배개도등온확증방법령민차역우조작.
To develop a cross-priming amplification (CPA) method for the detection of all serotypes of V.cholera on food.Specific primers and probe were designed,we developed cross-primer isothermal amplification method,using the immuno-gold standard test strip to test results.The specificity of the method was evaluated by 57 different bacterial strains.The sensitivity of the method was evaluated by testing the quantitative DNA,pure bacteria counted and the sample of adding V.cholera comparing with the loop-mediated isothermal amplification (LAMP) method.All of the V.cholera strains showed positive results,and 41 other types of bacteria gave negative results.The limit of detection of the CPA method was 79.28 fg of genomic DNA,4.2 × 102 CFU/mL for bacteria in pure culture,and 5.6 CFU per 25 g of sample with pre-enrichment.This method showed a higher sensitivity than the LAMP method did and was more convenient to perform.These results indicate that the CPA method can be used for the rapid preliminary screening of V.cholera.