跨越式滚环等温扩增双染料比色法检测食品中的沙门氏菌  

Colorimetric Detection of Salmonella in Foods by Dual-Dye Saltatory Rolling Circle Amplification

作　　者：孙艳玉 李子坤[2] 张蕴哲[1] 徐慧 卢鑫[3] 张伟[1,4,5] SUN Yanyu;LI Zikun;ZHANG Yunzhe;XU Hui;LU Xin;ZHANG Wei(College of Food Science and Technology,Hebei Agricultural University,Baoding 071000,Hebei;Hebei Agricultural University,Baoding 071000,Hebei;College of Science and Technology,Hebei Agricultural University,Cangzhou 061100,Hebei;College of Life Sciences,Hebei Agricultural University,Baoding 071000,Hebei;Hebei Provincial Key Laboratory of Analysis and Control for Zoonoses Microbial,Baoding 071000,Hebei)

机构地区：[1]河北农业大学食品科技学院,河北保定071000 [2]河北农业大学,河北保定071000 [3]河北农业大学理工系,河北沧州061100 [4]河北农业大学生命科学学院,河北保定071000 [5]河北省人畜共患病原微生物分析与防控重点实验室,河北保定071000

出　　处：《中国食品学报》2025年第1期400-410,共11页Journal of Chinese Institute Of Food Science and Technology

基　　金：国家自然科学基金项目(32172288,31371772);河北省自然科学基金重点项目(C2019204342);中央引导地方科技发展资金项目(216Z5501G,226Z5503G,236Z5502G);河北省外专百人计划项目(360-0803-JSN-3YGS);河北省博士后科研项目(B2021005007);河北省重点研究开发计划项目(18275501D);河北省教育厅科研项目(QN2022073);河北省自然科学基金项目(C2019204284)。

摘　　要：沙门氏菌是导致腹泻病的四大原因之一,对人类健康危害极大。本研究基于甲酚红(CR)和羟基萘酚蓝(HNB)的不同作用模式和显色原理,建立一种跨越式滚环等温扩增(SRCA)双染料比色法,用于沙门氏菌的可视化检测。首先针对沙门氏菌设计SRCA引物组,然后优化双染料添加量,建立SRCA双染料比色检测方法。通过特异性、灵敏度和检出限试验,对该方法进行评估。通过检测60份实际样品,评价该方法的实际应用性能。结果表明:HNB/CR最佳浓度比为0.6,即HNB浓度为75μmol/L,CR浓度为125μmol/L。用该方法检出8株沙门氏菌为阳性,其余15株非沙门氏菌菌株均为阴性。该方法灵敏度为6.8 CFU/mL,是PCR方法(6.8×10^(2)CFU/mL)的100倍。检出限为9.5 CFU/mL,比PCR方法(9.5×10^(2)CFU/mL)低100倍。检测实际样品,计算其敏感性、特异性和符合率分别为100%,98.2%,98.3%。本研究建立的SRCA双染料比色方法能够特异、准确、灵敏地检测沙门氏菌。与使用单一染料相比,双染料混合使用使阴性(紫色)与阳性(绿色)样品间具有更高的色相对比度和更广的颜色变化范围。此外,比色染料可预先加入反应体系之中,能有效避免二次污染。Salmonella is among the four leading culprits of diarrhoeal afflictions and poses a considerable risk to human health.Based on the various action modes and colour rendering principles of cresol red(CR)and hydroxynaphthol blue(HNB),a colorimetric detection of Salmonella in food by doul-dye SRCA has been created.The first step involved designing the SRCA primers for the invA gene of Salmonella,followed by optimization of the double-dye addition to establish a doul-dye SRCA colorimetric method.The method underwent evaluation via specificity,sensitivity,and detection limit tests,and its practical application performance was assessed by detecting 60 actual samples.The study identified that the ideal concentration ratio of HNB/CR was 0.6,which equated to 75μmol/L HNB and 125μmol/L CR.The test yielded positive results for 8 Salmonella strains and negative results for other strains.The sensitivity of the method was 6.8 CFU/mL,which was 100 times higher than that of PCR(6.8×10^(2)CFU/mL),and the detection limit was 9.5 CFU/mL,which was 100 times lower than that of PCR(9.5×10^(2)CFU/mL).Importantly,the sensitivity,specificity,accuracy of this method were 100%,98.2%,and 98.3%.Therefore,the doul-dye SRCA colorimetric method can detect Salmonella quickly,specifically and accurately.On the one hand,the combination of two dyes produces a higher hue contrast and a wider range of color variation between negative(purple)and positive(green)samples than using a single dye.On the other hand,the colorimetric dyes can be pre-added to the reaction system,which can effectively avoid secondary pollution.

关 键 词：跨越式滚环等温扩增 双染料 比色检测 沙门氏菌 

分 类 号：O657.3[理学—分析化学] TS207.4[理学—化学]