pH响应比色酶联免疫吸附法检测牛奶中猪霍乱沙门氏菌

pH Responded Colorimetric ELISA for Sensitive Detection of Salmonella Choleraesuis in Milk

人类常常因误食被猪霍乱沙门氏菌感染的食物而引发中毒。因此,急需构建适用于快速、灵敏检测食物中猪霍乱沙门氏菌污染的新方法。鉴于此,本文构建了一种pH响应比色酶联免疫吸附法灵敏检测牛奶中猪霍乱沙门氏菌。该方法利用葡萄糖氧化酶催化底物葡萄糖产生葡萄糖酸,导致溶液pH下降,进而引发溴甲酚紫(Bromocresol purple,BCP)溶液颜色由紫色变成亮黄色,随后通过记录BCP颜色变化(OD 430/OD 590)实现目标菌的定量检测。当菌浓度为2.54×103~6.17×10^5 CFU/mL,该方法定量检测猪霍乱沙门氏菌可用方程一表述:y 1=0.1051ln(x)+0.7024(R 2=0.7513);当菌浓度为6.17×10^5~1.67×10^7 CFU/mL,该方法定量检测猪霍乱沙门氏菌可用方程二表述:y2=1.3216ln(x)-15.797(R^(2)=0.9711);当菌浓度大于1.67×10^7 CFU/mL时,BCP溶液呈现明显亮黄色,OD 430/OD 590值趋于稳定,无法实现猪霍乱沙门氏菌定量检测。将六个不同浓度的猪霍乱沙门氏菌(2.5×103~5.6×10^6 CFU/mL)加标至牛奶中,检测结果显示回收率介于72.16%~103.58%,相对标准偏差介于7.54%~15.30%。总之,本研究所构建的比色ELISA方法适用于牛奶中不同浓度的猪霍乱沙门氏菌快速、灵敏定量检测。

Human maybe get poisoning after eating Salmonella choleraesuis-infected food.Thus,developing a new analytical technology for rapid and sensitive detection of Salmonella choleraesuis in food is urgently needed.Hence,a pH responded colorimetric enzyme-linked immunosorbent assay(pH-ELISA)was developed for sensitive detection of Salmonella choleraesuis in milk.In this method,glucose oxidase(GOx)was used to catalyze glucose for the generation of gluconic acid.The color of bromocresol purple(BCP)was changed from purple to bright yellow because the pH of sample solution decreased.Under the optimal conditions,the proposed method achieved two linear independent regression equations.When Salmonella choleraesuis concentration increased from 2.54×10^3 to 6.17×10^5 CFU/mL,the first regression equation was described as y1=0.1051ln(x)+0.7024(R^(2)=0.7513).With the increase of Salmonella choleraesuis concentration from 6.17×10^5 to 1.67×10^7 CFU/mL,the second regression equation was expressed as y2=1.3216ln(x)-15.797(R^(2)=0.9711).The average recoveries of the proposed method for spiked milk samples at Salmonella choleraesuis concentrations ranging from 2.5×10^3~5.6×10^6 CFU/mL were 72.16%~103.58%with the relative standard deviations ranging from 7.54%~15.30%.In conclusion,the developed colorimetric ELISA is suitable for the rapid and sensitive quantitation of Salmonella choleraesuis in milk.