奶牛乳房炎四种致病菌PCR核酸免疫层析试纸条快速检测方法的建立及应用

Establishment and application of PCR nucleic acid immunochromatographic strip for rapid detection of four pathogenic bacteria in dairy cow mastitis

为了开发检测导致奶牛乳房炎的无乳链球菌、金黄色葡萄球菌、大肠杆菌和停乳链球菌这四种重要致病菌的PCR核酸免疫层析试纸条方法,本研究采用柠檬酸还原法制备10 nm的胶体金溶液,选用优化的10μL 1 mol/L碳酸钾溶液调节pH和5μL异硫氰酸荧光素抗体偶联金颗粒,并使用优化的封闭液(10%BSA、0.25%PEG2000、0.05%吐温-20、0.05%Triton-X100)进行封闭,将地高辛抗体和山羊抗鼠二抗分别喷涂在检测线和质控线上,烘干并组装成试纸条,进行细菌的纯培养物和鲜牛奶加样检测。结果显示,所制备的PCR核酸免疫层析试纸条和凝胶电泳均具有较好的特异性,最低检测限要比凝胶电泳高10倍,在模拟污染牛奶样品测试中,试纸条仍然具有很好的特异性且灵敏度比凝胶电泳的高。上述结果表明,本研究建立的PCR核酸免疫层析试纸条检测方法具有良好的临床应用潜力。

A PCR nucleic acid immunochromatographic stripmethod for detection of four important pathogens of mastitis including Streptococcus aureus,Staphylococcus aureus,Escherichia coli and Streptococcus dysgalactia in dairy cow was established in this study.Colloidal gold nanoparticles with diameter of 10 nm were firstly prepared by the citric acid reduction method.Secondly,5 μL fluoresce isothiocyanate antibody was coupled with gold particles,and the optimized concentration of blocking liquid was established for blocking.Thirdly,anti-digoxin antibody and sheep anti-mouse antibody were sprayed on the detection line and the quality control line respectively,and dried for detecting the standard bacteria samples and the spiked fresh milk samples,respectively.The trial results indicated that, the prepared PCR nucleic acid immunochromatographic strip had a good specificity,and the detection limit of the strip was about ten-fold better than that of the gel electrophoresis.For the artificially polluted milk samples,the test strip also had a good specificity and the sensitivity was much higher than that of the gel electrophoresis.We concludethat the PCR nucleic acid immunochromatographic strip test method established in this study will have great application potential in various fields.