2种食源性微生物胶体金银染技术的检测方法研究  被引量:2

Study on the detection of two kinds of food borne microorganisms by colloidal gold and silver staining

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作  者:马生龙[1] 李云霞[1] 马莉萍[1] 聂莹莹[1] 韩根亮[1] MA Sheng - long, LI Yun - xia, MA Li - ping, NIE Ying - ying, HAN Gen - liang(Gansu Provincial Key Laboratory of Sensor and Sensing Technology, Institute of Sensor Technology, Gansu Academy of Sciences, Lanzhou, Gansu 730000, Chin)

机构地区:[1]甘肃省科学院传感技术研究所甘肃省传感器与传感技术重点实验室,甘肃兰州730000

出  处:《中国卫生检验杂志》2018年第4期385-389,共5页Chinese Journal of Health Laboratory Technology

基  金:甘肃省科学院青年科技创新基金(2015QN-05);甘肃省科学院应用开发项目(2016JK-07);甘肃省科学院应用开发项目(2016JK-05);甘肃省科学院院地合作项目(2014-CG-25);甘肃省自然基金项目(1506RJZA191)

摘  要:目的建立一种可视化快速检测食源性微生物的新方法。方法设计与沙门菌Inva基因,大肠杆菌uid A基因5'端和3'端互补的巯基化探针和氨基化探针以及靶探针。氨基化探针连接醛基化玻片,并与提取的靶DNA或靶探针互补连接,靶DNA或靶探针另一端再与巯基化探针连接形成复合结构;利用胶体金生物亲和性好的特点,让胶体金和巯基化探针连接,银染放大信号,检测微生物。结果靶探针和靶DNA与两种探针杂交时分别选择1 mmol/L、100 nmol/L、1 nmol/L、100 pmol/L、1 pmol/L 5种浓度,并用不同靶DNA作对照;探针最低浓度为1 pmol/L,细菌靶DNA最低浓度为1 pmol/L,银染结果肉眼清晰可见。进一步验证实验结果,扩增Inva基因和uid A基因,分别得到400 kb和300 kb PCR产物,并与探针杂交银染,也得到了肉眼可见的清晰结果。结论该方法可以快速、简便的检测食源性微生物,市场前景广阔。Objective To establish a new method of visual fast detection of foodborne microorganisms. Methods Thiolation and amination probes complementary to the 5' and 3' ends of the Salmonella Inva gene,E. coli uid A gene,and a target probe were designed. The amination probe was connected to the aldehyde-based slide and complementary to the extracted target DNA or the target probe. The other end of the target DNA or the target probe was further connected with the thiolated probe to form a composite structure. The use of colloidal gold bio-affinity was good,so that the gold and thiolated colloidal gold probe linked to amplify silver staining signal for the detection of microorganisms. Results When the target probe and the target DNA were hybridized with the two probes,five concentrations of 1 mmol/L,100 nmol/L,1 nmol/L,100 pmol/L and 1 pmol/L were selected,with different targeted DNA as control. The lowest concentration of probe was 1 pmol/L and the lowest concentration of bacterial target DNA was 1 pmol/L. The results of silver staining were clearly visible. Further validation of the experimental results showed that the Inva gene and the uid A gene were amplified,and PCR products of 400 kb and 300 kb were obtained,respectively,and hybridized with a probe for silver-stained,and clear results were also obtained. Conclusion This method could detect foodborne microbes quickly and easily with wide prospect of market.

关 键 词:胶体金 银染 食源性微生物 

分 类 号:R446.5[医药卫生—诊断学]

 

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