多种食源性致病菌的基因芯片检测技术.pdfVIP

[]PCRClustal X Oligo 6.0 DNA PCR 164DNA DNA9 2 3 CV DNA10 10 pg/ l ( )15% 3.54 105 cfu/ml PCR []DNA []R378 []A []0577-7402(2012)08-0765-05 1 2 2 2 1 1 1 1* GAO Xing , QU Xian-feng , SUN Wei , LI Hua-yu , XIN Wen-wen , GAO Shan , KANG Lin , WANG Jing-lin 1State Key Laboratory of Pathogen and Biosecurity, Institute of Microbiology and Epidemiology, Academy of Military Medical Scien ces, Beijing 100071, China 2Institute of Environment Protection and Inspection, No. 63850 Troops, Baicheng , Jilin 137001, China * Corresponding author, E-mail: wangjlin@bmi.ac.cn This work was supported by Infection Diseases Special Project, Minister of Health of China (2008ZX10004-001) [Abstract]ObjectiveTo study the application of DNA microarray technique for screening and identifying multiple food- borne pathogens. MethodsThe oligonucleotide probes were designed by Clustal X and Oligo 6.0 at the conserved regions of specific genes of multiple food-borne pathogen s, and then were validated by bioinformatic analyses. The 5 end of each probe was modified by amino-group and 10 Poly-T, and the optimized probes were synthesized and spotted on aldehyde-coated slides. The bacteria DNA template incubated with Klenow enzyme was amplified by arbitrarily primed PCR, and PCR products incorporated into Aminoallyl-dUTP were coupled with fluorescent dye. After hybridization of the purified PCR products with DNA microarray, the hybridization image and fluorescence intensity analysis was acquired by ScanArray and GenePix Pro 5.1 software. A series of detection conditions such as ar