摘要
通过不对称PCR技术和基因芯片技术建立一种准确、快速和高敏感性的检测肉中常见食源性致病菌的方法。用FTA滤膜从食品样品中直接提取模板DNA,用玻片作基因芯片的固相载体。选择常见的可引起食物中毒的15株菌的16SrDNA基因的一个片段作为目的基因。设计这段基因的一对通用引物,下游引物用Cy5标记。设计25条种属的特异性探针,将它们氨基化修饰并点到玻片上,将不对称PCR的15种扩增产物与制作好的基因芯片杂交,用genepix4分析杂交结果。结果表明:10株食源性致病菌(金黄色葡萄球菌、肉毒梭状芽孢杆菌、产气荚膜梭菌、宋内氏志贺氏菌、霍乱弧菌、普通变形杆菌、拟态弧菌、单核增生李斯特菌、小肠结肠炎耶尔森氏菌、腊样芽孢杆菌)的检测具有高敏感性和特异性;副溶血性弧菌的敏感性相对较低;河流弧菌同副溶血性弧菌存在很弱的错杂交;乙型溶血性链球菌的检测同单核增生李斯特菌和腊样芽孢杆菌存在很弱的错杂交,但是均不影响检测结果。大肠杆菌O157:H7和沙门氏菌由于同源性非常高,可以通过多重PCR方法检测出来。整个样品的检测耗时6h。基因芯片与其它检测方法相比有很大的优势,它不仅准确、快速、高效,而且高通量,可广泛应用于食源性致病菌感染的诊断、应对和控制。
An accurate, rapid and sensitive method for detection of common foodborne pathogen in food samples was established by using asy-PCR and gene chip technology FTA filter was used as template preparation for asy-PCR. Glass slide was used as the array support. A mutation region of the 16SrDNA gene was selected as the target gene from 15 species of bacteria causing foodborne infections. A pair of universal primers was designed for asy-PCR amplification of the 16SrDNA gene. The backward primer was labeled by Cy5. Twenty-five species （genera）-specific oligonucleotide de- tection probes were synthesized and spotted onto the glass slide, The 16SrDNA gene amplification products of 15 species of pathogenic bacteria were hybridized to the oligonucleotide array, Hybridization results were analyzed with geneplx4. Results indicate that ten species of pathogenic bacteria （Staphylococcus aureus, Clostridium botulinum, Clostridium perfringens , Shigella sonnei, Vibrio cholerae , Proteus vulgaris , Vibrio mimicus , Listeria monocytogenes , Yersinia enteracolitica, Bacillus cereus） show high sensitivity and specificity for the oligonucleotide array; Vibrio parahaemolyticus showed low sensitivity for the oligonucleotide array; Vibrio fluvialis gave weak cross-reaction with Vibrio parahaemolyticus; Streptococcus hemolytic--β gave weak cross-reaction with Listeria monocytogenes and Bacillus cereus; but the reaction did not affect their detection. There was high identity between Escherichia coli O157：H7 and Salmonella, they could be detected through multiplex PCR. The process of the detection took six hours from template preparation to the result obtained , The superiority of gene chip method over other tests lies on its accuracy, rapidity and efficiency in the diagno- sis, treatment and control of foodborne pathogenic infections.
出处
《中国食品学报》
EI
CAS
CSCD
2008年第4期113-122,共10页
Journal of Chinese Institute Of Food Science and Technology
