【作者】 周巍；

【导师】 张伟；

【作者基本信息】 河北农业大学 ， 微生物学， 2008， 硕士

【摘要】 通过不对称PCR技术和基因芯片技术建立了一种准确、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。对两轮不对称PCR反应体系中退火温度、Mg²⁺浓度进行了优化,以确定适宜PCR反应体系和PCR扩增程序。第一轮适宜反应体系分别为:5uL的10×PCR buffer,0.5uL的20mmol/LdNTP,引物F1、R2各1uL（25umol/L）,0.5uL的Taq酶（2.5U）,含有基因组DNA模板的滤膜,用去离子水补足到50 uL。循环的条件为:94℃变性5min,从94℃变性30s、58.8℃退火1 min到72℃延伸30s共25个循环,最后72℃延伸5 min。第二轮适宜反应体系为:5uL的10×PCR buffer,0.5uL的20 mmol/L dNTP,引物R2 1uL（25umol/L）,0.5uL的Taq酶（2.5U）,第一轮的PCR产物1.5uL,用去离子水补足到50uL。循环的条件为:94℃变性5 min,从94℃变性30s、55.6℃退火30s到72℃延伸30s共30个循环,最后72℃延伸5min。设计二十五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对杂交结果进行分析。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。整个样品的检测过程耗时6h。本方法检测灵敏度是10²CFU/mL。针对在线BLAST的不足,建立了16SrDNA基因核苷酸序列本地数据库,并在此基础上进行寡核苷酸探针的本地BLAST以确保其特异性。通过与在线BLAST比较,本地BLAST在准确性、冗余信息的去除以及使用简便性等方面要优于在线BLAST。基因芯片相比较其它检测方法有很大的优势,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。更多还原

【Abstract】 A accurate rapid and sensitive method for detection for common foodborne pathogenic in food samples was established by using asy-PCR and DNA microarray technology.FTA Filter was used as template preparation for asy-PCR.Glass was used as the array support.A mutation region of the 16SrDNA gene was selected as the discrimination target 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.The PCR system was optimized including annealing temperature and the quantities of the Mg²⁺.The first reaction mixture consisted of 5uL 10×PCR buffer,0.5uL 20mmol/L dNTP,1uL of primer F1、R2（25umol/L）,0.5uL Taq （2.5U）（TaKaRa）,FTA filtering,42μL of double-distilled water,the whole system was 50μL;The reaction was run under the following conditions:DNA pre-denaturation at 94℃for 5 min;DNA denaturation at 94℃for 30 s,primer annealing at 58.8℃for 1min, extension at 72℃for 30s,for 25 cycles,and extension at 72℃for 5min.The second reaction mixture consisted of 5uL 10×PCR buffer,0.5uL 20mmol/L dNTP,1uL of primer R2（25umol/L）,0.5uL Taq（2.5U）（TaKaRa）,1.5uL product of the first PeR reaction, 43μL of double-distilled water,the whole system was 50μL;The reaction was run under the following conditions:DNA pre-denaturation at 94℃for 5 min;DNA denaturation at 94℃for 30 s,primer annealing at 55.6℃for 1min,extension at 72℃for 30s,for 25 cycles, and extension at 72℃for 5min.Twenty-five species（genera）-specific oligonucleotide detection probes were synthesized and spotted onto the glass.The 16SrDNA gene amplification products of 15 species of pathogenic bacteria were hybridized to the oligonucleotide array.Hybridization results were analyzed with genepix4.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 enterocolitica,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-βgave weak cross-reaction with Listeria monocytogenes and Bacillus cereus;but the reaction did not affect their detection.There is high identity between Escherichia coli O157:H7and Salmonella,they can be detected through muLtiplex PCR.The process of the detection took six hours from template preparation to the resuLt obtained.The sensitivity of the study was 10²CFU/mL.To overcome the shortages of on-line BLAST,the local nucleotide database of cry genes from Bt was established,and local BLAST was run based on it to ensure the specificity for each cry genes of oligonucleotide probes.By comparing local BLAST with on-line BLAST,advantages such as veracity,removing redundant and convenience were observed in local BLAST.The superiority of oligonucleotide array over other tests lies on its accuracy,rapidity and efficiency in the diagnosis,treatment and control of foodborne infections.更多还原