IPaH-1基因在志贺氏菌表达的检测及PCR条件的优化

Detection of IPaH1 gene in the Shigella and optimization of PCR conditions

目的检测IPaH1基因在志贺氏菌特异性表达水平并对PCR条件进行优化。方法应用设计的针对IPaH1基因的特异性引物,通过PCR法检测IPaH1基因在鲍氏志贺氏菌、野生志贺氏菌、大肠埃希氏菌、出血性大肠埃希氏菌(O157:H7)、肠炎沙门氏菌、阪崎肠杆菌、普通变形杆菌、蜡样芽孢杆菌、粪肠球菌、金黄色葡萄球菌、单增生李斯特菌、副溶血性弧菌、产气荚膜梭菌的表达水平,并对PCR反应中退火温度、最佳引物浓度等进行优化。结果①在志贺氏菌检测到IPaH1基因特异性表达,而大肠埃希氏菌、出血性大肠埃希氏菌(O157:H7)、肠炎沙门氏菌、阪崎肠杆菌、普通变形杆菌、蜡样芽孢杆菌、粪肠球菌、金黄色葡萄球菌、单增生李斯特菌、副溶血性弧菌、产气荚膜梭菌未见表达;②PCR法扩增志贺氏菌IPaH1基因时,第一对引物IPaH1-1的最适退火温度为55.0℃-59.0℃,第二对引物IPaH1-2为51.0℃-55.0℃;两对引物理想终浓度均为0.1μM。结论 IPaH1基因特异表达于志贺氏菌;应用设计的引物通过PCR法扩增志贺氏菌IPaH1基因的最适退火温度分别为55.0℃-59.0℃、51.0℃-55.0℃,最适引物终浓度均为0.1μM。

Objective To detect the expression of IPaH1 genes in Shigella and PCR conditions optimized.Methods The expression of ipaH gene levels in Shigella boydii,wild Shigella,Escherichia coli,Enterohemorrhage E.coli（O157：H7）,Salmonella enterica subsp,Enterobacter sakazakii,Proteus vulgaris,Bacillus cereus was determined by PCR,and the optimization of PCR conditions on annealing temperature and primer concentration was evoluated.Results ① IPaH1 gene specific expression were detected in Shigella while no expression were detected in Escherichia coli,Enterohemorrhage E.coli（O157：H7）,Salmonella enterica subsp,Enterobacter sakazakii,Proteus vulgaris,Bacillus cereus,Enterococcus faecalis,Staphylococcus aureus,Listeria monocytogenes,Vibrio parahaemolyticus,Clostridium perfringens;②Amplification of Shigella ipaH1 gene by PCR,the first primers of IPaH1-1 optimal annealing temperature is 55.0℃-59.0℃,and the second ones of IPaH1-2 is 51.0℃-55.0℃;the final optimal concentration of primers is 0.1 μM.Conclusion IPaH1 gene specifically expressed in Shigella and its optimal annealing temperature was respectively 56°C,52°C,the optimal final concentration of primers is 0.1 μM.