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IPaH-1基因在志贺氏菌表达的检测及PCR条件的优化 被引量:2

Detection of IPaH1 gene in the Shigella and optimization of PCR conditions
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摘要 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.
出处 《中国实验诊断学》 2012年第10期1770-1773,共4页 Chinese Journal of Laboratory Diagnosis
基金 国家质检总局支撑项目项目编号2006IK145:吉林省科技厅20090721
关键词 志贺氏菌 毒力基因 PCR Shigella virulence genes PCR
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