多重PCR快速检测两种致泻性大肠杆菌方法的建立
Establishment of a multiplex PCR method for rapid detection of two kinds of diarrheagenic Escherichia coli
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摘要: 本研究以肠出血性大肠杆菌O157∶H7及肠侵袭性大肠杆菌为目标菌,针对大肠杆菌共同基因uid A、肠出血性大肠杆菌O157∶H7的特异基因O-antigen、肠侵袭性大肠杆菌的特异基因ipa H设计3对引物,建立并优化了检测两种菌的多重PCR检测方法,进行了特异性验证和灵敏度分析,并应用于人工接种新鲜莴苣的检测中。实验结果表明,本研究建立的三重PCR快速检测两种致病菌的检出限为6.3×103CFU/m L,具有较好的灵敏度和特异性。利用所建立的多重PCR方法对人工接种肠出血性大肠杆菌O157∶H7和肠侵袭性大肠杆菌的新鲜莴苣进行检测,检出限为7.8×104CFU/m L。此方法能够对肠出血性大肠杆菌O157∶H7和肠侵袭性大肠杆菌两种食源性致病菌进行快速检测。Abstract: To establish a rapid multiplex PCR method, which can simultaneously detect Enterohemorrhagic Escherichia coli O157∶ H7 and Enteroinvasive Escherichia coli, three pairs of primers had been designed based on the O- antigen gene in Enterohemorrhagic Escherichia coli O157 ∶ H7, ipa H gene in Enteroinvasive Escherichia coli and uid A gene in all Escherichia coli.The method was established and optimized, and the specificity and sensitivity were also analyzed. This method was applied to the detection of the artificially infected fresh lettuce with two pathogens.The results showed that the triplex PCR could detect two kinds of diarrheagenic Escherichia coli. The sensitivity was 6.3 × 103 CFU / m L.This method had high specificity and sensitivity.The sensitivity of the detection of artificial lettuce infected with two pathogens was 7.8 × 104 CFU / m L.It was suggested that the method was suitable for rapid detection of Enterohemorrhagic Escherichia coli O157 ∶ H7 and Enteroinvasive Escherichia coli in fresh agricultural products.
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