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目的探讨多重实时PCR(multiplex real-time PCR,MRP)和脉冲场凝胶电泳(pulse field gel elec-trophoresis,PFGE)技术在食源性疾病检测中的应用。方法利用多重实时PCR技术对39份样本增菌培养物进行沙门菌侵袭蛋白A基因(invA)、肠产毒素性大肠埃希菌不耐热肠毒素基因(elt)、志贺菌和肠侵袭性大肠埃希菌侵袭力基因(ipaH)的检测,并根据多重实时PCR的结果有针对性的开展了常规的分离培养鉴定工作。应用PFGE对10株分离菌株进行分子分型。结果 10份患者肛拭检出沙门菌侵袭蛋白A(invA)基因,并从这10份样本中分离到10株都柏林血清型沙门菌。39份样本均未检出肠产毒素性大肠埃希菌不耐热肠毒素基因(elt)、志贺菌和肠侵袭性大肠埃希菌侵袭力基因(ipaH)。10株都柏林血清型沙门菌PFGE条带完全一致。结论多重实时PCR有助于提高细菌性食源性疾病的判明率,指导病原菌的常规分离培养,PFGE可用于细菌性食源性疾病的追踪溯源。
Objective To investigate the application of multiplex real-time PCR (MRP) and pulse field gel electrophoresis (PFGE) in the detection of food-borne diseases. Methods Multiplex real-time polymerase chain reaction (PCR) was used to detect the invA, invitrogen, enterotoxigenic Escherichia coli enterotoxigenic (elt), Shigella and intestinal invasive Escherichia coli invasiveness gene (ipaH) detection, and based on the results of multiple real-time PCR targeted conventional isolation and culture identification. Ten isolates were isolated by PFGE. Results In 10 patients, the invA gene of salmonella was detected by anus swab and ten strains of S.pneumoniae were isolated from these ten samples. None of the 39 samples detected the enterotoxigenic Escherichia coli heat-labile enterotoxin gene (elt), Shigella and enteroinvasive Escherichia coli invasion gene (ipaH). Ten strains of S. typhimurium PFGE bands were identical. Conclusion Multiple real-time PCR is helpful to improve the identification rate of bacterial food-borne diseases and to guide the routine isolation and culture of pathogenic bacteria. PFGE can be used for the tracing of bacterial food-borne diseases.