一种快速、简便的模板提取方法用于PCR检测粪便中的金黄色葡萄球菌的研究

An new template preparation method for rapid and sensitive PCR detection of Staphylococcus aureus in feces

目的建立一种快速、简便的模板提取方法用于PCR检测粪便中的金黄色葡萄球菌。方法利用FTA滤膜,无需增菌,直接从人工污染金黄色葡萄球菌的粪便中提取DNA模板,以金黄色葡萄球菌耐热核酸酶基因(nuc)为靶基因,经过PCR扩增得到279 bp的产物。通过凝胶电泳,和凝胶成像技术观察结果。结果使用FTA滤膜可有效的提取DNA模板,消除了样本中的PCR抑制因子,通过PCR方法检测人工污染的粪便中的金黄色葡萄球菌,检出限为102 cfu/ml,可在6 h内完成检测。结论 FTA滤膜用于PCR检测粪便中的金黄色葡萄球菌操作简便、快速。使用FTA滤膜法制备模板DNA,为食源性致病菌快速检测构建了一个技术平台。

Objective To establish an new template preparation method for rapid and sensitive PCR detection of Staphylococcus aureus in feces .Methods FTA filter membrane was used to extract DNA in Staphylococcus aureus from artificially contaminated feces.Primers targeting the thermostable nuclease gene （nuc） were used to amplify a 279 bp DNA fragment which was confirmed by DNA sequencing .Results Application of FTA filter membrane could effectively extract DNA template and removed PCR inhibitive factor in specimens .In the detection of Staphylococcus aureus from artificially contaminated feces by PCR,the detection limit was 102cfu/ml,which could be finished within 6 hours.Conclusion PCR amplification using FTA filter membrane provides a faster and more sensitive method of detecting Staphylococcus aureus in feces.At the same time,which establishes a technological platform for rapid detection of foodborne pathogenic bacteria .