3种分子生物学方法检测金黄色葡萄球菌的 比较研究

Comparative study on 3 kinds of molecular amplification methods for rapid detection of Staphyloccocus aureus

中文关键词:  金黄色葡萄球菌  常规PCR  实时荧光定量PCR  环介导等温扩增技术

英文关键词:Staphylococcus aureus  conventional PCR  real-time quantitative PCR  loop-mediated isothermal amplification

中文摘要:

      目的 比较常规聚合酶链反应 (polymerase chain reaction, PCR)、实时荧光定量PCR (real-time quantitative PCR, qPCR)和环介导等温扩增反应(loop-mediated isothermal amplification, LAMP)对金黄色葡萄球菌的检测效果。方法 以金黄色葡萄球菌(Staphyloccocus aureus)标准菌株(ATCC6538)DNA为模板, 以耐热核酸酶(Nuc)基因为靶标, 采用常规PCR、qPCR和LAMP对其进行扩增并比较其检测灵敏度, 同时对鸡蛋中金黄色葡萄球菌的检出率进行比较。结果 常规PCR对S. aureus的检测灵敏度为400个基因拷贝数, 低于实时荧光定量PCR和LAMP的40个基因拷贝数的检出限。在实际样品检测中常规PCR对50份鸡蛋外壳样品中S. aureus的检出率为6%, qPCR和LAMP的检出率分别为10%, 而在鸡蛋内容物中均未检测到。结论 3种检测方法检测S. aureus均具有较高的灵敏度, 但常规PCR的灵敏度低于qPCR和LAMP检测方法, qPCR需要昂贵的仪器, 不适合基层现场应用, 而LAMP方法简单、快捷, 具有良好的应用前景。

英文摘要:

      Objective To compare the detection effect of Staphylococcus aureus by conventional polymerase chain reaction (PCR), real-time quantitative PCR (qPCR) and loop-mediated isothermal amplification (LAMP). Methods Genomic DNA of S. aureus reference strain ATCC6538 was used as the template for 3 kinds of molecular methods with thermonuclease (Nuc) gene as target gene. The conventional PCR, qPCR and LAMP assay were established, and then the sensitivity and detection rate of S. aureus in eggs were compared. Results DNA copy number of conventional PCR was 400 for detection of S. aureus, which was lower than the detection limits of LAMP and qPCR with DNA copy number of 40. In the assay of 50 egg samples, the detection rate of S. aureus in the eggshells by LAMP was 10%, which was equal to qPCR, but higher than conventional PCR (6%). S. aureus was not detected in the egg content. Conclusion Three kinds of molecular amplification methods for detection of S. aureus all have high sensitivity. But the sensitivity of conventional PCR is lower than qPCR and LAMP. qPCR needs expensive instrument, which is not suitable for the field application. LAMP is simple, fast and inexpensive, which had good application prospect in the field.