实验动物金黄色葡萄球菌LAMP法快速检测

Rapid Detection of Staphylococcus aureus in Laboratory Animals by Loop-mediated Isothermal Technique

目的建立一种灵敏、高效,可用于实验动物金黄色葡萄球菌检测的环介导等温扩增技术(Loop-mediated isothermal amplification,LAMP)。方法针对金黄色葡萄球菌nuc基因(V01281.1)参考设计4条LAMP扩增引物,并选取常见实验动物致病菌进行引物特异性检测;通过优化LAMP反应体系中Mg^(2+)浓度、dNTPs浓度及LAMP反应时间,确立LAMP反应最佳条件;本文采用LAMP法和PCR法对不同浓度梯度的金黄色葡萄球菌以及用不同浓度金黄色葡萄球菌人工感染的粪样进行检测,比较两种方法的检测灵敏度。结果通过条件优化,本实验建立的LAMP法检测实验动物金黄色葡萄球菌的最佳反应温度为60℃,反应时间为50 min,优化后的LAMP反应体系为dNTP浓度2.0 mmol/L、Mg^(2+)浓度8.0 mmol/L、引物对FIP/BIP浓度1.6μmol/L、引物对F3/B3浓度0.2μmol/L,且与其它常见实验动物致病菌无交叉反应;对金葡菌纯培养物进行检测,LAMP法检测灵敏度为9CFU/反应管(9×10~3CFU/mL),PCR法检测灵敏度为9×10~1CFU/反应管(9×10~4CFU/mL),对人工接种金黄色葡萄球菌的SD大鼠粪样进行检测,LAMP法检测灵敏度为4.49×10~4CFU/0.1 g粪样,PCR法检测灵敏度为4.49×10~5CFU/0.1 g粪样。结论本实验建立的实验动物金黄色葡萄球菌LAMP检测方法,与PCR法,免疫学法和传统检测方法比较,具有特异性强、灵敏度高、操作简单等特点,可应用于实验动物金黄色葡萄球菌的快速检测。

Objective To establish a sensitive and efficient method for detecting Staphylococcus aureus in laboratory animals.Method A set of four primers targeting the heat-stable nuclease （nuc） gene （V01281.1） of S.aureus were designed and the specifity of the Lamp assay was evaluated by common pathogenic bacteria in laboratory animals.The reaction conditions of LAMP were optimized including Mg2＋ concentration、dNTPs concentration and time.Using LAMP and PCR method to detect S.aureus in pure culture and artificially contaminated feces and compared their sensitivity.Result No cross reaction was found in non-S.aureus bacteria and the optimal reaction condition of LAMP were 1.6 μmol/L of each inner primer,0.2 μmol/L of each outer primer,2.0 mmol/L of each dNTP,8.0 mmol/L Mg2＋ and incubated at 60 ℃ for 50 min.In pure culture,the detection limit of LAMP was 9 CFU/test （9 × 103 CFU/mL） compared with 9 × 101 CFU/test （9 × 104 CFU/mL） of PCR.In artificially contaminated feces,the detection limit of LAMP was 4.49 × 104 CFU/0.1 g feces compared with 4.49 × 105 CFU/0.1 g feces of PCR.Conclusion The developed LAMP method is simple、sensitive and specific,and is available for rapid detection of S.aureus in laboratory animals.