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目的建立简单、快速环介导恒温扩增技术(LAMP)检测鼠疫耶尔森菌的方法。方法基于鼠疫菌染色体上特异的核酸序列3a设计引物;应用浊度仪和目视法对反应结果进行检测;通过对鼠疫菌和其他近源菌同时进行检测评价方法的特异性;通过对不同稀释浓度的鼠疫菌DNA模板进行LAMP和PCR检测以确定方法的灵敏度。结果用建立的LAMP法对与鼠疫菌近源30种其他菌株进行扩增,结果均为阴性,该方法具有很高的特异性。LAMP法检测鼠疫菌DNA的灵敏度可达到20 pg,比常规PCR法高10倍。检测反应在25 min以内完成。结论该方法具有快速、灵敏、特异、操作简单的特点,有望发展成为现场快速检测鼠疫菌的有效手段。
Objective To establish a simple and rapid method for the detection of Yersinia pestis with ring-mediated isothermal amplification (LAMP). Methods Primers were designed based on the specific nucleic acid sequence 3a on the chromosome of Y. pestis. The reaction results were detected by turbidimetry and visual inspection. The specificity of the method was evaluated by simultaneous detection of Y. pestis and other near-source bacteria. Concentration of Yersinia pestis DNA template for LAMP and PCR assays to determine the sensitivity of the method. Results Using the established LAMP method, 30 other strains of Yersinia pestis were amplified, and the result was negative. The method was highly specific. The sensitivity of LAMP method to detect Yersinia pestis DNA is up to 20 pg, which is 10 times higher than that of conventional PCR. Detection reaction is completed within 25 min. Conclusion The method is rapid, sensitive, specific and easy to operate. It is expected to be an effective method for rapid detection of Yersinia pestis in the field.