基于核酸预混室温储运技术的荧光定量PCR快速检测鼠疫耶尔森氏菌

Yersinia pestis detection through rapid fluorescence quantitative PCR based on a nucleic acid premix,and storage and transportation at room temperature

目的实现荧光定量PCR技术在鼠疫现场和基层的应用。方法本试验将已建立的基于核酸预混室温储运技术的荧光定量PCR方法应用于我国各鼠疫疫源地野生菌株的检测,进行特异性评价。选取分布于我国境内不同鼠疫疫源地野生鼠疫耶尔森氏菌株、鼠疫减毒菌株、耶尔森菌属近缘菌株假结核菌及小肠结肠炎菌进行荧光定量PCR检测。结果55株鼠疫耶尔森氏菌及鼠疫减毒菌株扩增阳性,假结核耶尔森氏菌、小肠结肠炎耶尔森氏菌无阳性扩增,冻干试剂在室温25℃和37℃条件下可保存6个月,敏感性与冷冻保存无差异,核酸扩增诊断可在1 h内完成。结论应用基于核酸预混室温储运技术的荧光定量PCR方法对我国各鼠疫疫源地55株鼠疫耶尔森氏菌检测呈现高度特异性,本试验冻干试剂具有可室温保存、便于运输、检测结果精准、快速等特点,具有较好的鼠疫现场应用前景。

To enable fluorescence quantitative PCR detection for field and grass-roots applications,a fluorescence quantitative PCR method based on a nucleic acid premix,and room temperature storage and transportation was used to detect wild strains in plague foci in China,and the specificity was evaluated.A total of 55 wild Yersinia pestis strains,attenuated plague strains,Yersinia pseudotuberculosis strains and Yersinia enterocolitis strains distributed in different plague foci in China were detected by fluorescence quantitative PCR.A total of 55 strains of Yersinia pestis and attenuated strains were positive for amplification,but no positive amplification was found for pseudo tuberculosis Yersinia pestis and Yersinia enterocolitica.After storage for 6 months at 25℃and 37℃at room temperature,the lyophilized reagents yielded a sensitivity no different from that with cryopreservation.The nucleic acid amplification could be completed within 1 h.The fluorescence quantitative PCR method based on a nucleic acid premix,and storage and transportation at room temperature had high specificity for the detection of 55 strains of Yersinia pestis in various plague foci China.The lyophilized reagent can be stored at room temperature,which is also convenient temperature for transportation;and the detection results are accurate and rapid.Therefore,this method has good prospects for field application in plague foci.