致病性小肠结肠炎耶尔森氏菌实时荧光RPA检测方法的建立

本研究建立了致病性小肠结肠炎耶尔森氏菌的实时荧光重组酶聚合酶扩增(real-timeRecombinasePolymerase Amplification,real-time RPA)检测方法。根据致病性小肠结肠炎耶尔森氏菌黏附侵袭位点ail致病基因序列设计特异性引物和exo探针,在37℃恒温下20 min内即可完成检测,方法特异性高,对目的DNA的检测限为0.1 ng/μL。在稳定性实验中,对10 ng/μL、0.1ng/μL、0.01 ng/μL、0.001 ng/μL的目的DNA各进行8个平行的测试,0.1 ng/μL及以上浓度的DNA均可稳定检出;当DNA为0.1 ng/μL时,8个平行的阈值时间和最大荧光值的相对标准偏差(relative standard deviation,RSD)最低且≤7.06%。致病性小肠结肠炎耶尔森氏菌在奶粉和牛肉的人工污染样品中初始污染量为3 CFU/25 g时,培养20 h后,可用本方法检出。对20份各种类实际样品进行检测,本方法与国标方法结果一致。本方法对致病性小肠结肠炎耶尔森氏菌的常温现场快速定性检测具有重要意义。

Development of a Real-time RPA Assay for Pathogenic Yersinia enterocolitica Detection

A real-time recombinase polymerase amplification(RPA) assay for pathogenic Yersinia enterocolitica detection was established in this work. According to the sequences of Yersinia enterocolitica pathogenic ail(Attachment Invasion Locus) gene, the specific primer and exo probe were designed and the detection could be finished within 20 mins at 37 ℃. This real-time RPA assay showed a good specificity and the detection limit of the target DNA was 0.1 ng/μL. In stability study, the assay was performed at the DNA concentration of 10 ng/μL, 0.1 ng/μL, 0.01 ng/μL and 0.001 ng/μL in octuplicate. The concentration of 0.01 ng/μL and above could be detected. The relative standard deviation(RSD) of threshold time and maximum fluorescence values of the eight parallels at 0.1 ng/μL DNA were the lowest(≤7.06%). In artificially contaminated powdered milk and beef samples with an initial bacterial concentration of 3 CFU/25 g, the pathogen can be detected using this real-time RPA method after incubation for 20 hours. The detection of 20 different real samples showed that the results of real-time RPA assay were consistent with that of GB 4789.8-2016, which is the national standard of China to detect Yersinia enterocolitica. This real-time RPA assay is helpful for the rapid field inspection at room temperature when conducting qualitative detection of pathogenic Yersinia enterocolitica.