Key Laboratory of Zoonosis Research, Ministry of Education, Institute of Zoonosis, Jilin University, Changchun, 130062, China.
Key Laboratory of Zoonosis Research, Ministry of Education, Institute of Zoonosis, Jilin University, Changchun, 130062, China.
Anal Biochem. 2021 May 15;621:114157. doi: 10.1016/j.ab.2021.114157. Epub 2021 Mar 9.
Pathogenic Yersinia (Y.) enterocolitica is the primary causative agent of Yersiniosis, with outbreaks in numerous countries around the world, and causes diarrhea and vomiting in animals and humans. Therefore, an instrument-free and convenient nucleic acid visualization method, RPA-SYBR Green I, was established, which combines recombinase polymerase amplification (RPA) with the fluorescent dye SYBR Green I for the detection of the adhesion gene ail in pathogenic Y. enterocolitica. After optimization of a series of conditions such as primer concentration, the detection of pathogenic Y. enterocolitica could be finally completed within about 20 min (from DNA extraction to observation of results) at an isothermal temperature of 39°C. RPA-SYBR Green I had no cross-reactivity with other bacteria and the detection limit was 10 CFU/μL, with sensitivity equal to that of conventional PCR. The method established in this paper and conventional PCR identified a total of 5 spiked samples and 15 meat samples stored in refrigerated, and it was concluded that there was 100% consistency between the two methods. Overall, RPA-SYBR Green I is a visual and facilitate detection assay that can accurately discover pathogenic Y. enterocolitica.
致病性耶尔森菌（Y.）是肠结肠炎耶尔森菌的主要病原体，在世界上许多国家都有爆发，并导致动物和人类腹泻和呕吐。因此，建立了一种无仪器和方便的核酸可视化方法 RPA-SYBR Green I，它将重组酶聚合酶扩增（RPA）与荧光染料 SYBR Green I 结合，用于检测致病性 Y. enterocolitica 的粘附基因 ail。经过一系列条件（如引物浓度）的优化，最终可以在 39°C 的等温温度下，大约 20 分钟内（从 DNA 提取到观察结果）完成对致病性 Y. enterocolitica 的检测。RPA-SYBR Green I 与其他细菌无交叉反应，检测限为 10 CFU/μL，与常规 PCR 的灵敏度相当。本文建立的方法和常规 PCR 共鉴定了 5 个冷藏添加样本和 15 个肉类样本，两种方法的一致性为 100%。总的来说，RPA-SYBR Green I 是一种可视化且方便的检测方法，可以准确发现致病性 Y. enterocolitica。
