Duan Zhixiong, Wang Shan, Xie Niqi, Zhao Junying, Dong Jian, Li Jin
Department of Laboratory Medicine, The Affiliated Dazu's Hospital of Chongqing Medical University, Chongqing, China.
Department of Laboratory Medicine, The Chen Jia qiao Hospital of Sha Ping Ba District, Chongqing, China.
Heliyon. 2024 Aug 30;10(17):e37050. doi: 10.1016/j.heliyon.2024.e37050. eCollection 2024 Sep 15.
Our objective was to establish a rapid and precise method for detecting hypervirulent (hvKP) by utilizing a duplex real-time multienzyme isothermal rapid amplification (real-time MIRA) and to evaluate its performance in clinical spiked blood specimens.
The research comprised two phases: an initial pilot study to establish the methodology and a clinical validation study to assess its effectiveness. In the pilot phase, we designed specific primers and probes targeting the hvKP and genes and subsequently developed a duplex real-time MIRA assay to evaluate its detection limits, specificity, and efficiency. In the clinical validation phase, we analyzed thirty-three spiked blood specimens using the duplex real-time MIRA assay.
The duplex real-time MIRA assay demonstrated no cross-reactivity with other strains. Sensitivity experiments confirmed that the assay had a detection limit as low as 8 × 10 CFU per reaction for hvKP. The analysis of clinical spiked blood specimens indicated that the sensitivity and specificity of the duplex real-time MIRA assay were on par with those of duplex real-time PCR.
These findings confirm that the duplex real-time MIRA assay is a fast, straightforward, and dependable method for detecting hvKP.
我们的目标是利用双链实时多酶等温快速扩增技术（实时MIRA）建立一种快速、精确检测高毒力肺炎克雷伯菌（hvKP）的方法，并评估其在临床加标血液标本中的性能。
本研究包括两个阶段：建立方法的初步预试验研究和评估其有效性的临床验证研究。在预试验阶段，我们设计了针对hvKP的 和 基因的特异性引物和探针，随后开发了一种双链实时MIRA检测方法，以评估其检测限、特异性和效率。在临床验证阶段，我们使用双链实时MIRA检测方法分析了33份加标血液标本。
双链实时MIRA检测方法与其他菌株无交叉反应。敏感性实验证实，该检测方法对hvKP的检测限低至每个反应8×10 CFU。对临床加标血液标本的分析表明，双链实时MIRA检测方法的敏感性和特异性与双链实时PCR相当。
这些结果证实，双链实时MIRA检测方法是一种快速、简便且可靠的检测hvKP的方法。
