Yan Chao, Zhou Yao, Du Shuheng, Du Bing, Zhao Hanqing, Feng Yanling, Xue Guanhua, Cui Jinghua, Gan Lin, Feng Junxia, Fan Zheng, Fu Tongtong, Xu Ziying, Zhang Qun, Zhang Rui, Cui Xiaohu, Tian Ziyan, Chen Yujie, Zhang Ting, Huang Lei, Yuan Jing
Department of Bacteriology, Capital Institute of Pediatrics, Beijing, China.
Children's Hospital Capital Institute of Pediatrics, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China.
Microbiol Spectr. 2023 Mar 13;11(2):e0398422. doi: 10.1128/spectrum.03984-22.
Hypervirulent Klebsiella pneumoniae (hvKp) is a major human pathogen associated with liver abscess, pneumonia, meningitis, and endophthalmitis. It is challenging to differentiate hvKp from classical Klebsiella pneumoniae (cKp) using conventional methods, necessitating the development of a rapid, sensitive, and convenient assay for hvKp detection. In this study, we constructed a recombinase-aided amplification (RAA) method targeting hvKp genes and , and also analyzed the pathogenic characteristics of hvKp. We optimized the reaction temperature and system, and evaluated its sensitivity, specificity, and clinical application. The primer and probe sets -set1 and -set2 delivered significant fluorescent signals at 39°C with the shortest gene amplification times (sensitivity: 20 copies/reaction). This RAA assay showed no cross-reactivity with 15 other common pathogenic bacteria. Its applicability was confirmed by the evaluation of 208 clinical specimens, of which 45 were confirmed to be hvKp. The sensitivity and specificity of the RAA assay were both 100% compared with real-time PCR as the reference standard. To verify the assay, we also assessed the diversity of molecular characteristics among the hvKp isolates and identified serotype K1 and sequence type ST23 as the dominant clone. Virulence factors and were highly associated with virulence level. In conclusion, our novel RAA assay is a powerful tool for early diagnosis and epidemiological surveillance of hvKp. Klebsiella pneumoniae is the most common opportunistic bacterial species and a major threat to public health. Since the 1990s, hvKp has received increasing attention from public health officials and infectious disease specialists. Hypervirulent strains differ from classical strains in terms of phenotypic features and clinical outcomes. It is hard to identify hvKp from cKp using the conventional methods including colony morphology analysis, serum killing assays, mouse lethality assays, string tests, and real-time PCR. In this study, we established a rapid, sensitive and convenient recombinase-aided amplification assay for hvKp detection targeting virulence genes and . Our RAA assay provides an important tool for the rapid diagnosis of infectious diseases caused by hvKp, particularly in primary laboratories.
高毒力肺炎克雷伯菌（hvKp）是一种主要的人类病原体，与肝脓肿、肺炎、脑膜炎和眼内炎有关。使用传统方法将hvKp与经典肺炎克雷伯菌（cKp）区分开来具有挑战性，因此需要开发一种快速、灵敏且便捷的检测方法来检测hvKp。在本研究中，我们构建了一种针对hvKp基因 和 的重组酶辅助扩增（RAA）方法，并分析了hvKp的致病特征。我们优化了反应温度和体系，并评估了其灵敏度、特异性和临床应用。引物和探针组-set1和-set2在39°C时发出显著的荧光信号，基因扩增时间最短（灵敏度：20拷贝/反应）。该RAA检测方法与其他15种常见病原菌无交叉反应。通过对208份临床标本的评估证实了其适用性，其中45份被确认为hvKp。以实时荧光定量PCR作为参考标准，RAA检测方法的灵敏度和特异性均为100%。为了验证该检测方法，我们还评估了hvKp分离株之间分子特征的多样性，并确定血清型K1和序列型ST23为优势克隆。毒力因子 和 与毒力水平高度相关。总之，我们的新型RAA检测方法是早期诊断和监测hvKp流行病学的有力工具。肺炎克雷伯菌是最常见的机会致病菌，对公众健康构成重大威胁。自20世纪90年代以来，hvKp越来越受到公共卫生官员和传染病专家的关注。高毒力菌株在表型特征和临床结果方面与经典菌株不同。使用包括菌落形态分析、血清杀菌试验、小鼠致死试验、拉丝试验和实时荧光定量PCR在内的传统方法很难从cKp中鉴定出hvKp。在本研究中，我们建立了一种快速、灵敏且便捷的重组酶辅助扩增检测方法，用于靶向毒力基因 和 的hvKp检测。我们的RAA检测方法为快速诊断由hvKp引起的传染病提供了重要工具，特别是在基层实验室。
