Department of Respiratory Medicine, Zhejiang Provincial People's Hospital, People's Hospital of Hangzhou Medical College, Hangzhou, Zhejiang, People's Republic of China.
Graduate School of Clinical Medicine, Bengbu Medical College, Bengbu, China.
BMC Microbiol. 2022 Jan 8;22(1):20. doi: 10.1186/s12866-021-02363-3.
Legionella pneumophila is an opportunistic waterborne pathogen of significant public health problems, which can cause serious human respiratory diseases (Legionnaires' disease). Multiple cross displacement amplification (MCDA), a isothermal nucleic acid amplification technique, has been applied in the rapid detection of several bacterial agents. In this report, we developed a MCDA coupled with Nanoparticles-based Lateral Flow Biosensor (MCDA-LFB) for the rapid detection of L. pneumophila.
A set of 10 primers based on the L. pneumophila specific mip gene to specifically identify 10 different target sequence regions of L. pneumophila was designed. The optimal time and temperature for amplification are 57 min and 65 °C. The limit of detection (LoD) is 10 fg in pure cultures of L. pneumophila. No cross-reaction was obtained and the specificity of MCDA-LFB assay was 100%. The whole process of the assay, including 20 min of DNA preparation, 35 min of L. pneumophila-MCDA reaction, and 2 min of sensor strip reaction, took a total of 57 min (less than 1 h). Among 88 specimens for clinical evaluation, 5 (5.68%) samples were L. pneumophila-positive by MCDA-LFB and traditional culture method, while 4(4.55%) samples were L. pneumophila-positive by PCR method targeting mip gene. Compared with culture method, the diagnostic accuracy of MCDA-LFB method was higher.
In summary, the L. pneumophila-MCDA-LFB method we successfully developed is a simple, fast, reliable and sensitive diagnostic tool, which can be widely used in basic and clinical laboratories.
嗜肺军团菌是一种具有重要公共卫生问题的机会性病原体，可引起严重的人类呼吸道疾病（军团病）。多重交叉置换扩增（MCDA）是一种等温核酸扩增技术，已应用于多种细菌的快速检测。在本报告中，我们开发了一种与纳米粒子侧向流动生物传感器（MCDA-LFB）相结合的 MCDA 方法，用于快速检测嗜肺军团菌。
设计了一组基于嗜肺军团菌特异性 mip 基因的 10 条引物，用于特异性识别 10 个不同的嗜肺军团菌靶序列区域。最佳扩增时间和温度为 57 分钟和 65°C。在纯培养的嗜肺军团菌中，检测限（LoD）为 10 fg。未发生交叉反应，MCDA-LFB 检测方法的特异性为 100%。整个检测过程包括 20 分钟的 DNA 制备、35 分钟的 L. 嗜肺军团菌-MCDA 反应和 2 分钟的传感器条带反应，总耗时 57 分钟（不到 1 小时）。在 88 份临床评估标本中，5（5.68%）份标本通过 MCDA-LFB 和传统培养方法检测为嗜肺军团菌阳性，而 4（4.55%）份标本通过针对 mip 基因的 PCR 方法检测为嗜肺军团菌阳性。与培养方法相比，MCDA-LFB 方法的诊断准确性更高。
综上所述，我们成功开发的嗜肺军团菌-MCDA-LFB 方法是一种简单、快速、可靠和敏感的诊断工具，可广泛应用于基础和临床实验室。
