Multiple Cross Displacement Amplification Coupled with Lateral Flow Biosensor (MCDA-LFB) for rapid detection of Legionella pneumophila

BMC Microbiology

Xiaojun Huang¹  Luxi Jiang¹  Meijun Song¹  Rumeng Gu²  Xiaomeng Li³  Deguang Mu⁴ 

[1] Department of Respiratory Medicine, Zhejiang Provincial People’s Hospital, People’s Hospital of Hangzhou Medical College, Hangzhou, Zhejiang, People’s Republic of China;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;Department of Respiratory and Critical Care Medicine, Shanghai Fourth People’s Hospital Affiliated to Tongji University, 1279 Sanmen Road, 200080, Shanghai, People’s Republic of China;Department of respiratory and critical care medicine, Xi’an Daxing Hospital, Xi’an, China;

关键词: L. pneumophila;    Multiple cross displacement amplification;    Lateral flow biosensor;    MCDA-LFB;    Limit of detection;   

DOI  :  10.1186/s12866-021-02363-3

来源: Springer

【 摘 要 】

BackgroundLegionella 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.ResultsA 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.ConclusionsIn 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.

【 授权许可】

CC BY