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

Luxi Jiang,Xiaojun Huang,Meijun Song,Xiaomeng Li,Deguang Mu,Rumeng Gu

doi:10.1186/s12866-021-02363-3

Abstract

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.

Highlights

Legionella pneumophila is an opportunistic waterborne pathogen of significant public health problems, which can cause serious human respiratory diseases (Legionnaires’ disease)
Demonstration and detection of L. pneumophila‐Multiple cross displacement amplification (MCDA) products In order to determine the effectiveness of the L. pneumophila MCDA primers (Table 1), the MCDA assay with Deoxyribo Nucleic Acid (DNA) from pure cultures of L. pneumophila were carried out at 65 °C for 35 mins
The DNA from L. pneumophila (ATCC 33152) was significantly amplified, while no amplification was observed from the DNA of Klebsiella pneumoniae (ATCC 12657), Listeria monocytogenes (ATCC 49593) and the double distilled water (DW) control

Summary

Introduction

Legionella pneumophila is an opportunistic waterborne pathogen of significant public health problems, which can cause serious human respiratory diseases (Legionnaires’ disease). Legionella species are opportunistic pathogens that are widely found in moist soil, freshwater environment and compost materials [1]. It is the causative agent of Legionnaires’ disease (LD), which has two clinical. In 2015, a report showed that there were 1.3 deaths from Legionnaires’ disease per 100,000 individuals in Europe [6]. For high-risk groups, rapid detection of L. pneumophila infection is critical to clinical prognosis. It is urgent to establish a rapid and accurate diagnostic method to guide the treatment of early infection of L. pneumophila

Methods

Results

Conclusion