Abstract
Legionella, a waterborne pathogen, is the main cause of Legionnaires’ disease. Therefore, timely and accurate detection and differentiation of Legionella pneumophila and non-Legionella pneumophila species is crucial. In this study, we develop an easy and rapid recombinase polymerase amplification assay combined with EuNPs-based lateral flow immunochromatography (EuNPs-LFIC-RPA) to specifically distinguish Legionella pneumophila and non-Legionella pneumophila. We designed primers based on the mip gene of Legionella pneumophila and the 5S rRNA gene of non-Legionella pneumophila. The recombinase polymerase amplification reaction could go to completion in 10 min at 37°C, and the amplification products could be detected within 5 min with EuNPs-LFIC strips. Using a florescent test strip reader, the quantitative results were achieved by reading the colored signal intensities on the strips. The sensitivity was 1.6 × 101 CFU/ml, and a linear standard linear curve plotted from the test strip reader had a correlation coefficient for the determination of Legionella pneumophila (R 2 = 0.9516). Completed concordance for the presence or absence of Legionella pneumophila by EuNPs-LFIC-RPA and qPCR was 97.32% (κ = 0.79, 95% CI), according to an analysis of practical water samples (n = 112). In short, this work shows the feasibility of EuNPs-LFIC-RPA for efficient and rapid monitoring of Legionella pneumophila and non-Legionella pneumophila in water samples.
Highlights
Legionella is widely found in natural and artificial waters, which can form an aerosol to cause acute infection of the respiratory tract and legionellosis (Albert-Weissenberger et al, 2007)
We present an Europium nanoparticle (EuNP)-lateral flow immunochromatography (LFIC)-recombinase polymerase amplification (RPA) assay to detect Legionella pneumophila and non-Legionella pneumophila
After the RPA amplification products passed through the two detection lines in the EuNPs-LFIC that contained an anti-FAM antibody or an antibiotin antibody, the double-stranded DNA labeled with FAM or biotin would be captured by the corresponding antibody
Summary
Legionella is widely found in natural and artificial waters, which can form an aerosol to cause acute infection of the respiratory tract and legionellosis (Albert-Weissenberger et al, 2007). Legionellosis is an infectious disease with a high recessive infection rate and mortality rates, and it presents a great hazard to human health. Patients with the disease usually have fever, chills, and a cough, which may Detection of Legionella by EuNPs-LFIC-RPA be dry or may produce sputum (Steinert et al, 2007). Since the first confirmed cases of Legionella in Nanjing in 1982, both epidemic and sporadic cases of the disease have been reported in China (Kang et al, 1982). In 2003, the Chinese inspection and quarantine system developed the industry and national standards for Legionella detection and counting based on a conventional culture method in China (HG/T 43232012, 2012). An international standard method that could detect and count live cells of pathogens by culturing on selective agar plates was developed to test whether water samples met the microbial standards (ISO, 2017). Rapid and accurate determination of the pathogen is the key to the diagnosis and control of the disease
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