Detection Of Environmental Pseudomonas Aeruginosa Isolates Using Loop-Mediated Isothermal Amplification (Lamp) Assay

Abstract

Pseudomonas aeruginosa is ubiquitous in natural settings but can also cause human diseases. Rapid detection of this bacteria will contribute to the early diagnosis of bacterial infection. In this work, we aimed to develop a rapid detection for environmental P. aeruginosa strains using loop-mediated isothermal amplification (LAMP) assay targeting a gene encoding for a hypothetical protein gene (GenBank ID: 882161). The experiment consisted of two parts: 1) Isolating P. aeruginosa-like bacteria from the environment and 2) Evaluating established LAMP assay on P. aeruginosa-like isolates. Fifty-two samples were collected from different geographical regions in Ho Chi Minh City (HCMC). Pseudomonas isolates were subjected to duplex polymerase chain reaction (PCR) targeting P. aeruginosa oprL-algD gene. Five of fourteen P. aeruginosa-like environmental strains were randomly selected for 16S rRNA sequencing. For evaluating the LAMP assay, the primer sequences were both obtained from a previous study and designed using NEB LAMP Primer Design. PCR using F3 and B3 was tested to check the presence of a hypothetical protein gene (GenBank ID: 882161) used for LAMP assay. Next, LAMP at 60, 63, and 65oC for 30 min and 45 min was optimized and LAMP was tested on all five confirmed P. aeruginosa following the instruction of the WarmStart® LAMP Kit. The sensitivity and specificity in identifying P. aeruginosa were evaluated to analyze the detection ability of the LAMP method. Among fifty-two isolates, there was a high prevalence of P. aeruginosa-like morphology in our population (96% of total samples). Regarding duplex-PCR, the five randomly selected isolates were P. aeruginosa which was confirmed by 16S rRNA sequencing. Interestingly, the LAMP assay showed 100% specificity, and the limitation of this assay was 10-3 ng/ul, the findings of which were equivalent to PCR when compared. In conclusion, the LAMP assay was a potential method to identify environmental P. aeruginosa isolates, which was better than the culture method and comparable to conventional PCR without the requirement of a thermal cycler. With more research, LAMP can become a rapid test for environmental P. aeruginosa in the future.