A Loop-mediated Isothermal Amplification With a Nanoparticle-Based Lateral Flow Biosensor Assay to Detect Pseudomonas aeruginosa in Endophthalmitis.

PURPOSE

Pseudomonas aeruginosa is the most common bacteria causing endophthalmitis after cataract surgery. Vitreous fluid culture and molecular studies are commonly used in clinical diagnoses, but have disadvantages, such as a long culture cycle and low detection sensitivity. Here, we report a loop-mediated isothermal amplification (LAMP) method combined with the nanoparticles-lateral flow biosensor (LFB) method for rapid and specific detection of P. aeruginosa.

METHODS

A set of six primers was designed to target the OprL gene of P. aeruginosa. Genomic DNA extracted from several gram-negative and gram-positive bacteria was used to determine the sensitivity and specificity of the analysis. LAMP reactions were conducted at 65 °C for 50 minutes, and results were reported using the LFB method.

RESULTS

The DNA template of P. aeruginosa was specifically recognized by the P. aeruginosa-LAMP-LFB (PA-LAMP-LFB) method as no cross reactions were observed for non-P. aeruginosa templates. The analytical sensitivity of our assay was 100 fg per test for the pure cultured DNA template, and the result obtained using the LFB was consistent with that of colorimetric indicator detection. The whole test could be completed within 1h. This method was used to detect P. aeruginosa, Staphylococcus aureus, and Klebsiella pneumoniae; only P. aeruginosa was positive. The positive rates of P. aeruginosa detected by a traditional culture method, the LAMP-LFB method, and the fluorescence quantitative polymerase chain reaction method were 17.7%, 17.7%, and 13.3%, respectively.

CONCLUSIONS

The P. aeruginosa-LAMP-LFB method established here is a rapid, specific, and sensitive method for the detection of P. aeruginosa, which can be widely used.