Loop Mediated Isothermal Amplification as a Rapid Molecular Method for Pseudomonas aeruginosa T3SS ExoY Gene Septicemia Detection in Beta- Lactamase Species Co-Infections

Abstract

Background: Pseudomonas aeruginosa is among the most important causative agent of infection in chronically ill patients admitted in hospitals globally. Coupled with its, mixed symptomatology, rapid drug resistance tendency and its causation of severe disease, a fast, reliable and affordable diagnostic technique is required to enable healthcare providers expeditiously mitigate its progression and eventual treatment. The Loop-Mediated Isothermal Amplification (LAMP) technique has the potential to serve as a simple, rapid, specific, sensitive and cost-effective point-of-care diagnostic tool. Broad Objective: To investigate Loop Mediated Isothermal Amplification as a molecular technique for microbial diagnostic and prognostic predictor.   Study Design: This study was aimed at evaluating LAMP assay against Simple Polymerase chain reaction and Multiplex PCR on the diagnosis of P. aeruginosa in mixed clinical samples. Materials and Methods: This study developed P. aeruginosa Loop Mediated Isothermal Amplification (PaLAMP) assay to target the ExoY gene with appropriate primer testing and validation procedures. Culture of patient bacterial samples was done on MHA and MHB medium, grown overnight in an Incubator and a incubating shaker at 37oc respectively. Housekeeping gene were identified through online bioinformatics and blasted against known sequences. A set of 6 primers, comprising 2 outer primers (F3 and B3), 2 inner primers (FIP and BIP), and 2 loop primers (FLP and BLP), were designed. Microbial DNA extraction was done followed by PCR amplification as a classical identification using LAMP outer primers 9(F3 and B3). LAMP amplicons were detected by real time turbidimetry (LA-500) at 64°C for 40 minutes as well as under UV light with 1.0 μl of 1/10-diluted original SYBR Green I. Results: LAMP validation against traditional PCR shows a high limit of detection at 10-6ng/µl compared to 10-5ng/µl for PCR. The findings are consistent with outcomes for real time turbidimetric outcomes. Real time LAMP turbidimetric results was cross validated by direct observation through SYBR fluorescence under UV light for positive P. aeruginosa detection through positive amplification. Conclusion: Thus far, Loop mediated isothermal amplification show significantly high limit of detection comparable to standard PCR, its use in field based diagnosis offers an opportunity for a cheap, reliable and faster method to determine disease trends and therapy approaches. This method can be applied in primary care to enhance accuracy in diagnosis and thereby prompt initiation of mitigation treatment regimens.