Authors: Tesalona S, Felizardo AR, Katigbak JS, Ludovice MM, Ongto NC, Juan Pardiñas JC, Rosales JMR,De Castro DL, Pineda-Cortel MR, Lagamayo E
Background: Carbapenem-resistant Klebsiella pneumoniae (CRKP), carbapenem-resistant Pseudomonas aeruginosa (CRPA), and carbapenem-resistant Acinetobacter baumannii (CRAB) have become endemic in Southern Europe and elsewhere and has been recognized as a global health phenomenon because some of these strains have acquired broad spectrum antibiotic resistance. This study optimized a loop-mediated isothermal amplification (LAMP) assay in the detection of the blaOXA-23 gene, which are critical components of carbapenem resistance.
Methodology: Fourteen (14) strains CRPA, CRAB, and CRKP, were used in this study. Then the strains positive for target genes using conventional PCR were then further investigated using LAMP assay. The optimal primer ratio and reaction temperature were determined by using five strains of CRKP, three strains of CRPA and one strain of CRAB that were pre-determined positive for blaOXA-23 gene using conventional PCR.
Results: In this study, the detection of the blaOXA-23 gene for CRKP, CRPA, and CRAB is optimal at 61ºC. Since only two primer preparations were compared in this study, it is safe to say that the 1:4 primer ratio gives better visual results than the 1:1 primer ratio. Manipulation of the temperatures and primer ratios lead us to the conclusion that changes in these factors did increase reaction speed and improve sensitivity, thus presenting optimization. With all things considered, the study presents that the antimicrobial resistance gene marker, blaOXA-23, was best detected using the LAMP assay when a temperature of 61°C and primer ratio of 1:4 was utilized.
Conclusion: The results demonstrate the usefulness of LAMP techniques for the diagnosis of CRKP, CRPA, and CRAB that are positive for blaOXA-23. The LAMP assay is a sensitive, rapid, and practical method in the detection of blaOXA-23.View/Download pdf