Estandarización de un protocolo para la rápida detección del Gen Bla-KPC en Bacterias Gramnegativas resistentes a Carbapenémicos mediante el uso de LAMP (Loop Mediated Isothermal Amplification)

Abstract

In Ecuador there is a high prevalence of bacteria carrying carbapenemase enzymes that represent the main mechanism of resistance against beta-lactam antibiotics, used in the treatment of respiratory infections, central nervous system, intra-abdominal, urinary, endocarditis and sepsis of unclassified origin. The gene coding for carbapenemases, bla-KPC-2, is the highest incidence in health institutions in the country, according to reports from the INSPI - CZ9, National Reference Center for Resistance to Antimicrobials (CNR-RAM). Currently hospitals or basic health centers lack a low cost nucleic acid amplification technique, rapid detection, high sensitivity, specificity and ability to discriminate resistance gene sequences. Through this research, a protocol for the rapid detection of the bla-KPC gene in Enterobacteria was standardized, using LAMP as a clinical diagnostic technique that can be used in hospitals and basic health centers in the future. 40 bacterial strains were selected from the CNR-RAM strain: 20 positive for bla-KPC; 11 strains with different resistance genes to carbapenemics (ESBL, bla-IMP, bla-NDM, bla-OXA-48, bla-Ctx-m) and 9 that did not present any resistance gene, all previously confirmed by microbiology and molecular biology. The amplification conditions of the bla-KPC gene region, which allowed to obtain a good quality amplicon were: temperature at 63 ° C, internal oligonucleotide concentrations: FIP / BIP 0.4 μM; MgSO 4 from 4mM; Betaine at 0.8M; dNTPs at 0.8mM; DNA volume 1μL; Amplification time was established in 60 minutes at the indicated temperature. LAMP presented a high sensitivity and specificity (> 99%).