Development of a Loop-Mediated Isothermal Amplification Assay for Sensitive and Rapid Detection ofCronobacter sakazakii

Abstract

Cronobacter sakazakii is an emerging pathogen associated with the ingestion of contaminated reconstituted formula, which causes necrotizing enterocolitis, sepsis, and meningitis in low-birth-weight preterm neonatal infants. Sensitive and specific detection methods are needed to better control C. sakazakii infections. This study aims to develop a highly specific and sensitive loop-mediated isothermal amplification (LAMP) assay for detecting C. sakazakii in powdered infant formula (PIF). A set of four LAMP primers were designed based on the published C. sakazakii ompA gene sequence. Specificity of the assay was evaluated using a panel of 22 C. sakazakii, 27 Enterobacteriaceae family except C. sakazakii, and 25 other strains. Assay sensitivity was determined using serial dilutions of C. sakazakii American Type Culture Collection 51329 culture ranging from 10(6) colony-forming units (CFU)/mL to extinction. The assay was also tested in experimentally inoculated PIF samples. The ompA-based LAMP assay was able to detect specifically all of the 22 C. sakazakii strains without amplification from 52 non-C. sakazakii strains. The detection limit was 10(1) CFU/mL in pure culture, up to 10-fold more sensitive than that of the ompA-polymerase chain reaction (PCR). When applied to PIF, sensitivity was 10(2) CFU/mL, up to 10-fold that of the ompA-PCR. The ompA-based LAMP assay developed in this study was sensitive, specific, and low cost with great potential for future field detection of C. sakazakii in PIF.