Development of a loop-mediated isothermal amplification assay for sensitive and rapid detection of Cronobacter...

Area of Science:

• Microbiology
• Food Safety
• Molecular Diagnostics

Background:

• Cronobacter sakazakii is an emerging pathogen linked to severe infant illnesses, including necrotizing enterocolitis, sepsis, and meningitis, particularly in low-birth-weight preterm infants.
• Contaminated reconstituted infant formula is a primary vehicle for C. sakazakii transmission, necessitating sensitive and specific detection methods for effective control.
• Existing detection methods may lack the sensitivity or specificity required for routine screening of powdered infant formula (PIF).

Purpose of the Study:

• To develop and validate a highly specific and sensitive loop-mediated isothermal amplification (LAMP) assay for the rapid detection of Cronobacter sakazakii.
• To evaluate the performance of the developed LAMP assay in detecting C. sakazakii in pure cultures and experimentally inoculated powdered infant formula (PIF) samples.
• To compare the sensitivity and specificity of the LAMP assay against conventional ompA-polymerase chain reaction (PCR) methods.

Main Methods:

• Design of a set of four loop-mediated isothermal amplification (LAMP) primers targeting the C. sakazakii ompA gene.
• Specificity testing using a diverse panel of 22 C. sakazakii strains, 27 Enterobacteriaceae strains (excluding C. sakazakii), and 25 other bacterial strains.
• Sensitivity determination through serial dilutions of C. sakazakii cultures and analysis of experimentally inoculated PIF samples, with comparisons to ompA-PCR.

Main Results:

• The ompA-based LAMP assay demonstrated high specificity, accurately detecting all 22 C. sakazakii strains while showing no amplification with 52 non-C. sakazakii strains.
• In pure culture, the assay achieved a detection limit of 10(1) colony-forming units (CFU)/mL, which is up to 10-fold more sensitive than ompA-PCR.
• When applied to PIF samples, the LAMP assay exhibited a sensitivity of 10(2) CFU/mL, also demonstrating up to 10-fold greater sensitivity than ompA-PCR.

Conclusions:

• The developed ompA-based LAMP assay is a sensitive, specific, and cost-effective tool for detecting Cronobacter sakazakii in powdered infant formula (PIF).
• This LAMP assay holds significant potential for routine field application and enhanced surveillance of C. sakazakii contamination in PIF.
• The assay provides a valuable advancement in food safety diagnostics, contributing to the prevention of infant infections caused by C. sakazakii.