Development of a loop-mediated isothermal amplification assay for detection of Cronobacter spp. (Enterobacter sakazakii)

Abstract

Contamination of Cronobacter spp. (Enterobacter sakazakii) in infant formulas and other food products is a severe problem. Here a loop-mediated isothermal amplification (LAMP) assay was developed for rapidly detecting Cronobacter spp. in powdered infant formula. Sequences of 16S/23S rDNA internal intergenic spacer of Cronobacter spp. were used as the target template to design LAMP primers. The detection outcome can be evaluated by the white precipitate or the fluorescence intensity under ultraviolet irradiation, both visible to naked eyes. The sensitivity and specificity of the LAMP assay was further analyzed in comparison with that of regular PCR and real time quantitative PCR. The results showed that all of Cronobacter spp. strains display positive reaction to the detections while all of the non-Cronobacter spp. strains were negative, and that the LAMP assay exhibits a high sensitivity of 9.1 fg/μL (The sensitivity of regular PCR and real time quantitative PCR is 91 and 9.1 pg/μL, respectively.). The amplified reaction could be accomplished in about 1 h, with the results visible to naked eyes. Hence, the LAMP assay developed by this study can provide a rapid and simple approach for the detection of Cronobacter spp. in infant formula.

Rapid Detection and Simultaneous Genotyping of Cronobacter spp. (formerly Enterobacter sakazakii) in Powdered Infant Formula Using Real-time PCR and High Resolution Melting (HRM) Analysis

“…A number of alternative molecular methods for Cronobacter spp. have been investigated, including conventional PCR assay using 16S rRNA [6], OmpA or rpoB genes [15], [16], real time PCR using TaqMan and SYBR Green [3], [9], and the loop-mediated isothermal amplification [17]. However, real-time PCR requires expensive labelled probe, and LAMP may have high risk of amplicon contamination.…”

Loop‐Mediated Isothermal Amplification Assays for Detecting Yersinia pseudotuberculosis in Milk Powders

“…By using sequences of 16S‐23S rDNA ITS region as a template to design primers, numerous other studies have reported the superior sensitivity of LAMP in comparison with PCR. A LAMP assay was developed to detect C. sakazakii and it showed a higher sensitivity (9.1 fg/μL) compared to conventional PCR (9.1 pg/μL) and real‐time PCR (9.1 pg/μL) (Liu and others ). In another study, detection limit of developed LAMP assay for A. acidoterrestris was 4.5×10 −2 CFU per tube, while the detection limit of conventional PCR assay was 6.75×10 0 CFU per tube (Chen and others ).…”

“…Therefore, it is one of the most popular targets for microbiological taxonomy (Chiu and others ). Sequences of the 16S‐23S rDNA ITS have been used as target template to design LAMP primers for detection of different bacteria in food systems, such as Alicyclobacillus acidoterrestris (Chen and others ) and Cronobacter sakazakii (Liu and others ). We validated that this currently developed LAMP assay can rapidly and precisely detect Y. pseudotuberculosis in commercial milk powder products.…”

Recent Progress of Nanozymes in the Detection of Pathogenic Microorganisms

“…[81] The proposed method by Zhang et al was based on propidium monoazide (PMA), loop-mediated isothermal amplification (LAMP), and nanozyme strip. LAMP is a gene amplification method for both live and dead cells, [82,83] whereas PMA is a DNA-binding dye that can penetrate dead or membrane-damaged bacteria, thereby inhibiting the subsequent amplification of DNA molecules in dead cells. [84] Thus, a combination of PMA and LAMP has been widely used to detect the bacterial viability.…”