Screening of specific nucleic acid targets for Cronobacter sakazakii and visual detection by loop-mediated isothermal amplification and lateral flow dipstick method in powdered infant formula

Abstract

Due to the lack of specific genes for rapid detection methods of Cronobacter sakazakii in food samples, whole genome sequence analysis was performed in this investigation using the basic local alignment search tool. Forty-two DNA fragments unique to C. sakazakii were mined, then primers were designed and screened by PCR and loop-mediated isothermal amplification (LAMP). Two primer sets, CS1 and CS31, were found as specific and stable primers, with their corresponding nucleic acid targets the CSK29544_00235 gene and CSK29544_03484 gene, respectively. Furthermore, compared with 3 genes reported previously, these 2 genes were verified as more specific to C. sakazakii among Cronobacter species, by sequence similarity alignment using Cronobacter MLST databases (http: / / pubmlst .org/ cronobacter). The specificity of the LAMP reaction approached 100% by using 48 bacterial strains, which included 22 C. sakazakii strains. Subsequently, LAMP was combined with visual lateral flow dipstick (LFD) based on the above 2 nucleic acid targets, and was demonstrated as a rapid, efficient method with high specificity. Finally, the detection sensitivity of this assay system for pure cultures and artificially contaminated milk was measured as 4.5 × 10 0 cfu/mL and 5.7 × 10 1 cfu/g, respectively. Total time to detection for this assay was within 2 h. Thus, the establishment of this LAMP-LFD method shows great significance and potential for rapid detection of C. sakazakii in powdered infant formula.

Detection of respiratory pathogenic bacterial nucleic acid detection by Loop-mediated Isothermal Amplification in patients with bacterial pulmonary infections

“…In recent years, the amplification of bacterial-specific DNA fragments by PCR technique can accurately identify the presence of bacteria, which is characterized by easy operation, short detection cycle, high sensitivity and specificity [ 19 ]. Therefore, PCR technology used to identify bacteria will likely replace the traditional sputum culture technique in the future.…”

Rapid and Visualized Detection of Virulence-Related Genes of Vibrio cholerae in Water and Aquatic Products by Loop-Mediated Isothermal Amplification

“…Different sensitivities of the LAMP technique have been reported in the detection of foodborne pathogens. For example, Fu et al (12) reported an LOD value of 4.50 CFU/ mL targeting specific nucleic acid targets of C. sakazakii in powdered infant formula samples by LAMP. LOD values of 20 and 10 CFU per reaction targeting ompW (28) and ctxA (10) genes of V. cholerae have also been reported by LAMP.…”

Desiccation and osmotic resistance mechanism of Cronobacter spp. isolated from powdered infant formula

“…In recent years, the Cronobacter spp. has been divided into seven species and three subspecies [1,2]. Among them, C. sakazakii and C. malonaticus are the main pathogenic bacteria in clinic [3].…”