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Rapid detection and simultaneous genotyping of Cronobacter spp. (formerly Enterobacter sakazakii) in powdered infant
Xian-Quan Cai1, Hai-Qiong Yu, Zhou-Xi Ruan
1State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, Gansu Province, People's Republic of China.
Insights
A new assay using real-time PCR and high-resolution melting (HRM) accurately detects Cronobacter spp. in infant formula within 24 hours. This rapid method also genotypes Cronobacter species, improving food safety.
Area of Science:
- Food Microbiology
- Molecular Diagnostics
- Pathogen Detection
Background:
- Cronobacter spp. is an emerging pathogen linked to severe infant illnesses like meningitis and sepsis.
- Accurate and rapid detection of Cronobacter in powdered infant formula is crucial for public health.
Purpose of the Study:
- To develop and validate a real-time PCR and high-resolution melting (HRM) assay for Cronobacter spp. detection.
- To enable rapid identification and simultaneous genotyping of Cronobacter species in powdered infant formula.
Main Methods:
- Development of a real-time PCR assay targeting the OmpA gene.
- Application of high-resolution melting (HRM) analysis for species identification.
- Validation using Cronobacter isolates, reference strains, and 89 actual powdered infant formula samples.
Main Results:
- The assay achieved 100% accuracy compared to conventional methods.
- High sensitivity was demonstrated with a detection limit of 10(2) CFU/ml without pre-enrichment.
- 100% concordance with ISO-IDF 22964 standard in real samples; detection time reduced to under 24 hours.
Conclusions:
- The established real-time PCR-HRM assay provides a rapid, sensitive, and accurate tool for Cronobacter spp. detection in powdered infant formula.
- The method facilitates simultaneous genotyping of Cronobacter species, aiding in source tracking and risk assessment.
- This probe-free assay reduces the risk of PCR carryover and significantly shortens the detection time compared to traditional methods.
Abstract:
Cronobacter spp. is an emerging pathogen that causes meningitis, sepsis, bacteremia, and necrotizing enterocolitis in neonates and children. The present study developed an assay integrating real-time PCR and high resolution melting (HRM) analysis targeting the OmpA gene for the specific detection and rapid identification of Cronobacter spp. (formerly Enterobacter sakazakii) in powdered infant formula. Eleven Cronobacter field isolates and 25 reference strains were examined using one pair of primers, having the accuracy of 100% in reference to conventional methods. The assay was proved to be highly sensitive with a detection limit of 10(2) CFU/ml without pre-enrichment, and highly concordant (100%) when compared with ISO-IDF 22964 in 89 actual samples. The method performed for Cronobacter spp. detection was less than 24 h, drastically shortened, compared to several days using standard culturing method, it is probe-free and reduces a risk of PCR carryover. Moreover, all Cronobacter strains examined in this study were genotyped into two species according to their HRM profiles. The established method should provide a molecular tool for direct detection and simultaneous genotyping of Cronobacter spp. in powdered infant formula.
