Researchers in China have developed a test that can quickly detect and identify Cronobacter species in powdered infant formula, with significantly greater sensitivity and much more rapid results than polymerase chain reaction (PCR), and without producing false positives.

Cronobacter sakazakii is a significant risk to powdered infant formula safety, necessitating fast, sensitive, easy-to-use, and accurate methods for Cronobacter testing and analysis. Several commercial kits are currently available for Cronobacter detection; specifically, PCR kits boast high sensitivity but require 24-hour enrichment, while loop-mediated isothermal amplification (LAMP) kits are more rapid and budget-friendly but lack in specificity. Additionally, immunoassays are cost-effective but encounter a 15 percent false negative rate in powdered infant formula.

Hoping to develop a well-rounded solution for detection and identification of viable Cronobacter in powdered infant formula, the researchers chose fluorescence RNA-targeted isothermal amplification assay (SAT) technology to provide a reliable, onsite, and resource-efficient food safety monitoring kit.

After optimizing SAT reaction conditions to enhance the test’s efficiency, the researchers validated its specificity against seven different Cronobacter sakazakii strains and 24 non-Cronobacter strains (including Citrobacter, Enterobacter, Salmonella, Shigella, Escherichia coli, and others). The SAT assay was also tested on powdered milk samples purchased from local farmers markets in China. Its performance was compared to that of a real-time PCR method and ISO 22964.

The SAT assay showed ten times greater sensitivity than PCR, with a detection limit of 2 log¹⁰ colony forming units per milliliter (CFU/ml) without pre-enrichment, 2 log CFU/10 ml with four hours enrichment, and 2 log CFU/1,000 ml with seven hours pre-enrichment. In comparison, the sensitivity of real-time PCR was 3 log CFU/ml without pre-enrichment, log CFU/ml with four hours pre-enrichment, and 2 log CFU/10 ml with eight hours pre-enrichment.

Moreover, the SAT test produced results in four hours, which is significantly faster than the real−time PCR method with 24-hour enrichment.

The SAT assay could also distinguish between dead and viable C. sakazakii, eliminating false positive results. In contrast, the real-time PCR assay exhibited a detection limit equivalent to that for detecting viable bacteria.

When compared against the internationally validated ISO 22964 for dairy matrices, the results of the ISO 22964 method and the SAT assay were in 100 percent agreement, while significantly reducing the detection timeframe to fewer than 72 hours. Additionally, less equipment is needed for SAT because the assay is carried out at a constant temperature and is therefore suitable for rapid, onsite testing.

Overall, the researchers believe their SAT assay, especially combined with enrichment, offers a rapid, sensitive, and easy-to-use method for onsite detection and identification for Cronobacter in infant formula and other foods.

The study, published in the Journal of Food Protection, was conducted by researchers at Guangzhou Customs Technology Center, the Heilongjiang Green Food Science Research Institute, Jianyuan Science and Technology Co. Ltd., the CAU Dong Jun Laboratory Testing Technology Co. Ltd., Gongbei Customs Technology Center, and Guangdong Maoming Agriculture and Forestry Technical College. The work was funded by the China Scientific Research Project of the General Administration of Customs; the Joint Guidance Project of the Natural Science Foundation of Heilongjiang Province, China; and the Key Research and Development Project for Tackling Key Common Technologies in the High-Quality Development of Agriculture in Hebei Province, China.