Rapid and sensitive detection of Enterobacter sakazakii by cross-priming amplification combined with immuno-blotting analysis.

Enterobacter sakazakii is a widespread and life-threatening bacterium especially in polluted powdered infant milk formula. Several methods have been developed for detection of E. sakazakii such as physiological and biochemical methods, PCR and loop-mediated isothermal amplification. However, these procedures were disadvantages due to a long assay time, low sensitivity or the use of toxic reagents. Our method of cross-priming amplification (CPA) under isothermal conditions combined with immuno-blotting analysis made the whole detection procedure more sensitivity and lower time-consuming. A set of specific displacement primers, cross primers and testing primers were designed based on six specific sequences in E. sakazakii 16S-23S rDNA internal transcribed spacer. Under isothermal condition at 63 °C for 60 min, the specific amplification and hybridization steps were processed simultaneously. The specificity of the CPA was tested in panel of 54 different bacterial strains and 236 milk powder products. Two red signal lines were developed on the BioHelix Express strip in all of positive E. sakazakii strains, and only one signal line was demonstrated by non- E. sakazakii bacterial strains. The limit of decetion of CPA was 6.3 ± 2.7277 fg for the genomic DNA, 88 ± 8.7892 cfu/ml for pure bacterial culture, and 3.2 ± 2.0569 cfu per 100 g milk powder with pre-enrichment. The current study demonstrated that the assay method of CPA combined with immuno-blotting analysis was a specific and sensitive detection for the rapid detection of E. sakazakii.