High-throughput suspension array for detecting four pathogens

Abstract

A new suspension array technology is proposed for the simultaneous quantitative determination of four pathogens in food. Four sets of primers and species-specific capture probes were designed based on the 16S rDNA gene sequences of Escherichia coli, Staphylococcus aureus, Vibrio parahaemolyticus, and Salmonella downloaded from GenBank. The specific nucleic acid probes were covalently bound to the surface of fluorescent microspheres for liquid suspension hybridization. The biotin-labeled PCR products obtained from the samples were hybridized with their complementary nucleic acid probes, which were detected after the addition of streptavidin phycoerythrin. No cross-reaction occurred among the products, and the sensitivity of the probe in the single-channel method was 5 × 10⁻⁶ mM. In the multi-channel method, the sensitivities were as follows: 5 × 10⁻⁴ mM for E. coli and Salmonella, and 5 × 10⁻⁵ mM for S. aureus and V. parahaemolyticus. The multi-channel method achieved multidetection of several pathogens. For real sample detection, the methods showed a sensitivity of 10³ CFU per g to 10⁰ CFU per g of vegetables after enrichment for 24 h at 37 °C. This study demonstrates the utility of the suspension array specific for the 16S rDNA gene for determining the presence of the four pathogens in food samples.