Immunochromatographic strip for rapid detection of Cronobacter in powdered infant formula in combination with silica-coated magnetic nanoparticles separation and 16S rRNA probe

摘要

Here we developed a sensitive, specific, and rapid immunochromatographic strip test for the detection of Cronobacter. Silica-coated magnetic nanoparticles were used to separate nucleic acid from Cronobacter lysate and eliminate the interference of food matrices successfully. A couple of 5'-end labeled probes, which was complementary to the 16S ribosomal DNA of Cronobacter, was used to hybridize with the nucleic acid. The hybrid product, labeled with digoxigenin on one side and biotin on the other side, was directly submitted to the immunochromatographic strip test and the anti-digoxigenin monoclonal antibody was immobilized on nitrocellulose membrane in the test line. The visualization was achieved by gold nanoparticles conjugated to streptavidin, and double red bands appearing in both test and control line indicated a positive result of the presence of Cronobacter in testing sample. The detection limit was 10~7 cfumL~(_1) in pure culture. After silica-coated magnetic nanoparticles treatment, the detection limit was 10~5 and 10~6 cfumL~(_1) in pure culture and powdered infant formula, respectively, and maintained stable even under the interference of 10~8 cfumL~(_1) Salmonella typhimurium. Furthermore, 100 positive powdered infant formula samples spiked 10~8 cfu mL~(_1) Cronobacter and 20 negative samples with none bacteria were tested by the strip, and the sensitivity and specificity of the test were both as high as 100%. This approach showed promise for microbial detection concerning food safety or clinical diagnosis.