Detection of food contaminants by SERS and nanopore technology
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[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] With growing consumption of aquaculture products, there is increasing demand on rapid and sensitive techniques that can detect prohibited substances in the seafood products. In this study, a novel surface-enhanced Raman spectroscopy (SERS) method was developed coupled with simplified extraction method and a novel gold nanorod substrate to detect banned aquaculture biocides (malachite green (MG) and crystal violet (CV)) and their mixture (1:1) in aqueous solution and fish samples. PCA results demonstrate that SERS can distinguish MG, CV and their mixture (1:1) in aqueous solution and in fish samples. Transformed spectra of MG and CV and two-sample t-test results show that the detection limit is 1 ppb both for MG and CV using standing gold nanorod substrates, which meet the maximum residue level of FDA. Satisfactory R values (R > 0.95) of PLS were obtained, indicating a good linear relationship between the actual concentration and predicted concentration of samples. The results demonstrate that the SERS method developed in this study can be used for rapid and accurate detection and quantification of MG and CV in fish samples. Escherichia coli O157:H7 is a serotype of the bacterial species E. coli and is one of the Shiga-toxin producing E. coli. Infection of E. coli O157:H7 will come up with several typical clinical symptoms, including bloody or non-bloody diarrhea, hemorrhagic colitis (HC), the hemolytic-uremic syndrome (HUS). Thus, quick and efficient detection methods of this pathogen are crucial in the food safety and diagnosis. Conventional methods, such as plate counting and PCR ensure high accuracy yet are time-consuming and labor intensive. Here, we demonstrate a method to distinguish this pathogenic serotype from others by single-nucleotide difference facilitated by nanopore technology. The synthetic DNA sequences, which mimic the target of E. coli O157:H7 or other serotypes, were mixed with DNA probes (with or without a locked nucleic acid (LNA) substitution) to form double-strand duplexes. Results show single LNA substitution of the probe can efficiently discriminate E. coli O157:H7 and others by magnifying the ratio of durations of the events from 1.6 to 12.2 fold. Besides, the probe can identify the pathogenic targeting sequence in different ratio mixtures with the corresponding nonpathogenic sequence, which successfully prevents interference, false positive or negative result.
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