A Rapid Method for Detection of Salmonella in Milk Based on Extraction of mRNA Using Magnetic Capture Probes and RT-qPCR

Yalong Bai; Yalong Bai; Yan Cui; Yujuan Suo; Chunlei Shi; Dapeng Wang; Xianming Shi

doi:10.3389/fmicb.2019.00770

Frontiers in Microbiology (Apr 2019)

A Rapid Method for Detection of Salmonella in Milk Based on Extraction of mRNA Using Magnetic Capture Probes and RT-qPCR

Yalong Bai,
Yalong Bai,
Yan Cui,
Yujuan Suo,
Chunlei Shi,
Dapeng Wang,
Xianming Shi

Affiliations

Yalong Bai: MOST-USDA Joint Research Center for Food Safety, School of Agriculture and Biology and State Key Laboratory of Microbial Metabolism, Shanghai Jiao Tong University, Shanghai, China
Yalong Bai: Institute for Agri-food Standards and Testing Technology, Shanghai Academy of Agricultural Sciences, Shanghai, China
Yan Cui: MOST-USDA Joint Research Center for Food Safety, School of Agriculture and Biology and State Key Laboratory of Microbial Metabolism, Shanghai Jiao Tong University, Shanghai, China
Yujuan Suo: Institute for Agri-food Standards and Testing Technology, Shanghai Academy of Agricultural Sciences, Shanghai, China
Chunlei Shi: MOST-USDA Joint Research Center for Food Safety, School of Agriculture and Biology and State Key Laboratory of Microbial Metabolism, Shanghai Jiao Tong University, Shanghai, China
Dapeng Wang: MOST-USDA Joint Research Center for Food Safety, School of Agriculture and Biology and State Key Laboratory of Microbial Metabolism, Shanghai Jiao Tong University, Shanghai, China
Xianming Shi: MOST-USDA Joint Research Center for Food Safety, School of Agriculture and Biology and State Key Laboratory of Microbial Metabolism, Shanghai Jiao Tong University, Shanghai, China

DOI: https://doi.org/10.3389/fmicb.2019.00770
Journal volume & issue: Vol. 10

Abstract

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Magnetic separation is an efficient method for target enrichment and elimination of inhibitors in the molecular detection systems for foodborne pathogens. In this study, we prepared magnetic capture probes by modifying oligonucleotides complementary to target sequences on the surface of amino-modified silica-coated magnetic nanoparticles and optimized the conditions and parameters of probe synthesis and hybridization. We innovatively put the complexes of magnetic capture probes and target sequences into qPCR without any need for denaturation and purification steps. This strategy can reduce manual steps and save time. We used the magnetic capture probes to separate invA mRNA from Salmonella in artificially contaminated milk samples. The detection sensitivity was 104 CFU/ml, which could be increased to 10 CFU/ml after a 12 h enrichment step. The developed method is robust enough to detect live bacteria in a complex environmental matrix.

Published in Frontiers in Microbiology

ISSN: 1664-302X (Online)
Publisher: Frontiers Media S.A.
Country of publisher: Switzerland
LCC subjects: Science: Microbiology
Website: http://www.frontiersin.org/journals/microbiology

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