Development of rapid assays to detect enterotoxin genes of Staphylococcus aureus in foods

• Main Authors: Hsin-Yi Yin, 尹心怡
• Other Authors: 溫曉薇
• Format: Others
• Language: en_US
• Published: 2017
• Online Access: http://ndltd.ncl.edu.tw/handle/73209604239394491787

博士 === 國立中興大學 === 食品暨應用生物科技學系所 === 105 === In Taiwan, among various staphylococcus enterotoxins (SEs) produced by Staphylococcus aureus, SEA is the most related to food poisoning outbreaks, followed by SEB. In the United States, annually there are 185,000 cases of foodborne illnesses caused by SEs. Until now, more than 20 kinds of SEs have been reported. SEs have the superantigenic activities and can resist to heat and proteases in human gastrointestinal tract. The syndromes caused by SEs include diarrhea, vomiting, retching, abdominal cramping and prostration, and a toxin dose of less than 1 μg will cause these symptoms. Therefore, the purpose of this study is to develop rapid assays for detecting enterotoxic S. aureus in food. First, we developed an immunomagnetic bead (IMB) based method with the use of NeutrAvidin-tagged liposomal nanovesicles (NA-LNs) which encapsulated fluorescent dyes as the detection reagent to detect S. aureus containing sea gene. Through a PCR reaction, the target DNA was amplified and labeled with digoxigenin (Dig) and biotin. The amplified target DNA was then captured by IMB modified with anti-Dig-antibody and detected by NA-LNs. The developed assay could detect S. aureus and differentiate it from Salmonella enterica and Escherichia coli, with a limit of detection (LOD) of 101 CFU mL-1 without pre-enrichment. With a 2-hour pre-enrichment, this developed assay could detect as little as 1 CFU in 25 mL of milk. Furthermore, we developed a multiplex loop-mediated isothermal amplification (m-LAMP) combined with a lateral flow assay (LFA) for simultaneously detecting the sea and seb genes of enterotoxic S. aureus. The LOD of this assay was 102 CFU mL-1 S. aureus, which was 10-fold lower than that of a multiplex PCR; and this assay did not show any cross-reactivity as detecting other enterotoxic S. aureus strains or other food pathogens. After 4~6-hour enrichment, this developed assay could detect as low as 1 CFU mL-1 of S. aureus in four different food matrixes - milk, apple juice, cheese, and rice. Conclusively, these two developed methods can be completely finished within a workday, which can provide an alternative way to easily and quickly screen the contamination of enterotoxic S. aureus in food products or in food supply chains.