Studies On The Establishment And Application Of Immunomagnetic Methods To Rapid Detection Of Staphylococcus Aureus And SEB In Shrimp

Seafood processing enterprise had been plagued by seafood which polluted severely from Staphylococcus aureus(S.aureus) and its enterotoxins.Therefore, the research established rapid immunomagnetic methods to detection S.aureus and SEB in shrimp, then methods were used to evalution the control effects of the two microbial agents.The two microbial agents are Bacillus antimicrobial peptides and Cordyceps militaris. The main contents are as follows:Made immuno-magnetic beads(IMB) using chemical bond method. IMBsaureus was made by magnetic beads coupling with S.aureus. Polyclonal antibody was obtained by immunizing rabbits. The antiserum titer of the SEB was1:16. IMBSEB was made by optimizing the additive concentration(5mg/mL), coupling times(24h and27h), the Maximum saturation adsorption of magnetic bead(293.27μg and318.56μg).According to the principle of immunomagnetic separation technology, we establish IMBS.aureus rapid detection for S.aureus with conventional bacteria technology method, and IMBSEB rapid detection for SEB with indirect competitive ELISA.IMBS.aureus rapid detection:we reached the max capture rate by optimizing human IgG amount(combined250μg human IgG), incubated temperature(37℃), incubated time(60min).We confirmed that the detection limit of IMBS.aureus was26cfu/10mL, the accuracy rating was92.81%, coefficient of variation were less than15%. Compared with GB method, IMBS.aureus had high sensitivity and high efficiency(26.5h) when it detected in shrimp. IMBSEB-ELISA rapid detection:we reached the max capture amount by optimizing antibody incubation time(75min).We got clear cut (5-500ng/mL) and IC5o(31.04ng/mL) under the best condition(the optimal coating concentration was0.625μg/mL, antibody dilution degrees was1:3000, incubation condition was4℃during20h using carbonate buffer solution, the blockage was BSA, the optimal proportion was7:3between competition antigenic). We made sure that its cross reaction was less than0.01%and the supreme blocking rate was71.92%.The lowest detectable limit was3.99ng/mL and the lowest quatitation limit was5ng/lOmL. The average recovery rate was95.13%and coefficient of variation were less than15%. IMBSEB had high sensitivity, high veracity (the average recovery rate was101.66%) and accuracy(the coefficient of variation was12.06%).IMB were used to evalution the control effects of the two microbial agents. The two kinds of agents were added in different growth periods of S.aureus.IMBs were uesd to detection bacteria and toxins content. It turned out that the MIC value of Bacillus antimicrobial peptides and Cordyceps militaris were0.675mg/mL and25mg/mL. The two preparations could control the growth of S.aureus, which were growed to20h. Bacillus antimicrobial peptides could control the mount of toxin. Cordyceps militaris could control SEB content when S.aureus was on avascular growth.In conclusion, immunomagnetic rapid assay provides a new perspective for foodborne pathogenic bacteria and its toxins. The methods provides the safeguard for the effective evaluation on seafood green security control.