P-073. Development of a New Lysis Method and Background Reduction Technology for Rapid Detection of Yeast and Mold

M. Pressel, S. Rouillon, M. Hohnadel, F. Olivieri, B. Engel, P. Guedon, S. Ribault;

Background. One major limitation of the ATP bioluminescence technology is the presence of free ATP that generates false positive results. A solution proposed here is the pretreatment of the sample filtered on a membrane with a dedicated reagent to remove free ATP before microorganisms detection. As the sensitivity of detection is directly linked to the efficiency of microorganisms lysis, a new system using microwaves was evaluated and compared to existing chemical lysis reagent. Method. The performance of pretreatment step is evalutated by spotting free ATP on the membrane. The reagent is homogeneously sprayed and the spot intensity is observed using Milliflex rapid platform. Once the free ATP is removed and before detection of intracellular ATP, the enzyme used for pretreatment has to be inactivated and cells lysed. A microwave cavity was specifically developed to optimize both performances. Enzyme inactivation by microwaves is controlled by spraying ATP. To determine lysis performances, a known quantity of microorganisms was filtered on membrane. Microwaves from 300 to 900W, temperature between 100 and 130°C, different cavity size were tested. After lysis step, membranes were sprayed with bioluminescence reagent and spots intensity and number were recorded. Results were compared with household microwaves, chemical lysis and no lysis. Traditional microbiology was performed to control the number of CFU. Results. Pretreatment of the membrane is able to remove free ATP: we observe a 60% intensity decrease in 1 min. Microwaves inactivated 100% of the reagent used for pretreatment. The best lysis is obtained with a direct plugged cavity, 800W, 2450MHz. All the micro-organisms on the membrane are lysed and the cells light intensity observed with a Milliflex Rapid imaging system equivalent to the signal obtained with a chemical lysis. Conclusion. The pretreatment reduces the false positive level in flavored water and alcoholic beverage. The use of microwaves is a high performance solution for the universal lysis of microorganisms on membrane and is compatible with the bioluminescence technology.

247/P. Virulence Factors and Toxins

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