Manual Food-Borne Pathogens: Methods and Protocols

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This protocol has primarily been standardized to screen individual wild-caught flies for the presence of Cronobacter spp.

Foodborne Pathogens -- Sandra Gompf, MD

However, this protocol was also easily adapted to screen body parts of single flies for the presence of other foodborne pathogens such as enterohemorrhagic E. Also, this protocol can potentially be adapted to detect foodborne pathogens from other insects that are known vectors of diseases cockroaches and ants , but more research in this area is needed. Foodborne illness outbreak investigations are very dynamic and comprise a multi-step process that may vary according to the specific situation and the local environment being investigated 12, These investigations are important because they provide immediate public health protection by preventing future illnesses.

Additionally, these investigations can elucidate new mechanisms by which foodborne microorganisms are spread, and raise important questions that lead to new areas for research Investigative techniques as well as standardized, rapid, and sensitive protocols are necessary for detecting foodborne pathogens from individual insects. This standardized protocol opens the opportunity to aseptically collect insects like flies, which can vector the foodborne bacterial pathogen, as part of an environmental sampling program.

The epidemiological information that can be gained from this would be of use in constructing an accurate picture of the mechanisms of transmission of foodborne pathogens by insects i. Finally, even though the commercial PCR-based detection system described here is practical to use and simplifies PCR amplification and visualization of a genus-level amplicon, it is by no means the only appropriate system.

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The lysate from enriched samples could alternatively be used to screen for the presence of foodborne pathogens by using publically available species-specific primer pairs. However, detection sensitivity should be demonstrated prior to their use. The use of specified instrumentation is not an endorsement by the U. Food and Drug Administration. The authors certify that there is no conflict of interest with any financial organization regarding the material discussed in this article.

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If you want more info regarding data storage, please contact gdpr jove. Collection of flies Collect individual flies using sterile entomological sweep nets. Put the nets in a cooler and transfer them to the lab. Mix the tube gently by inversion for 2 min. It is essential that the whole body of the fly be in contact with the media so that the microbiota present on the body surface S of the fly will be transferred to the BPW BPW-S.

Label the tube with a number and body part of the fly i. Using sterile forceps remove the fly from the BPW-S media and transfer it to an empty and clean 2 ml tube to surface disinfect the fly. Rinse 3 times with sterile distilled water. Transfer water from the last rinse to an autoclaved 2 ml tube. Mix gently by inversion at each step of the surface-disinfection process. After incubation, register the presence of any bacterial colonies.

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If this occurs, the presence of foodborne pathogens should only be reported on the body surface of the fly because cross-contamination between the body surface and the alimentary canal cannot be ruled out. After surface-disinfecting the fly, transfer it to a piece of autoclaved paper towel to remove excess water and then to a sterile 60 mm disposable Petri dish. Place the Petri dish under a dissecting scope and identify the fly to the species level using dichotomous keys for dipteran families 20, Label the tube with the same number selected for the individual fly and the body part of the fly i.


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  6. Mix the tube containing the BPW-A thoroughly for 5 - 10 min using a cell disruptor. Primary and Secondary Enrichment Label all primary and secondary enrichment tubes containing media according to the sample number and the body part of the fly. Incubate in a recirculating water bath at For L.

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    No secondary enrichment is required for the detection of L. Repeat steps 3. Turn on the automated heating block selecting the specific program for the target pathogen. Label and arrange cluster tubes containing the lysis reagent in the rack, according to the rack file. Initialize the PCR-based detection system instrument.

    Use new pipette tips for each sample. Cap cluster tubes and secure tightly using the capping tool. Place the rack of cluster tubes in the automated heating block after selecting the specific program for the target pathogen. Perform Lysis for the Detection of L. Use the mixture within 4 hr. Perform part 2 of lysis as follows: Prepare the lysis reagent as instructed in steps 5.

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