I recently attended the International Production and Processing Expo (IPPE). With more than 1,000 exhibitors, hundreds of talks, meetings and posters, there was a tremendous amount of information to process. As with any large gathering associated with any industry, buzz words and catchy phrases were tossed around. Sometimes these words, products or practices catch on and become reality, other times they have a relatively short life and an industry moves on. At IPPE, a very popular buzz word was “Limits Testing.” Okay, that is technically a couple of words, so let’s take it step further by adding Salmonella to the mix to produce a buzz phrase, “Salmonella Limits Testing.” Now we have not only a very hot topic of discussion, but a very real approach to monitoring Salmonella levels in fresh poultry products.
Salmonella Limits Testing in poultry presents some very real and very exciting advancement for food safety. Limits Testing is based upon the understanding that Salmonella occurs as a commensal organism in poultry. Sanitation, intervention, flock management, and GMP are all employed to control and keep this potential pathogen out of commerce as much as possible. Limits Testing is a semi-quantitative approach to discovering and diverting products that may present higher levels of Salmonella, thus averting the potential of these products moving into the food supply. Last but not least, the microbiological method utilized must be a nucleic acid based technology, it must be robust, affordable, sensitive, specific and produce an actionable result in less than 8 hours. 36 months ago, these analytical requirements would have been unobtainable. Today it is not only obtainable, it is routinely employed with success.
Roka Bioscience was asked by our client to develop a protocol for Limits Testing that would allow the client to detect approximately 1 cfu of Salmonella per gram in a 375 gram sample of fresh ground turkey. In addition, the protocol was required to produce a negative result in samples containing .1 cfu per gram of Salmonella in a 375 gram sample. Historically less sensitive DNA based methods or immunochemical methods require an enrichment or growth step to grow the target to a detectable number of cfu/gram (usually 10³ to 10⁴ is required). The Atlas instrument and assay for Salmonella species is a very specific and sensitive assay which utilizes rRNA as its target instead of DNA. The Roka Salmonella assay is routinely 1 to 2 log more sensitive than other assays commercially available, so earlier detection of the Salmonella target is achieved. The Roka team was able to develop a shortened enrichment protocol of 4 to 4.5 hours of enrichment in a 1:2 ratio of BPW at an enrichment temperature of 37°C. At the 4 hour mark, the client simply transfers 400 ul of enrichment into a barcoded lysis tube and loads onto the fully automated Atlas instrument for analysis. The assay time on the instrument is just under 3 hours and samples are loaded and run continuously. In addition, the continuous access feature of the instrument allows an operator to load samples as they become ready with no impact to the cost per sample or limiting access to the instrument.
The results produced with Limits Testing differ significantly from classical approaches that are qualitative presence/absence assay approaches. Results in this particular application are semi-quantitative results that give more information to the client. The client knows that a negative result on the Atlas indicates the level of Salmonella to be either not present or somewhere between a starting concentration of ≥.1cfu/gram and ≤1cfu/gram. A positive result is interpreted as ≥1 cfu/gram concentration of Salmonella.
When considering a Limits Testing approach it is very important to understand the capabilities and limitation of the assay approach of choice. Some questions to consider might be:
What is my established Limits Testing and sensitivity in matrix?
If attempting a PCR method with no pre-amplification clean up step, at what time point will my target be detectable above the background or noise associated with the matrix?
Is the Limits Testing value established meaningful with regards to a finished product or human health impact? For example, does an in-process sample such as a swab or rinsate translate to a potential concentration of target in finished goods and if so, at what prevalence?
If the goal is to use semi-quantitative analysis in a pre-harvest situation to determine order of harvest (lower level flocks to higher level flocks) has a prevalence rate for Salmonella and acceptable Limits Testing target been established?
If you are considering a Limits Testing approach as part of your microbial testing regime, it is important to understand the expectation and work with a solid partner in science. Generating a buzz around any novel or different method or approach is critical to the messaging, but even more critical is understanding how a technology aids production, reduces risk and ultimately impacts the safety of the products placed into commerce. Clients who have begun to employ a Salmonella Limts Testing approach gain more confidence each day and are able to divert or treat products with higher levels of Salmonella. A Limits Testing approach has not only proven beneficial to the entire risk assessment model, it is having real time impact on how the plant responds and reacts to higher concentrations of Salmonella. The 8 hour same day result generated by the Roka technology does not interrupt commerce and results in more complete, earlier knowledge that allows confident informed decisions to occur in a seamless fashion. Please contact us if you would like to learn more about Limits testing and the semi-quantitative detection of Salmonella.