Monitoring flocks, feed, raw meat and environmental samples for pathogens is critical to the poultry industry. In a recent application note, scientists describe how molecular testing can boost microbial detection capabilities.
Traditional testing methods use a combination of microbial culture and biochemical and serological strategies to isolate and detect individual microbial colonies. While these methods are standard practice and proven successful, identifying microbes using PCR offers several advantages.
With traditional methods, it can be difficult to isolate and distinguish the target organism when present amongst high levels of other organisms, which is why a secondary, selective enrichment step is usually required following initial enrichment using traditional plating methods. Waiting up to several days for a result can have a real financial impact on a business. PCR is highly sensitive and can identify relatively low amounts of microbes (typically about 1,000 CFU/mL) from a culture that may contain much higher levels of background flora, decreasing the time needed to grow up and purify cultures. This means results are available much sooner with PCR.
Taking these advantages further, real-time PCR is an improvement over end-point PCR, with no need to handle samples after the reaction is complete. Furthermore, real-time PCR methods that utilize a TaqMan™ probe, such as MicroSEQ™ and TaqMan™ assays (Thermo Fisher Scientific), show increased specificity over standard PCR methods because real-time PCR requires hybridization of three oligonucleotides to the target DNA, instead of just two oligonucleotides that are required for standard PCR.
Both end-point PCR and real-time PCR are completely customizable, however, real-time PCR can be more easily multiplexed. Many real-time PCR instruments can detect multiple dyes, individually as well as simultaneously, in the same PCR reaction mix. For example, the Applied Biosystems™ 7500 Fast Real-Time PCR System (Thermo Fisher Scientific) can detect up to five dyes in a single reaction. By doing so, investigators can design assays to simultaneously identify targets such as multiple species within a genus or a specific serovar within a single species. For example, one PCR assay can detect whether any Salmonella species are within the sample, while another PCR assay can detect Salmonella enterica subspecies enterica serovar Enteritidis.
Real-time PCR kits also simplify the process. For example, the TaqMan™ Salmonella spp. Ultimate Assay is a multiplex real-time PCR assay developed with input from the FDA. It was designed against two gene targets namely apeE and invA, both of which are highly specific for Salmonella. Scientists with Thermo Fisher Scientific recently validated the effectiveness of this kit by demonstrating 100% detection of both gene targets in 261 Salmonella isolates tested, and no detection in 20 non-Salmonella isolates.
As technology improves, so do advances in microbial testing. Learn more about poultry testing from farm to fork and download the application note to read more about how the move to molecular could improve your poultry testing.