ACTIVITY 3. IMPROVED RAPID DIAGNOSTICS FOR SALMONELLA SPP. THROUGH USE OF GENOMICS
Activity Leader: Lawrence Goodridge, McGill University, firstname.lastname@example.org, T. (514) 398-7921
The goal of Activity 3 is twofold: 1) to improve the specificity of already existing commercially available immunoassays and RT-PCR assays; and 2) develop a new sequence-based molecular assay (Ampliseq) capable of determining the pathogenic status of Salmonella isolates, and delivering confirmed Salmonella testing results more rapidly than the current confirmatory testing.
The low concentration of pathogens in foods requires an enrichment step to first grow (enrich) the bacteria to a detectable level from samples containing complex microflora. Even when sensitive molecular assays such as RT-PCR are used to detect bacteria in complex matrices such as food, enrichment is still needed to overcome the presence of substances (fat, protein, DNases) that can inhibit the PCR assay. Thus, enrichment should be considered as the first step of any rapid method, and it is where improvements in specificity can be achieved first. A common issue with enrichment and rapid detection of Salmonella from fresh produce samples is the presence of other closely related bacteria, principally Citrobacter spp., Proteus spp., and Hafnia spp. that can cause false positive test results when immunoassays or RT-PCR assays are used. This leads to a high percentage (up to 35%) of false positive test results. Another issue is the potential for false negative test results.
The following sub-objectives will be instrumental to reach the main goal:
Obj. 3.1 Develop more selective methods to reduce the presence of closely related bacteria during enrichment of Salmonella from fresh produce
It is anticipated that the best prospects for improving the specificity of immunoassays will be based around the enrichment media, since the similar nature of the cell surface between Salmonella and Proteus spp., Citrobacter spp., and Hafnia spp., mean that more specific surface exposed antigens are unlikely to be identified.
Obj. 3.2 Adapt WGS data to improve the specificity of existing commercially available RT-PCR assays for detection of foodborne Salmonella
Approximately 200 to 300 DNA targets that are informative for Salmonella pan-serovar detection will be identified from the Salmonella WGS from Activity 1. These targets will be analyzed against the WGS of closely related bacteria (also from Activity 1) to ensure that they are specific. The chosen sequences will then be assessed for suitable primer pair and probe development using an online suite of primer development and analysis tools.
Obj. 3.3 Develop a new sequence-based assay for faster confirmatory testing and assessing the pathogenic potential of Salmonella isolates
Due to the high false positive results, any positive test results obtained with immunological or molecular rapid assays are termed as presumptive results, and must be confirmed using more detailed and lengthy procedures. These techniques, based on the phenotype of bacteria, are labour-intensive and can take up to seven (7) days to confirm suspected contamination by biochemical and serological tests. It is expected the development of this new assay will decrease the time to achieve a confirmed test result for Salmonella by at least 48-72 hours.
1) Better Salmonella enrichment methods that decrease the presence of Citrobacter, Proteus and Hafnia spp. that are known to cause false positive test results, for incorporation into downstream diagnostic assays.
2) Improvement of two commercially existing diagnostic assays by focusing on the development of more specific enrichment for immunoassays, and more specific enrichment and PCR primers and probes for RT-PCR based detection of Salmonella.
3) Development of a sequence-based confirmatory assay for Salmonella that will identify the virulence of individual Salmonella isolates, from a pure colony, while reducing the time to confirmation by 48-72 hours.