Simultaneous detection of multiple Salmonella serovars from milk and chicken meat by real-time PCR using unique genomic target regions.

Authors: AFROJ, SAYMA - Tuskegee University; ALDAHAMI, KHALED - Tuskegee University; REDDY, GOPAL - Tuskegee University; Guard, Jean; ADESIYUM, ABIODUN - University Of The West Indies; SAMUEL, TEMESGEN - Tuskegee University; ABDELA, WOUBIT - Tuskegee University

Submitted to: Journal of Food Protection
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/19/2017
Publication Date: 11/1/2017
Citation: Afroj, S., Aldahami, K., Reddy, G., Guard, J.Y., Adesiyum, A., Samuel, T., Abdela, W. 2017. Simultaneous detection of multiple Salmonella serovars from milk and chicken meat by real-time PCR using unique genomic target regions. Journal of Food Protection. 800(11):1944-1957.

Interpretive Summary: A plateau in the reduction of Salmonella enterica associated with food borne illness in people has been in evidence since 2010 in the United States and the European Union. To break through the plateau, typing methods for rapidly processing food samples to identify disease-causing Salmonella serotypes are in development. This manuscript describes use of real time polymerase chain reaction (PCR) optimized to detect within a single reaction four serotypes that commonly cause disease and one serotype that is often associated with farm environments. The typing method is especially applicable to dairy and poultry products.

Technical Abstract: Background: A highly sensitive and specific novel genomic and plasmid target-based PCR platform was developed to detect multiple Salmonella serovars (S. Heidelberg, S. Dublin, S. Hadar, S. Kentucky and S. Enteritidis). Through extensive genome mining of protein databases of these serovars and comparative genomic analysis, unique genome loci were identified and utilized to develop PCR-based assays for multiplexed detection of the serovars. V-NTI Advanced-11 was used for the design and modification of primers. The designed primers were validated by in-silico PCR, and further blasted using NCBI and PATRIC databases. Initial validation of inclusivity and exclusivity assays involved testing of these primers with the genomic DNA of corresponding Salmonella serovars, 96 non-target Salmonella serovars, and 27 closely related bacterial species by conventional and real time PCR. Results: Assays targeting Salmonella serovars Hadar, Heidelberg, Kentucky and Dublin yielded 100% specificity and sensitivity, while those for serovar Enteritidis yielded 97% specificity and 88% sensitivity. The limits of detection of the five serovars were found to be 58.8 fg/µl for S. Heidelberg, 42.2 fg/µl for S. Kentucky, 200 fg/µl for S. Hadar, 63.4 fg/µl for S. Enteritidis, and 26 pg/µl for S. Dublin. Sensitivity assay was also performed by using milk artificially inoculated with pooled Salmonella serovars, yielding a detection limit of 1-10 CFU per 25 ml of milk samples. The multiplex assay from food was further validated with TaqMan assay containing internal amplification control. The minimum DNA detected using this multiplexed Real-Time PCR was 75.8 fg (1.53×101) for Heidelberg, 140.8 fg (2.8×101) for Enteritidis and 3.48 pg (6.96×102) for Dublin serovars. PCR efficiencies were 89.8% for S. Heidelberg, 94.5%, for S. Enteritidis and 75.5% for S. Dublin. Thirty different types of pasteurized milk samples purchased from different grocery shops were all negative by cultural techniques as well as with Salmonella specific invA gene PCR. Among thirty chicken samples similarly tested, twelve (40%) were positive by both cultural technique as well as conventional PCR with Salmonella specific invA gene. Testing of the twelve samples by serovar-specific PCR detected single as well as mixed contaminations with S. Kentucky, S. Enteritidis and S. Heidelberg. Conclusion: The diagnostic assays developed in this study could be used as tools for routine detection of Salmonella as well as for epidemiological investigations of foodborne disease outbreaks.