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ARS Home » Plains Area » Clay Center, Nebraska » U.S. Meat Animal Research Center » Meat Safety and Quality » Research » Publications at this Location » Publication #389450

Research Project: Holistic Tactics to Advance the Microbiological Safety and Quality of the Red Meat Continuum

Location: Meat Safety and Quality

Title: Universal hydrolysis probe-based approach for specific detection and genotyping of foodborne pathogens

item VALEZ, FRANK - Florida State University
item Bosilevac, Joseph - Mick
item MISHRA, ABHINAV - University Of Georgia
item SINGH, PRASHANT - Florida State University

Submitted to: Journal of Microbiological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/25/2022
Publication Date: 1/1/2023
Citation: Valez, F., Bosilevac, J.M., Mishra, A., Singh, P. 2023. Universal hydrolysis probe-based approach for specific detection and genotyping of foodborne pathogens. Journal of Microbiological Methods. 204. Article 106632.

Interpretive Summary: One of the most common methods to test food for contaminating pathogens is to test for the specific segments of DNA of a pathogen. This DNA test requires the use of special reagents for each pathogen. While many of the reagents are inexpensive, the tests that require fluorescent labeled DNA probes are costly and make the DNA tests expensive. We developed and validated a method that uses a universal replacement for the expensive fluorescent labeled probes. A different fluorescent probe is normally needed for each different real time DNA tests, but the universal replacement can be used in multiple different tests. This universal reagent approach can simplify pathogen test development when looking for different DNA targets and offers an economical approach that can make all real time DNA tests less expensive.

Technical Abstract: Real-time PCR assays are the method of choice for the specific detection of DNA targets. Multiple real-time PCR chemistries are used for developing pathogen detection assays. Among them, a hydrolysis probe is a preferred choice for pathogen detection assays. Two known limitations of hydrolysis probes are high cost and limited storage life. Therefore, this study aimed to develop and validate a universal hydrolysis probe (UHP)-based approach with high-resolution melt (HRM) analysis capabilities. The approach can be used for the detection and genotyping of target DNA. The approach described in this study was validated by standardizing nine UHP assays for detecting seven Shiga toxin-producing Escherichia coli serogroups, Listeria monocytogenes, and Salmonella strains. These nine assays were validated with 141 pure culture bacterial strains. Additionally, the HRM capability of the developed approach was validated for three UHP assays targeting E. coli O26, O111, and O121 using 96 DNAs isolated from enriched food samples. The nine assays specifically detected the target bacterial strains, and the three assays showed single nucleotide polymorphism (SNP) identification capability and no crossreactivity with non-target strains. The developed approach can be performed in singleplex or multiplex format and combined with HRM analysis. The data from this study demonstrate that the UHP real-time PCR approach is a robust method for detecting any deoxyribonucleic acid target.