TickPath Layerplex: adaptation of a real-time PCR methodology for the simultaneous detection and molecular surveillance of tick-borne pathogens

Authors: MODARELLI, JOSEPH - Texas A&M University; FERRO, PAMELA - Texas A&M University; Perez De Leon, Adalberto - Beto; ESTEVE-GASENT, MARIA - Texas A&M University

Submitted to: Scientific Reports
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/23/2019
Publication Date: 5/6/2019
Citation: Modarelli, J.J., Ferro, P.J., Perez De Leon, A.A., Esteve-Gasent, M.D. 2019. TickPath Layerplex: adaptation of a real-time PCR methodology for the simultaneous detection and molecular surveillance of tick-borne pathogens. Scientific Reports. 9:6950. https://doi.org/10.1038/s41598-019-43424-y.
DOI: https://doi.org/10.1038/s41598-019-43424-y

Interpretive Summary: Tick-borne diseases (TBD) are common across the United States and can result in debilitating or even lethal diseases in a variety of animal species including humans. TBD are caused by viruses, bacteria, or parasites transmitted to a susceptible host through the bite of an infected tick. Rapid, sensitive and specific molecular diagnostic techniques are needed for TBD. This research publication describes the development of patented technology called TickPath Layerplex. This is a molecular diagnostic assay designed to detect and characterize rapidly and accurately microbes causing common TBD in dogs, some of which can also make humans sick. TickPath Layerplex was evaluated experimentally testing samples from dogs and ticks, and shown to be compatible with common instruments utilized in most molecular diagnostic laboratories. This novel technology showed high sensitivity, specificity, and screening capability for TBD that could be adapted to test samples from other susceptible hosts.

Technical Abstract: Tick-borne diseases (TBD) are common across the United States and can result in critical and chronic diseases in a variety of veterinary patients. Moreover, borreliosis, anaplasmosis, rickettsiosis, ehrlichiosis, and babesiosis are zoonotic and have been cited as the most common TBDs. Molecular diagnostic methodologies utilized for screening domestic dogs for these causative agents include real-time PCR (qPCR) assays in both singleplex and multiplex formats. However, current limitations of qPCR instruments restrict the number of fluorogenic labels that can be differentiated by the instrument for a given reaction. This study describes the development of the TickPath Layerplex, a diagnostic assay based on qPCR methodology that was adapted for the simultaneous detection and characterization of 11 pathogens responsible for causing 5 common TBDs in domestic dogs. The analytical and diagnostic performance of the layerplex assay was evaluated and shown to be compatible with common instruments utilized in molecular diagnostic laboratories. Test results revealed no inhibition or reduction in sensitivity during validation of the layerplex assay, and the limit of detection was determined to be near 16 genome copy equivalents per microliter. Overall, the high sensitivity, specificity, and screening capability of the assay demonstrate its utility for broadly screening dogs for common TBDs.