Rapid Sequence Identification of Foot-and-Mouth Disease Virus Utilizing FMDV-ONTAPS: The Oxford Nanopore Technologies Amplicon P1 Sequencing Protocol

Diagnostic testing of foot-and-mouth disease virus (FMDV) currently utilizes reverse transcription quantitative PCR (RT-qPCR) to detect the presence of viral RNA and double antibody sandwich ELISAs (DAS-ELISAs) to determine viral serotype. Serotype identification is critical to support informed vaccine selection to combat outbreaks. While DAS-ELISAs are capable of serotype identification, the test suffers from low sensitivity and requires a viral isolate for successful detection. In this study, we developed FMDV-ONTAPS: an Oxford Nanopore Technologies Amplicon P1 Sequencing protocol involving reverse transcription-PCR to amplify P1 of the FMDV genome, and Nanopore sequencing of the amplicons to provide genetic data for serotype and subtype/topotype identification. FMDV isolates representing all seven serotypes were successfully sequenced with this method. Additionally, the protocol successfully provided serotype identification from a variety of specimen matrices obtained from experimentally infected animals that included milk, serum, oral and nasal swabs, tissue suspensions, vesicular fluid, and oral fluid. The limit of detection for FMDV cell culture isolates was comparable for both sequencing and RT-qPCR detection. RT-qPCR Cq values for clinical samples evaluated ranged from 8 to 28.21. Sequencing was successful for all samples except for a single tissue suspension sample (Cq of 28.21). Identification of FMDV serotype in clinical samples is critical for effective outbreak response, and Nanopore sequencing offers a timelier and more sensitive alternative to DAS-ELISAs.