Enhancement of loop-mediated isothermal amplification (LAMP) with guanidine hydrochloride for the detection ofsubspecies(Strangles).

Abstract

subspecies, commonly referred to as "strangles", poses a significant biosecurity challenge across equine farms worldwide. The continuous prevalence and highly transmissibility of strangles necessitates a rapid and accurate diagnostic procedure. However, current "gold-standard" techniques, such as cultures and quantitative polymerase chain reaction (qPCR), are unreliable or inaccessible, and require lengthy periods between sample collection and results. Moreover, the lack of a standardized detection protocol can lead to variations in results. This study aimed to develop a reproducible and field-deployable diagnostic assay to detect strangles in real-time. Utilising the rapid technique loop-mediated isothermal amplification (LAMP), we developed an assay targeting a conserved region of thespecific M gene (SeM). Additionally, we optimised our assay with guanidine hydrochloride (GuHCl) to enhance the assay's performance and detection capabilities. The Str-LAMP was able to detectwithin 13 minutes and 20 seconds for both synthetic DNA and clinical isolates, with a limit of detection (LOD) of  53 copies/µl. Our assay demonstrated high repeatability with the inter-coefficient of variation ranging from 0.17% to 3.93%. Furthermore, the clinical sensitivity and specificity was calculated at 91.3% and 93.3%, respectively, with a correct classification rate of 91.8%. The implementation of this newly developed strangles assay can be employed as an efficient aid for in-field surveillance programs. The assay's reproducibility can allow for equine managers to undertake routine self-surveillance on their properties, without the requirement of specialised training. The Str-LAMP assay has the potential to be a valuable tool to help mitigate potential strangles outbreaks.