Effective control and monitoring of foot‐and‐mouth disease (FMD) relies upon rapid and accurate disease confirmation. Currently, clinical samples are usually tested in reference laboratories using standardized assays recommended by The World Organisation for Animal Health (OIE). However, the requirements for prompt and serotype‐specific diagnosis during FMD outbreaks, and the need to establish robust laboratory testing capacity in FMD‐endemic countries have motivated the development of simple diagnostic platforms to support local decision‐making. Using a portable thermocycler, the T‐COR™ 8, this study describes the laboratory and field evaluation of a commercially available, lyophilized pan‐serotype‐specific real‐time RT‐PCR (rRT‐PCR) assay and a newly available FMD virus (FMDV) typing assay (East Africa‐specific for serotypes: O, A, Southern African Territories [SAT] 1 and 2). Analytical sensitivity, diagnostic sensitivity and specificity of the pan‐serotype‐specific lyophilized assay were comparable to that of an OIE‐recommended laboratory‐based rRT‐PCR (determined using a panel of 57 FMDV‐positive samples and six non‐FMDV vesicular disease samples for differential diagnosis). The FMDV‐typing assay was able to correctly identify the serotype of 33/36 FMDV‐positive samples (no cross‐reactivity between serotypes was evident). Furthermore, the assays were able to accurately detect and type FMDV RNA in multiple sample types, including epithelial tissue suspensions, serum, oesophageal–pharyngeal (OP) fluid and oral swabs, both with and without the use of nucleic acid extraction. When deployed in laboratory and field settings in Tanzania, Kenya and Ethiopia, both assays reliably detected and serotyped FMDV RNA in samples (n = 144) collected from pre‐clinical, clinical and clinically recovered cattle. These data support the use of field‐ready rRT‐PCR platforms in endemic settings for simple, highly sensitive and rapid detection and/or characterization of FMDV.

Peste des petits ruminants (PPR) is a disease of small ruminants caused by peste des petits ruminants virus (PPRV), and is endemic in Asia, the Middle East and Africa. Effective control combines the application of early warning systems, accurate laboratory diagnosis and reporting, animal movement restrictions, suitable vaccination and surveillance programs, and the coordination of all these measures by efficient veterinary services. Molecular assays, including conventional reverse transcription-polymerase chain reaction (RT-PCR) and real-time RT-PCR (RT-qPCR) have improved the sensitivity and rapidity of diagnosing PPR. However, currently these assays are only performed within laboratory settings; therefore, the development of field diagnostics for PPR would improve the fast implementation of control policies, particularly when PPR has been targeted to be eradicated by 2030. Loop-mediated isothermal amplification (LAMP) assays are simple to use, rapid, and have sensitivity and specificity within the range of RT-qPCR; and can be performed in the field using disposable consumables and portable equipment. This study describes the development of a novel RT-LAMP assay for the detection of PPRV nucleic acid by targeting the N-protein gene. The RT-LAMP assay was evaluated using cell culture propagated PPRVs, field samples from clinically infected animals and samples from experimentally infected animals encompassing all four lineages (I-IV) of PPRV. The test displayed 100% concordance with RT-qPCR when considering an RT-qPCR cut-off value of C_T >40. Further, the RT-LAMP assay was evaluated using experimental and outbreak samples without prior RNA extraction making it more time and cost-effective. This assay provides a solution for a pen-side, rapid and inexpensive PPR diagnostic for use in the field in nascent PPR eradication programme.

The COVID-19 pandemic has illustrated the importance of rapid, accurate diagnostic testing for the effective triaging and cohorting of patients and timely tracking and tracing of cases. However, a surge in diagnostic testing quickly resulted in worldwide competition for the same sample preparation and real-time RT-PCR diagnostic reagents (rRT-PCR). Consequently, Hampshire Hospitals NHS Foundation Trust, UK sought to diversify their diagnostic portfolio by exploring alternative amplification chemistries including those that permit direct testing without RNA extraction. This study describes the validation of a SARS-CoV-2 RT-LAMP assay, which is an isothermal, autocycling, strand-displacement nucleic acid amplification technique which can be performed on extracted RNA (RNA RT-LAMP) or directly from swab (Direct RT-LAMP). Analytical specificity (ASp) of this new RT-LAMP assay was 100% and analytical sensitivity (ASe) was between 1⨯10 ¹ and 1⨯10 ² copies when using a synthetic DNA target. The overall diagnostic sensitivity (DSe) and specificity (DSp) of RNA RT-LAMP was 97% and 99% respectively, relative to the standard of care (SoC) rRT-PCR. When a C _T cut-off of 33 was employed, above which increasingly evidence suggests there is a very low risk of patients shedding infectious virus, the diagnostic sensitivity was 100%. The DSe and DSp of Direct-RT-LAMP was 67% and 97%, respectively. When setting C _T cut-offs of ≤33 and ≤25, the DSe increased to 75% and 100%, respectively. Time from swab-to-result for a strong positive sample (C _T < 25) was < 15 minutes. We propose that RNA RT-LAMP could replace rRT-PCR where there is a need for increase in throughput, whereas Direct RT-LAMP could be used as a screening tool for triaging patients into appropriate hospitals wards, at GP surgeries and in care homes, or for population screening to identify super shedders. Direct RT-LAMP could also be used during times of high prevalence to save critical extraction and rRT-PCR reagents by screening out those strong positives from diagnostic pipelines.

Epizootic haemorragic disease (EHD) is an important disease of white-tailed deer and can cause a bluetongue-like illness in cattle. A definitive diagnosis of EHD relies on molecular assays such as real-time RT-qPCR or conventional PCR. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a cost-effective, specific, and sensitive technique that provides an alternative to RT-qPCR. We designed two sets of specific primers targeting segment-9 of the EHD virus genome to enable the detection of western and eastern topotypes, and evaluated their performance in singleplex and multiplex formats using cell culture isolates (n = 43), field specimens (n = 20), and a proficiency panel (n = 10). The limit of detection of the eastern and western RT-LAMP assays was estimated as ~24.36 C_T and as ~29.37 C_T in relation to real-time RT-qPCR, respectively, indicating a greater sensitivity of the western topotype singleplex RT-LAMP. The sensitivity of the western topotype RT-LAMP assay, relative to the RT-qPCR assay, was 72.2%, indicating that it could be theoretically used to detect viraemic cervines and bovines. For the first time, an RT-LAMP assay was developed for the rapid detection of the EHD virus that could be used as either a field test or high throughput screening tool in established laboratories to control the spread of EHD.