The development of two field-ready reverse transcription loop-mediated isothermal amplification assays for the rapid detection of Seneca Valley virus 1.

Bryony Armson,Veronica L Fowler,Nick Morant,Charlotte Walsh,Nick J Knowles,Duncan Clark

doi:10.1111/tbed.13051

Abstract

Seneca Valley virus 1 (SVV‐1) has been associated with vesicular disease in swine, with clinical signs indistinguishable from those of other notifiable vesicular diseases such as foot‐and‐mouth disease. Rapid and accurate detection of SVV‐1 is central to confirm the disease causing agent, and to initiate the implementation of control processes. The development of rapid, cost‐effective diagnostic assays that can be used at the point of sample collection has been identified as a gap in preparedness for the control of SVV‐1. This study describes the development and bench validation of two reverse transcription loop‐mediated amplification (RT‐LAMP) assays targeting the 5′‐untranslated region (5′‐UTR) and the VP3‐1 region for the detection of SVV‐1 that may be performed at the point of sample collection. Both assays were able to demonstrate amplification of all neat samples diluted 1/100 in negative pig epithelium tissue suspension within 8 min, when RNA was extracted prior to the RT‐LAMP assay, and no amplification was observed for the other viruses tested. Simple sample preparation methods using lyophilized reagents were investigated, to negate the requirement for RNA extraction. Only a small delay in the time to amplification was observed for these lyophilized reagents, with a time from sample receipt to amplification achieved within 12 min. Although diagnostic validation is recommended, these RT‐LAMP assays are highly sensitive and specific, with the potential to be a useful tool in the rapid diagnosis of SVV‐1 in the field.

Highlights

Seneca Valley virus 1 (SVV‐1) is the only known virus belonging to the species Senecavirus A, genus Senecavirus, within the familyPicornaviridae (Knowles et al, 2012)
This study describes the development of two Reverse transcription loop‐mediated isothermal amplification (RT‐LAMP) assays using lyophilized reagents, targeting the 5′‐untranslated region (5′UTR) and virus protein (VP) 3‐1 regions for the detection of SVV‐1, and performed on a portable real‐time fluorometer suitable for field use
Samples used in this study (Table 1) were archival samples previously submitted to the World Reference Laboratory for foot‐and‐mouth disease (FMD) (WRLFMD; The Pirbright Institute, UK)

Summary

| INTRODUCTION

Seneca Valley virus 1 (SVV‐1) is the only known virus belonging to the species Senecavirus A, genus Senecavirus, within the family. Picornaviridae (Knowles et al, 2012) It is a non‐enveloped, single‐ stranded, positive‐sense RNA virus recently associated with vesicular disease in swine in Brazil, the USA, China, Canada, Colombia and Thailand A number of accurate and sensitive rRT‐PCR methods have been developed, targeting the viral polymerase 3D region (Fowler et al, 2017), the VP1 coding region (Bracht et al, 2016), and the 5′ untranslated region (5′‐UTR) (Gimenez‐Lirola et al, 2016) Diagnosis via these methods relies on the transport of samples under appropriate conditions from the point of collection to centralized laboratory settings, which may add a significant time delay and favour the spread of disease, considering that modes of transmission have not yet been fully elucidated (Yoon, 2015). This study describes the development of two RT‐LAMP assays using lyophilized reagents, targeting the 5′‐untranslated region (5′UTR) and virus protein (VP) 3‐1 regions for the detection of SVV‐1, and performed on a portable real‐time fluorometer suitable for field use

| MATERIALS AND METHODS

| RESULTS

| DISCUSSION