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

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.

Keywords: Seneca Valley virus-1; point-of-care diagnostics; rapid detection; reverse transcription loop-mediated isothermal amplification.

Figure 1

Oligonucleotide primers used for RT‐LAMP amplification of SVV‐1. (a) Primer set 1 (P1) targeting the 5′UTR region; (b) Primer set 2 (P2), targeting the VP3‐1 region. Nucleotide positions of the primers in both primer sets (P1 and P2) are mapped to GenBank accession number DQ641257 [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 2

Comparison of ‘wet’ and lyophilized reagents using direct detection by RT‐LAMP with primer set 1 (a) and primer set 2 (b). Black bars represent ‘wet’ reagents and grey bars represent lyophilized reagents. Neat: SVV‐1 sample NC‐88‐23626 diluted 1/100 in negative pig epithelium tissue suspension to simulate a natural original suspension sample. This ‘neat’ sample was then diluted ½, ¼, 1/8, 1/10, 1/16 and 1/20 in nuclease‐free water (NFW) and compared to extracted RNA from the ‘neat’ sample as a positive control

Figure 3

A box‐plot to compare Tp values of ‘wet’ and lyophilized reagents for primer sets 1 (P1) and 2 (P2) using extracted RNA from the seven SVV‐1 samples. SVV‐1 samples were diluted 1/100 in negative pig epithelium tissue suspension prior to RNA extraction