Table 1. Viral RNA used for testing sensitivity and specificity of RT-LAMP primers.
| RT-LAMP primer set (100 PFU target) | ||||
|---|---|---|---|---|
| Virus | Strain or Isolate Designation | SLEV 3’-UTR | WNV E gene | WEEV nsP4 |
| Flaviviruses | ||||
| St. Louis encephalitis virus (SLEV)1 | Kern217 | + | – | – |
| Bfs1750 (Ct 17.7) | + | – | – | |
| Ruls (Ct 12.9) | + | – | – | |
| Coav750 (Ct 23.9) | + | – | – | |
| 69M1143 (Ct 13.7) | + | – | – | |
| BeAn246407 (Ct 20.4) | + | – | – | |
| CorAn9124 (PCR neg.) | + | – | – | |
| CorAn9275 (PCR neg.) | + | –2 | – | |
| West Nile virus (WNV) | L-CA-04 SAC-04-7168 | – | + | – |
| Yellow fever virus (YFV) | 17D | – | – | – |
| Rocio virus (ROCV) | SP H 34675 | – | – | – |
| Usutu virus (USV) | SA AR 1776 | – | – | – |
| Ilheus virus (ILHV) | Ilheus B44532 | – | – | – |
| Dengue virus serotype 1 (DENV-1) | BC-796 | – | – | – |
| Dengue virus serotype 2 (DENV-2) | BC-122-94 | – | – | – |
| Dengue virus serotype 3 (DENV-3) | BC 156–97 | – | – | – |
| Alphaviruses | ||||
| Western equine encephalitis virus (WEEV)1 | KERN 5547 | – | – | + |
| Lake43 (Ct 14.9) | – | – | + | |
| Sindbis virus (SINV) | EDS-14 | – | – | – |
| Ross River virus (RRV) | SW 38457 | – | – | – |
| Chikungunya virus (CHIKV) | Ross | – | – | – |
| Venezuelan equine encephalitis virus (VEEV) | TC-83 | –2 | – | – |
| Barmah Forest virus (BFV) | Barmah Forest (TVP-4119) | – | – | – |
| Highlands J virus (HJV) | WC-431 | – | – | – |
| Eastern equine encephalitis virus (EEEV)3 | EEEV/X/USA/A15072/2003 | – | – | – |
1 Isolatesof SLEV other than Kern 217, and WEEV isolate Lake43 were not quantitated by plaque assay. Extracted RNA was quantitated by RT-qPCR and used undiluted as a template in RT-LAMP. RT-qPCR Ct values are indicated. CorAn 9124 and CorAn9275 were not detected by RT-qPCR primers.
2 A single reaction showed positive amplification within 45 minutes of incubation (1 of 11 replicates for SLEV CorAn 9275 with WNV primers; 1 of 18 replicates for VEEV TC-83 with SLEV primers). Melt curve analysis indicates that these are isolated occurrences of cross-contamination with positive-control RNA.
3 Cross-reactivity testing for EEEV was performed with 106 copies of an in vitro transcribed fragment of the EEEV nsP4 gene corresponding to the WEEV RT-LAMP target site including an additional 46 nt upstream and 65 nt downstream of the outer primer (F3 and B3) binding sites. The in vitro transcribed RNA represents nucleotides 5980–6329 (350 bases) of Genbank # KJ469613.1.