Abstract
As one of the top 10 plant viruses, the severity of losses to crop productivity caused by the tomato spotted wilt virus (TSWV) has resulted in an urgent need to develop a more sensitive and rapid method of detection. In this study, we developed a CRISPR/Cas13a-based detection system to diagnose TSWV in tomato and western flower thrips (Frankliniella occidentalis). The detection system relies on recombinase polymerase amplification and Cas13a-mediated collateral cleavage activity. Positive results can be distinguished after 20 min by a significantly enhanced fluorescence signal. We tested the sensitivity of CRISPR/Cas13a-based detection system and found that the detection system that we developed has limits of detection that reaches 2.26 × 102 copies/μl and a 10-fold increase compared with the sensitivity of using RT-PCR to detect the virus. Furthermore, the CRISPR/Cas13a-based detection system has a high selectivity for the TSWV without interference from other viruses. The CRISPR/Cas13a-based detection system was utilized to detect the TSWV in samples of tomato leaves and the transmission vector F. occidentalis that were fully consistent with the results when RT-PCR was used to detect the virus.
Highlights
Plant diseases caused by infection with tospoviruses have been causing enormous crop losses worldwide (Oliver and Whitfield, 2016)
The specimens of F. occidentalis were detected as non-viruliferous vectors by an RT-PCR assay that uses specific primers for the tomato spotted wilt virus (TSWV) (Supplementary Table 1), and they were maintained on healthy kidney bean pods at 26◦C, 70% relative humidity, and a 16h:8-h light:dark photoperiod
The results showed that other viruses, such as the tomato zonate spot virus (TZSV), tomato mosaic virus (TMV), cucumber mosaic virus (CMV), and potato virus Y (PVY), displayed almost unchanged fluorescence intensity compared with the TSWV sample, and that after only 15 min (30 kinetics cycle), the fluorescence intensity of the TSWV differed significantly from that of the other viruses (Figure 4)
Summary
Plant diseases caused by infection with tospoviruses have been causing enormous crop losses worldwide (Oliver and Whitfield, 2016). The tomato spotted wilt virus (TSWV), the type species of the genus Orthotospovirus, the family Tospoviridae, and the order Bunyavirales, is a tripartite negative/ambisense ssRNA virus. Based on their molecular weight, the three ssRNAs have been designated RNA-S, RNA-L, and RNA-M. RNA-M and RNA-S are ambisense RNAs, which encode the movement protein NSm and the glycoprotein Gn/Gc, respectively. Thrips acquire the virus during larval stages, and only thrips adults that acquired the virus as larvae can transmit the pathogen (Oliver and Whitfield, 2016).
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