Development and Evaluation of ELISA and qRT-PCR for Identification of Squash vein yellowing virus in Cucurbits.

Squash vein yellowing virus (SqVYV) causes viral watermelon vine decline. To facilitate detection of SqVYV, enzyme linked-immunosorbent assay (ELISA) and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) diagnostic methods were developed. Both methods were capable of detecting SqVYV in a wide range of cucurbit hosts. ELISA was able to detect virus in infected host tissue diluted to at least 1:2,560, which was sufficient for detection in symptomatic squash and watermelon plants. The qRT-PCR method was capable of reliably detecting as few as 3.4 copies of a cloned fragment of SqVYV genomic RNA with an average cycle threshold (C) value of 36.4. The sensitivities and specificities for each detection method were estimated by latent class analysis for a set of inoculated squash and watermelon plants at two sampling scales. The scales were hierarchical, with individual plants representing the upper scale and samples from the plant representing the lower scale. The number of samples per plant varied from 1 to 8, and a plant was diagnosed positive if any of its samples tested positive. For all analyses, a cutoff C of 35 was chosen for qRT-PCR, which is approximately 2.5 cycles lower than the lowest C value achieved for mock-inoculated plants (presumed to be a false positive). qRT-PCR showed high sensitivities (≥0.99) at both sampling scales for squash and watermelon, whereas the sensitivities for ELISA ranged from 0.58 to 0.76. The specificities for both tests were very similar (≥0.94), with ELISA sometimes outperforming qRT-PCR. These diagnostic methods provide additional tools for the identification of SqVYV and management of SqVYV-induced watermelon vine decline.