Detection and quantitation of the new world Squash leaf curl virus by TaqMan real-time PCR

• Development of a TaqMan real-time PCR assay for specific detection of SLCV.
• This qPCR assay is 1000 times more sensitive than conventional PCR.
• Absolute quantitation of viral load in different squash organs and in viruliferous whiteflies.
• In silico analysis shows suitability for detection of the western hemisphere SLCV.

Squash leaf curl diseases are caused by distinct virus species that are separated into two major phylogenetic groups, western and eastern hemisphere groups. The western group includes the new world Squash leaf curl virus (SLCV) which causes major losses to cucurbit production and induces severe stunting and leaf curl in squash plants. A TaqMan-based real time polymerase chain reaction (qPCR) assay has been developed for detection and quantitation of SLCV. Designed primers and probe targeted the AV1 (coat protein) gene and in silico analysis showed that they detect a large number of SLCV isolates. The developed assay could detect the virus in 18 fg of total nucleic acid and 30 genomic units. The qPCR assay was about 1000 times more sensitive than PCR and amplified successfully SLCV from a wide range of cucurbit hosts and from viruliferous whiteflies. The developed qPCR assay should be suitable for detection and quantitation purposes for all reported SLCV isolates of the western hemisphere.