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Title: A duplex real-time RT-PCR system with an internal control offers sensitive and reliable broad spectrum detection of Squash mosaic virus variants

Author
item Li, Rugang
item BERENDSEN, SVEN - Rijk Zwaan Breeding Bv
item Ling, Kai-Shu

Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/13/2015
Publication Date: 3/1/2016
Citation: Li, R., Berendsen, S., Ling, K. 2016. A duplex real-time RT-PCR system with an internal control offers sensitive and reliable broad spectrum detection of Squash mosaic virus variants. Plant Disease. 100:625-629.

Interpretive Summary: Squash mosaic virus (SqMV) is a seed borne and beetle transmitted virus infecting cucurbit productions worldwide. Development and application of a sensitive and reliable seed health assay is an important means to ensure safe production and trading of certified SqMV-tested cucurbit seeds. With the knowledge that there are diverse genetic variants, a new set of primers and corresponding TaqMan probes were designed and proven to produce a broad spectrum reaction to all known genotypes of SqMV. Through incorporation of an internal amplification control using 18S rRNA, a duplex quantitative reverse transcription polymerase chain reaction (qRT-PCR) was developed to offer a sensitive and reliable seed health assay for commercial seeds lots. The broader adaptation of this newly developed technology will improve the reliability of detecting all known SqMV genotypes and allow for profitable productions of cucurbit crops in the U.S. and around the world.

Technical Abstract: Squash mosaic virus (SqMV) is a seed-borne virus, belonging to the genus Commovirus in the subfamily Comoviridae of family Secoviridae. SqMV has a bipartite single-stranded ribonucleic acid (RNA) genome (RNA1 and RNA2) encapsidated separately with two capsid proteins. Two serotypes (genotypes) of SqMV with only 88-89% sequence identity are recognized and a broad spectrum qRT-PCR was established. However, that qRT-PCR failed to detect an emerging isolate RZ isolate of squash mosaic virus (RZ-SqMV) belonging to a third genotype, that was recently isolated on squash. To develop a new qRT-PCR that would offer a sensitive and reliable detection to all SqMV variants in different genotypes, from a multiple sequence alignment of available SqMV sequences, a conserved sequence region in the 3’-terminal RNA2 was identified and a new TaqMan RT-PCR developed. Unlike the original qRT-PCR-2011 which reacted to SqMV isolates only in two genotypes, this new qRT-PCR-2015 offered broad spectrum detection to SqMV isolates in all three genotypes. Furthermore, a duplex qRT-PCR system using an internal control with an endogenous gene sequence (18S rRNA) was successfully developed to improve the reliability in interpreting the cycle threshold reading for disease diagnosis.