A Duplex Real-Time RT-PCR System with an Internal Control Offers Sensitive and Reliable Broad-Spectrum Detection of...

Squash mosaic virus (SqMV), a seedborne virus, belongs to the genus Comovirus in the subfamily Comovirinae of the family Secoviridae. SqMV has a bipartite single-stranded RNA genome (RNA1 and RNA2) encapsidated separately with two capsid proteins. With the recent identification of a third genotype in SqMV, a greater genetic diversity with only 88 to 89% sequence identity among them are recognized. With the existence of genetic diversity, a previously developed quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) failed to detect isolates in this new genotype. Therefore, it was necessary to create a new qRT-PCR that would react with all SqMV isolates in three different genotypes. From a multiple sequence alignment of the available SqMV sequences in GenBank, a conserved sequence segment in the 3' untranslated region of RNA2 was identified for primer and probe design. A new qRT-PCR was developed, which provided broad-spectrum reactions to SqMV isolates, including those from the newly recognized third genotype. To improve the reliability in determining the sample quality and result interpretation, an internal amplification control with an endogenous gene sequence (18S ribosomal RNA) was successfully incorporated to develop a duplex qRT-PCR system that was useful for seed health test.