A One-Step Real-Time RT-PCR Assay for the Detection and Quantitation of Sugarcane Streak Mosaic Virus

Abstract EN

Sugarcane mosaic disease is caused by the Sugarcane streak mosaic virus (SCSMV; genus Poacevirus, family Potyviridae) which is common in some Asian countries.

Here, we established a protocol of a one-step real-time quantitative reverse transcription PCR (real-time qRT-PCR) using the TaqMan probe for the detection of SCSMV in sugarcane.

Primers and probes were designed within the conserved region of the SCSMV coat protein (CP) gene sequences.

Standard single-stranded RNA (ssRNA) generated by PCR-based gene transcripts of recombinant pGEM-CP plasmid in vitro and total RNA extracted from SCSMV-infected sugarcane were used as templates of qRT-PCR.

We further performed a sensitivity assay to show that the detection limit of the assay was 100 copies of ssRNA and 2 pg of total RNA with good reproducibility.

The values obtained were approximately 100-fold more sensitive than those of the conventional RT-PCR.

A higher incidence (68.6%) of SCSMV infection was detected by qRT-PCR than that (48.6%) with conventional RT-PCR in samples showing mosaic symptoms.

SCSMV-free samples were verified by infection with Sugarcane mosaic virus (SCMV) or Sorghum mosaic virus (SrMV) or a combination of both.

The developed qRT-PCR assay may become an alternative molecular tool for an economical, rapid, and efficient detection and quantification of SCSMV.