title: A One-Step Real-Time RT-PCR Assay for the Detection and Quantitation of Sugarcane Streak Mosaic Virus.
abstract: 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.
authors.affiliation: Key Laboratory of Sugarcane Biology and Genetic Breeding, Ministry of Agriculture, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, China ; College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, China.
authors.forename: Wei-Lin
authors.initials: WL
authors.lastname: Fu
authors.affiliation: Key Laboratory of Sugarcane Biology and Genetic Breeding, Ministry of Agriculture, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, China.
authors.forename: Sheng-Ren
authors.initials: SR
authors.lastname: Sun
authors.affiliation: Key Laboratory of Sugarcane Biology and Genetic Breeding, Ministry of Agriculture, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, China.
authors.forename: Hua-Ying
authors.initials: HY
authors.lastname: Fu
authors.affiliation: Key Laboratory of Sugarcane Biology and Genetic Breeding, Ministry of Agriculture, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, China.
authors.forename: Ru-Kai
authors.initials: RK
authors.lastname: Chen
authors.affiliation: College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, China.
authors.forename: Jin-Wei
authors.initials: JW
authors.lastname: Su
authors.affiliation: Key Laboratory of Sugarcane Biology and Genetic Breeding, Ministry of Agriculture, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, China ; Guangxi Collaborative Innovation Center of Sugarcane Industry, Guangxi University, Nanning, Guangxi 530005, China.
authors.forename: San-Ji
authors.initials: SJ
authors.lastname: Gao
mesh_terms: D009029:Mosaic Viruses / Q000235:genetics* / Q000302:isolation & purification*; D012367:RNA, Viral / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D031786:Saccharum / Q000821:virology*; D012680:Sensitivity and Specificity
chemical_list: D012367:RNA, Viral