title: Increased sensitivity of RT-PCR for Potato virus Y detection using RNA isolated by a procedure with differential centrifugation.
abstract: The sensitivity of reverse transcription-polymerase chain reaction (RT-PCR) for virus detection is influenced by many factors such as specificity of primers and quality of templates. These factors become extremely important for successful detection when virus concentration is low. Total RNA isolated from Potato virus Y (PVY)-infected potato plants using the sodium sulfite RNA isolation method or RNeasy plant mini kit contains a high proportion of host RNA and may also contain trace amount of phenolic and polysaccharide residues, which may inhibit RT-PCR. The goal of this study was to enhance the sensitivity of PVY detection by reducing host RNA in the extract by differential centrifugation followed by extraction using an RNeasy mini kit (DCR method). One-step RT-PCR had relatively low amplification efficiency for PVY RNA when a high proportion of plant RNA was present. SYBR Green-based real time RT-PCR showed that the RNA isolated by the DCR method had a higher cycle threshold value (Ct) for the elongation factor 1-α mRNA (Ef1α) of potato than the Ct value of the RNA extracted using the RNeasy plant mini kit, indicating that the DCR method significantly reduced the proportion of potato RNA in the extract. The detectable amount of RNA extracted using the DCR method was <0.001ng when plant sap from 10 PVY-infected and PVY-free potato leaflets in a 1.5:100 fresh weight ratio was extracted, compared with 0.01 and 0.02ng of RNA using the RNeasy plant mini kit and sodium sulfite RNA isolation methods, respectively.
authors.affiliation: Department of Biology, University of New Brunswick, PO Box 4400, Fredericton, New Brunswick E3B 5A3, Canada.
authors.forename: Jianhua
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Potato Research Center, 850 Lincoln Rd, Fredericton, New Brunswick E3B 4Z7, Canada.
authors.forename: Xianzhou
authors.initials: X
authors.lastname: Nie
authors.affiliation: S.I.P.R.E - Comité Nord, Rue des Champs Potez, 62217 Achicourt, France.
authors.forename: Sébastien
authors.initials: S
authors.lastname: Boquel
authors.affiliation: Potato Research Center, 850 Lincoln Rd, Fredericton, New Brunswick E3B 4Z7, Canada.
authors.forename: Fadi
authors.initials: F
authors.lastname: Al-Daoud
authors.affiliation: Potato Research Center, 850 Lincoln Rd, Fredericton, New Brunswick E3B 4Z7, Canada. Electronic address: pelletieryvan@gmail.com.
authors.forename: Yvan
authors.initials: Y
authors.lastname: Pelletier
mesh_terms: D002498:Centrifugation / Q000379:methods*; D010935:Plant Diseases / Q000821:virology*; D017800:Potyvirus / Q000235:genetics / Q000302:isolation & purification*; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D011198:Solanum tuberosum / Q000821:virology
chemical_list: D012367:RNA, Viral
keywords: PVY; RNA isolation; RT-PCR; Virus detection