title: Rapid and Quantitative Detection ofLeifsonia xylisubsp.xyliin Sugarcane Stalk Juice Using a Real-Time Fluorescent (TaqMan) PCR Assay.
abstract: Ratoon stunting disease (RSD) of sugarcane, one of the most important diseases seriously affecting the productivity of sugarcane crops, was caused by the bacterial agentLeifsonia xylisubsp.xyli(Lxx). A TaqMan probe-based real-time quantitative polymerase chain reaction (qPCR) assay was established in this study for the quantification ofLxxdetection in sugarcane stalk juice. A pair of PCR primers (Pat1-QF/Pat1-QR) and a fluorogenic probe (Pat1-QP) targeting thePart1gene ofLxxwere used for the qPCR assay. The assay had a detection limit of 100 copies of plasmid DNA and 100 fg ofLxxgenomic DNA, which was 100-fold more sensitive than the conventional PCR. Fifty (28.7%) of 174 stalk juice samples from two field trials were tested to be positive by qPCR assay, whereas, by conventional PCR, only 12.1% (21/174) were tested to be positive with a published primer pair CxxITSf#5/CxxITSr#5 and 15.5% (27/174) were tested to be positive with a newly designed primer pair Pat1-F2/Pat1-R2. The new qPCR assay can be used as an alternative to current diagnostic methods forLxx, especially when dealing with certificating a large number of healthy cane seedlings and determining disease incidence accurately in commercial fields.
authors.affiliation: National Engineering Research Center for Sugarcane, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, China.
authors.forename: Hua-Ying
authors.initials: HY
authors.lastname: Fu
authors.affiliation: National Engineering Research Center for Sugarcane, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, China.
authors.forename: Sheng-Ren
authors.initials: SR
authors.lastname: Sun
authors.affiliation: National Engineering Research Center for Sugarcane, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, China.
authors.forename: Jin-Da
authors.initials: JD
authors.lastname: Wang
authors.affiliation: National Engineering Research Center for Sugarcane, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, China.
authors.forename: Kashif
authors.initials: K
authors.lastname: Ahmad
authors.affiliation: National Engineering Research Center for Sugarcane, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, China.
authors.forename: Heng-Bo
authors.initials: HB
authors.lastname: Wang
authors.affiliation: National Engineering Research Center for Sugarcane, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, China.
authors.forename: Ru-Kai
authors.initials: RK
authors.lastname: Chen
authors.affiliation: National Engineering Research Center for Sugarcane, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, China.
authors.forename: San-Ji
authors.identifier: 0000-0003-3442-8470
authors.initials: SJ
authors.lastname: Gao
mesh_terms: D001628:Beverages / Q000382:microbiology*; D005453:Fluorescence; D008835:Micrococcaceae / Q000737:chemistry / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012015:Reference Standards; D031786:Saccharum / Q000382:microbiology*; D012680:Sensitivity and Specificity