title: Multiplex reverse transcription loop-mediated isothermal amplification for the simultaneous detection of CVB and CSVd in chrysanthemum.
abstract: A multiplex reverse transcription loop-mediated isothermal amplification (mRT-LAMP) assay was developed for the simultaneous detection of Chrysanthemum Virus B (CVB) and Chrysanthemum stunt viroid (CSVd), which are the major viral pathogens of chrysanthemum worldwide. Two sets of mRT-LAMP primers were designed for the coat protein gene of CVB and the complete nucleotide sequence of CSVd, and a restriction enzyme cleavage site was inserted into two pairs of species-specific primers. The mRT-LAMP assay was designed by combining these two sets for a total of eight primers. The mRT-LAMP method distinguished between CVB and CSVd due to the subsequent restriction enzyme analysis. The sensitivity of the mRT-LAMP method was 10(3) times higher than classical PCR regarding the detection limits for CVB and CSVd. No positive results were observed when RNA from other chrysanthemum pathogens were used as mRT-LAMP templates. The method was verified by testing chrysanthemum samples collected from Beijing and Henan Province and showed high reliability and sensitivity. The developed mRT-LAMP assay also offers an efficient, convenient, and rapid tool for screening chrysanthemum virus and viroid, especially CVB and CSVd, and can be diagnosed in a single reaction. These results suggest that the new mRT-LAMP method may be used routinely for virus and viroid surveys.
authors.affiliation: Department of Ornamental Horticulture, College of Agronomy and Biotechnology, China Agricultural University, Beijing 100193, China; Beijing Entry-exit Inspection and Quarantine Bureau, Beijing 100026, China.
authors.forename: Xing-Liang
authors.initials: XL
authors.lastname: Liu
authors.affiliation: Department of Biological Science, College of Life Science, Henan Normal University, Xinxiang 453007, China.
authors.forename: Xi-Ting
authors.initials: XT
authors.lastname: Zhao
authors.affiliation: Department of Ornamental Horticulture, College of Agronomy and Biotechnology, China Agricultural University, Beijing 100193, China.
authors.forename: Imtiaz
authors.initials: I
authors.lastname: Muhammad
authors.affiliation: State Key Laboratory of Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China.
authors.forename: Bei-Bei
authors.initials: BB
authors.lastname: Ge
authors.affiliation: Department of Ornamental Horticulture, College of Agronomy and Biotechnology, China Agricultural University, Beijing 100193, China. Electronic address: hongbo1203@cau.edu.cn.
authors.forename: Bo
authors.initials: B
authors.lastname: Hong
mesh_terms: D017788:Carlavirus / Q000235:genetics / Q000302:isolation & purification*; D031202:Chrysanthemum / Q000821:virology*; D017931:DNA Primers / Q000235:genetics; D057230:Limit of Detection; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D010935:Plant Diseases / Q000821:virology*; D048348:Reverse Transcription; D012680:Sensitivity and Specificity; D014772:Viroids / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D017931:DNA Primers
keywords: Chrysanthemum Virus B; Chrysanthemum stunt viroid; Multiplex; Restriction enzyme; Reverse transcription loop-mediated isothermal amplification