title: Reverse transcription loop-mediated isothermal amplification assay for detecting tomato chlorosis virus.
abstract: A betaine-free reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed and optimised for detecting tomato chlorosis virus (ToCV), one of the most important viruses that infect tomato crops worldwide. A set of four specific primers was designed against the RNA-dependent RNA polymerase (RdRp) gene. The betaine-free RT-LAMP procedure could be completed within 40 min under isothermal conditions at 60 °C without a thermal cycler, and no cross-reactivity was seen with other tomato viral pathogens. Sensitivity analysis showed that RT-LAMP could detect viral dilutions up to 2.0×10(-7)ng, which is 100-times more sensitive than reverse transcription-polymerase chain reaction (RT-PCR). In addition, naked-eye observation after staining in-tube RT-LAMP products with SYBR Green I facilitated detection of ToCV by avoiding the requirement for ethidium staining following gel electrophoresis. These results suggest that ToCV RT-LAMP is a rapid, sensitive, and affordable diagnostic tool that is more suitable than RT-PCR for the detection and surveillance of ToCV in field samples.
authors.affiliation: State Key Laboratory of Crop Biology, College of Plant Protection, Shandong Agricultural University, Tai'an 271018, Shandong, China.
authors.forename: Li-ming
authors.initials: LM
authors.lastname: Zhao
authors.affiliation: State Key Laboratory of Crop Biology, College of Plant Protection, Shandong Agricultural University, Tai'an 271018, Shandong, China.
authors.forename: Gang
authors.initials: G
authors.lastname: Li
authors.affiliation: State Key Laboratory of Crop Biology, College of Plant Protection, Shandong Agricultural University, Tai'an 271018, Shandong, China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Gao
authors.affiliation: College of Food Science and Engineering, Shandong Agricultural University, Tai'an 271018, Shandong, China.
authors.forename: You-rong
authors.initials: YR
authors.lastname: Zhu
authors.affiliation: State Key Laboratory of Crop Biology, College of Plant Protection, Shandong Agricultural University, Tai'an 271018, Shandong, China.
authors.forename: Jin
authors.initials: J
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Crop Biology, College of Plant Protection, Shandong Agricultural University, Tai'an 271018, Shandong, China. Electronic address: zhuxp@sdau.edu.cn.
authors.forename: Xiao-ping
authors.initials: XP
authors.lastname: Zhu
mesh_terms: D052160:Benzothiazoles; D029383:Crinivirus / Q000235:genetics / Q000302:isolation & purification*; D003429:Cross Reactions; D003959:Diamines; D018551:Solanum lycopersicum / Q000821:virology*; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D009930:Organic Chemicals / Q000378:metabolism; D010935:Plant Diseases / Q000821:virology*; D011804:Quinolines; D012367:RNA, Viral / Q000235:genetics; D048348:Reverse Transcription; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D013194:Staining and Labeling / Q000379:methods; D013696:Temperature; D013997:Time Factors
chemical_list: D052160:Benzothiazoles; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; D012367:RNA, Viral; C098022:SYBR Green I
keywords: Detection; Isothermal amplification; RT-LAMP; RT-PCR; Tomato chlorosis virus