title: Development of Reverse Transcription Thermostable Helicase-Dependent DNA Amplification for the Detection of Tomato Spotted Wilt Virus.
abstract: A protocol for the reverse transcription-helicase-dependent amplification (RT-HDA) of isothermal DNA was developed for the detection of tomato spotted wilt virus (TSWV). Specific primers, which were based on the highly conserved region of the N gene sequence in TSWV, were used for the amplification of virus's RNA. The LOD of RT-HDA, reverse transcriptase-loop-mediated isothermal amplification (RT-LAMP), and reverse transcriptase-polymerase chain reaction (RT-PCR) assays were conducted using 10-fold serial dilution of RNA eluates. TSWV sensitivity in RT-HDA and RT-LAMP was 4 pg RNA compared with 40 pg RNA in RT-PCR. The specificity of RT-HDA for TSWV was high, showing no cross-reactivity with other tomato and Tospovirus viruses including cucumber mosaic virus (CMV), tomato black ring virus (TBRV), tomato mosaic virus (ToMV), or impatiens necrotic spot virus (INSV). The RT-HDA method is effective for the detection of TSWV in plant samples and is a potential tool for early and rapid detection of TSWV.
authors.affiliation: Technology Center of the Shandong Entry-Exit Inspection and Quarantine Bureau, 70 Qutangxia Rd, Qingdao, Shandong 266002, China.
authors.forename: Xinghai
authors.initials: X
authors.lastname: Wu
authors.affiliation: Technology Center of the Shandong Entry-Exit Inspection and Quarantine Bureau, 70 Qutangxia Rd, Qingdao, Shandong 266002, China.
authors.forename: Chanfa
authors.initials: C
authors.lastname: Chen
authors.affiliation: Technology Center of the Shandong Entry-Exit Inspection and Quarantine Bureau, 70 Qutangxia Rd, Qingdao, Shandong 266002, China.
authors.forename: Xizhi
authors.initials: X
authors.lastname: Xiao
authors.affiliation: Technology Center of the Shandong Entry-Exit Inspection and Quarantine Bureau, 70 Qutangxia Rd, Qingdao, Shandong 266002, China.
authors.forename: Ming Jun
authors.initials: MJ
authors.lastname: Deng
mesh_terms: D017931:DNA Primers / Q000235:genetics; D018551:Solanum lycopersicum / Q000821:virology*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D012367:RNA, Viral / Q000032:analysis / Q000235:genetics*; D048348:Reverse Transcription; D013696:Temperature; D017865:Tospovirus / Q000235:genetics* / Q000302:isolation & purification*
chemical_list: D017931:DNA Primers; D012367:RNA, Viral