title: One-step detection of Bean pod mottle virus in soybean seeds by the reverse-transcription loop-mediated isothermal amplification.
abstract: BACKGROUND
  Bean pod mottle virus (BPMV) is a wide-spread and destructive virus that causes huge economic losses in many countries every year. A sensitive, reliable and specific method for rapid surveillance is urgently needed to prevent further spread of BPMV.
  
  
  METHODS
  A degenerate reverse-transcription loop-mediated isothermal amplification (RT-LAMP) primer set was designed on the conserved region of BPMV CP gene. The reaction conditions of RT-LAMP were optimized and the feasibility, specificity and sensitivity of this method to detect BPMV were evaluated using the crude RNA rapidly extracted from soybean seeds.
  
  
  RESULTS
  The optimized RT-LAMP parameters including 6 mM MgCl2, 0.8 M betaine and temperature at 62.5-65°C could successfully amplify the ladder-like bands from BPMV infected soybean seeds. The amplification was very specific to BPMV that no cross-reaction was observed with other soybean viruses. Inclusion of a fluorescent dye makes it easily be detected in-tube by naked eye. The sensitivity of RT-LAMP assay is higher than the conventional RT-PCR under the conditions tested, and the conventional RT-PCR couldn't be used for detection of BPMV using crude RNA extract from soybean seeds.
  
  
  CONCLUSIONS
  A highly efficient and practical method was developed for the detection of BPMV in soybean seeds by the combination of rapid RNA extraction and RT-LAMP. This RT-LAMP method has great potential for rapid BPMV surveillance and will assist in preventing further spread of this devastating virus.
authors.affiliation: Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of Education, Department of Plant Pathology, Nanjing Agricultural University, Nanjing 210095, People's Republic of China.
authors.forename: Qi-Wei
authors.initials: QW
authors.lastname: Wei
authors.forename: Cui
authors.initials: C
authors.lastname: Yu
authors.forename: Shu-Ya
authors.initials: SY
authors.lastname: Zhang
authors.forename: Cui-Yun
authors.initials: CY
authors.lastname: Yang
authors.forename: Karwitha
authors.initials: K
authors.lastname: Miriam
authors.forename: Wen-Na
authors.initials: WN
authors.lastname: Zhang
authors.forename: Dao-Long
authors.initials: DL
authors.lastname: Dou
authors.forename: Xiao-Rong
authors.initials: XR
authors.lastname: Tao
mesh_terms: D001483:Base Sequence; D017798:Comovirus / Q000145:classification* / Q000302:isolation & purification*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D012367:RNA, Viral / Q000235:genetics; D012639:Seeds / Q000821:virology*; D012680:Sensitivity and Specificity; D013025:Glycine max / Q000821:virology*; D013696:Temperature; D013997:Time Factors
chemical_list: D012367:RNA, Viral