title: Simultaneous detection and differentiation of three genotypes of Brassica yellows virus by multiplex reverse transcription-polymerase chain reaction.
abstract: BACKGROUND
  Brassica yellows virus (BrYV), proposed to be a new polerovirus species, three distinct genotypes (BrYV-A, BrYV-B and BrYV-C) have been described. This study was to develop a simple, rapid, sensitive, cost-effective method for simultaneous detection and differentiation of three genotypes of BrYV.
  
  
  RESULTS
  In this study, a multiplex reverse transcription-polymerase chain reaction (mRT-PCR) was developed for simultaneous detection and differentiation of the three genotypes of BrYV. The three genotypes of BrYV and Tunip yellows virus (TuYV) could be differentiated simultaneously using six optimized specific oligonucleotide primers, including one universal primer for detecting BrYV, three BrYV genotype-specific primers, and a pair of primers for specific detection of TuYV. Primers were designed from conserved regions of each virus and their specificity was confirmed by sequencing PCR products. The mRT-PCR products were 278 bp for BrYV-A, 674 bp for BrYV-B, 505 bp for BrYV-C, and 205 bp for TuYV. Amplification of three target genotypes was optimized by increasing the PCR annealing temperatures to 62 °C. One to three fragments specific for the virus genotypes were simultaneously amplified from infected samples and identified by their specific molecular sizes in agarose gel electrophoresis. No specific products could be amplified from cDNAs of other viruses which could infect crucifer crops. Detection limits of the plasmids for multiplex PCR were 100 fg for BrYV-A and BrYV-B, 10 pg for BrYV-C, and 1 pg for TuYV, respectively. The mRT-PCR was applied successfully for detection of three BrYV genotypes from field samples collected in China.
  
  
  CONCLUSIONS
  The simple, rapid, sensitive, and cost-effective mRT-PCR was developed successfully for detection and differentiation of the three genotypes of BrYV.
authors.affiliation: State Key Laboratory for Agrobiotechnology and Ministry of Agriculture Key Laboratory for Plant Pathology, China Agricultural University, Beijing, China.
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Zhang
authors.affiliation: State Key Laboratory for Agrobiotechnology and Ministry of Agriculture Key Laboratory for Plant Pathology, China Agricultural University, Beijing, China.
authors.forename: Yanmei
authors.initials: Y
authors.lastname: Peng
authors.affiliation: State Key Laboratory for Agrobiotechnology and Ministry of Agriculture Key Laboratory for Plant Pathology, China Agricultural University, Beijing, China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Wang
authors.affiliation: State Key Laboratory for Agrobiotechnology and Ministry of Agriculture Key Laboratory for Plant Pathology, China Agricultural University, Beijing, China.
authors.forename: Zongying
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, China.
authors.forename: Dawei
authors.initials: D
authors.lastname: Li
authors.affiliation: State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, China.
authors.forename: Jialin
authors.initials: J
authors.lastname: Yu
authors.affiliation: State Key Laboratory for Agrobiotechnology and Ministry of Agriculture Key Laboratory for Plant Pathology, China Agricultural University, Beijing, China. Hanchenggui@cau.edu.cn.
authors.forename: Chenggui
authors.initials: C
authors.lastname: Han
mesh_terms: D001937:Brassica / Q000821:virology; D002681:China; D003365:Costs and Cost Analysis; D017931:DNA Primers / Q000235:genetics; D004586:Electrophoresis; D005838:Genotype; D052679:Luteoviridae / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D060885:Multiplex Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D010935:Plant Diseases / Q000821:virology*; D010957:Plasmids; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D012680:Sensitivity and Specificity; D013696:Temperature; D013997:Time Factors
chemical_list: D017931:DNA Primers
keywords: Brassica yellows virus; Crucifer crops; Detection; Genotype; Multiplex RT-PCR