title: A one-step multiplex RT-PCR assay for simultaneous detection of four viruses that infect peach.
abstract: A multiplex reverse transcription polymerase chain reaction (mRT-PCR) assay was developed to enable the simultaneous detection and differentiation of four viruses that infect peach, namely Apple chlorotic leaf spot virus (ACLSV), Cherry green ring mottle virus (CGRMV), Prunus necrotic ringspot virus (PNRSV) and Apricot pseudo-chlorotic leaf spot virus (APCLSV). In this study, four pairs of primers, one specific for each virus, were designed; the corresponding PCR products were 632, 439, 346 and 282 bp in length for ACLSV, CGRMV, PNRSV and APCLSV, respectively, and the fragments could be distinguished clearly by agarose gel electrophoresis. The sensitivity and specificity of the method were tested using individual RT-PCR and enzyme-linked immunosorbent assay (ELISA), and the identity of the RT-PCR amplification products was also confirmed by DNA sequencing. The results of RT-PCR and ELISA, along with batch detection using samples collected from peach orchards, revealed that this rapid and simple technique is an effective way to identify the four viruses simultaneously.
  
  
  CONCLUSIONS
  The mRT-PCR assay described in this study was developed for the simultaneous detection of four peach viruses from infected peach samples is reliable and sensitive. In contrast to conventional uniplex RT-PCR, mRT-PCR is more efficient, reducing costs, time and handling when testing large numbers of samples. This rapid and simple method is useful for large-scale surveys of viruses that infect peach.
authors.affiliation: State Key Laboratory of Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China; Institute of Plant Virology, Fujian Agriculture and Forestry University, Fuzhou, China.
authors.forename: Y
authors.initials: Y
authors.lastname: Yu
authors.forename: Z
authors.initials: Z
authors.lastname: Zhao
authors.forename: D
authors.initials: D
authors.lastname: Jiang
authors.forename: Z
authors.initials: Z
authors.lastname: Wu
authors.forename: S
authors.initials: S
authors.lastname: Li
mesh_terms: D017931:DNA Primers / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods; D010935:Plant Diseases / Q000821:virology*; D027861:Prunus / Q000821:virology*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014780:Viruses / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D017931:DNA Primers
keywords: ELISA; detection assays; multiplex RT-PCR; peach viruses