title: High-throughput multiplex real-time PCR assay for the simultaneous quantification of DNA and RNA viruses infecting cassava plants.
abstract: OBJECTIVE
  To develop a multiplex TaqMan-based real-time PCR assay (qPCR) for the simultaneous detection and quantification of both RNA and DNA viruses affecting cassava (Manihot esculenta) in eastern Africa.
  
  
  RESULTS
  The diagnostic assay was developed for two RNA viruses; Cassava brown streak virus (CBSV) and Uganda cassava brown streak virus (UCBSV) and two predominant DNA viruses; African cassava mosaic virus (ACMV) and East African cassava mosaic virus (EACMV), which cause the economically important cassava brown streak disease (CBSD) and cassava mosaic disease (CMD) respectively. Our method, developed by analysing PCR products of viruses, was highly sensitive to detect target viruses from very low quantities of 4-10 femtograms. Multiplexing did not diminish sensitivity or accuracy compared to uniplex alternatives. The assay reliably detected and quantified four cassava viruses in field samples where CBSV and UCBSV synergy was observed in majority of mixed-infected varieties.
  
  
  CONCLUSIONS
  We have developed a high-throughput qPCR diagnostic assay capable of specific and sensitive quantification of predominant DNA and RNA viruses of cassava in eastern Africa.
  
  
  CONCLUSIONS
  The qPCR methods are a great improvement on the existing methods and can be used for monitoring virus spread as well as for accurate evaluation of the cassava varieties for virus resistance.
authors.affiliation: Natural Resources Institute, University of Greenwich, Chatham Maritime, Kent, UK.
authors.forename: G
authors.initials: G
authors.lastname: Otti
authors.affiliation: Natural Resources Institute, University of Greenwich, Chatham Maritime, Kent, UK.
authors.forename: S
authors.initials: S
authors.lastname: Bouvaine
authors.affiliation: Naliendele Agricultural Research Institute, Naliendele, Tanzania.
authors.forename: B
authors.initials: B
authors.lastname: Kimata
authors.affiliation: Naliendele Agricultural Research Institute, Naliendele, Tanzania.
authors.forename: G
authors.initials: G
authors.lastname: Mkamillo
authors.affiliation: International Institute of Tropical Agriculture, Ibadan, Nigeria.
authors.forename: P L
authors.initials: PL
authors.lastname: Kumar
authors.affiliation: Natural Resources Institute, University of Greenwich, Chatham Maritime, Kent, UK.
authors.forename: K
authors.initials: K
authors.lastname: Tomlins
authors.affiliation: Natural Resources Institute, University of Greenwich, Chatham Maritime, Kent, UK.
authors.forename: M N
authors.initials: MN
authors.lastname: Maruthi
mesh_terms: D053839:Begomovirus / Q000235:genetics*; D004267:DNA Viruses / Q000235:genetics; D002365:Manihot / Q000821:virology*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods; D010935:Plant Diseases / Q000821:virology*; D019176:Potyviridae / Q000235:genetics*; D012328:RNA Viruses / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods
keywords: TaqMan assays; begomovirus; cassava diseases; ipomovirus; real-time PCR; virus detection and quantification