title: Detection and quantitation of Citrus leaf blotch virus by TaqMan real-time RT-PCR.
abstract: A real-time RT-PCR assay based on the TaqMan chemistry was developed for reliable detection and quantitation of Citrus leaf blotch virus (CLBV) in citrus plants. Detection by this method was highly specific and about one thousand times more sensitive than detection by conventional RT-PCR. An external standard curve using in vitro synthesized RNA transcripts of the selected target allowed a reproducible quantitative assay, with a wide dynamic range (seven logarithmic units of concentration) and very low variation coefficient values. This protocol enabled detection of as little as 100 copies of CLBV RNA in various tissues and citrus varieties infected with CLBV sources from different geographical origins. The new assay greatly improves current detection methods for CLBV and it has been most helpful for the Spanish citrus sanitation, quarantine and certification programs, and fitness evaluation of infectious cDNA clones of CLBV, useful potentially as viral vectors for citrus.
authors.affiliation: Centro de Protección Vegetal y Biotecnología, Instituto Valenciano de Investigaciones Agrarias, Moncada, Valencia, Spain.
authors.forename: Susana
authors.initials: S
authors.lastname: Ruiz-Ruiz
authors.forename: Silvia
authors.initials: S
authors.lastname: Ambrós
authors.forename: María del Carmen
authors.initials: Mdel C
authors.lastname: Vives
authors.forename: Luis
authors.initials: L
authors.lastname: Navarro
authors.forename: Pedro
authors.initials: P
authors.lastname: Moreno
authors.forename: José
authors.initials: J
authors.lastname: Guerri
mesh_terms: D002957:Citrus / Q000821:virology*; D052677:Flexiviridae / Q000235:genetics / Q000302:isolation & purification*; D015345:Oligonucleotide Probes / Q000235:genetics; D010935:Plant Diseases / Q000821:virology*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D015345:Oligonucleotide Probes