Rapid Differentiation and Identification of Potential Severe Strains of Citrus tristeza virus by Real-Time Reverse Transcription-Polymerase Chain Reaction Assays

Abstract

A multiplex Taqman-based real-time reverse transcription (RT) polymerase chain reaction (PCR) assay was developed to identify potential severe strains of Citrus tristeza virus (CTV) and separate genotypes that react with the monoclonal antibody MCA13. Three strain-specific probes were developed using intergene sequences between the major and minor coat protein genes (CPi) in a multiplex reaction. Probe CPi-VT3 was designed for VT and T3 genotypes; probe CPi-T36 for T36 genotypes; and probe CPi-T36-NS to identify isolates in an outgroup clade of T36-like genotypes mild in California. Total nucleic acids extracted by chromatography on silica particles, sodium dodecyl sulfate-potassium acetate, and CTV virion immunocapture all yielded high quality templates for real-time PCR detection of CTV. These assays successfully differentiated CTV isolates from California, Florida, and a large panel of CTV isolates from an international collection maintained in Beltsville, MD. The utility of the assay was validated using field isolates collected in California and Florida.

Development of real-time PCR based assays for simultaneous and improved detection of citrus viruses

“…A valuable advantage is in the multiplexing capability, which allows for high throughput pathogens screening. To date, multiplex RT-qPCR has been successfully used to detect woody plant-infecting viruses and to differentiate CTV and PPV strains (Varga and James 2006;Pallas et al, 2008;Ruiz-Ruiz et al 2009;Ananthakrishnan et al 2010;Yokomi et al 2010), whereas, none of these protocols were aimed at identifying multiple viruses infections. To our knowledge, this is the first one step multiplex RT-qPCR successfully developed to detect three of the most common citrus viruses.…”

“…To overcome these limitations and improve the sensitivity and detection limits of the assays, PCR-based and molecular hybridization techniques have been developed in the last ten years for CTV, CPsV and CVV detection (Hilf and Garnsey 2000;Bennani et al 2002;Martín et al 2004;Roy et al 2005;Barbarossa and Savino 2006;Rosa et al 2007;Loconsole et al 2009). As an alternative to traditional PCR, several real-time (q) RT-PCR protocols have been successfully used to detect and differentiate CTV strains (Ruiz-Ruiz et al 2007Bertolini et al 2008;Saponari et al 2008;Yokomi et al 2010).…”

A rapid detection tool for VT isolates of Citrus tristeza virus by immunocapture-reverse transcriptase loop-mediated isothermal amplification assay

“…In planta CTV isolates CA-VT-AT39, CA-T30-AT4, CA-RB-115, CA-RB-AT35, CA-S1-L, CCTEA 11661 (T36 genotype) [20,22] were maintained at the USDA-ARS in Parlier, California. Lyophilized citrus leaves infected with B165 and T68 were obtained from Beltsville, Maryland [23]. RNA extraction was done from two hundred mg of leaf petioles using RNeasy Plant Mini Kit (Qiagen) to compare the sensitivity of the RT-LAMP assay with the one step RT-ddPCR assay.…”

“…However, implementation of this regulatory program requires these mild strains to be removed. Currently, RT-qPCR using CTV genotype specific probes are available to detect VT and other CTV genotypes [4,20,23,[27][28][29]. Another method combining sequential enzyme immunoassays and capillary electrophoreses-single strand conformation polymorphisms can be used to characterize CTV isolates [30].…”

Epidemiología de Plum pox virus y Citrus tristeza virus en bloques de plantas de vivero. Métodos de control

“…Para la caracterización molecular de aislados de CTV se han utilizado varios procedimientos entre ellos: 1) el patrón de los dsRNA (Dodds et al, 1987;Moreno et al, 1990;Guerri et al, 1991;, 2) hibridación tomando como patrón ADNc o ARNc de varias regiones del ARNg de CTV (Rosner y Bar-Joseph, 1984;Rosner et al, 1986;Albiach-Martí et al, 2000;Narváez et al, 2000), 3) utilización de la técnica RFLP ("Restriction Fragment Length Polymorphism") para el análisis del gen que codifica la proteína de la cápsida (Gillings et al, 1993;Roy et al, 2003), 4) utilización de patrones de amplificación mediante RT-PCR con iniciadores de PCR específicos de diferentes genotipos de CTV (Hilf et al, 1999. Más recientemente se han publicado trabajos que proponen la técnica RT-PCR a tiempo real para distinguir aislados de CTV suaves de agresivos (Ruiz-Ruiz et al, 2009;Yokomi et al, 2010).…”