Development of a one tube-one step RT-PCR protocol for the detection of seven viroids in four genera: Apscaviroid, Hostuviroid, Pelamoviroid and Pospiviroid.
A one tube-one step RT-PCR was developed for the detection of seven viroids (Apple scar skin viroid, Apple dimple fruit viroid, Pear blister canker viroid, Hop stunt viroid, Chrysanthemum stunt viroid, Citrus exocortis viroid and Peach latent mosaic viroid) in four genera that infect eight plant species. The efficiency and specificity of this method were optimized by the use of Moloney-murine leukemia virus (M-MLV) reverse-transcriptase and HotStarTaq DNA polymerase which allowed increase sensitivity of viroid detection. The method was assessed with 56 viroid-infected field plants. The multiplex one tube-one step RT-PCR has the advantage of requiring less hands-on time to set up an assay than standard multiplex one, it also reduces the possibility of false positive tests because all steps are performed in the same tube thus, avoiding cross-contamination. The method may be used routinely for viroid detection in sanitary and certification programmes.
Ragozzino
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E
Istituto Sperimentale per la Patologia Vegetale, Via C. G. Bertero, 22-00156 Rome, Italy.
Faggioli
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F
Barba
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M
D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D005189:False Positive Reactions; D010935:Plant Diseases / Q000821:virology*; D010944:Plants / Q000821:virology; D012367:RNA, Viral / Q000032:analysis / Q000302:isolation & purification; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014772:Viroids / Q000235:genetics* / Q000302:isolation & purification*
D012367:RNA, Viral; D012194:RNA-Directed DNA Polymerase; D004259:DNA-Directed DNA Polymerase