title: Development of a rapid diagnostic assay for the detection of tomato chlorotic dwarf viroid based on isothermal reverse-transcription-recombinase polymerase amplification.
abstract: A molecular diagnostic assay utilizing reverse transcription-recombinase polymerase amplification (RT-RPA) at an isothermal constant temperature of 39°C and target-specific primers and probe were developed for the rapid, sensitive, and specific detection of tomato chlorotic dwarf viroid (TCDVd) in infected leaf and seed tissues. The performance of the AmplifyRP(®) Acceler8™ RT-RPA diagnostic assay, utilizing a lateral flow strip contained within an amplicon detection chamber, was evaluated and the results were compared with a standard RT-PCR assay. The AmplifyRP(®) Acceler8™ assay was specific for TCDVd in leaf and seed tissues, its sensitivity was comparable to conventional RT-PCR in leaf tissues, and it does not require extensive sample purification, specialized equipment, or technical expertise. This is the first report utilizing an RT-RPA assay to detect viroids and the assay can be used both in the laboratory and in the field for TCDVd detection.
authors.affiliation: USDA ARS Molecular Plant Pathology Laboratory, Beltsville, MD 20705, United States. Electronic address: rose.hammond@ars.usda.gov.
authors.forename: Rosemarie W
authors.initials: RW
authors.lastname: Hammond
authors.affiliation: Agdia Inc., 52642 County Road 1, Elkhart, IN 46514, United States.
authors.forename: Shulu
authors.initials: S
authors.lastname: Zhang
mesh_terms: D017931:DNA Primers / Q000235:genetics; D018551:Solanum lycopersicum / Q000821:virology*; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D015345:Oligonucleotide Probes / Q000235:genetics; D010935:Plant Diseases / Q000821:virology*; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D045522:Recombinases / Q000378:metabolism; D012680:Sensitivity and Specificity; D013696:Temperature; D013997:Time Factors; D014772:Viroids / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D017931:DNA Primers; D015345:Oligonucleotide Probes; D045522:Recombinases; D012194:RNA-Directed DNA Polymerase
keywords: AmplifyRP(®) acceler8™; Detection; Petunia; RT-RPA; Seed; TCDVd; Tomato; Tomato chlorotic dwarf viroid