1. Rapid diagnostic detection of tomato apical stunt viroid based on isothermal reverse transcription-recombinase polymerase amplification
- Author
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Natalia Kovalskaya and Rosemarie W. Hammond
- Subjects
biology ,Serial dilution ,Viroid ,Total rna ,food and beverages ,RNA ,Recombinase Polymerase Amplification ,Reverse Transcription ,biology.organism_classification ,Sensitivity and Specificity ,Molecular biology ,Viroids ,Reverse transcriptase ,Plant Viruses ,Recombinases ,Virology ,Tomato apical stunt viroid ,biology.protein ,Polymerase - Abstract
Tomato apical stunt viroid (TASVd) is a serious threat to tomato plants that can cause a considerable yield loss. In the present study, two isothermal molecular diagnostic assays based on reverse transcription-recombinase polymerase amplification (RT-RPA) utilizing the AmplifyRP® platform for plant pathogen detection were developed. The results of this research demonstrated distinct specificity of both developed assays, AmplifyRP® Acceler8TM and AmplifyRP® XRT, expressed in the absence of any cross-reaction activity to all total RNA extracts obtained from plants infected with other pospiviroids. The RT-RPA assays detected viroid RNA in 81- and 27-fold dilutions of the original TASVd-infected crude extract for AmplifyRP® Acceler8TM and AmplifyRP® XRT, respectively. The sensitivity tests in serial water dilutions showed the ability of AmplifyRP® Acceler8TM and AmplifyRP® XRT to detect 8 and 80 fg of pure TASVd RNA transcript, respectively. The influence of crude extract on viroid RNA transcript detection was also examined and a decrease of sensitivity of approximately 100-fold for both RT-RPA assays was revealed. To our knowledge, this is the first report describing development of RT-RPA assays to detect TASVd in plants using the AmplifyRP® platform that can be further employed both in laboratory conditions and in the field for on-site diagnosis.
- Published
- 2022