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Apple Scar Skin Viroid Case Study

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Apple Scar Skin Viroid (ASSVd), a non-protein coding, single-stranded, circular RNA pathogen limits the detection based on the immunoassay. In this study, a visible nanobarcoding assay for one-step Reveres transcription loop-mediated isothermal amplification (RT-LAMP) was developed to improve the LAMP selectivity and promote its diagnostic use in detecting ASSVd in plants. ASSVd LAMP was accelerated by the use of loop primers and highly sensitive with a detection limit of 103 copies of ASSVd cDNA per reaction within 30 min. The real-time LAMP and melting curve analysis could differentiate between the true-positive LAMP amplicons and false-positive non-specific primer amplification products. The optimized RT-LAMP was then followed by the…show more content…
Thus, they cannot be available for rapid on-site detection, because of long time ((>2 h) (5).
In particular, there have been an increased number of reports of viroid infection, which is mostly latent and symptomless in plants and caused by the smallest RNA. Unlike viruses, viroids are not coated with proteins, thus limiting the detection methods based on the immunoassay. Diagnosis and discrimination of viroids are challenging, because a large number of nursery stock are introduced frequently and distributed rapidly among growers. Apple scar skin viroids (ASSVds) in apple cause the plant diseases including scar skin (reddish brown patches with brownish scar-like tissue), cracking, and spotting symptoms. Because of these disease symptoms, viroid has caused serious economic losses in the apple industry (6, 7).
Loop-mediated isothermal amplification (LAMP) and reverse transcription (RT)-LAMP have been used to detect several types of infectious reagents with high sensitivity, efficiency, and rapidity under isothermal conditions (5, 8, 9). The need for simple, visible, and rapid detection of LAMP/or RT-LAMP amplicons has led to a great variety of end point and real-time/on-line monitoring for field applications (10, 11). According to the general principles of LAMP amplification, 4 to 6 primers are designed to react to specific regions on the target sequences. When appropriate initiating structure
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