Apple Scar Skin Viroid (ASSVd), a nonprotein coding, circular RNA pathogen is relatively difficult to detect by immunoassay. We report here a one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay to improve selectivity for diagnostic use in detecting ASSVd in plants. ASSVd RT-LAMP was accelerated using loop primers and was found to be highly sensitive with a detection limit of 10⁴ copies of cDNA-ASSVd within 30 min. Real-time LAMP and melting curve analysis could differentiate between the true-positive LAMP amplicons and false-positive nonspecific primer amplification products. The optimized RT-LAMP was then followed by the addition of nonthiolated AuNP:poly-adenine (A10)-ASSVd LAMP barcodes, showing a high authentication capacity with colorimetric changes. This type of barcoding assay is a potential alternative for rapid and multiple viroid diagnosis, providing for visible sensing in the field that can be applied to viroid-free planting.

中文翻译：

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聚腺嘌呤偶联的LAMP条形码检测苹果疤痕皮肤类病毒

苹果疤痕皮肤类病毒（ASSVd）是一种非蛋白质编码的环状RNA病原体，相对较难通过免疫测定法检测到。我们在这里报告了一步一步的逆转录环介导的等温扩增（RT-LAMP）分析，以提高诊断中用于检测植物中ASSVd的选择性。使用环引物加速了ASSVd RT-LAMP的合成，并发现其高度敏感，检测极限为10 ⁴30分钟内复制cDNA-ASSVd。实时LAMP和解链曲线分析可以区分真阳性LAMP扩增子和假阳性非特异性引物扩增产物。经过优化的RT-LAMP然后添加非硫醇化的AuNP：聚腺嘌呤（A10）-ASSVd LAMP条形码，显示出具有比色变化的高验证能力。这种类型的条形码测定法是快速和多重类病毒诊断的潜在替代方法，提供了可用于无类病毒种植的现场可见感测。