Citrus Pest Detection Program, Central California Tristeza Eradication Agency, Tulare, CA, USA.
Department of Microbiology and Plant Pathology, University of California, Riverside, CA, USA.
Methods Mol Biol. 2022;2316:153-162. doi: 10.1007/978-1-0716-1464-8_14.
Viroids are the smallest known infectious pathogens. They are nonprotein-encoding, single-stranded, circular, naked RNA molecules that can cause several diseases in economically important crops. With the advent of thermal cyclers incorporating fluorescent detection, reverse transcription coupled to the quantitative polymerase chain reaction (RT-qPCR) has transformed the way the viroids are detected. The method involves using sequence-specific primers that anneal to the viroid RNA of interest. The viroid RNA serves as a template during reverse transcription, in which the enzyme reverse transcriptase generates a cDNA copy of a portion of the target RNA molecule. After first-strand cDNA synthesis, RNA template from cDNA:RNA hybrid molecule is removed by digestion with RNase H to improve the sensitivity of PCR step. This cDNA is then be used as a template for amplification of viroid sequence in PCR.
类病毒是已知最小的传染性病原体。它们是非蛋白编码的、单链的、环状的、裸露的 RNA 分子，可导致一些重要经济作物发生多种疾病。随着带有荧光检测的热循环仪的出现，逆转录定量聚合酶链反应（RT-qPCR）改变了类病毒的检测方式。该方法涉及使用与感兴趣的类病毒 RNA 退火的序列特异性引物。在逆转录过程中，类病毒 RNA 作为模板，逆转录酶生成目标 RNA 分子一部分的 cDNA 拷贝。在第一链 cDNA 合成后，通过用 RNase H 消化 cDNA:RNA 杂交分子来去除 RNA 模板，以提高 PCR 步骤的灵敏度。然后，将该 cDNA 用作 PCR 中类病毒序列扩增的模板。
