利用内标为基础的苹果锈果类病毒RT-PCR检测技术

为开发准确、灵敏的苹果锈果类病毒(Apple scar skin viroid,ASSVd)RT-PCR检测方法,以田间染病苹果组织为试材,提取高质量总RNA为模板,在常规RT-PCR检测体系中引入苹果线粒体nad5基因作为内标基因,对RT-PCR反应体系和程序进行优化,并测定其灵敏度和稳定性,最后利用优化的检测体系确定苹果果实着色期最佳检测部位。结果表明:以RNA提取改良法提取总RNA为模板合成cDNA后,进行PCR扩增,优化后的PCR体系(25μL)中cDNA模板2μL、ASSVd(20pmol/μL)正反向引物各1.0μL,nad5(20pmol/μL)正反向引物各0.1μL;扩增程序中退火温度为60.9℃,循环次数为35次;检测灵敏度为37.5ng新鲜样本;果实着色期,染病植株的幼嫩叶片、1a生枝的韧皮部和木质部、果实及部分种子样本均能检测到病毒,最佳检测部位为幼嫩韧皮部。研究结果可以为ASSVd高效、快速、准确的检测提供可靠方法,并为田间病害诊断及无毒苗木繁育提供依据和借鉴。

Internal reference gene-based RT-PCR assay for the detection of apple scar skin viroid

In order to develop an accurate and sensitive method to detect apple scar skin viroid (ASSVd),a RT-PCR assay based on internal standard was developed.The total RNA with high quality was extracted from diseased apple branch.The primers of ASSVd and apple mitochondria gene nad5 were added to the assay and the RT-PCR detection method was optimized.In fruit coloring period,the optimal detection tissue was also determined.The total RNA was extracted by improved RNA-extraction method and eDNA was synthesed.Our results showed that the optimized reaction system contained cDNA template 2 μL,the forward and reverse primers of ASSVd(20 pmol/μL) both for 1.0μL,and the forward and reverse primer of nad5(20 pmol/μL) both for 0.1 μL.The PCR annealing temperature was 60.9℃ and the cycle was 35.The sensitivity of the optimized RT-PCR method could reach as less as 37.5 ng fresh sample.The tissues,including young leaves,phloem,xylem,fruit and part of seed could be used to detect ASSVd in fruit coloring period,and the young phloem was the best test tissue in this special period.The method provided a reliable way for efficient,rapid and accurate detection of ASSVd and lay a foundation for disease diagnosis and detoxification seedling breeding.