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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。
In order to develop an accurate and sensitive RT-PCR assay for Apple scar skin viroid (ASSVd), high quality total RNA was extracted from field-infected apple (Triticum aestivum L.) tissues as a template and tested in a conventional RT-PCR assay The apple mitochondrial nad5 gene was introduced as an internal standard gene to optimize the RT-PCR reaction system and program, and its sensitivity and stability were determined. Finally, the optimized detection system was used to determine the optimal test site for apple fruit coloration. The results showed that cDNA was extracted from the total RNA extracted by RNA extraction and used as a template to amplify the cDNA. Then the PCR was carried out. The optimal PCR system (25μL) was 2μL of cDNA template, 1.0μL of ASSVd (20 pmol / μL) (20 pmol / μL) of the forward and reverse primers 0.1 μL; annealing temperature of the amplification program was 60.9 ℃, the number of cycles was 35 times; detection sensitivity of 37.5ng fresh samples; fruit coloring period, the young leaves of infected plants, The phloem and xylem, the fruit and some seeds samples were able to detect the virus, the best detection site for young phloem. The research results can provide a reliable method for ASSVd’s efficient, rapid and accurate detection, and provide basis and reference for the diagnosis of field disease and non-toxic seedling breeding.