摘要
为简化苹果锈果类病毒(apple skin scar viroid,ASSVd)的常规RT-PCR检测步骤,找到更为快速准确的检测方法,以感染ASSVd的田间苹果枝条为试验材料,根据基因库中ASSVd的基因序列设计合成特异性引物,选用国产试剂盒,对RT-PCR体系进行优化。结果表明,总RNA和反转录产物的用量分别为37.4～74.8ng、1.0～3.0μL,退火温度为63.8℃,可以获得良好的两步RT-PCR扩增;而总RNA用量为3.74～7.48ng、退火温度在50.1～62.6℃范围内时,一步RT-PCR的扩增效果较好。采用优化的RT-PCR检测体系对山东采集的样品进行检测,2种检测体系的检测结果完全一致。研究建立的两步和一步RT-PCR优化体系为ASSVd的批量快速检测奠定了基础。
In order to simplify the conventional RT-PCR of apple skin scar viroid and find faster and more accurate detection methods, the apple shoots in field infected by apple skin scar viroid were used as materials to optimize the RT-PCR systems. Specific primers of apple skin scar viroid were designed and synthesized according to the gene sequences in Genebank and domestic kit was selected in this study. The results showed that under the conditions of the amount of total RNA 37.4-74.8 ng, reverse transcription products 1.0-3.0 μL and the annealing temperature 63.8℃, two-step RT-PCR system got a good amplification. While the amount of total RNA 3.74-7.48 ng and the annealing temperature from 50.1 to 62.6℃, the detection systems of one-step RT-PCR got the better amplification. The two RT-PCR detection systems were tested with samples collected from Shandong Province, and the results were completely consistent. This study established the quick and reliable detection method of apple scar skin viroid by the two-step and the one-step RT-PCR by kit.
出处
《中国农学通报》
CSCD
2012年第19期254-259,共6页
Chinese Agricultural Science Bulletin
基金
现代农业产业技术体系专项资金项目"砧木评价与利用"(CARS-28)
