库尔勒香梨脉黄病毒RT-PCR检测技术研究

Studies on RT-PCR Detection Technology of Viral Diseases in Kuala Pear

以库尔勒香梨叶片和皮层为材料 ,对总RNA提取方法进行了研究 ,从中筛选出了适合库尔勒香梨总RNA的提取方法。结果表明 ,要获得良好的RT PCR扩增 ,RT体系中dNTPs浓度、互补引物、AMV、模板的浓度要分别达到 0 .1mmol·L-1、0 .2 μmol·L-1、0 .0 5U·μl-1、0 .0 1μg·μl-1以上。此外 ,使用RNasin能有效抑制RT体系中RNase对病毒RNA的降解作用 ,可使RT过程顺利进行 ;PCR体系中dNTPs浓度至少要达到 0 .1mmol·L-1,0 .2～ 0 .3mmol·L-1可以获得最佳的扩增效果 ;TaqE浓度要达到 0 .0 15U·μl-1以上 ;引物浓度适宜范围 0 .3～ 0 .7μmol·L-1;Mg2 + 要达到 1.2 6mmol·L-1以上。

Using Kuala pear leaves and cortexes as materials, total RNA was extracted using three methods, and these methods were studied. The fittest methods for Kuala pear were screened out. Because pear tissues are abundant in amylase and hydroxybenzene, these methods were modified in order to obtain high quality products. Based on the effective RT PCR detection system of two viruses, the concentration of RT reaction ingredients dNTPs, primer, AMV, template and RNasin were optimized. The concentration of RCR reaction ingredients dNTPs, primer, Mg 2+ ; Taq E and cDNA were also optimized. Influences of denaturalization temperature, and RT time on PCR were studied. Results indicated that concentration of dNTPs, primer, AMV, template should not lower than 0.1 mmol·L -1 , 0.2 μmol·L -1 , 0.05 u·μl -1 , 0.01 μg·μl -1 in RT system. RNasin restrained the action of RNase, insured the process of RT go on wheels. Denaturalization temperature, time of dsRNA should be at 95℃ for 3～5 min. RT time should no less than 40 min; Concentration of dNTPs and primer should be 0.2～0.3 mmol·L -1 , 0.3～0.7 μmol·L -1 in PCR system, respectively. The proper concentration of Mg 2+ should be more than 1.26 mmol·L -1 .