关键词：
苹果病毒;苹果茎沟病毒;苹果茎痘病毒;苹果褪绿叶斑病毒;两步多重RT-PCR
摘 要：
【目的】为建立检测3种苹果潜隐性病毒更为快速准确的方法,【方法】以携带苹果茎沟病毒(ASGV)、苹果褪绿叶斑病毒(ACLSV)和苹果茎痘病毒(ASPV)的成龄苹果树韧皮部为试材,根据基因库中苹果茎痘病毒(Apple stempitting virus,ASPV)的外壳蛋白基因序列,设计合成了2对特异性引物,分别与合成的苹果茎沟病毒(ASGV)引物和苹果褪绿叶斑病毒(ACLSV)的引物组合配伍,筛选出最佳的引物对组合。对cDNA的合成所用引物和总RNA模板进行遴选和量化,对PCR过程中cDNA的用量、引物对的终浓度、退火温度等主要影响因素进行优化。【结果】结果表明,反转录引物为Oligo(dT)18、总RNA 0.1～3.0μL时,可以得到较好的cDNA;ASPV-FR与ASGV-Pch、ACLSV-Pch引物组合优于ASPV-Pch,并且筛选出4组最佳的终浓度处理组合;cDNA用量为2.0~4.0μL、退火温度为50.0~52.9℃、循环数为35时,扩增效果较好。采用优化的多重RT-PCR体系对采自陕西和山东两省的样品进行检测,并用3种病毒的单重RT-PCR体系进行验证。【结论】结果呈现高度一致性,充分印证了该多重RT-PCR体系的准确性,适用于大量样品的快速检测。
译 名：
Quick detection of apple latent viruses by two-step multiplex RT-PCR assay
作 者：
CHEN Hong-xia,SHAO Jian-zhu,SUN Jian-she,CAO Xiao-feng,QIAO Xue-hua(College of Horticulture,Agricultural University of Hebei,Baoding 071000 China)
关键词：
Apple virus;ASGV;ASPV;ACLSV;Two-step multiplex PCR
摘 要：
【Objective】 The objective of the study is to find a faster method for detecting three apple latent viruses accurately.【Method】 Taking the phloem of mature apple trees infected by Apple stem grooving virus(ASGV),Apple stem pitting virus(ASPV) and Apple chlorotic leaf spot virus(ACLSV) as materials,the specific primers of ASPV-FR were designed according to the coat protein gene sequences of ASPV(Apple stem pitting virus) in Genebank,and the primer sets of ASGV-Pch,ACLSV-Pch and ASPV-Pch were synthesized according to the references.Two pairs of specific ASPV primers were respectively combined with ASGV-Pch and ACLSV-Pch for screening out the best combination.The primer of first-strand cDNA synthesis and the total RNA amount were tested.The amount of cDNA,the final concentration of three sets primers,the annealing temperature and other major factors affecting the test were optimized.【Result】 The results showed that under the conditions of total RNA 0.1 to 3.0 μL and Oligo(dT) 18 primer,the better cDNA could be obtained.The best primer combination was ASPV-FR,ASGV-Pch and ACLSV-Pch,and four valuable pairs were chosen.While reverse transcription products 2.0 to 4.0 μL and the annealing temperature from 50.0 to 52.9 ℃ and the cycles of 35,the multiplex RT-PCR system could get a good amplification.The multiplex RT-PCR detection results of samples collected from Shaanxi and Shandong provinces were consistent with the single one,which showed the higher accuracy.【Conclusion】The studies suggested that the optimal method with simplicity and rapidity can be applied to detection of apple latent viruses in large amount samples.
