Barley yellow dwarf virus and Cereal yellow dwarf virus quantification by real-time polymerase chain reaction in resistant and susceptible plants
Barley yellow dwarf virus and Cereal yellow dwarf virus quantification by real-time polymerase chain reaction in resistant and susceptible plants
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DOI：
10.1094/phyto.2003.93.11.1386
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发表时间：
2003-11-01
期刊：
影响因子：
3.2
通讯作者：
Anderson, JM
中科院分区：
文献类型：
--
作者：
Balaji, B;Bucholtz, DB;Anderson, JM
大麦和谷类黄矮病毒（YDV）的可靠检测和定量是管理小粒谷类作物黄矮病的关键组成部分。目前使用的方法是酶联免疫吸附试验（ELISA），使用抗血清对外壳蛋白，是具体的每一个不同的YDV。最近，定量实时逆转录聚合酶链反应（Q-RT-PCR）已被用于检测细菌和病毒病原体，并研究基因表达。利用RT-PCR的高灵敏度和实时荧光定量PCR的优势，我们利用大麦黄矮病毒PAV（BYDV-PAV）和禾谷类黄矮病毒RPV（CYDV-RPV）外壳蛋白基因特异性引物对YDV进行了检测和定量。该Q-RT-PCR用于检测BYDV和CYDV，并检查抗病冰草、抗病小麦系、感病小麦系和感病燕麦系中的病害发展。在感病燕麦中，BYDV-PAV和CYDV-RPV分别在接种后2和6 h检测到，而ELISA在接种后4和10 d检测到。在燕麦和小麦中，BYDV-PAV RNA比CYDV-RPV RNA积累得更快、水平更高，这可能是PAV比CYDV更普遍、引起更严重病毒疾病的原因。Q-RT-PCR具有重复性好、灵敏度高的特点，有望用于黄矮病的快速诊断。
Reliable detection and quantification of barley and cereal yellow dwarf viruses (YDVs) is a critical component in managing yellow dwarf diseases in small grain cereal crops. The method currently used is enzyme-linked immunosorbent assay (ELISA), using antisera against the coat proteins that are specific for each of the various YDVs. Recently, quantitative real-time reverse-transcription polymerase chain reaction (Q-RT-PCR) has been used to detect bacterial and viral pathogens and to study gene expression. We applied this technique to detect and quantify YDVs using primers specific for Barley yellow dwarf virus-PAV (BYDV-PAV) and Cereal Yellow dwarf virus-RPV (CYDV-RPV) coat protein genes because of the higher sensitivity of RT-PCR and the advantage of using a real-time PCR instrument. This Q-RT-PCR was used to detect BYDV and CYDV, and to examine disease development in a resistant wheatgrass, a resistant wheat line, a susceptible wheat line, and a susceptible oat line. BYDV-PAV and CYDV-RPV were detected as early as 2 and 6 h, respectively, in susceptible oat compared with detection by ELISA at 4 and 10 days postinoculation. BYDV-PAV RNA accumulated more rapidly and to a higher level than CYDV-RPV RNA in both oat and wheat, which may account for PAV being more prevalent and causing more severe viral disease than CYDV. Q-RT-PCR is reproducible, sensitive, and has the potential to be used for examining yellow dwarf disease and as a rapid diagnostic tool for YDVs.