一种快速同步检测小麦黄花叶病毒和中国小麦花叶病毒的方法

A Simplified Method for Quick Detection of Wheat Yellow Mosaic Virus and Chinese Wheat Mosaic Virus

针对小麦黄花叶病毒(WYMV)和中国小麦花叶病毒(CWMV)在症状、传播途径、发生规律等方面高度相似、难以用常规手段鉴定的问题,根据两种病毒基因序列的异同点设计了三条引物CWWY-R2、CW1-F2和WY1-F2,以植物总RNA为模板、CWWY-R2为引物反转录合成cDNA。通过优化建立了一种同步检测两种病毒的反应体系:cDNA1.6μL,引物(10μmol.L-1)各0.4μL,10×PCR Buffer(不含Mg2+)2μL,dNTPs(10mmol.L-1 each)0.4μL,Taq DNA聚合酶(5U.μL-1)0.8μL,MgCl2(25mmol.L-1)0.8μL,ddH2O 13.2μL,合计20μL;PCR反应条件:94℃预变性5min,94℃50s,50℃50s,72℃90s,共30个循环,72℃延伸10min。WYMV和CWMV的PCR扩增预期目的片段分别为508和918bp。利用该方法对江苏高邮、扬州和大丰的样本进行检测,它们分别为WYMV、WYMV、CWMV单一侵染,证明该方法准确性较高,可用于两种病害同步检测。

Wheat yellow mosaic virus（WYMV）and Chinese wheat mosaic virus（CWMV）are similar in symptoms,transmission routes and occurrence rule and it is difficult to detect them with the traditional methods.Aiming at this problem,multiplex RT-PCR was established to detect WYMV and CWMV at the same time.Three degenerate primers CWWY-R2,CW1-F2 and WY1-F2 were designed according to variation in nucleotide sequence between CWMV and WYMV.The cDNAs were reverse transcripted with total plant RNA as template and CWWY-R2 as the reverse transcription primer.Then a fast detection method of these two viruses was established through optimizing the concentrations of the main ingredients and PCR conditions.The optimal PCR reaction system was 1.6 μL of diluted cDNA,0.4 μL of each primer set（10 μmol·L-1 forward and reverse primer each）,2 μL of 10×PCR Buffer（without Mg2＋）,0.4 μL of dNTPs（10 mmol·L-1 each）,0.8 μL of Taq DNA polymerase（5 U·μL-1）,0.8 μL of MgCl2（25 mmol·L-1） and 13.2 μL ddH2O.The PCR Reaction profile was as denaturation at 94℃ for 5 min,30 cycles of 94℃ for 50 s,50℃ for 50 s and 72℃ for 90 s,a final extension at 72℃ for 10 min.Expected fragments of 508 bp for WYMV and 918 bp for CWMV were successfully amplified by the multiplex RT-PCR method.The samples from Gaoyou,Yangzhou and Dafeng County of Jiangsu Province were detected with this method,and the results revealed that they were infected by WYMV,WYMV and CWMV,respectively.Expreimental results showed that this method was with high efficiency and accuracy.