葫芦科作物3种主要病毒的多重RT-PCR方法的建立

Multiplex RT-PCR assay for simultaneous detection of three viruses of Zucchini

小西葫芦黄花叶病毒(Zucchini yellowmosaic virus,ZYMV)、西瓜花叶病毒(Watermelon mosaic virus,WMV)和黄瓜花叶病毒(Cucumber mosaic virus,CMV)是危害葫芦科作物最严重、最广泛的3种病毒。研究建立了以18SrRNA为内标检测ZYMV、CMV和WMV侵染葫芦科作物3种主要病毒的多重RT-PCR方法,并对影响多重RT-PCR扩增的退火温度、Taq聚合酶浓度、dNTPs浓度和镁离子浓度4种主要因素进行优化;对多重RT-PCR的灵敏性进行了测定,并与单重RT-PCR灵敏性进行比较。结果表明:最佳的退火温度、Taq聚合酶量、dNTPs浓度和Mg2+浓度分别为60.7℃、1.0U、0.8mmol/L和2.5mmol/L。多重RT-PCR各病毒的灵敏度与单重RT-PCR相同。

Zucchini yellow mosaic virus （ZYMV）, Watermelon mosaic virus （WMV）and Cucumber mosaic virus （CMV） are most important pathogens infecting cucurbitaceous crops. A multiplex reverse transcription-polymerase chain reaction （mRT-PCR） was developed for detection of three cucurbitaceous vriuses ： ZYMV, WMV and CMV at the same tube with 18S rRNA of the plant as internal control. The main impact factors of mRT-PCR： annealing temperature （Tin）, Taq DNA Poly- merase, dNTPs and Mg^2＋ were optimized, and compared with sensitivity of single RT-PCR （sRT-PCR）. The mRT-PCR for detecting the three viruses, the appropriate Tin, Taq DNA Polymerase concentration,dNTPs concentration and Mg^2＋ concentration were 60.7 ℃, 1.0 U, 0.8 mmol/L and 2.5 mmol/L respectively. The resuhs of the multiplex RT-PCR were consistent with sRT-PCR to detect each of the viruses