李坏死环斑病毒检测方法的比较研究

Comparative Studies on Detection Methods of Prunus necrotic ringspot virus

针对感染李坏死环斑病毒(Prunus necrotic ringspot virus,PNRSV)植株病毒的特异性检测,将DAS- ELISA、RT-PCR方法综合改进、优化,建立了免疫捕获反转录PCR(IC-RT-PCR)检测PNRSV的方法.该方法采用PNRSV抗体特异性免疫捕获病毒,并根据GenBank中PNRSV序列设计的特异性引物对PN5/PN3进行PCR扩增,得到约760 bp大小的特异性基因片段.扩增产物经克隆测序后,结果表明:产物序列与PNRSV的外壳蛋白编码基因存在94%～98%的同源性.检测广泛性以及灵敏度比较研究显示.IC-RT-PCR方法能从含量很低的受病样品中检出PNRSV,检测灵敏度与RT-PCR相近,但所检测样品的范围更广,适用于从植物各部位检测低含量的病毒.IC-RT-PCR检测灵敏度比DAS-ELISA方法高出1000倍.

For the purpose of detection PNRSV in rose, IC - RT - PCR （immunocapture-reverse transcriptease-polymerase chain reaction）was established based on optimized double-antibody sandwich （DAS）- ELISA and RT- PCR to detect Prunus necrotic ringspot virus （PNRSV） from infected rose plants. Capture antiserum of PNRSV for virus immunocapture was conducted and a 760 bp fragment was amplified from diseased tissues with specific primers, designed on the basis of the coat protein gene of PNRSV. The fragment was cloned and sequenced. Sequence analysis result showed it shared 94%-98 % nueleotide identity with the coat protein gene of PNRSV. The detection sensitivity of IC - RT - PCR is identical to that of RT- PCR. While IC- RT- PCR could detect PNRSV-infected samples more widely, especially from plant samples with low virus concentration in different tissues. And the detection sensitivity of IC- RT- PCR is 1000 times higher than that of DAS- ELISA.