目的 建立一种高效、快速、准确地同步检测侵染三七Panax notoginseng的2种病毒——三七Y病毒（Panax notoginsengvirus Y，PnVY）和中国番茄黄化曲叶病毒（tomato yellow leaf curl China virus，TYLCCNV）的双重PCR技术。方法 分别以单一感染PnVY或TYLCCNV及复合侵染了这2种病毒的染病三七植株为样品，CTAB法提取叶片总核酸作为模板，根据GenBank上已登录的PnVY和TYLCCNV这2种病毒核酸序列设计特异性检测引物。通过单一PCR技术明确染病三七样品PnVY和TYLCCNV的发生情况，筛选出可用于一步法双重PCR检测PnVY和TYLCCNV的引物组合及引物浓度，并对反应条件进行优化。对扩增产物进行克隆测序，验证一步法双重PCR检测的准确性和特异性。结果 建立的一步法双重PCR检测技术能同步有效地检测三七病样中的PnVY和TYLCCNV，检测灵敏度高，最低检测限点为核酸原液的0.01稀释倍数，特异性强。结论 所建立的一步法双重PCR检测技术可以高效、快速、准确、特异地同步检测三七样品中的PnVY、TYLCCNV2种病毒。

Objective To establish an efficient, rapid and accurate method for the simultaneous detection of panax virus Y (PnVY) and tomato yellow leaf curl China virus (TYLCCNV) in Panax notoginseng by dual PCR. Methods Diseased P. notoginseng leaves of single infection of PnVY, or TYLCCNV, and co-infection of PnVY and TYLCCNV were selected as test samples. Total RNAs were extracted using CTAB method. Specific detection primers were designed according to the nucleic acid sequences of the two viruses registered on GenBank. The occurrence of PnVY and TYLCCNV in P. notoginseng samples were confirmed by conventional PCR, then the appropriate primer combination and concentration were screened for the dual detection assay about PnVY and TYLCCNV by one-step PCR, and the amplification conditions were also optimized. The accuracy and specificity of the PCR assay were tested by cloning and sequencing the amplified DNA fragments. Results The established one-step dual PCR detection technology could simultaneously and effectively detect PnVY and TYLCCNV in the diseased P. notoginseng samples with higher detection sensitivity and specificity. The detection limits of the dual PCR method were 10^-2 dilution for both PnVY and TYLCCNV. Conclusion The established one-step dual PCR detection technique can detect PnVY and TYLCCNV viruses simultaneously in the diseased P. notoginseng samples efficiently, quickly, accurately and specifically.

R286.2

国家自然科学基金资助项目(31660039);云南省专家工作站(202005AF150040)