摘要
为了能够快速且同时大批量检测甘薯样本是否感染甘薯曲叶病毒(SPLCV),本研究建立了SPLCV cp基因的PCR-层析试纸条快速检测方法。首先,本研究构建SPLCV cp基因的重组质粒作为阳性对照,并对标记的特异性引物的扩增条件进行优化。结果显示,退火温度为57℃时,特异性扩增效果最佳;特异性检测结果表明,该引物对与甘薯无症状1号病毒(SPSMV-1)、甘薯杆状DNA病毒B(SPBV-B)、甘薯羽状斑驳病毒(SPFMV)、甘薯褪绿矮化病毒(SPCSV)和甘薯潜隐病毒(SPLV)均无交叉反应,表明该引物对具有较高的特异性。其次,本研究构建了PCR-层析试纸条快速检测系统,当抗体包被磁粒时,20μg地高辛单克隆抗体为0.1 mg羧基磁粒的最佳抗体标记量。特异性扩增产物在新构建的层析系统中3 min左右进行可视化检测;灵敏度检测结果表明,该层析系统的最低检测限为0.0001 ng·μL^(-1)基因组DNA,灵敏度是普通凝胶检测的10倍,说明该系统灵敏度较高。综上,本研究建立的PCR-层析试纸条检测系统具有可视性、灵敏度高、简便快速的特点,且可以同时对大批量的样本进行SPLCV检验,满足了脱毒薯苗上市前检测量大的需求。
To rapidly and simultaneously detect sweet potato leaf curl virus(SPLCV)in a large number of samples,a rapid detection method for PCR-chromatographic strip based on cp gene of SPLCV was established in this paper.Firstly,we constructed the recombinant plasmid of the cp gene of SPLCV as the positive control and optimized the amplification conditions of the labeled specific primers.The results showed that 57℃was the optimum annealing temperature.The results of the specificity test showed that SPLCV could be specifically detected by this method,and there was no cross-reactions with other sweet potato viruses such as Sweet potato symptomless virus 1(SPSMV-1),Sweet potato badnavirus B(SPBV-B),Sweet potato feathery mottle virus(SPFMV),Sweet potato chlorotic stunt virus(SPCSV)and Sweet potato latent virus(SPLV),that indicated the primers had good specificity.Secondly,we constructed a PCR-chromatography rapid detection system.The results showed that 20μg digoxin antibodies were the optimal antibody labeling amount for 0.1 mg carboxyl magnetic particles.The specific amplified products were visualized in the newly constructed chromatographic system after about 3 min.The sensitivity results showed that the minimum detection limit was 0.0001 ng·μL^(−1)of SPLCV genomic DNA,which was 10 times lower than that of normal gel detection.That indicated this system is highly sensitive.In summary,the tomographic strip detection system established in this study has the characteristics of visibility,high sensitivity,and rapidity,and can be used to detect a large number of samples at the same time,which meets the demand of detecting a large number of virus-free sweet potato seedlings before put on the market.
作者
张小梅
李广录
刘宇
侯文邦
ZHANG Xiaomei;LI Guanglu;LIU Yu;HOU Wenbang(Henan University of Science and Technology,Luoyang,Henan 471000)
出处
《核农学报》
CAS
CSCD
北大核心
2023年第5期955-961,共7页
Journal of Nuclear Agricultural Sciences
基金
河南省科技惠民专项项目(182207310015)。
关键词
甘薯
甘薯曲叶病毒
PCR-层析试纸条
灵敏性
Ipomoea batata
sweet potato leaf curl virus
PCR-based lateral flow assay
sensitivity
