中国农业科学2024，Vol.57Issue(14)：2781-2790,10.DOI:10.3864/j.issn.0578-1752.2024.14.007
甘薯褪绿矮化病毒西非株系RT-RPA-LFD检测方法的建立与应用
Establishment and Application of RT-RPA-LFD Detection Method for Sweet Potato Chlorotic Stunt Virus WA Strain
摘要
Abstract
[Objective]This study aims to establish a novel technique for detecting the west African strain of sweet potato chlorotic stunt virus(SPCSV-WA)by combining reverse transcriptase recombinase polymerase amplification and lateral flow dipstick(RT-RPA-LFD).[Method]Primers and probes with good amplification results and strong specificity were designed and screened on the basis of the conserved sequences of the SPCSV-WA coat protein gene and heat shock protein gene.Then,the conditions such as primer and probe concentrations,amplification system,temperature,and reaction time were optimized to detect SPCSV-WA using RT-RPA-LFD.This method was used to detect common viruses on sweet potato such as the east African strain of sweet potato chlorotic stunt virus(SPCSV-EA),sweet potato feathery mottle virus(SPFMV),sweet potato latent virus(SPLV)and sweet potato virus G(SPVG)to verify the specificity.Total RNA from SPCSV-infected sweet potato leaves was diluted in a 10-fold gradient,and RT-PCR and RT-RPA-LFD were performed to compare the sensitivity of the two methods.Field sweet potato samples and test tube seedling samples were subjected to RT-RPA,RT-RPA-LFD,and RT-PCR detection to validate the practicality of the method.[Result]The RT-RPA-LFD detection method for SPCSV-WA was established using the optimal primer CSV357F/R and the CSV-CP-Probe(47 bp).The working concentrations of the primer and probe were 0.2 and 0.06 μmol·L-1,respectively.The reaction conditions were set to 42℃for 5 min.This method could specifically detect SPCSV-WA and had no cross-reaction with other common viruses on sweet potato.The RT-RPA-LFD could detect viruses up to 10-4 dilution,whereas RT-PCR could only detect up to 10-3 dilution,making the sensitivity of RT-RPA-LFD 10 times higher than that of RT-PCR.Of the 22 field-gathered sweet potato samples tested by RT-PCR,RT-RPA,and RT-RPA-LFD,11 positive samples were consistently found across the three methods.The testing results of 28 test tube seedling samples showed that RT-PCR and RT-RPA-LFD consistently detected five positive samples.[Conclusion]The RT-RPA-LFD detection method for SPCSV-WA has been established and it is characterized by its speed,simplicity,specificity,sensitivity,and visibility.This method can be used for testing virus-free test tube seedling samples of sweet potato as well as for on-site rapid detection of field sweet potato samples in basic level units.关键词
甘薯褪绿矮化病毒西非株系/逆转录酶重组酶聚合酶扩增/侧向流层析/快速检测/甘薯Key words
west African strain of sweet potato chlorotic stunt virus(SPCSV-WA)/reverse transcriptase recombinase polymerase amplification(RT-RPA)/lateral flow dipstick(LFD)/rapid detection/sweet potato引用本文复制引用
王永江,乔奇,王爽,赵付枚,田雨婷,张德胜,张振臣..甘薯褪绿矮化病毒西非株系RT-RPA-LFD检测方法的建立与应用[J].中国农业科学,2024,57(14):2781-2790,10.基金项目
国家甘薯产业技术体系(CARS-10-GW15)、河南省科技攻关项目(232102111098)、河南省农业科学院自主创新项目(2023ZC047,2024ZC058) （CARS-10-GW15）
