Establishment of RT-RPA for Citrus yellow vein clearing virus (CYVCV) detection.
Establishment of RT-RPA for Citrus yellow vein clearing virus (CYVCV) detection.
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DOI：
10.3864/j.issn.0578-1752.2021.15.009
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发表时间：
2021-01-01
影响因子：
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通讯作者：
Song, Z.
中科院分区：
文献类型：
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作者：
Ma, Zhi-min;Xu, Jian-jian;Song, Z.
目的：利用逆转录重组酶聚合酶扩增（RT-RPA）结合侧流量测仪（LFD），建立一种快速、简便、准确、肉眼可见的柑橘黄脉清病毒（CYVCV）检测新方法。方法：根据CYVCV外壳蛋白基因保守序列设计5对引物。通过对不同样品的检测，选择扩增效率和特异性最好的引物对。对所选引物进行修饰，并设计相应的特异性探针。通过设置6个反应梯度时间（5、10、20、30、40和50 min）和8个反应梯度温度（37、38、39、40、41、42、43和44℃），优化RT-RPA检测CYVCV的体系。通过检测CYVCV、柑桔叶斑疹病毒（CLBV）、柑桔tristeza病毒（CTV）、柑桔碎叶病毒（CTLV）、柑桔类表皮病毒（CEVd）、柑桔牛皮癣病毒（CPV）、蜜桔小病毒（SDV）、亚洲解放候选菌（CLas）和柑桔黄单胞菌亚种感染的样品，评价所建立的RT-RPA的特异性。柠檬酸（Xcc）。将感染CYVCV的柑橘总RNA样品稀释10倍。以原始RNA溶液和10-1、10-2、10-3、10-4、10-5、10-6、10-7稀释液为模板检测RT-RPA的敏感性，并与RT-PCR的敏感性进行比较。在田间随机采集不同柑橘品种的叶片。同时采用RT-RPA和RT-PCR检测方法，检验所建立的RT-RPA检测方法的适用性。结果：建立了CYVCV RT-RPA检测体系，引物对为CY1- f /R，探针对为CY1 （47 bp）。特异性扩增CYVCV靶片段，全长177bp。最佳反应条件为39℃，30 min。结果可通过LFD试纸直接判断。在特异性检测中，只有感染CYVCV的样品呈阳性，其余样品均为阴性。灵敏度检测中，10-4稀释度为RT-RPA和RT-PCR检测灵敏度最低。两种方法的灵敏度相当。随机抽取的45份柑橘田间样品中，RT-PCR和RT-RPA检测阳性37份，阳性率均为82.2%，表明本研究建立的RT-RPA检测方法稳定可靠。结论：建立了一种RT-RPA检测CYVCV的方法。该方法简便、快速、直观。适用于基础条件不足的实验室或植保检疫站的现场快速检测。
Objective: The objective of this study is to establish a fast, simple, accurate and visualized with naked eyes new detection method for citrus yellow vein clearing virus (CYVCV) using reverse transcription-recombinase polymerase amplification (RT-RPA) combined with lateral flow dipstick (LFD). Method: Five pairs of primers were designed according to the conservative sequence of the coat protein gene of CYVCV. By detecting different samples, the pair of primers with the best amplification efficiency and specificity was selected. The selected primers were modified and its corresponding specific probe was designed. According setting 6 reaction gradient times (5, 10, 20, 30, 40 and 50 min) and 8 reaction gradient temperatures (37, 38, 39, 40, 41, 42, 43 and 44°C), the RT-RPA system for CYVCV detection was optimized. The specificity of the established RT-RPA was evaluated by detecting the samples infected with CYVCV, citrus leaf blotch virus (CLBV), citrus tristeza virus (CTV), citrus tatter leaf virus (CTLV), citrus exocortis viroid (CEVd), citrus psorosis virus (CPV), satsuma dwarf virus (SDV), Candidatus Liberibacter asiaticus (CLas) and Xanthomonas citri subsp. citri (Xcc), respectively. The citrus total RNA samples infected with CYVCV was diluted by 10 times. The original RNA solution and 10-1, 10-2, 10-3, 10-4, 10-5, 10-6, 10-7 dilutions were used as templates for testing the sensitivity of RT-RPA, and the sensitivity was compared with RT-PCR. Leaves of different citrus varieties were randomly collected from the field. RT-RPA and RT-PCR were used at the same time to test the applicability of the established RT-RPA detection method. Result: A RT-RPA detection system for CYVCV was established, with primer pairs CY1-F/R and corresponding probe CY1 (47 bp). It could specifically amplify the target fragment of CYVCV with a size of 177 bp. The best reaction conditions were 39°C, 30 min. The result could be judged by the LFD test strip directly. In the specific test, only samples infected with CYVCV were positive, and the rest were negative. In the sensitivity detection, 10-4 dilution was the lowest detection sensitivity of RT-RPA and RT-PCR. The sensitivity of the two methods was equivalent. Among the 45 field citrus samples taken randomly, 37 samples were positive by RT-PCR and RT-RPA, and the positive rate was both 82.2%, indicating that the RT-RPA method established in this study was stable and reliable. Conclusion: A RT-RPA detection method for CYVCV is established. The method is convenient, rapid, and visualized. It can be applied to on-site rapid detection for the labs with insufficient basic conditions or plant protection and quarantine station.