柑橘黄化脉明病毒RT-RPA检测方法的建立

[目的]利用逆转录重组酶聚合酶扩增技术(reverse transcription-recombinase polymerase amplification,RT-RPA),结合侧向流层析(lateral flow dipstick,LFD)试纸条,建立一种快速简便、特异、灵敏、裸眼可视的柑橘黄化脉明病毒(citrus...

Establishment of RT-RPA for Citrus Yellow Vein Clearing Virus (CYVCV) Detection

【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℃), 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℃, 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.