柑橘叶斑驳病毒的逆转录重组酶聚合酶扩增检测

doi:10.3864/j.issn.0578-1752.2021.09.008

摘要/Abstract

摘要：

【目的】建立柑橘叶斑驳病毒（citrus leaf blotch virus,CLBV）逆转录重组酶聚合酶扩增（reverse transcription-recombinase polymerase amplification,RT-RPA）的快速检测体系,为该病毒提供快速、简便的检测方法。【方法】以CLBV ORF1保守序列为靶标,通过设计、筛选特异性检测引物、优化反应温度和反应时长等条件建立CLBV的RT-RPA检测体系。分别以感染CLBV、柑橘衰退病毒（citrus tristeza virus,CTV）、柑橘黄化脉明病毒（citrus yellow vein clearing virus,CYVCV）、柑橘裂皮类病毒（citrus exocortis viroid,CEVd）、柑橘碎叶病毒（citrus tatter leaf virus,CTLV）、柑橘鳞皮病毒（citrus psorosis virus,CPsV）、温州蜜柑萎缩病毒（satsuma dwarf virus,SDV）、柑橘溃疡病菌（Xanthomonas citri subsp. citri,Xcc）和柑橘黄龙病菌亚洲种（Candidatus Liberibacter asiaticus,CLas）的柑橘核酸为模板,应用该体系进行RT-RPA扩增,评价检测特异性;利用CLBV阳性样品核酸的10倍浓度梯度稀释模板,比较RT-RPA、RT-PCR和RT-qPCR的检测灵敏度;通过比较上述3种方法对72份柑橘样品的CLBV检出率,检验所建立RT-RPA法的适用性。【结果】以感染了CLBV的柑橘核酸样品为模板,通过对设计的3对引物筛选试验、6个温度和5个反应时长的梯度试验,建立了CLBV的RT-RPA检测体系,明确引物RCLBV-F/RCLBV-R2能够特异扩增CLBV ORF1序列,且扩增效果良好,目的片段大小为144 bp,反应最适温度为40℃,反应最佳时长为30 min。在感染CLBV、CTV、CYVCV、CEVd、CTLV、CPsV、SDV、Xcc和CLas的柑橘样品、健康对照和空白对照中,该体系仅在CLBV侵染样品中扩增出预期大小的特异性目的条带,说明建立的RT-RPA检测体系特异性强;灵敏度检测结果表明,RT-RPA和RT-PCR的检测灵敏度相当,但低于RT-qPCR;所建立的RT-RPA方法在72份柑橘样品中检测出11份样品为CLBV阳性,检出率为15.28%,该结果与普通RT-PCR和RT-qPCR检测方法的检测结果一致。【结论】建立了CLBV的RT-RPA检测体系,该体系具有耗时短、操作简便、特异性强等优点,适用于田间一定规模柑橘样品的检测。

关键词: 柑橘叶斑驳病毒, 逆转录重组酶聚合酶扩增, 快速检测

Abstract:

【Objective】The objective of this study is to establish a rapid reverse transcription-recombinase polymerase amplification (RT-RPA) detection system for citrus leaf blotch virus (CLBV), and to provide an efficient and simple detection method for CLBV. 【Method】RT-RPA primers for CLBV detection were designed on conserved sequence of the ORF1, and their specificity was verified. The reaction temperature and reaction time were then optimized to establish RT-RPA system for CLBV detection. Nucleic acids from samples infected with citrus tristeza virus (CTV), citrus yellow vein clearing virus (CYVCV), citrus exocortis viroid (CEVd), citrus tatter leaf virus (CTLV), citrus psorosis virus (CPsV), satsuma dwarf virus (SDV), Xanthomonas citri subsp. citri (Xcc) and Candidatus Liberibacter asiaticus (CLas), respectively, were used to evaluate the specificity of the established RT-RPA method. The 10-fold dilution of CLBV positive samples was used to compare the detection sensitivity of RT-RPA, RT-PCR and RT-qPCR. In order to evaluate the applicability of the RT-RPA, 72 citrus samples were parallelly detected by the above three methods. 【Result】In order to establish a RT-RPA method for CLBV detection, a series of experiments have been approached. Firstly, RCLBV-F/RCLBV-R2 that selected out of three designed primer pairs could specifically amplify the ORF1 fragments of CLBV with the aim size of 144 bp by RT-RPA, whereas no specific band could be obtained from samples infected with CTV, CYVCV, CEVd, CTLV, CPsV, SDV, Xcc or CLas, showing the strong specificity of the RT-RPA method. Secondly, through gradient test of 6 temperatures and 5 reaction times, the optimal reaction temperature and time of the RT-RPA system were determined to be 40℃ and 30 min, respectively. Thirdly, the sensitivity of the RT-RPA was compared to that of RT-PCR and RT-qPCR, which is similar to the former but less than the latter. The RT-RPA established in this study was addressed to detect 72 citrus samples, 11 samples were detected to be CLBV positive with positive rate of 15.28%, which was consistent with that of RT-PCR and RT-qPCR. 【Conclusion】A RT-RPA system for CLBV detection was established and optimized, which has advantages of short time consuming and easy operation with potential usage for a relatively large scale of virus monitoring in field.

Key words: citrus leaf blotch virus (CLBV), RT-RPA, rapid detection

段玉,许建建,马志敏,宾羽,周常勇,宋震. 柑橘叶斑驳病毒的逆转录重组酶聚合酶扩增检测[J]. 中国农业科学, 2021, 54(9): 1904-1912.

DUAN Yu,XU JianJian,MA ZhiMin,BIN Yu,ZHOU ChangYong,SONG Zhen. Detection of Citrus Leaf Blotch Virus by Reverse Transcription- Recombinase Polymerase Amplification (RT-RPA)[J]. Scientia Agricultura Sinica, 2021, 54(9): 1904-1912.

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检测方法 Method	引物名称 Primer	引物序列 Primer sequence (5′-3′)	引物序列位置 Primer sequence position (bp)
RT-RPA	RPACF1	TTAGCACTTTAACCTCAGACCCCTTACTTGG	2460-2491
	RPACR1	GATAAAAGCCGCCTTCCTTCCTATGAACTGG	2560-2590
	RCLBV-F	ATGAACACTCACGGCGATGAAATTCCCACA	2108-2137
	RCLBV-R1	TTCTGCAAGTGAAACATAGGATCCCCCATT	2240-2269
	RCLBV-R2	GGATCCCCCATTAAATTCCATAAGCCAGTC	2222-2251
RT-PCR	CLBV1F	AGCCATAGTTGAACCATTCCTC	7159-7180
RT-PCR	CLBV5R	GCAGATCATTCACCACATGC	7564-7583
RT-qPCR	CLBVF	CAATTGCATGAACACTCACGG	2101-2121
RT-qPCR	CLBVR	GGACCCCCCATTAAATTCCA	2251-2231