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

Abstract

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

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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References 30

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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

Detection of Citrus Leaf Blotch Virus by Reverse Transcription- Recombinase Polymerase Amplification (RT-RPA)

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