Scientia Agricultura Sinica ›› 2021, Vol. 54 ›› Issue (9): 1904-1912.doi: 10.3864/j.issn.0578-1752.2021.09.008

• PLANT PROTECTION • Previous Articles     Next Articles

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

DUAN Yu(),XU JianJian,MA ZhiMin,BIN Yu,ZHOU ChangYong(),SONG Zhen()   

  1. Citrus Research Institute of Southwest University/Chinese Academy of Agricultural Sciences, Chongqing 400712
  • Received:2020-07-08 Accepted:2020-08-04 Online:2021-05-01 Published:2021-05-10
  • Contact: ChangYong ZHOU,Zhen SONG E-mail:982432080@qq.com;zhoucy@cric.cn;songzhen@cric.cn

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

Table 1

Primers sequences for the detection of CLBV"

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
CLBV5R GCAGATCATTCACCACATGC 7564-7583
RT-qPCR CLBVF CAATTGCATGAACACTCACGG 2101-2121
CLBVR GGACCCCCCATTAAATTCCA 2251-2231

Fig. 1

Specificity of three RT-RPA primer pairs for CLBV detection"

Fig. 2

CLBV detection by RT-RPA at different temperatures"

Fig. 3

CLBV detection by RT-RPA at different reaction times"

Fig. 4

Specificity of RT-RPA for CLBV detection"

Fig. 5

Sensitivity of RT-RPA for CLBV detection"

Fig. 6

CLBV detection for some citrus samples by RT-PCR, RT-RPA and RT-qPCR"

[1] VIVES M C, GALIPIENSO L, NAVARRO L, MORENO P, GUERRI J. Characterization of two kinds of subgenomic RNAs produced by citrus leaf blotch virus. Virology, 2002,295(2):328-336.
[2] RUBINO L, RUSSO M, DE STRADIS A, MARTELLI G P. Tepovirus, a novel genus in the family Betaflexiviridae. Archives of Virology, 2012,157(8):1629-1633.
[3] 朱晨熹, 王国平, 郑亚洲, 杨作坤, 王利平, 徐文兴, 洪霓. 来源于猕猴桃的柑橘叶斑驳病毒的RT-PCR检测及外壳蛋白基因序列分析. 植物病理学报, 2016,46(1):11-16.
ZHU C X, WANG G P, ZHENG Y Z, YANG Z K, WANG L P, XU W X, HONG N. RT-PCR detection and sequence analysis of coat protein gene of citrus leaf blotch virus infecting kiwifruit trees. Acta Phytopathologica Sinica, 2016,46(1):11-16. (in Chinese)
[4] NAVARRO L, PINA J A, BALLESTAROLMOS J F, MORENO P, CAMBRA M. A new graft transmissible disease found in Nagami kumquat//Ninth IOCV Conference Proceedings. Riverside Springer, 1984: 234-240.
[5] CHE X B, PIESTUN D, MAWASSI M, YANG G, SATYANARAYANA T, GOWDA S, DAWSON W O, BAR-JOSEPH M. 5′-Coterminal subgenomic RNAs in citrus tristeza virus-infected cells. Virology, 2001,283(2):374-381.
[6] 林尤剑, 高日霞. 福建猕猴桃病害调查与鉴定. 福建农业大学学报, 1995,24(1):49-53.
7 LIN Y J, GAO R X. Survey and identification of Actinidia spp. diseases in Fujian, China. Journal of Fujian Agricultural University, 1995,24(1):49-53. (in Chinese)
[7] GALIPIENSO L, VIVIES M C, MORENO P, MILNE R G, NAVARRO L, GUERRI J. Partial characterisation of citrus leaf blotch virus, a new virus from Nagami kumquat. Archives of Virology, 2001,146(2):357-368.
[8] LIU H, SONG S, WU W, MI W, SHEN C, BAI B X, WU Y F. Distribution and molecular characterization of citrus leaf blotch virus from Actinidia in Shaanxi Province, China. European Journal of Plant Pathology, 2019,154(3):855-862.
[9] GUERRI J, PINA J A, VIVES M C, NAVARRO L, MORENO P. Seed transmission of citrus leaf botch virus: Implications in quarantine and certification programs. Plant Disease, 2004,88(8):906.
[10] GALIPIENSO L, VIVES M C, NAVARRO L, MORENO P, GUERRI J. Detection of citrus leaf blotch virus using digoxigenin-labeled cDNA probes and RT-PCR. European Journal of Plant Pathology, 2004,110(2):175-181.
[11] PENG Q D, QIU L, YANG T, NING J C, XU Q Y, DONG J H, XI D H. A multiple reverse transcription PCR assay for simultaneous detection of four main viruses in kiwifruit. European Journal of Plant Pathology, 2020,156(4):1207-1212.
[12] 黄爱军, 王莹, 丁敏, 卢占军, 易龙. 柑橘4种病毒多重PCR检测技术的建立及应用. 园艺学报, 2019,46(8):1616-1622.
HUANG A J, WANG Y, DING M, LU Z J, YI L. Establishment and application of multiple PCR rapid detection of four citrus viruses. Acta Horticulturae Sinica, 2019,46(8):1616-1622. (in Chinese)
[13] 刘欢, 米伟丽, 刘斐, 吴薇, 吴宽, 吴云锋. 猕猴桃植株中柑橘叶斑驳病毒实时荧光定量PCR检测技术的建立及应用. 植物病理学报, 2019,49(2):167-173.
LIU H, MI W L, LIU F, WU W, WU K, WU Y F. Development and evaluation of a real-time fluorescent quantitative PCR assay for detection of citrus leaf blotch virus in kiwifruit plants. Acta Phytopathologica Sinica, 2019,49(2):167-173. (in Chinese)
[14] RUIZ-RUIZ S, AMBROS A, VIVES M D G, NAVARRO L, MORENO P, GUERRI J. Detection and quantitation of citrus leaf blotch virus by TaqMan real-time RT-PCR. Journal of Virological Methods, 2009,160(1/2):57-62.
[15] OSMAN F, HODZIC E, KWON S J, WANG J B, VIDALAKIS G. Development and validation of a multiplex reverse transcription quantitative PCR (RT-qPCR) assay for the rapid detection of citrus tristeza virus, citrus psorosis virus, and citrus leaf blotch virus. Journal of Virological Methods, 2015,220:64-75.
[16] LIU H, WU W, TAN J Q, LI Y, MI W L, JIANG L J, WU Y F. Development and evaluation of a one-step reverse transcription loop-mediated isothermal amplification for detection of citrus leaf blotch virus. Journal of Virological Methods, 2019,270:150-152.
[17] PIEPENBURG O, WILLIAMS C H, STEMPLE D L, ARMES N A. DNA detection using recombination proteins. PLoS Biology, 2006,4(7):1115-1121.
[18] LIU H, WANG J B, LI P, BAI L, JIA J W, PAN A H, LONG X Q, CUI W D, TANG X M. Rapid detection of P-35S and T-nos in genetically modified organisms by recombinase polymerase amplification combined with a lateral flow strip. Food Control, 2020,107:106775.
[19] DESHIELDS J B, MOROZ N, MORA-ROMERO G A, TANAKA K. Recombinase polymerase amplification (RPA) for the rapid isothermal detection of Spongospora subterranea f. sp. subterranea and potato mop-top virus. American Journal of Potato Research, 2019,96(6):617-624.
[20] KAPOOR R, SRIVASTAVA N, KUMAR S, SAEITHA R K, SHARMA S K, JAIN R K, BARANWEL V K. Development of a recombinase polymerase amplification assay for the diagnosis of banana bunchy top virus in different banana cultivars. Archives of Virology, 2017,162(9):2791-2796.
[21] QIAN W J, LU Y, MENG Y Q, YE Z Z, WANG L, WANG R, ZHENG Q Q, WU H, WU J. Field detection of citrus Huanglongbing associated with ‘Candidatus Liberibacter asiaticus’ by recombinese polymerase amplification within 15 min. Journal of Agricultural and Food Chemistry, 2018,66(22):5473-5480.
[22] WANG Y, CHEN R H, NIE X Z, ZHONG Z Y, LI C Y, LI K, HUANG W, FU X Y, LIU J, NIE B H. Rapid and sensitive detection of potato virus Y by isothermal reverse transcription-recombinase polymerase amplification assay in potato. Molecular and Cellular Probes, 2020,50:101505.
[23] KIM N Y, LEE H J, JEONG R D. A portable detection assay for apple stem pitting virus using reverse transcription-recombinase polymerase amplification. Journal of Virological Methods, 2019,274:113747.
[24] SILVA G, BÖMER M, NKERE C, KUMAR P L, SEAL S E. Rapid and specific detection of yam mosaic virus by reverse-transcription recombinase polymerase amplification. Journal of Virological Methods, 2015,222:138-144.
[25] BABU B, WASHBURN B K, MILLER S H, PODUCH K, SARIGUL T, KNOX G W, OCHOA-CORONA F M, PARET M L. A rapid assay for detection of rose rosette virus using reverse transcription- recombinase polymerase amplification using multiple gene targets. Journal of Virological Methods, 2017,240:78-84.
[26] JIAO Y B, XU C T, LI J L, GU Y, XIA C, XIE Q, XIE Y B, AN M N, XIA Z, WU Y H. Characterization and a RT-RPA assay for rapid detection of chilli veinal mottle virus (ChiVMV) in tobacco. Virology Journal, 2020,17:33.
[27] IVANOV A V, SAFENKOVA I V, ZHERDEV A V, DZANTIEV B B. Nucleic acid lateral flow assay with recombinase polymerase amplification: Solutions for highly sensitive detection of RNA virus. Talanta, 2020,210:120616.
[28] HARPER S J, CHOOI K M, PEARSON M N. First report of citrus leaf blotch virus in New Zealand. Plant Disease, 2008,92(10):1470.
[29] JIAO Y B, JIANG J Y, AN M N, XIA Z H, WU Y H. Recombinase polymerase amplification assay for rapid detection of maize chlorotic mottle virus in maize. Archives of Virology, 2019,164(10):2581-2584.
[30] ZENG R, LUO J Y, GAO S G, XU L H, SONG Z W, DAI F M. Rapid detection of cucumber green mottle mosaic virus by reverse transcription recombinase polymerase amplification. Molecular and Cellular Probes, 2019,43:84-85.
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