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,Citrus Research Institute of Southwest University/Chinese Academy of Agricultural Sciences, Chongqing 400712
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. Keywords:citrus leaf blotch virus (CLBV);RT-RPA;rapid detection
PDF (1804KB)元数据多维度评价相关文章导出EndNote|Ris|Bibtex收藏本文 本文引用格式 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 doi:10.3864/j.issn.0578-1752.2021.09.008 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 Acricultura Sinica, 2021, 54(9): 1904-1912 doi:10.3864/j.issn.0578-1752.2021.09.008
M:DNA分子质量标记DL2000 Marker;1、2、5、6、9、10:CLBV阳性核酸CLBV positive nucleic acid;3、7、11:健康对照CLBV negative control;4、8、12:ddH2O Fig. 1Specificity of three RT-RPA primer pairs for CLBV detection
M:DNA分子质量标记DL2000 Marker;CTV—CLBV:不同柑橘病害阳性样本Different citrus disease positive samples;ck:健康对照Health control;H2O:水对照Water control Fig. 4Specificity of RT-RPA for CLBV detection
A:RT-PCR;B:RT-RPA;C:RT-qPCR。M:DNA分子质量标记DL2000 Marker;1—7:柑橘样品核酸Sample nucleic acid Fig. 6CLBV detection for some citrus samples by RT-PCR, RT-RPA and RT-qPCR
VIVESM C, GALIPIENSOL, NAVARROL, MORENOP, GUERRIJ. Characterization of two kinds of subgenomic RNAs produced by citrus leaf blotch virus , 2002,295(2):328-336. [本文引用: 1]
RUBINOL, RUSSOM, DESTRADIS A, MARTELLIG P. Tepovirus, a novel genus in the family Betaflexiviridae , 2012,157(8):1629-1633. [本文引用: 1]
ZHUC X, WANGG P, ZHENGY Z, YANGZ K, WANGL P, XUW X, HONGN. 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) [本文引用: 3]
NAVARROL, PINAJ A, BALLESTAROLMOSJ F, MORENOP, CAMBRAM. A new graft transmissible disease found in Nagami kumquat//Ninth IOCV Conference Proceedings , 1984: 234-240. [本文引用: 1]
LINY J, GAOR X. Survey and identification of Actinidia spp. diseases in Fujian, China , 1995,24(1):49-53. (in Chinese) [本文引用: 1]
GALIPIENSOL, VIVIESM C, MORENOP, MILNER G, NAVARROL, GUERRIJ. Partial characterisation of citrus leaf blotch virus, a new virus from Nagami kumquat , 2001,146(2):357-368. [本文引用: 1]
LIUH, SONGS, WUW, MIW, SHENC, BAIB X, WUY F. Distribution and molecular characterization of citrus leaf blotch virus from Actinidia in Shaanxi Province, China , 2019,154(3):855-862. [本文引用: 1]
GUERRIJ, PINAJ A, VIVESM C, NAVARROL, MORENOP. Seed transmission of citrus leaf botch virus: Implications in quarantine and certification programs , 2004,88(8):906. [本文引用: 1]
GALIPIENSOL, VIVESM C, NAVARROL, MORENOP, GUERRIJ. Detection of citrus leaf blotch virus using digoxigenin-labeled cDNA probes and RT-PCR , 2004,110(2):175-181. [本文引用: 1]
PENGQ D, QIUL, YANGT, NINGJ C, XUQ Y, DONGJ H, XID H. A multiple reverse transcription PCR assay for simultaneous detection of four main viruses in kiwifruit , 2020,156(4):1207-1212. [本文引用: 1]
HUANGA J, WANGY, DINGM, LUZ J, YIL. Establishment and application of multiple PCR rapid detection of four citrus viruses Acta Horticulturae Sinica, 2019,46(8):1616-1622. (in Chinese) [本文引用: 1]
LIUH, MIW L, LIUF, WUW, WUK, WUY 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) [本文引用: 2]
RUIZ-RUIZS, AMBROSA, VIVESM D G, NAVARROL, MORENOP, GUERRIJ. Detection and quantitation of citrus leaf blotch virus by TaqMan real-time RT-PCR , 2009,160(1/2):57-62. [本文引用: 3]
OSMANF, HODZICE, KWONS J, WANGJ B, VIDALAKISG. 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 , 2015,220:64-75. [本文引用: 1]
LIUH, WUW, TANJ Q, LIY, MIW L, JIANGL J, WUY F. Development and evaluation of a one-step reverse transcription loop-mediated isothermal amplification for detection of citrus leaf blotch virus , 2019,270:150-152. [本文引用: 1]
PIEPENBURGO, WILLIAMSC H, STEMPLED L, ARMESN A. DNA detection using recombination proteins , 2006,4(7):1115-1121. [本文引用: 1]
LIUH, WANGJ B, LIP, BAIL, JIAJ W, PANA H, LONGX Q, CUIW D, TANGX M. Rapid detection of P-35S and T-nos in genetically modified organisms by recombinase polymerase amplification combined with a lateral flow strip , 2020,107:106775. [本文引用: 1]
DESHIELDSJ B, MOROZN, MORA-ROMEROG A, TANAKAK. Recombinase polymerase amplification (RPA) for the rapid isothermal detection of Spongospora subterranea f. sp. subterranea and potato mop-top virus , 2019,96(6):617-624. [本文引用: 1]
KAPOORR, SRIVASTAVAN, KUMARS, SAEITHAR K, SHARMAS K, JAINR K, BARANWELV K. Development of a recombinase polymerase amplification assay for the diagnosis of banana bunchy top virus in different banana cultivars , 2017,162(9):2791-2796. [本文引用: 1]
QIANW J, LUY, MENGY Q, YEZ Z, WANGL, WANGR, ZHENGQ Q, WUH, WUJ. Field detection of citrus Huanglongbing associated with ‘Candidatus Liberibacter asiaticus’ by recombinese polymerase amplification within 15 min , 2018,66(22):5473-5480. [本文引用: 1]
WANGY, CHENR H, NIEX Z, ZHONGZ Y, LIC Y, LIK, HUANGW, FUX Y, LIUJ, NIEB H. Rapid and sensitive detection of potato virus Y by isothermal reverse transcription-recombinase polymerase amplification assay in potato , 2020,50:101505. [本文引用: 1]
KIMN Y, LEEH J, JEONGR D. A portable detection assay for apple stem pitting virus using reverse transcription-recombinase polymerase amplification , 2019,274:113747. [本文引用: 1]
SILVAG, B?MERM, NKEREC, KUMARP L, SEALS E. Rapid and specific detection of yam mosaic virus by reverse-transcription recombinase polymerase amplification , 2015,222:138-144. [本文引用: 1]
BABUB, WASHBURNB K, MILLERS H, PODUCHK, SARIGULT, KNOXG W, OCHOA-CORONAF M, PARETM L. A rapid assay for detection of rose rosette virus using reverse transcription- recombinase polymerase amplification using multiple gene targets , 2017,240:78-84. [本文引用: 1]
JIAOY B, XUC T, LIJ L, GUY, XIAC, XIEQ, XIEY B, ANM N, XIAZ, WUY H. Characterization and a RT-RPA assay for rapid detection of chilli veinal mottle virus (ChiVMV) in tobacco , 2020,17:33. [本文引用: 1]
IVANOVA V, SAFENKOVAI V, ZHERDEVA V, DZANTIEVB B. Nucleic acid lateral flow assay with recombinase polymerase amplification: Solutions for highly sensitive detection of RNA virus , 2020,210:120616. [本文引用: 1]
HARPERS J, CHOOIK M, PEARSONM N. First report of citrus leaf blotch virus in New Zealand , 2008,92(10):1470. [本文引用: 2]
JIAOY B, JIANGJ Y, ANM N, XIAZ H, WUY H. Recombinase polymerase amplification assay for rapid detection of maize chlorotic mottle virus in maize , 2019,164(10):2581-2584. [本文引用: 1]
ZENGR, LUOJ Y, GAOS G, XUL H, SONGZ W, DAIF M. Rapid detection of cucumber green mottle mosaic virus by reverse transcription recombinase polymerase amplification , 2019,43:84-85. [本文引用: 1]