Scientia Agricultura Sinica ›› 2021, Vol. 54 ›› Issue (15): 3241-3249.doi: 10.3864/j.issn.0578-1752.2021.15.009

• PLANT PROTECTION • Previous Articles     Next Articles

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

MA ZhiMin(),XU JianJian,DUAN Yu,WANG ChunQing,SU Yue,ZHANG Qi,BIN Yu,ZHOU ChangYong(),SONG Zhen()   

  1. Citrus Research Institute, Southwest University/National Citrus Engineering Research Center, Chongqing 400712
  • Received:2020-11-12 Accepted:2020-12-28 Online:2021-08-01 Published:2021-08-10
  • Contact: ChangYong ZHOU,Zhen SONG E-mail:mazhimin02@163.com;zhoucy@cric.cn;songzhen@cric.cn

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Abstract:

【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.

Key words: citrus, citrus yellow vein clearing virus (CYVCV), RT-RPA, lateral flow dipstick test strip, rapid detection

Table 1

Primers used in this study"

体系
System		 引物名称
Primer name		 引物序列
Primer sequence (5′-3′)		 引物序列位置
Primer sequence position
RT-RPA
CY1-F GTCACTAACT CCATAGCTAA CCCTAAACAGAC 6561-6737
CY1-R [biotin]-GTTGAGATCTTCGTTCGCTCCAATAATATCAG
CY1-Probe [6-FAM]CT ATCGGGAAAG CTTGGGTCCG CATCTTG[THF]AC ATCGATCCTG CCAAC-C3 Spacer 6598-6644
CY2-F AAAACCCACA ACAATGGGGT CTCTCCACCT GCC 6462-6652
CY2-R AAGAACACGTTGGCAGGATCGATGTTCAAGATGCG
CY3-F GTCTACCGACGACAACAAGGGCAAACAACC 6287-6435
CY3-R TACGCTTTTCGATGGGCTCTTGGTTTTCCT
CY4-F AACAAGGGCAAACAACCACTTCACCCGACA 6300-6435
CY4-R TACGCTTTTCGATGGGCTCTTGGTTTTCCT
CY5-F GCCCTAACGACACGACCCCGAAACCTATCC 6337-6439
CY5-R GTGATACGCTTTTCGATGGGCTCTTGGTTT
RT-PCR 614F TACCGCAGCTATCCATTTCC 6174-6785
614R GCAGAAATCCCGAACCACTA

Fig. 1

RT-RPA primer pairs screening for CYVCV detection M： Standard molecular weight. The same as below。A：1： Positive sample;2：ddH2O。B：1：Young leaf;2：Young bark;3： Old leaf;4：Old bark"

Fig. 2

Sequence alignment of RT-RPA products"

Fig. 3

Screening of RT-RPA reaction time for CYVCV detection"

Fig. 4

Screening of RT-RPA reaction temperature for CYVCV detection"

Fig. 5

RT-RPA specificity test for CYVCV detection"

Fig. 6

Sensitivity of RT-RPA and RT-PCR for CYVCV detection"

Fig. 7

CYVCV detection using RT-RPA and RT-PCR for citrus samples from orchard"

Table 2

CYVCV detection using RT-RPA and RT-PCR for different citrus varieties"

品种
Variety		 样品株数
Number of samples		 阳性样品数（阳性率） Positive number (Positive rate)
RT-RPA RT-PCR
尤力克柠檬 Eureka lemon 5 5 (100%) 5 (100%)
纽荷尔脐橙 Newhall navel orange 5 5 (100%) 5 (100%)
塔罗科血橙 Tarocco 5 5 (100%) 5 (100%)
冰糖橘 Bingtangju 5 5 (100%) 5 (100%)
默科特橘 Murcott angerine 5 5 (100%) 5 (100%)
沙田柚 Shatianyou 5 2 (40%) 2 (40%)
强德勒柚 Chandler pummelo 5 0 (0) 0 (0)
沃柑 Orah 5 5 (100%) 5 (100%)
爱媛38 Ehime 38 hybrid citrus 5 5 (100%) 5 (100%)
总计 Total 45 37 (82.2%) 37 (82.2%)
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