Scientia Agricultura Sinica ›› 2024, Vol. 57 ›› Issue (14): 2781-2790.doi: 10.3864/j.issn.0578-1752.2024.14.007

• PLANT PROTECTION • Previous Articles     Next Articles

Establishment and Application of RT-RPA-LFD Detection Method for Sweet Potato Chlorotic Stunt Virus WA Strain

WANG YongJiang(), QIAO Qi, WANG Shuang, ZHAO FuMei, TIAN YuTing, ZHANG DeSheng, ZHANG ZhenChen()   

  1. Institute of Plant Protection, Henan Academy of Agricultural Sciences/Henan Key Laboratory of Crop Pest Control/IPM Key Laboratory in Southern Part of North China, Ministry of Agriculture and Rural Affairs, Zhengzhou 450002
  • Received:2024-02-28 Accepted:2024-04-20 Online:2024-07-16 Published:2024-07-24
  • Contact: ZHANG ZhenChen

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Abstract:

【Objective】 This study aims to establish a novel technique for detecting the west African strain of sweet potato chlorotic stunt virus (SPCSV-WA) by combining reverse transcriptase recombinase polymerase amplification and lateral flow dipstick (RT-RPA-LFD).【Method】 Primers and probes with good amplification results and strong specificity were designed and screened on the basis of the conserved sequences of the SPCSV-WA coat protein gene and heat shock protein gene. Then, the conditions such as primer and probe concentrations, amplification system, temperature, and reaction time were optimized to detect SPCSV-WA using RT-RPA-LFD. This method was used to detect common viruses on sweet potato such as the east African strain of sweet potato chlorotic stunt virus (SPCSV-EA), sweet potato feathery mottle virus (SPFMV), sweet potato latent virus (SPLV) and sweet potato virus G (SPVG) to verify the specificity. Total RNA from SPCSV-infected sweet potato leaves was diluted in a 10-fold gradient, and RT-PCR and RT-RPA-LFD were performed to compare the sensitivity of the two methods. Field sweet potato samples and test tube seedling samples were subjected to RT-RPA, RT-RPA-LFD, and RT-PCR detection to validate the practicality of the method.【Result】 The RT-RPA-LFD detection method for SPCSV-WA was established using the optimal primer CSV357F/R and the CSV-CP-Probe (47 bp). The working concentrations of the primer and probe were 0.2 and 0.06 μmol·L-1, respectively. The reaction conditions were set to 42 ℃ for 5 min. This method could specifically detect SPCSV-WA and had no cross-reaction with other common viruses on sweet potato. The RT-RPA-LFD could detect viruses up to 10-4 dilution, whereas RT-PCR could only detect up to 10-3 dilution, making the sensitivity of RT-RPA-LFD 10 times higher than that of RT-PCR. Of the 22 field-gathered sweet potato samples tested by RT-PCR, RT-RPA, and RT-RPA-LFD, 11 positive samples were consistently found across the three methods. The testing results of 28 test tube seedling samples showed that RT-PCR and RT-RPA-LFD consistently detected five positive samples.【Conclusion】 The RT-RPA-LFD detection method for SPCSV-WA has been established and it is characterized by its speed, simplicity, specificity, sensitivity, and visibility. This method can be used for testing virus-free test tube seedling samples of sweet potato as well as for on-site rapid detection of field sweet potato samples in basic level units.

Key words: west African strain of sweet potato chlorotic stunt virus (SPCSV-WA), reverse transcriptase recombinase polymerase amplification (RT-RPA), lateral flow dipstick (LFD), rapid detection, sweet potato

Table 1

Primers used in this study"

体系
System		 编号
Number		 引物名称
Primer name		 引物和探针序列
Primer and probe sequence (5′-3′)		 大小
Size (bp)		 来源
<BOLD>S</BOLD>ource
RT-RPA
 C1 CSV170F ATGGCTGATAGCACTAAAGTCGAAGAGAAGAAC 170 本试验This study
CSV170R GTAAGAATACCACTRGTTARCTCCATATCTC
C2 CSV216F GRCTTCCAACTATACTCTTTCCCGT 216 本试验This study
CSV216R GATATTATGTCAAAGAGTCAAGAGGATG
C3 CSV237F GGCRTGGGCAAATCAGAGTACGTCCGAAAAGAAT 237 本试验This study
CSV237R TTTCGCCTGYAAATGGTAATCGGGTTTAACAATAC
C4 CSV253F TTTGGGAAGACGAGATATGGAGYTAACTAGTGGT 253 本试验This study
CSV253R TTTCGGACGTACTCTGATTTGCCCATGCCTGW
C5 SCV259F YGGCTAGTTTGGGAAGACGAGATATGGAGYTAA 259 本试验This study
CSV259R TCGGACGTACTCTGATTTGCCCAYGCCTGW
C6 CSV357F [6-Fam]-TTTGGGAAGACGAGATATGGAGYTAACYAGTG 357 本试验This study
CSV357R Biotin-GGCATGTTAGRTTGAATATGGTTTATGAAGTTGT
CSV-CP-Probe [6-Fam]-CAGATATTATGTCAAAGAGTCAAGAGGAT[THF] AGTTTAAGCGTCATATG-C3Spacer
C7 CSV405F CGGRTCGGARTTTACYCCMACGTGTYGTCYRTYA 405 本试验This study
CSV405R CCKAACGATTCACATACAGACTTCATAAACATYC
C8 CSV313F WCAGGCRTGGGCAAATCAGAGTACGTCCGAAAA 313 本试验This study
CSV313R CTGCGGTTGTCGCCAAATCATTACCTCTCATG
RT-PCR CP-F ATGGCTGATAGCACTAAAGTCGA 774 文献[7] Reference [7]
CP-R TCAACAGTGAAGACCTGTTCCAG
SPCSV-EA-SPSP1-T7 TAATACGACTCACTATAATGRMTACTGRCAAAGTAAACGATG 791 文献[10] Reference [10]
SPCSV-EA-SPSP4 TCAACAGTGAAGACCRGYACCRGTCAA
SPVG-P5-T7 TAATACGACTCACTATAACTGAATTCTCYRCTGAAGARAYATAYGATGC 1017 文献[28] Reference [28]
SPVG-P3 ACTAAGCTTTTACTGCACACCCCTCATACC
SPLV-P5-T7 TAATACGACTCACTATAGCCGAY GAAACCATCMTCGAT 1093 文献[29] Reference [29]
SPLV-P3 GTCTC Y GGTATAAGACG

Table 2

Combinations of the reaction system"

编号
Number		 引物和探针反应浓度
Concentration of primer and probe (μmol·L-1)		 镁离子浓度
Concentration of Mg2+ (μmol·L-1)
A CSV357F (2×10-1)/CSV357R (2×10-1)+CSV-CP-Probe (6×10-2) 2.10×104
B CSV357F-fam (2×10-1)/CSV357R (2×10-1) 2.10×104
C CSV357F (4×10-1)/CSV357R (4×10-1)+CSV-CP-Probe (8×10-2) 1.68×104
D CSV357F-fam (2×10-1)/CSV357R (2×10-1) 1.68×104
E CSV357F (2×10-1)/CSV357R (2×10-1)+CSV-CP-Probe (6×10-2) 1.68×104
F CSV357F (1.6×10-1)/CSV357R (1.6×10-1)+CSV-CP-Probe (6×10-2) 1.68×104

Fig. 1

RT-RPA primer pairs screening for SPCSV-WA detection"

Fig. 2

Screening of primers and probes concentration"

Fig. 3

Screening of RT-RPA-LFD reaction time for SPCSV-WA detection"

Fig. 4

Screening of RT-RPA-LFD reaction temperature for SPCSV-WA detection"

Fig. 5

Sensitivity of RT-RPA-LFD (A) and RT-PCR (B) for SPCSV-WA detection"

Fig. 6

RT-RPA-LFD specificity for SPCSV-WA detection"

Fig. 7

SPCSV detection results of field sweet potato samples"

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