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作物学报 ›› 2025, Vol. 51 ›› Issue (5): 1248-1260.doi: 10.3724/SP.J.1006.2025.44113

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

基于闭合哑铃介导等温扩增可视化检测大豆花叶病毒SC15方法的建立及应用

殷丛丛1(), 李睿琦2, 岳霈尧2, 李晨1, 牛景萍3, 赵晋忠1, 杜维俊2, 岳爱琴2,*()   

  1. 1山西农业大学基础部, 山西晋中 030801
    2山西农业大学农学院, 山西晋中 030801
    3山西农业大学生命科学学院, 山西晋中 030801
  • 收稿日期:2024-07-09 接受日期:2025-02-17 出版日期:2025-05-12 网络出版日期:2025-02-20
  • 通讯作者: *岳爱琴, E-mail: yueaiqinnd@126.com
  • 作者简介:E-mail: yincongcong@sxau.edu.cn
  • 基金资助:
    山西省科技重大专项计划揭榜挂帅项目子课题(202201140601025-3-06);山西省农业关键核心技术攻关子课题(NYGG27-04-01);山西省“1331”工程—作物学一流学科建设项目

Establishment and application of a visual detection method for soybean mosaic virus SC15 based on closed dumbbell mediated isothermal amplification

YIN Cong-Cong1(), LI Rui-Qi2, YUE Pei-Yao2, LI Chen1, NIU Jing-Ping3, ZHAO Jin-Zhong1, DU Wei-Jun2, YUE Ai-Qin2,*()   

  1. 1Department of Basic Sciences, Shanxi Agricultural University, Jinzhong 030801, Shanxi, China
    2College of Agronomy, Shanxi Agricultural University, Jinzhong 030801, Shanxi, China
    3College of Life Sciences, Shanxi Agricultural University, Jinzhong 030801, Shanxi, China
  • Received:2024-07-09 Accepted:2025-02-17 Published:2025-05-12 Published online:2025-02-20
  • Contact: *E-mail: yueaiqinnd@126.com
  • Supported by:
    Sub-project of the Shanxi Province Science and Technology Major Special Plan Unveiling Project(202201140601025-3-06);Sub-project on Key Core Technology Breakthroughs in Agriculture of Shanxi Province(NYGG27-04-01);Shanxi “1331 Project” Crop Science First-Class Discipline Construction Project

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摘要：

大豆花叶病是一种由大豆花叶病毒(soybean mosaic virus, SMV)引起的最为普遍和严重的全球性大豆病害, 可导致大豆产量和种子品质大幅降低, 我国大豆产区均受其影响。在我国, SMV被划分为22个株系(SC1~SC22), 其中SMV-SC15毒性最强。但是, 目前尚无有效的早期诊断方法, 本研究基于闭合哑铃介导等温扩增(closed dumbbell mediated isothermal amplification, CDA), 建立了一种可视化快速检测SMV-SC15的方法, 实现了对SC15的高效特异检测与鉴定。根据SMV不同株系CP基因组序列的多态性设计了CDA方法的引物对(MF/MR), 建立并优化了检测SMV-SC15的反应体系, 确定了最佳反应条件: 反应温度63℃、Bst DNA聚合酶用量4.8 U以及引物浓度0.6 μmol L-1。以溴百里酚蓝(BTB)和SYBR Green I为指示剂实现了检测结果的可视化。对比分析CDA体系和加环引物CDA体系(L-CDA)检测SMV-SC15的稳定性、特异性和灵敏度发现, L-CDA体系实时荧光扩增曲线达到阈值的时间比CDA缩短5~6 min, 其最低检出浓度低至1×10-4 ng μL-1, 灵敏度为CDA体系的10倍。本研究通过L-CDA体系检测了200份不同品种的田间大豆叶片样本, 显色结果对应于RT-qPCR检测的Ct值约为32, 其灵敏度和特异性分别为100%和96.3%。

关键词: 大豆花叶病毒, 可视化检测, 闭合哑铃介导等温扩增, 核酸检测, 体系优化

Abstract:

Soybean mosaic virus (SMV) disease is one of the most widespread and serious global soybean diseases, which can cause significant reductions in soybean yields and seed quality, and affect all soybean-producing regions in China. SMV is divided into 22 strains (SC1-SC22) in China, among which SMV-SC15 has stronger toxicity. However, there is currently no effective early diagnostic method. In this study, a visual and rapid method for detecting SMV-SC15 was established based on closed dumbbell mediated isothermal amplification (CDA), achieving highly specific and sensitive detection of SC15. Primer pairs (MF/MR) for CDA method was designed based on the polymorphism of CP sequences in different strains of SMV, and the reaction system for detecting SMV-SC15 was optimized. The optimal reaction conditions were obtained as follows: reaction temperature was 63℃, the dose of Bst DNA polymerase was 4.8 U, and primer concentration was 0.6 μmol L-1. The detection results were visualized by using BTB and SYBR Green I as color developing agents. A comparative analysis was conducted on the stability, specificity and sensitivity of the CDA system and the CDA system with ring primers (L-CDA) for detecting SMV-SC15. The results showed that the time for real-time fluorescence amplification curve of L-CDA system to reach the threshold was 5-6 min shorter than that of CDA system, and the lowest detection limit was 1×10-4 ng μL-1, which was 10 times lower than that of CDA system. The L-CDA system was used to detect 200 soybean leaf samples of different varieties in the field, and the colorimetric result corresponds to a Ct value of approximately 32 in RT-qPCR detection, with a sensitivity of 100% and a specificity of 96.3%.

Key words: soybean mosaic virus, visual detection, closed dumbbell mediated isothermal amplification, nucleic acid detection, system optimization

附表1

200份样本RT-qPCR和L-CDA检测结果"

编号
Number		 品种
Cultivar		 RT-qPCR (Ct值)
Ct value of
RT-qPCR		 L-CDA 编号
Number		 品种
Cultivar		 RT-qPCR (Ct值)
Ct value of
RT-qPCR		 L-CDA
1 SZ-17 30.43 + 101 SZ-1185 N/A −
2 SZ-60 28.29 + 102 SZ-1186 35.74 −
3 SZ-106 N/A − 103 SZ-1195 35.65 −
4 SZ-220 20.08 + 104 SZ-1197 21.81 +
5 SZ-288 21.73 + 105 SZ-1199 22.01 +
6 SZ-292 31.05 + 106 SZ-1202 N/A −
7 SZ-319 40.98 − 107 SZ-1225 30.29 +
8 SZ-350 32.75 − 108 SZ-1228 N/A −
9 SZ-351 N/A − 109 SZ-1229 18.71 +
10 SZ-353 24.70 + 110 SZ-1230 N/A −
11 SZ-355 N/A − 111 SZ-1239 N/A −
12 SZ-356 36.41 − 112 SZ-1244 19.06 +
13 SZ-357 N/A − 113 SZ-1248 N/A −
14 SZ-358 N/A − 114 SZ-1251 28.58 +
15 SZ-359 19.46 + 115 SZ-1277 35.13 −
16 SZ-693 29.53 + 116 SZ-1278 20.14 +
17 SZ-699 N/A − 117 SZ-1283 23.40 +
18 SZ-747 34.42 − 118 SZ-1284 33.34 +
19 SZ-750 22.54 + 119 SZ-1293 34.79 −
20 SZ-829 19.22 + 120 SZ-1294 N/A −
21 SZ-844 19.32 + 121 SZ-1295 21.17 +
22 SZ-847 N/A − 122 SZ-1296 N/A −
23 SZ-850 22.33 + 123 SZ-1297 22.96 +
24 SZ-854 22.01 + 124 SZ-1298 30.68 +
25 SZ-855 30.95 + 125 SZ-1299 23.65 +
26 SZ-856 21.90 + 126 SZ-1300 30.23 +
27 SZ-864 32.41 − 127 SZ-1303 20.18 +
28 SZ-865 31.45 + 128 SZ-1308 31.42 +
29 SZ-874 22.19 + 129 SZ-1310 22.30 +
30 SZ-882 N/A − 130 SZ-1312 N/A −
31 SZ-883 36.26 − 131 SZ-1317 26.93 +
32 SZ-884 N/A − 132 SZ-1322 24.13 +
33 SZ-885 28.33 + 133 SZ-1326 N/A −
34 SZ-888 N/A − 134 SZ-1327 25.20 +
35 SZ-907 26.65 + 135 SZ-1328 31.01 +
36 SZ-908 23.65 + 136 SZ-1331 N/A −
37 SZ-910 32.35 − 137 SZ-1344 27.51 +
38 SZ-913 28.28 + 138 SZ-1346 23.02 +
39 SZ-915 25.65 + 139 SZ-1347 20.41 +
40 SZ-916 19.11 + 140 SZ-1348 21.83 +
41 SZ-919 24.56 + 141 SZ-1351 32.98 +
42 SZ-922 33.77 − 142 SZ-1353 36.45 −
43 SZ-923 21.66 + 143 SZ-1355 N/A −
44 SZ-924 28.87 + 144 SZ-1356 N/A −
45 SZ-928 22.66 + 145 SZ-1357 N/A −
46 SZ-929 29.84 + 146 SZ-1358 22.57 +
47 SZ-931 33.43 − 147 SZ-1359 27.95 +
48 SZ-933 40.14 − 148 SZ-1360 22.94 +
49 SZ-954 27.51 + 149 SZ-1361 N/A −
50 SZ-962 N/A − 150 SZ-1362 22.56 +
51 SZ-967 19.48 + 151 SZ-1363 20.06 +
52 SZ-981 31.85 + 152 SZ-1398 25.21 +
53 SZ-983 38.09 − 153 SZ-1402 32.63 +
54 SZ-989 22.77 + 154 SZ-1404 29.92 +
55 SZ-993 22.74 + 155 SZ-1407 32.66 −
56 SZ-1021 26.90 + 156 SZ-1408 28.80 +
57 SZ-1022 38.75 − 157 SZ-1409 37.08 −
58 SZ-1023 21.23 + 158 SZ-1410 25.51 +
59 SZ-1040 22.23 + 159 SZ-1411 30.74 +
60 SZ-1041 21.50 + 160 SZ-1413 N/A −
61 SZ-1042 27.02 + 161 SZ-1417 26.97 +
62 SZ-1050 N/A − 162 SZ-1418 N/A −
63 SZ-1053 N/A − 163 SZ-1419 26.91 +
64 SZ-1067 N/A − 164 SZ-1421 26.96 +
65 SZ-1069 36.98 − 165 SZ-1424 N/A −
66 SZ-1070 27.73 + 166 SZ-1429 25.29 +
67 SZ-1072 21.06 + 167 SZ-1455 33.76 −
68 SZ-1073 25.13 + 168 SZ-1456 39.25 −
69 SZ-1080 N/A − 169 汾豆98 Fendou 98 N/A −
70 SZ-1081 22.96 + 170 汾豆99 Fendou 99 26.02 +
71 SZ-1083 21.50 + 171 河北5号 Hebei 5 22.15 +
72 SZ-1084 36.69 − 172 荷豆13 Hedou 13 24.13 +
73 SZ-1111 18.32 + 173 晋大53 Jinda 53 21.45 +
74 SZ-1112 N/A − 174 晋大88 Jinda 88 N/A −
75 SZ-1113 21.85 + 175 晋大114 Jinda 114 32.18 −
76 SZ-1114 22.64 + 176 晋大116 Jinda 116 N/A −
77 SZ-1118 21.11 + 177 晋大117 Jinda 117 33.71 −
78 SZ-1123 36.55 − 178 晋大118 Jinda 118 N/A −
79 SZ-1128 23.60 + 179 晋大131 Jinda 131 24.37 +
80 SZ-1130 27.24 + 180 晋大133 Jinda 133 24.48 +
81 SZ-1132 23.30 + 181 晋大134 Jinda 134 24.61 +
82 SZ-1133 19.67 + 182 晋大136 Jinda 136 26.47 +
83 SZ-1135 N/A − 183 晋大140 Jinda 140 35.23 −
84 SZ-1136 36.10 − 184 晋大141 Jinda 141 35.95 −
85 SZ-1139 N/A − 185 晋大142 Jinda 142 N/A −
86 SZ-1144 25.04 + 186 晋大143 Jinda 143 34.50 −
87 SZ-1146 27.30 + 187 晋豆19 Jindou 19 N/A −
88 SZ-1147 25.28 + 188 晋豆37 Jindou 37 23.83 +
89 SZ-1152 N/A − 189 晋豆47 Jindou 47 36.34 −
90 SZ-1159 23.56 + 190 晋遗53 Jinyi 53 22.13 +
91 SZ-1160 30.25 + 191 开育12 Kaiyu 12 24.67 +
92 SZ-1164 N/A − 192 宁豆5240
Ningdou 5240		 20.23 +
93 SZ-1166 N/A − 193 秦豆2014
Qindou 2014		 31.21 +
94 SZ-1168 36.90 − 194 圣豆12 Shengdou 12 20.79 +
95 SZ-1171 22.61 + 195 圣豆25 Shengdou 25 24.31 +
96 SZ-1172 22.00 + 196 石豆14 Shidou 14 30.47 +
97 SZ-1175 26.32 + 197 石豆17 Shidou 17 24.15 +
98 SZ-1176 21.33 + 198 石885 Shi 885 21.31 +
99 SZ-1180 23.31 + 199 石936 Shi 936 19.18 +
100 SZ-1183 31.69 + 200 夏丰1号 Xiafeng 1 33.61 −

表1

CDA体系引物序列"

引物
Primer		 序列
Sequences (5′-3′)
MF GAGAGCTGCAGCCTTCATTTGTG-GTCACCTCCAAAACACCG
MR CGGGAGTTAACAACAAGCTGTTTG-CATCTCTTGCAGTGTGCC
F2 AGGGAGTTAGCCCGTTAT
R2 CCCAAAAGAGAATGCATGTT
LF TATTGCCTCTCTTGCCCTG
LR CATCTCGACCAACTCCGAA

图1

CDA引物设计示意图 A: SMV不同株系CP蛋白多序列对比结果; B: CDA检测SMV-SC15的引物设计原理; C: CDA检测SMV-SC15的引物序列及所在位置。"

图2

闭合哑铃介导等温扩增原理图"

图3

CDA检测SMV-SC15的条件优化 A: 不同温度条件下CDA的荧光扩增曲线; B: 不同酶用量CDA的荧光扩增曲线; C: 不同引物浓度下CDA的荧光扩增曲线。"

图4

CDA、L-CDA和OL-CDA方法比较 A: CDA、L-CDA和OL-CDA方法检测SMV-SC15的荧光扩增曲线; B, C: 基于BTB和SYBR Green I的CDA、L-CDA和OL-CDA方法检测SMV-SC15的可视化结果。NC-1: ddH2O; NC-2: 无引物体系。"

图5

CDA、L-CDA检测SMV-SC15的稳定性分析 A、C: CDA、L-CDA 3次重复的荧光扩增曲线; B、D: CDA、L-CDA 3次重复的熔解峰曲线; E、F: CDA、L-CDA检测SMV-SC15 3份阳性样本和3份阴性样本的可视化结果。"

图6

基于CDA和L-CDA方法检测SMV-SC15的特异性 A、B: 基于CDA检测SMV-SC3、SMV-SC7、SMV-SC15和SMV-SC18的荧光扩增曲线和可视化结果(以BTB、SYBR Green I为指示剂)。C、D: 基于L-CDA检测SMV-SC3、SMV-SC7、SMV-SC15和SMV-SC18的荧光扩增曲线和可视化结果(以BTB、SYBR Green I为指示剂)。"

图7

CDA和L-CDA方法检测SMV-SC15的灵敏度 A、B: CDA灵敏度检验的荧光扩增曲线及可视化结果; C、D: L-CDA灵敏度检验的荧光扩增曲线及可视化结果。"

图8

基于BTB的L-CDA方法检测200份样本的可视化结果分析 A: 基于BTB的L-CDA检测200份样本的可视化结果; B: L-CDA样品管显色的色块拼图; C: 基于L-CDA显色的LAB值与RT-qPCR的Ct值相比的散点图。"

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