生物技术通报 ›› 2021, Vol. 37 ›› Issue (1): 272-281.doi: 10.13560/j.cnki.biotech.bull.1985.2020-0389
李信申1(), 黄小梅2, 吴淑秀3, 黄瑞荣1, 魏林根4, 华菊玲1()
收稿日期:
2020-04-04
出版日期:
2021-01-26
发布日期:
2021-01-15
作者简介:
李信申,男,博士,研究方向:植物细菌病害防控;E-mail: 基金资助:
LI Xin-shen1(), HUANG Xiao-mei2, WU Shu-xiu3, HUANG Rui-rong1, WEI Lin-gen4, HUA Ju-ling1()
Received:
2020-04-04
Published:
2021-01-26
Online:
2021-01-15
摘要:
为实现植物青枯病的早期诊断,需要建立一种适于田间快速便捷检测青枯病菌的方法。以细胞色素C基因为靶标设计一套特异性引物,建立了植物青枯病的LAMP检测方法。此方法最低检测极限为1 pg,可在1 h 内完成,不依赖昂贵复杂的仪器,结果可经肉眼观察。利用此方法,在人工接种发病的茄子、番茄、花生、芝麻和凹头苋茎部浸出液和马铃薯病薯块茎组织液中均检测出青枯病菌的存在,尤其适用于田间疑似罹病的芝麻、花生、番茄、马铃薯和甘薯等植株的检测,且LAMP法的检出率远高于PCR法。应用LAMP技术检测青枯病菌快速高效、特异性强、灵敏度高,操作简单,适于在基层推广运用。
李信申, 黄小梅, 吴淑秀, 黄瑞荣, 魏林根, 华菊玲. 植物青枯病菌环介导等温扩增快速检测技术研究[J]. 生物技术通报, 2021, 37(1): 272-281.
LI Xin-shen, HUANG Xiao-mei, WU Shu-xiu, HUANG Rui-rong, WEI Lin-gen, HUA Ju-ling. Rapid Detection of Plant Bacterial Wilt by Loop-mediated Isothermal Amplification[J]. Biotechnology Bulletin, 2021, 37(1): 272-281.
菌株编号 | 菌株名称 | 寄主 | 演化型/生理小种 |
---|---|---|---|
1 | 青枯病菌菌株Ejxnc01 | 茄子 | Ⅰ/1 |
2 | 青枯病菌菌株Cjxnc01 | 辣椒 | Ⅰ/1 |
3 | 青枯病菌菌株Tjxjx01 | 番茄 | Ⅰ/1 |
4 | 青枯病菌菌株Pjxga01 | 花生 | Ⅰ/1 |
5 | 青枯病菌菌株Seppx05 | 芝麻 | Ⅰ/1 |
6 | 青枯病菌菌株JSja03 | 凹头苋 | Ⅰ/1 |
7 | 青枯病菌菌株Tjxga03 | 烟草 | Ⅰ/1 |
8 | 青枯病菌菌株Gjxjj01 | 姜 | Ⅰ/4 |
9 | 青枯病菌菌株Pjxnc01 | 马铃薯 | Ⅱ/3 |
10 | 青枯病菌菌株Pjxga02 | 马铃薯 | Ⅱ/3 |
11 | 青枯病菌菌株Pjxjj03 | 马铃薯 | Ⅱ/3 |
12 | 青枯病菌菌株Pjxda04 | 马铃薯 | Ⅱ/3 |
13 | 蒲桃雷尔氏菌R001 | 丁香树 | - |
14 | 皮氏伯克霍尔德氏菌Bp001 | 土壤 | - |
表1 用于检测引物特异性的青枯病菌和非青枯病菌菌株
菌株编号 | 菌株名称 | 寄主 | 演化型/生理小种 |
---|---|---|---|
1 | 青枯病菌菌株Ejxnc01 | 茄子 | Ⅰ/1 |
2 | 青枯病菌菌株Cjxnc01 | 辣椒 | Ⅰ/1 |
3 | 青枯病菌菌株Tjxjx01 | 番茄 | Ⅰ/1 |
4 | 青枯病菌菌株Pjxga01 | 花生 | Ⅰ/1 |
5 | 青枯病菌菌株Seppx05 | 芝麻 | Ⅰ/1 |
6 | 青枯病菌菌株JSja03 | 凹头苋 | Ⅰ/1 |
7 | 青枯病菌菌株Tjxga03 | 烟草 | Ⅰ/1 |
8 | 青枯病菌菌株Gjxjj01 | 姜 | Ⅰ/4 |
9 | 青枯病菌菌株Pjxnc01 | 马铃薯 | Ⅱ/3 |
10 | 青枯病菌菌株Pjxga02 | 马铃薯 | Ⅱ/3 |
11 | 青枯病菌菌株Pjxjj03 | 马铃薯 | Ⅱ/3 |
12 | 青枯病菌菌株Pjxda04 | 马铃薯 | Ⅱ/3 |
13 | 蒲桃雷尔氏菌R001 | 丁香树 | - |
14 | 皮氏伯克霍尔德氏菌Bp001 | 土壤 | - |
引物名称 | 引物序列(5'-3') | 长度/bp |
---|---|---|
F3 | AGCGGTGCCAATCCGTA | 17 |
B3 | TGCCATGGTCAGGTACTGAT | 20 |
FIP | AGCAATCCGAAGGTGCCGAATGTCGCGTA- CAACCAGGA | 38 |
BIP | TCGGTATCCCGACAACACCATGTGGGCGT- CGATCGCATA | 39 |
表2 用于环式扩增细胞色素C信号肽的引物序列
引物名称 | 引物序列(5'-3') | 长度/bp |
---|---|---|
F3 | AGCGGTGCCAATCCGTA | 17 |
B3 | TGCCATGGTCAGGTACTGAT | 20 |
FIP | AGCAATCCGAAGGTGCCGAATGTCGCGTA- CAACCAGGA | 38 |
BIP | TCGGTATCCCGACAACACCATGTGGGCGT- CGATCGCATA | 39 |
图1 LAMP反应体系的优化 (A)M:Marker DL2000;1:0 mmol/L;2:2 mmol/L;3:4 mmol/L;4:6 mmol/L;5:8 mmol/L;6:10 mmol/L;7:12 mmol/L;CK:阴性对照;(B)M:Marker DL2000;1-6.内引物浓度/外引物浓度分别为2∶1,4∶1,6∶1,8∶1,10∶1,12∶1;CK:阴性对照;(C)M:Marker DL2000;1:59℃;2:60℃;3:61℃;4:62℃;5:63℃;6:64℃;7:65℃;CK:阴性对照
来源植物 | 取样部位 | 样本数量 | LAMP检测 | PCR检测 | ||||
---|---|---|---|---|---|---|---|---|
检出总数 | 检出率 | 检出总数 | 检出率 | |||||
芝麻 | 茎杆 | 25 | 25 | 100% | 23 | 92.00% | ||
花生 | 茎杆 | 24 | 23 | 95.83% | 21 | 87.50% | ||
番茄 | 叶片 | 23 | 23 | 100% | 21 | 91.30% | ||
马铃薯 | 块茎 | 24 | 23 | 95.83% | 22 | 91.67% | ||
甘薯 | 块茎 | 22 | 21 | 95.45% | 20 | 90.91% |
表3 疑似罹病植株样本LAMP和PCR检测结果
来源植物 | 取样部位 | 样本数量 | LAMP检测 | PCR检测 | ||||
---|---|---|---|---|---|---|---|---|
检出总数 | 检出率 | 检出总数 | 检出率 | |||||
芝麻 | 茎杆 | 25 | 25 | 100% | 23 | 92.00% | ||
花生 | 茎杆 | 24 | 23 | 95.83% | 21 | 87.50% | ||
番茄 | 叶片 | 23 | 23 | 100% | 21 | 91.30% | ||
马铃薯 | 块茎 | 24 | 23 | 95.83% | 22 | 91.67% | ||
甘薯 | 块茎 | 22 | 21 | 95.45% | 20 | 90.91% |
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