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作物杂志,2019, 第6期: 168–176 doi: 10.16035/j.issn.1001-7283.2019.06.027

• 植物保护 • 上一篇    下一篇

基于qPCR和LAMP技术的马铃薯晚疫病菌快速检测方法

祝菊澧1,2,3,梁静思1,2,3,张佩1,王伟伟1,2,3,林桐司骐1,谢欣娱1,苏瑞1,唐唯1,2,3   

  1. 1云南师范大学生命科学学院,650500,云南昆明
    2云南师范大学马铃薯科学研究院,650500,云南昆明
    3云南省马铃薯生物学重点实验室,650500,云南昆明
  • 收稿日期:2019-05-24 修回日期:2019-08-26 出版日期:2019-12-15 发布日期:2019-12-11
  • 通讯作者: 唐唯
  • 作者简介:祝菊澧,硕士研究生,主要从事马铃薯病原菌快速检测研究;|梁静思为并列第一作者,硕士研究生,主要从事马铃薯病原物基因组学研究
  • 基金资助:
    国家自然科学基金(31660503);云南师范大学2019年度研究生科研创新基金(ysdy js2019132)

A Rapid Detection Method for Potato Late Blight Caused by Phytophthora infestans Based on qPCR and LAMP Assays

Zhu Juli1,2,3,Liang Jingsi1,2,3,Zhang Pei1,Wang Weiwei1,2,3,Lin Tongsiqi1,Xie Xinyu1,Su Rui1,Tang Wei1,2,3   

  1. 1School of Life Science, Yunnan Normal University, Kunming 650500, Yunnan, China
    2Joint Academy of Potato Science, Yunnan Normal University, Kunming 650500, Yunnan, China
    3Key Laboratory of Potato Biology of Yunnan Province, Kunming 650500, Yunnan, China
  • Received:2019-05-24 Revised:2019-08-26 Online:2019-12-15 Published:2019-12-11
  • Contact: Wei Tang

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摘要：

马铃薯晚疫病菌（Phytophthora infestans）能侵染多种茄科植物,它引起的马铃薯晚疫病,是马铃薯生产中的第一大病害。为了开发能在田间快速检测马铃薯晚疫病病原的方法,利用P. infestans T30-4基因组测序数据的contig 1.18131,设计qPCR和LAMP引物,优化扩增条件后得到引物的特异性和灵敏度,最后通过检测田间收获薯块,比较形态学传统方法、qPCR及LAMP的差异。特异性检测结果发现,qPCR和LAMP仅在含有P. infestans DNA模板的体系有阳性扩增,在寄主和其他微生物DNA中均无扩增;在优化的条件下,qPCR和LAMP的检测下限可达1×10 -6ng/μL,在有寄主和其他微生物DNA存在的条件下,引物的灵敏度没有显著差异。利用两种快速方法对在大理、丽江及昆明3个地区田间收获薯块上检测发现,qPCR和LAMP方法得到的检出率差异极为不显著（P=0.420）,两种快速检测方法和形态学鉴定方法检出率差异极显著（P=0.009）。在大理、丽江及昆明3个地区的薯块中,两种分子检测方法检出率均比形态学方法高。其中,qPCR检测方法比形态学方法分别提高了12.00%、2.00%、8.70%;LAMP检测方法比形态学方法分别提高了11.30%、2.00%、8.70%。

关键词: 定量PCR（qPCR）, 环介导等温扩增（LAMP）, 马铃薯晚疫病菌, 潜伏侵染, 检测

Abstract:

Phytophthora infestans can infect many kinds of Solanaceae plants. Potato late blight (PLB) caused by P. infestans is a most severe disease in the potato production. To better and fast diagnose PLB in the fields, quantitative PCR (qPCR) and Loop-mediated isothermal amplification (LAMP) primers were designed based on the P. infestans T30-4 whole genome shotgun contig 1.18131. After amplification conditions optimized, both specificity and sensitivity were detected. Finally, developed qPCR and LAMP methods were used to detect PLB incidence and compared with traditional isolation method on harvested potato tubers in the fields. Specific detection showed that qPCR and LAMP were positive amplification only in the presence of P. infestans DNA template, and were no amplification in the presence of host and other microorganism DNA. Moreover, as low as 1×10 -6ng/μL gDNA was detected using both qPCR and LAMP methods, while the sensitivity of primers showed no significant difference in the presence of P. infestans DNA template, host and other microorganism DNA. Detecting harvested tubers in the field using two methods in Dali, Lijiang and Kunming, qPCR and LAMP showed no significant difference but extremely significant difference between two fast detection methods and morphology based identification. Additionally, in Dali, Lijiang and Kunming, PLB incidence of the two molecular detection methods were both higher than morphology based identification method, in which qPCR detection method was 12.00%, 2.00% and 8.70% higher than morphology based identification method, and LAMP detection method was 11.30%, 2.00% and 8.70% higher than morphology based identification method, respectively.

Key words: Quantitative PCR (qPCR), Loop-mediated isothermal amplification (LAMP), Phytophthora infestans, Latent infection, Detection

表1

用于qPCR和LAMP检测卵菌及其他马铃薯相关病原菌菌株信息"

分类
Clossify		 编号
No.		 菌株
Strain		 种
Species		 寄主(分离部位)
Hosts (Isolation source)		 来源
Source		 ITS序列
ITS sequence
卵菌 1 110P Phytophthora infestans Solanum tuberosum (leaf) 中国云南 LS479123
Oomycetes 2 XD1213 P. infestans S. tuberosum(leaf) 中国云南 LS479124
3 XD15 P. infestans S. tuberosum(leaf) 中国云南 LS479193
4 JC1207 P. infestans S. tuberosum(leaf) 中国云南 LS479126
5 XD1314 P. infestans S. tuberosum(leaf) 中国云南 LS479127
6 XA-4 P. infestans S. tuberosum(tuber) 中国云南 LS479171
7 DN111 P. infestans S. tuberosum(unknown) 日本北海道 LS479169
8 80029 P. infestans S. tuberosum(unknown) 荷兰瓦格宁根 LS479173
9 88069
 P. infestans
 Lycopersicon esculentum(leaf) 荷兰瓦格宁根 LS479172
10 Pds13 P. sojae Glycine max(leaf) 中国云南 LS479895
11 Ddr1231 P. parasitica Nicotiana tabacum (leaf) 中国云南 LS479896
12 LL2480 P. capsica Capsicum annuum(fruit) 中国云南 LS479897
13 Fuz37 Pythium acanthicum Saccharum officiarum L. (root) 中国云南 LS479899
真菌 14 AT1289 Alternaria alternate S. tuberosum(leaf) 中国云南 LS479189
Fungus 15 Hz1177 Synchytrium endobioticum S. tuberosum(root) 中国云南 LS479191
16 Hz1608 Fusarium solani S. tuberosum(leaf) 中国云南 LS479188
细菌 17 Hh1503
 Clavibacter michiganensissubsp. Sepedonicus S. tuberosum(tuber) 中国云南 LS479190
Bacteria 18 Hhrf1
 Pectobacterium carotovorum subsp.Carotovorum S. tuberosum(tuber)
 中国云南 LS479898
19 Hhqk1 Ralstonia solanacearum S. tuberosum(stem) 中国云南 LS479192

表2

LAMP与实时荧光定量PCR的引物"

扩增类型PCR type 引物名称Primer name 序列Sequence (5'-3') 目标长度Target length (bp)
qPCR infnew1-F ACCACACCTAAAAACTTTCCACGT 241
infnew1-R CATCCACTGCTGAAAGTTGC
LAMP F3 ACCCAATAGTTGGGGGTCTT
B3 AGCGTTCTTCATCGATGTGC
BIP CTGTGGGGACGAAAGTCTCTGCGC
CTAGACATCCACTGCTGA
FIP CGAAGTCCAAACGCTCGCCTTGCT
TTATTGCTGGCGGCTA

图1

利用Neighbor-joining分析Blast结果 中括号位置为contig 1.18131所在位置"

图2

利用优化qPCR测得的致病疫霉种特异性检测结果"

图3

qPCR特异性和灵敏度检测 A：qPCR特异性检测,1：P. infestans gDNA;2：加入P. infestans gDNA的混合溶液;3：不加P. infestans gDNA的混合溶液;4：ddH2O。B：灵敏度检测,利用P. infestans（1×10-6~1×10-1ng/μL）DNA模板,建立标准曲线;E：扩增效率（103%）。C：在有寄主存在条件下的灵敏度检测,在有寄主存在条件下,利用引物infnew1,以P. infestans DNA浓度梯度（1×10-6~1×10-1ng/μL）建立标准曲线,E：扩增效率（108%）"

图4

LAMP特异性及灵敏度检测 A：19株菌株的LAMP反应;B：SYBR Green染色后在紫外线下观察阳性扩增的亮度变化;C：2.0%琼脂糖凝胶电泳检测LAMP产物"

表3

LAMP,qPCR及形态学方法检测薯块PLB检出率"

地区
Region		 PLB发病率a (%)
PLB incidencea		 样品数
Number of samples		 症状b
Disease symptomsb		 LAMP qPCR 形态学c
Morphologyc
大理Dali 32.50±10.80 150 77 (51.30%) 78 (52.00%) 79 (52.70%) 61 (40.70%)
丽江Lijiang 12.66±4.25 150 21 (14.00%) 21 (14.00%) 21 (14.00%) 18 (12.00%)
昆明Kunming 22.71±11.62 150 48 (32.00%) 46 (30.70%) 46 (30.70%) 33 (22.00%)

表4

样品中分离出微生物数量情况"

地点Site 致病疫霉
P. infestans		 腐皮镰孢霉菌
Fusarium solani		 枯草芽孢杆菌
Bacillus subtilis		 赖氏菌属
Leifsoniasp.		 类芽孢杆菌属
Paenibacillussp.		 副球菌属
Paracoccussp.
大理Dali 61 7 13 3 11 13
丽江Lijiang 18 0 10 0 1 5
昆明Kunming 33 5 18 7 5 10
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