黄瓜细菌性角斑病菌微滴数字PCR定量检测方法的建立与应用

doi:10.7668/hbnxb.20196026

华北农学报 ›› 2026, Vol. 41 ›› Issue (3): 198-207. doi: 10.7668/hbnxb.20196026

所属专题： 黄瓜；植物保护；热点论文；蔬菜专题；

• 资源环境•植物保护 • 上一篇下一篇

黄瓜细菌性角斑病菌微滴数字PCR定量检测方法的建立与应用

储天然¹, 申永俊¹, 王艺凯², 高苇³, 石延霞¹, 谢学文¹, 李磊¹, 范腾飞¹, 李宝聚¹, 柴阿丽¹

¹ 中国农业科学院蔬菜花卉研究所, 蔬菜生物育种国家重点实验室, 北京 100081
² 中国海关科学技术研究中心, 北京 100026
³ 天津市农业科学院植物保护研究所, 天津 300381

收稿日期:2025-11-27 出版日期:2026-06-28
通讯作者:
柴阿丽(1983-),女,山西运城人,研究员,博士,主要从事蔬菜病害诊断与监测预警研究。
李宝聚(1967-),男,黑龙江饶河人,研究员,博士,主要从事蔬菜病害诊断与防控研究。
作者简介:
储天然(2001-),女,安徽安庆人,硕士,主要从事蔬菜病害传播规律研究。
基金资助:
“十四五”国家重点研发计划(2023YFD1401200); 国家自然科学基金项目(32172653); 国家自然科学基金项目(31872159); 中国农业科学院科技创新工程项目(CAAS-ASTIP-IVFCAAS); 天津市重点研发计划科技支撑重点项目(23YFZCSN00150)

Establishment and Application of a Microdroplet Digital PCR Quantitative Detection Method for Quantification of Pseudomonas amygdali pv.lachrymans in Cucumber

CHU Tianran¹, SHEN Yongjun¹, WANG Yikai², GAO Wei³, SHI Yanxia¹, XIE Xuewen¹, LI Lei¹, FAN Tengfei¹, LI Baoju¹, CHAI Ali¹

¹ State Key Laboratory of Vegetable Biobreeding, Institute of Vegetables and Flowers,Chinese Academy of Agricultural Sciences, Beijing 100081, China
² Science and Technology Research Center of China Customs, Beijing 100026, China
³ Plant Protection Research Institute of Tianjin Academy of Agricultural Sciences, Tianjin 300381, China

Received:2025-11-27 Published:2026-06-28

语音导读.mp3(746KB)

摘要/Abstract

摘要：

为实现黄瓜细菌性角斑病菌(Pal)的精准定量检测,以甘油醛-3-磷酸脱氢酶基因(gap1)为靶基因,设计了特异性引物Pal-F/R和探针Pal-Pro,建立了微滴数字PCR(ddPCR)定量检测方法。通过对退火温度、引物和探针浓度等关键参数进行优化,确定最佳反应条件为退火温度61.2 ℃,反应体系中最佳引物和探针终浓度分别为600,250 nmol/L。该方法灵敏度高,对基因组DNA的检测下限为2.7拷贝/μL。利用建立的ddPCR方法,对32份采自发病和黄瓜棚室中的叶片菌脓、棚顶水滴及叶缘吐水样本进行检测,检出26份Pal阳性样本,较荧光定量qPCR方法(TaqMan qPCR)多检出3份,表明该方法在早期检测方面比qPCR具有更高的灵敏度。本研究建立的ddPCR检测方法具有特异性强、灵敏度高的特点,可为黄瓜细菌性角斑病菌的绝对定量检测提供可靠的技术支持,对病害早期诊断和高效预警具有重要意义。

关键词: 黄瓜细菌性角斑病菌, 微滴数字PCR, 探针法荧光定量PCR, gap1基因, 定量检测

Abstract:

A novel microdroplet digital PCR(ddPCR)assay was developed for rapid and quantitative detection of Pseudomonas amygdali pv.lachrymans(Pal),the causative agent of cucumber bacterial angular leaf spot disease.A specific primer pair Pal-F/R and probe Pal-Pro targeting the glyceraldehyde-3-phosphate dehydrogenase(gap1)gene were designed.The annealing temperature,primer and probe concentrations,and specificity and sensitivity tests of ddPCR were optimized.The results showed that the optimal annealing temperature was 61.2 ℃ and the optimal final concentrations of the primers and the probe were 600,250 nmol/L,respectively.The ddPCR assay demonstrated high sensitivity,with a detection limit of 2.7 copies/μL for genomic DNA.A total of 32 samples of bacterial ooze,greenhouse roof water droplets,and leaf margin guttation fluids collected from asymptomatic leaf lesions in diseased and healthy cucumber greenhouses were detected via ddPCR assay,Pal was detected in 26 samples,which was 3 more than the results obtained by the TaqMan qPCR,indicating that the ddPCR assay exhibits higher sensitivity for early detection compared to qPCR.The ddPCR assay developed in this study is highly specific and sensitive,providing a reliable technical approach for absolute quantitative detection of Pal and is of great significance for the early diagnosis warning of the cucumber bacterial angular spot disease.

Key words: Cucumber bacterial angular spot, Droplet digital PCR, TaqMan real-time PCR, gap1 gene, Quantitative testing

中图分类号:

储天然, 申永俊, 王艺凯, 高苇, 石延霞, 谢学文, 李磊, 范腾飞, 李宝聚, 柴阿丽. 黄瓜细菌性角斑病菌微滴数字PCR定量检测方法的建立与应用[J]. 华北农学报, 2026, 41(3): 198-207. doi: 10.7668/hbnxb.20196026.

CHU Tianran, SHEN Yongjun, WANG Yikai, GAO Wei, SHI Yanxia, XIE Xuewen, LI Lei, FAN Tengfei, LI Baoju, CHAI Ali. Establishment and Application of a Microdroplet Digital PCR Quantitative Detection Method for Quantification of Pseudomonas amygdali pv.lachrymans in Cucumber[J]. Acta Agriculturae Boreali-Sinica, 2026, 41(3): 198-207. doi: 10.7668/hbnxb.20196026.

http://www.hbnxb.net/CN/Y2026/V41/I3/198

图/表 8

参考文献 26

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物种 Species	菌株编号 Isolate code	寄主 Host	地区 Origin
扁桃假单胞流泪致病变种 Pseudomonas amygdali pv. lachrymans	MN002	黄瓜	中国
巴西果胶杆菌 Pectobacterium brasiliensis	SX309	黄瓜	中国
西瓜噬酸菌 Acidovorax citrulli	TG24060603	甜瓜	中国
野油菜黄单胞菌疮痂致病变种 Xanthomonas campestris pv. vesicatoria	LJ23080501	辣椒	中国
真泡黄单胞菌 Xanthomonas euvesicatoria	LJ23080502	辣椒	中国
丁香假单胞菌番茄致病变种 Pseudomonas syringae pv. tomato	ATCC BAA-871^T	番茄	英国
密执安棒形杆菌密执安亚种 Clavibacter michiganensis subsp. michiganensis	ATCC 10202	番茄	未知
茄科雷尔氏菌 Ralstonia solanacearum	ATCC 11696	番茄	美国
胡萝卜果胶杆菌 Pectobacterium carotovorum	ATCC 15713	马铃薯	德国

物种 Species	菌株编号 Isolate code	寄主 Host	地区 Origin
扁桃假单胞流泪致病变种 Pseudomonas amygdali pv. lachrymans	MN002	黄瓜	中国
巴西果胶杆菌 Pectobacterium brasiliensis	SX309	黄瓜	中国
西瓜噬酸菌 Acidovorax citrulli	TG24060603	甜瓜	中国
野油菜黄单胞菌疮痂致病变种 Xanthomonas campestris pv. vesicatoria	LJ23080501	辣椒	中国
真泡黄单胞菌 Xanthomonas euvesicatoria	LJ23080502	辣椒	中国
丁香假单胞菌番茄致病变种 Pseudomonas syringae pv. tomato	ATCC BAA-871^T	番茄	英国
密执安棒形杆菌密执安亚种 Clavibacter michiganensis subsp. michiganensis	ATCC 10202	番茄	未知
茄科雷尔氏菌 Ralstonia solanacearum	ATCC 11696	番茄	美国
胡萝卜果胶杆菌 Pectobacterium carotovorum	ATCC 15713	马铃薯	德国

引物名称 Primer	序列(5'-3') Sequence (5'-3')	目的基因 Target gene	长度/bp Length
Pal-F	5'-ACGCAATCAATGATCCCGAGCAAAA-3'	gap1	103
Pal-R	5'-CAGGTACGCGTACGGCCATGCCGG-3'
Pal-Pro	FAM-CACCAGACCCACCGCTTCGGCAGCAC-BHQ1

引物名称 Primer	序列(5'-3') Sequence (5'-3')	目的基因 Target gene	长度/bp Length
Pal-F	5'-ACGCAATCAATGATCCCGAGCAAAA-3'	gap1	103
Pal-R	5'-CAGGTACGCGTACGGCCATGCCGG-3'
Pal-Pro	FAM-CACCAGACCCACCGCTTCGGCAGCAC-BHQ1

样品编号 Sample	地区 Origin	温室编号 Greenhouse No.	病情指数 Disease index	样品类型 Sample type	菌含量/(拷贝/mL) Pal concentration
样品编号 Sample	地区 Origin	温室编号 Greenhouse No.	病情指数 Disease index	样品类型 Sample type	qPCR	ddPCR
1-1	天津武清	Ⅰ	55.6	菌脓	2.69×10⁷	1.72×10⁷
1-2		(116.96°E, 39.43°N)		吐水	1.75×10⁴	2.31×10⁴
1-3				棚顶水滴	5.44×10⁷	6.18×10⁷
2-1		Ⅱ	45.5	菌脓	4.25×10⁷	2.51×10⁷
2-2		(116.84°E, 39.41°N)		吐水	1.95×10⁴	2.20×10⁴
2-3				棚顶水滴	3.41×10⁵	5.04×10⁵
3-1	北京大兴	Ⅲ	40.3	菌脓	6.10×10⁷	3.01×10⁷
3-2		(116.24°E, 39.32°N)		吐水	4.28×10⁴	4.02×10⁴
3-3				棚顶水滴	1.03×10⁵	3.86×10⁵
4-1	山东寿光	Ⅳ	32.2	菌脓	4.64×10⁷	7.28×10⁷
4-2		(117.62°E, 35.78°N)		吐水	1.42×10³	1.76×10⁵
4-3				棚顶水滴	3.89×10⁴	5.55×10⁵
5-1		Ⅴ	40.2	菌脓	4.09×10⁸	2.26×10⁸
5-2		(118.94°E, 36.93°N)		吐水	6.18×10³	6.67×10³
5-3				棚顶水滴	4.94×10³	6.01×10⁴
6-1		Ⅵ	33.4	菌脓	2.20×10⁷	2.18×10⁸
6-2		(119.22°E, 36.74°N)		吐水	n/a	3.37×10²
6-3				棚顶水滴	6.23×10⁴	6.26×10⁵
7-1		Ⅶ	28.5	菌脓	2.98×10⁶	6.61×10⁷
7-2		(119.26°E, 36.75°N)		吐水	n/a	n/a
7-3				棚顶水滴	n/a	1.21×10²
8-1	河北馆陶	Ⅷ	38.1	菌脓	1.10×10⁸	8.34×10⁸
8-2		(115.23°E, 36.60°N)		吐水	6.61×10³	6.41×10⁴
8-3				棚顶水滴	4.94×10³	6.01×10³
9-1		Ⅸ	27.0	菌脓	7.43×10⁶	8.23×10⁶
9-2		(115.23°E, 36.60°N)		吐水	n/a	n/a
9-3				棚顶水滴	n/a	3.03×10¹
10-1		Ⅹ	18.6	菌脓	7.43×10⁷	1.28×10⁸
10-2		(115.23°E, 36.61°N)		吐水	n/a	n/a
10-3				棚顶水滴	n/a	n/a
11-1	河北青县	Ⅺ	0.0	吐水	n/a	n/a
11-2		(116.81°E, 38.58°N)		棚顶水滴	n/a	n/a

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黄瓜细菌性角斑病菌微滴数字PCR定量检测方法的建立与应用

Establishment and Application of a Microdroplet Digital PCR Quantitative Detection Method for Quantification of Pseudomonas amygdali pv.lachrymans in Cucumber

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黄瓜细菌性角斑病菌微滴数字PCR定量检测方法的建立与应用

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