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

doi:10.7668/hbnxb.20196026

Abstract

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

CLC Number:

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.

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References 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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