细小果胶杆菌与多样果胶杆菌实时荧光定量PCR与LAMP检测技术的建立

doi:10.7668/hbnxb.20195601

摘要/Abstract

摘要：

建立细小果胶杆菌和多样果胶杆菌快速灵敏的分子检测技术为马铃薯茎腐病的快速检测、早期诊断、传播途径与动态奠定技术基础。通过对细小果胶杆菌特异性位点基因10748与多样果胶杆菌特异性位点基因1602序列进行分析,利用 Primer 3与NEB网站引物设计软件进行实时荧光定量PCR与LAMP引物设计,经过特异性验证、反应条件优化与灵敏度验证对引物进行筛选并进行样品检测。设计并筛选出细小果胶杆菌与多样果胶杆菌实时荧光定量PCR引物XX3-F/R与DY1-F/R,对人工接种病原菌植物样本DNA检测灵敏度分别达到10,1 pg/μL,对病原菌gDNA检测灵敏度均达到0.1 pg/μL。设计出细小果胶杆菌与多样果胶杆菌LAMP引物,检测灵敏度与实时荧光定量PCR检测技术相同。建立起关于马铃薯气生茎腐病菌细小果胶杆菌与多样果胶杆菌的实时荧光定量PCR与LAMP检测技术,均可对细小与多样果胶杆菌侵染的马铃薯茎部与块茎以及带菌果蝇进行检测。

关键词: 马铃薯气生性茎腐病, 细小果胶杆菌, 多样果胶杆菌, PCR, LAMP

Abstract:

Establishing a rapid and sensitive molecular detection technology for Pectobacterium parvum and P.versatile will lay a technical foundation for the rapid detection,early diagnosis,as well as the transmission routes and dynamics of potato stem rot.Through the sequence analysis of the specific locus gene 10748 of P.parvum and the specific locus gene 1602 of P.versatile,the Real-time PCR and LAMP primer design software were carried out by using Primer 3 and NEB website primer design software.After specificity validation,reaction condition optimization,and sensitivity validation,primers were screened and sample detection was performed.Quantitative Real-time PCR primers XX3-F/R and DY1-F/R were designed and screened for P.parvum and P.versatile.The sensitivity for detecting DNA in artificially inoculated plant samples reached 10,1 pg/μL,respectively.The sensitivity for detecting gDNA of pathogenic bacteria reached 0.1 pg/μL.Design LAMP primers for P.parvum and P.versatile,with detection sensitivity similar to Quantitative Real-time PCR detection technology.Quantitative Real-time PCR and LAMP detection techniques had been established for the detection of potato aerial stem rot pathogens,such as P.parvum and P.versatile,which can detect potato stems and tubers infected by P.parvum and P.versatile,as well as drosophila carrying bacteria.

Key words: Potato aerial stem rot disease, P.parvum, P.versatile, PCR, LAMP

中图分类号:

王燕飞, 王金辉, 赵冬梅, 张岱, 潘阳, 李倩, 朱杰华, 杨志辉. 细小果胶杆菌与多样果胶杆菌实时荧光定量PCR与LAMP检测技术的建立[J]. 华北农学报, 2026, 41(2): 202-213. doi: 10.7668/hbnxb.20195601.

WANG Yanfei, WANG Jinhui, ZHAO Dongmei, ZHANG Dai, PAN Yang, LI Qian, ZHU Jiehua, YANG Zhihui. Establishment of Quantitative Real-time PCR and LAMP Detection Techniques for Pectobacterium parvum and P.versatile[J]. Acta Agriculturae Boreali-Sinica, 2026, 41(2): 202-213. doi: 10.7668/hbnxb.20195601.

http://www.hbnxb.net/CN/Y2026/V41/I2/202

图/表 22

表1 供试病原菌菌株信息

Tab.1 Information about the strains of the pathogenic bacteria tested

病原菌 Pathogens	菌株编号 Strain number	寄主(品种) Host(Variety)	采集地点 Collect site	采集时间(年-月) Collect time
细小果胶杆菌P.parvum	YT22221	马铃薯(荷兰十五)	河北唐山玉田	2022-06
多样果胶杆菌P.versatile	FN22511	马铃薯(希森六)	河北承德丰宁	2022-08
黑腐果胶杆菌P.atrosepticum	FN20412	马铃薯(品种未知)	河北唐山丰润	2020-08
帕曼蒂氏果胶杆菌P.parmentieri	YL22131	马铃薯(希森六)	陕西榆林	2022-07
极地果胶杆菌P.polaris	WW22521	马铃薯(希森六)	甘肃武威	2022-07
旁遮普果胶杆菌P.punjabense	XW22812	马铃薯(米拉)	云南宣威	2022-07
胡萝卜果胶杆菌P.carotovorum	FN22613	马铃薯(希森六)	河北承德丰宁	2022-08
巴西果胶杆菌P.brasiliensis	XW22111	马铃薯(V7)	云南宣威	2022-07
波兰果胶杆菌P.polonicum	BY21311	马铃薯(品种未知)	河北保定博野	2021-05
欧文氏菌Erwinia rhapontici	BYQ31	芹菜(品种未知)	河北保定博野	2021-05
成团泛菌Pantoea agglomerans	FN22611	马铃薯(希森六)	河北承德丰宁	2022-08
沙雷氏菌Serratia marcescens	GX23711	马铃薯(希森六)	广西	2023-03
假单胞菌Psedomonas straminea	GX23461	马铃薯(希森六)	广西	2023-03
肠杆菌Enterobacter soil	GX23381	马铃薯(希森六)	广西	2023-03

表2 细小果胶杆菌与多样果胶杆菌实时荧光定量PCR引物序列

Tab.2 Sequences of P.parvum and P.versatile Quantitative Real-time PCR primers

病原菌 Pathogens	引物名称 Primer name	引物序列(5'—3') Primer sequence(5'—3')
细小果胶杆菌	XX1-F	CCAAGCGGTAAAGGGTTT
P. parvum	XX1-R	AAGGGCATGTTGATGGTG
	XX2-F	GATGGAGAAGTATGCGGG
	XX2-R	CACCATCAACATGCCCTT
	XX3-F	AAAAATAAGATTGGTGGGGAA
	XX3-R	TGTCGTTGCTTATAAACTCTTG
多样果胶杆菌	DY1-F	AATGCGGACCTTTCATCC
P. versatile	DY1-R	CCGGGAACTTTCGTCAAT
	DY2-F	ACCGCGAAATAAGACCAC
	DY2-R	CCCGTGGGTAATGAAAGG
	DY3-F	GCTGATTCTCCCAACAGG
	DY3-R	TTTGAGGTCACCAGCATG

图1 细小果胶杆菌(A)与多样果胶杆菌(B)LAMP引物设计

Fig.1 LAMP primer design of P.parvum(A) and P.versatile(B)

表3 细小果胶杆菌与多样果胶杆菌LAMP引物序列

Tab.3 Sequences of P.parvum and P.versatile LAMP primers

病原菌 Pathogens	引物名称 Primer name	引物序列(5'—3') Primer sequence(5'—3')
细小果胶杆菌	F3	AAAAATAAGATTGGTGGGGAA
P.parvum	B3	TGTCGTTGCTTATAAACTCTTG
	FIP	ACGTCTCTATCTGATGTGAGTTTTTCCAAGCAATGAAATGTATTATGGT
	BIP	AAGGGCATGTTGATGGTGTAGGCCTACGATTACTCGACCT
多样果胶杆菌	F32	TCCAGACAGTACGGGATA
P.versatile	B32	GCTTGATCAATAGTAATGCCATA
	FIP2	GCTGTCGTTATAGCGGTGATAGCCCGGTGATAAACGTCAG
	BIP2	AACGCGACATATAGTCATTTAGACATTCTTATGCCTGTTAATTCATCA

图2 细小果胶杆菌实时荧光定量PCR 引物特异性验证的琼脂糖凝胶图与熔解曲线 M.2 000 bp Marker;1—16.细小果胶杆菌YT22221、多样果胶杆菌FN22511、黑腐果胶杆菌FN20412、帕曼蒂氏果胶杆菌YL22131、极地果胶杆菌WW22521、旁遮普果胶杆菌XW22812、胡萝卜果胶杆菌FN22613、巴西果胶杆菌XW22111、波兰果胶杆菌BY21311、欧文氏菌BYQ31、成团泛菌FN22611、沙雷士菌GX23711、假单胞菌GX23461、肠杆菌GX23381、马铃薯的植物DNA、ddH₂O。图10-A同。

Fig.2 Agarose gel diagram and melting curve of Quantitative Real-time PCR primer specificity verification of P.parvum M.2 000 bp Marker;1—16.P.parvum YT22221,P.versatile FN22511,P.atrosepticum FN20412,P.parmentieri YL22131,P.polaris WW22521,P.punjabense XW22812,P.carotovorum FN22613,P.brasiliensis XW22111,P.polonicum BY21311,Erwinia rhapontici BYQ31,Pantoea agglomerans FN22611, Serratia marcescens GX23711,Psedomonas straminea GX23461,Enterobacter soil GX23381,plant DNA of potato,ddH₂O.The same as Fig.10-A.

图3 多样果胶杆菌实时荧光定量PCR 引物特异性验证的琼脂糖凝胶图与熔解曲线 M.2 000 bp Marker;1—16.多样果胶杆菌FN22511、细小果胶杆菌YT22221、黑腐果胶杆菌FN20412、帕曼蒂氏果胶杆菌YL22131、极地果胶杆菌WW22521、旁遮普果胶杆菌XW22812、胡萝卜果胶杆菌FN22613、巴西果胶杆菌XW22111、波兰果胶杆菌BY21311、欧文氏菌BYQ31、成团泛菌FN22611、沙雷士菌GX23711、假单胞菌GX23461、肠杆菌GX23381、马铃薯的植物DNA、ddH₂O。图10-B同。

Fig.3 Agarose gel diagram and melting curve of Quantitative Real-time PCR primer specificity verification of P.versatile M.2 000 bp Marker;1—16.P.versatile FN22511, P.parvum YT22221,P.atrosepticum FN20412,P.parmentieri YL22131,P.polaris WW22521,P.punjabense XW22812,P.carotovorum FN22613,P.brasiliensis XW22111,P.polonicum BY21311,Erwinia rhapontici BYQ31,Pantoea agglomerans FN22611, Serratia marcescens GX23711,Psedomonas straminea GX23461,Enterobacter soil GX23381,Plant DNA of potato,ddH₂O.The same as Fig.10-B.

图4 细小果胶杆菌实时荧光定量PCR 引物浓度条件优化不同小写字母表示差异显著(P<0.05)。图5—7同。

Fig.4 Optimization of Quantitative Real-time PCR primer concentration conditions for P.parvum Different lowercase letters indicate significant differences(P<0.05).The same as Fig.5—7.

图5 多样果胶杆菌实时荧光定量PCR 引物浓度条件优化

Fig.5 Optimization of Quantitative Real-time PCR primer concentration conditions for P.versatile

图6 细小果胶杆菌实时荧光定量PCR 退火温度条件优化

Fig.6 Optimization of annealing temperature conditions for Quantitative Real-time PCR of P.parvum

图7 多样果胶杆菌实时荧光定量PCR 退火温度条件优化

Fig.7 Optimization of annealing temperature conditions for Quantitative Real-time PCR of P.versatile

图8 细小果胶杆菌实时荧光定量PCR引物灵敏度检测 A.引物XX3-F/R病原菌gDNA灵敏度检测。1—7.DNA浓度为100,10,1,0.1,1×10^-2,1×10^-3,1×10^-4 ng/μL;8.对照;图9-A、C同。B.引物XX3-F/R人工接种植物样本DNA灵敏度检测。1—6.DNA浓度为100,10,1,0.1,1×10^-2,1×10^-3 ng/μL;7.对照;图9-B、D同。

Fig.8 Quantitative Real-time PCR primer sensitivity detection of P.parvum A.Primer XX3-F/R pathogen gDNA sensitivity detection.1—7.DNA concentrations were 100,10,1,0.1,1×10^-2,1×10^-3,1×10^-4 ng/μL;8.Control;the same as Fig.9-A,C.B.Primer XX3-F/R sensitivity detection of DNA in artificially inoculated plant samples.1—6.DNA concentrations were 100,10,1,1×10^-2,1×10^-3 ng/μL;7.Control;the same as Fig.9-B,D.

图9 多样果胶杆菌实时荧光定量PCR引物灵敏度检测 A.引物DY1-F/R病原菌gDNA灵敏度检测;B.引物DY1-F/R人工接种植物样本DNA灵敏度检测;C.引物DY2-F/R病原菌gDNA灵敏度检测;D.引物DY2-F/R人工接种植物样本DNA灵敏度检测。

Fig.9 Quantitative Real-time PCR primer sensitivity detection of P.versatile A.Primer DY1-F/R pathogen gDNA sensitivity detection;B.Primer DY1-F/R sensitivity detection of DNA in artificially inoculated plant samples; C.Primer DY2-F/R pathogen gDNA sensitivity detection;D.Primer DY2-F/R sensitivity detection of DNA in artificially inoculated plant samples.

图10 细小果胶杆菌(A)与多样果胶杆菌(B)LAMP引物特异性验证结果与琼脂糖凝胶图

Fig.10 Validation results of specificity of LAMP primers and agarose gel diagram for P.parvum(A) and P.versatile(B)

图11 LAMP反应条件优化结果与琼脂糖凝胶图 M.2 000 bp Marker。A.细小果胶杆菌LAMP引物浓度条件优化结果;B.细小果胶杆菌LAMP反应时间优化结果;C.细小果胶杆菌LAMP反应温度优化结果;D.多样果胶杆菌LAMP引物浓度条件优化结果;E.多样果胶杆菌LAMP反应时间优化结果;F.多样果胶杆菌LAMP反应温度优化结果;1—4.引物的终浓度分别为内引物0.4 μmol/L(外引物0.1 μmol/L)、内引物0.8 μmol/L(外引物0.2 μmol/L)、内引物1.2 μmol/L(外引物0.3 μmol/L)、内引物1.6 μmol/L(外引物0.4 μmol/L)。

Fig.11 LAMP reaction conditions optimization results and agarose gel diagram M.2 000 bp Marker.A.Optimization results of LAMP primer concentration conditions for P.parvum;B.Optimization results of LAMP reaction time for P.parvum;C.Optimization results of LAMP reaction temperature of P.parvum;D.Optimization results of LAMP primer concentration conditions for P.versatile;E.Optimization results of LAMP reaction time for P.versatile;F.Optimization results of LAMP reaction temperature of P.versatile;1—4.The final concentrations of primers were 0.4 μmol/L for inner primers(0.1 μmol/L for outer primers),0.8 μmol/L for inner primers(0.2 μmol/L for outer primers),1.2 μmol/L for inner primers(0.3 μmol/L for outer primers),and 1.6 μmol/L for inner primers(0.4 μmol/L for outer primers).

图12 细小果胶杆菌LAMP灵敏度检测结果与琼脂糖凝胶图 M.2 000 bp Marker。A.病原菌gDNA灵敏度检测;B.人工接种植物样本DNA灵敏度检测。图13同。

Fig.12 LAMP sensitivity detection results and agarose gel diagram of P.parvum M.2 000 bp Marker.A.Pathogen gDNA sensitivity detection;B.Sensitivity detection of DNA in artificially inoculated plant samples.The same as Fig.13.

图13 多样果胶杆菌LAMP灵敏度检测结果与琼脂糖凝胶图

Fig.13 LAMP sensitivity detection results and agarose gel diagram of P.versatile

图14 实时荧光定量PCR检测接种12,24,36 h后茎部各区段

Fig.14 Samples from each stem segment after inoculation 12,24,36 h were detected by Quantitative Real-time PCR

图15 实时荧光定量PCR检测接种12,24,48 h后块茎各区段样品

Fig.15 Samples from each tuber segment after inoculation 12,24,48 h were detected by Quantitative Real-time PCR

图16 实时荧光定量PCR检测饲料培养基中混入不同OD₆₀₀值的菌液的果蝇样本

Fig.16 Quantitative Real-time PCR detection of drosophila samples mixed with different OD₆₀₀values in feed culture medium

图17 LAMP检测接种12,24,36 h后茎部各区段样品结果与凝胶电泳图 Marker.2 000 bp Marker。A.细小果胶杆菌LAMP;B.多样果胶杆菌LAMP。图18同。

Fig.17 Results and agarose gel diagram of samples from each stem segment after inoculation 12,24,36 h were detected by LAMP Marker.2 000 bp Marker.A.P.parvum LAMP;B.P.versatile LAMP.The same as Fig.18.

图18 LAMP检测接种12,24,48 h后块茎各区段样品结果与凝胶电泳图

Fig.18 Results and agarose gel diagram of samples from each tuber segment after inoculation 12,24,48 h were detected by LAMP

图19 LAMP检测饲料培养基中接种500 μL 不同OD₆₀₀值的菌液的果蝇样本 M.2 000 bp Marker;A.细小果胶杆菌LAMP;B.多样果胶杆菌LAMP;1.H₂O;2—6.OD₆₀₀分别为0.1,0.2,0.4,0.6,0.8。

Fig.19 LAMP detection drosophila samples feed medium inoculated with 500 μL of bacterial fluid with different OD₆₀₀values M.2 000 bp Marker;A.P.parvum LAMP;B.P.versatile LAMP;1.H₂O;2—6.OD₆₀₀ are 0.1,0.2,0.4,0.6,0.8.

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