玉米内州萎蔫病菌荧光重组酶介导等温扩增检测方法的建立

doi:10.3969/j.ISSN.1004-1524.2021.09.12

浙江农业学报 ›› 2021, Vol. 33 ›› Issue (9): 1676-1685.DOI: 10.3969/j.ISSN.1004-1524.2021.09.12

玉米内州萎蔫病菌荧光重组酶介导等温扩增检测方法的建立

单长林¹(), 周圆¹, 任琰², 季文彬¹, 李孝军¹

1.舟山海关综合技术服务中心,浙江舟山 316021
2.杭州海关技术中心,浙江杭州 310000

收稿日期:2020-12-09 出版日期:2021-09-25 发布日期:2021-10-09
作者简介:单长林(1987—),男,山东单县人,硕士,农艺师,从事植物检疫工作。E-mail: changlin_shan@163.com
基金资助:
浙江省基础公益研究计划(LGN18C140001)

Development of a fluorescent recombinase-aided amplification method for Clavibacter michiganensis subsp. nebraskensis detection

SHAN Changlin¹(), ZHOU Yuan¹, REN yan², JI Wenbin¹, LI Xiaojun¹

1. Comprehensive Technology Service Center of Zhoushan Customs, Zhoushan 316021, China
2. Hangzhou Customs Technology Center, Hangzhou 310000, China

Received:2020-12-09 Online:2021-09-25 Published:2021-10-09

摘要/Abstract

摘要：

玉米内州萎蔫病菌(Clavibacter michiganensis subsp. nebraskensis)是进境玉米重要检疫性病原物之一。通过分析比较玉米内州萎蔫病菌的纤维素酶基因celB,筛选出独特和保守的基因序列用于设计引物和探针,利用重组酶介导等温扩增技术(recombinase-aided amplification,RAA)建立玉米内州萎蔫病菌的荧光RAA检测方法。以celB基因序列作为靶标序列设计出引物2F、6R和探针P,建立了玉米内州萎蔫病菌荧光RAA检测方法,该方法在39 ℃恒温条件下,20 min内可完成检测反应,特异性好,且灵敏度高达130 fg·μL^-1,利用该方法检测4份玉米模拟样本阳性,8份玉米真实样品阴性,检测结果与参照国家标准GB/T 36840—2018一致。结果表明,建立的荧光RAA检测方法快速、特异、灵敏,能够满足现场检测要求。

关键词: 玉米内州萎蔫病菌, celB序列, 重组酶介导等温扩增, 荧光探针, 快速检测

Abstract:

Clavibacter michiganensis subsp. nebraskensis(Cmn) is one of the important quarantine pathogens of imported corn. Our research objective was to develop a robust and rapid fluorescent recombinase-aided amplification (RAA) to detect Cmn. Comparative analyses of cellulase gene were performed to identify unique and conserved gene regions for primer and probe design. The unique celB sequence was determined for specific detection of Cmn. After primers screening and specificity verification, the fluorescent RAA assay with primers 2F/6R and probe P was established. Under 39 ℃, the fluorescent RAA could detect Cmn specifically with the sensitivity at 130 fg·μL^-1 in 20 min. Simulated samples and actual samples were used to evaluate the reliability of fluorescent RAA, the detection result was the same as GB/T 36840—2018. Overall, the fluorescent RAA assay established in this study was high speed, specificity, and high sensitivity, which is proved to be a reliable method for Cmn detection.

Key words: Clavibacter michiganensis subsp. nebraskensis, celB, recombinase-aided amplification, fluorescent probe, rapid detection

中图分类号:

S435.131.4⁺9

单长林, 周圆, 任琰, 季文彬, 李孝军. 玉米内州萎蔫病菌荧光重组酶介导等温扩增检测方法的建立[J]. 浙江农业学报, 2021, 33(9): 1676-1685.

SHAN Changlin, ZHOU Yuan, REN yan, JI Wenbin, LI Xiaojun. Development of a fluorescent recombinase-aided amplification method for Clavibacter michiganensis subsp. nebraskensis detection[J]. Acta Agriculturae Zhejiangensis, 2021, 33(9): 1676-1685.

图/表 7

表1 试验所用玉米样品、荧光RAA和国家推荐标准(GB/T 36840—2018)反应结果

Table 1 Maize samples used in this study and the result detected by fluorescent RAA and GB/T 36840—2018

样品 Sample	编码 No.	反应结果Detection reasults		来源 Sourse
样品 Sample	编码 No.	Fluorescent RAA	GB/T 36840—2018	来源 Sourse
模拟样品Simulation sample	1-3103304-1	+	+	*
	1-3103304-2	+	+	*
	1-3103304-3	+	+	*
	1-3103304-4	+	+	*
玉米样品Imported maize	1-3103304	-	-	乌克兰Ukraine
	1-2218257	-	-	乌克兰Ukraine
	1-2677655	-	-	乌克兰Ukraine
	1-2958531	-	-	乌克兰Ukraine
	1-6368269	-	-	美国USA
	1-6367871	-	-	美国USA
	1-6551666	-	-	美国USA
	1-6551598	-	-	美国USA

表2 试验所用基因序列

Table 2 Genome sequences of bacterial strains used in this study

菌株 Bacterial species, strain	Genbank登录号 GenBank accession No.		目标序列CelB sequence						纤维素酶基因其他序列Other sequence of CelB
菌株 Bacterial species, strain	Genbank登录号 GenBank accession No.		位置 Position/nt		长度 Length/bp		一致性 Per.Ident/%		位置 Position/nt		长度 Length/bp		一致性 Per.Ident/%
C.michiganensis subsp. nebraskensis
NCPPB 2581^a	HE614873		2 525 339~2 525 815		477		100		2 525 816~2 526 973		1 158		100
61-1	CP033723		2 525 700~2 526 182		483		98.76		2 526 183~2 526 340		1 158		100
7580	CP033722		2 525 693~2 526 175		483		98.76		2 526 176~2 526 333		1 158		100
HF4	CP033721		2 526 095~2 526 553		459		95.18		2 526 554~2 527 711		1 158		100
C. michiganensis subsp. insidiosus
ATCC 10253	CP021038	2 629 864~2 630 140		276		70.14		2 630 217~2 631 401		1 188		83.08
C.michiganensis subsp. michiganensis
NCPPB 382	AM711867	2 753 512~2 753 816		304		70.28		2 753 902~2 754 939		1 038		84.55
C. michiganensis subsp. capsici
1101	CP048049	2 508 432~2 508 717		285		70.26		2 508 788~2 509 842		1 461		82.58
C. michiganensis subsp. sepedonicus
	AM849034	49 546~49 891		345		70.69		48 490~49 624		1 135		82.46

表3 荧光RAA引物、探针

Table 3 Primers and probe of fluorescent RAA used in this study

名称 Name	序列 Sequence(5'-3')	GC含量 Content/%	Tm/℃	长度 Length/bp
1F	GGTCAGTTACTGGTGAATGTGAGCACGG	53.6	64.4	28
2F	GTTACTGGTGAATGTGAGCACGGGGTTGC	55.2	68.3	29
3F	TCGGAGCCTGAGTTCCCGCGACGTTCAGAC	63.3	74.9	30
4F	CGTGCATGGTCTGTCCGGCCGGGATCTTG	65.5	76.4	29
5F	GGTGAATGTGAGCACGGGGTTGCTCGGAG	62.1	73.1	29
6F	GAATGTGAGCACGGGGTTGCTCGGAGCCT	62.1	73.5	29
7F	TGTGAGCACGGGGTTGCTCGGAGCCTGAG	65.5	75.3	29
1R	GTCGAGTTGGACGGTCTCCTGGGACAGCC	65.5	71.9	29
2R	GTCCGCGGTGTCGAGTTGGACGGTCTCCT	65.5	73.7	29
3R	GCGGTGTCGAGTTGGACGGTCTCCTGGGA	65.5	74.4	29
4R	GTCTGAGCTCGTCATCACTGCGAAGTCCG	58.6	69.1	29
5R	AGCTCGTCATCACTGCGAAGTCCGCGGTG	62.1	73.8	29
6R	CACGTCTGAGCTCGTCATCACTGCGAAGT	55.2	67.9	29
7R	CTACACGTCTGAGCTCGTCATCACTGCGA	55.2	67	29
cmn-P	ACGTGATGCGGCTCGACGCCACGGTGCACT[FAM-dT]	75.1	73.1	48
	C[dT-BHQ1]GCCCCAGCTGTTCG-block

图1 多序列分析及引物、探针位置

Fig.1 Multiple sequence analysis with location of primers/probe

图2 引物筛选结果 A,正向引物1F,2F,3F,4F与 1R,2R,3R,4R组合40循环后相对荧光强度;B,正向引物2F,5F,6F,7F与 4R,5R,6R,7R组合40循环后相对荧光强度;C,正向引物2F与3R,4R,6R组合40循环后相对荧光强度。

Fig.2 Results of primer screening A, △Rn of fluorescent RAA after 40 cycles using different primers combining 1F/2F/3F/4F with 1R/2R/3R/4R, respectively; B, △Rn of fluorescent RAA after 40 cycles using different primers combining 2F/5F/6F/7F with 4R/5R/6R/7R, respectively;C, △Rn of fluorescent RAA after 40 cycles using primers 2F/3R, 2F/4R, 2F/6R, respectively.

图3 荧光RAA灵敏性检测结果 A,引物1F/1R 荧光RAA最低检测浓度初测结果;B,引物2F/6R荧光RAA灵敏性检测结果。E0~E7的DNA浓度分别为26.1、2.6、2.6×10-1、2.6×10-2、2.6×10-3、2.6×10-4、2.6×10-5、2.6×10-6 ng·μL-1;N为阴性对照。

Fig.3 Sensitivity result of fluorescent RAA A, The minimum detectable concentration of fluorescent RAA with R1/F1 primers; B, Sensitivity result of fluorescent RAA with 2F6R. The concentration of DNA in E0-E7 was 26.1, 2.6, 2.6×10-1, 2.6×10-2, 2.6×10-3, 2.6×10-4, 2.6×10-5, 2.6×10-6 ng·μL-1. N wasa negative control.

图4 荧光RAA特异性实验 1~11,玉米细菌性枯萎病菌、丁香假单胞杆菌番茄致病变种、砖红色微杆菌、密执安棍状杆菌诡谲亚种、密执安棍状杆菌密执安、成团泛菌、菠萝泛菌、黄单胞菌、伯克霍尔德氏菌、玉米迪克氏菌、欧氏杆菌;N,无菌ddH2O;Cmn-1,浓度为26.1 ng·μL-1 Cmn基因组;Cmn-2,浓度为1.3×10-4ng·μL-1 Cmn基因组。

Fig.4 Specificity result of fluorescent RAA 1-11, P. stewartii subsp. stewarii, P. syringae pv. syringae,M. testaceum, C. michiganensis subsp. insidiosus, C. michiganensis subsp. michiganensis, P. agglomerans, P. ananatis, X. oryzae, B. andropogonis, D. zeae, E. chrysanthemi var. zea; N, Negtive control; Cmn-1, 26.1 ng·μL-1 Cmn DNA; Cmn-2, 1.3×10-4 ng·μL-1 Cmn DNA.

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玉米内州萎蔫病菌荧光重组酶介导等温扩增检测方法的建立

Development of a fluorescent recombinase-aided amplification method for Clavibacter michiganensis subsp. nebraskensis detection

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