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

doi:10.3969/j.ISSN.1004-1524.2021.09.12

Acta Agriculturae Zhejiangensis ›› 2021, Vol. 33 ›› Issue (9): 1676-1685.DOI: 10.3969/j.ISSN.1004-1524.2021.09.12

• Plant Protection • Previous Articles Next Articles

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

Abstract

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

CLC Number:

S435.131.4⁺9

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.

Figures/Tables 7

References 31

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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

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

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

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

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

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

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