Establishment of on-site rapid dual-mode fluorescence RPA detection method for bacterialfruit of blotch

doi:10.3969/j.issn.1004-1524.2022.07.20

Acta Agriculturae Zhejiangensis ›› 2022, Vol. 34 ›› Issue (7): 1519-1528.DOI: 10.3969/j.issn.1004-1524.2022.07.20

• Quality and Safety of Agricultural Products • Previous Articles Next Articles

Establishment of on-site rapid dual-mode fluorescence RPA detection method for bacterialfruit of blotch

WANG Chenwei¹^,²(), WANG Xiaofu²^,^*(), WEI Wei², CHEN Xiaoyun², SHEN Jie³, XU Junfeng², CAI Jian¹^,^*()

1. School of Biology and Food Engineering,Fuyang Normal University, Fuyang 236037, Anhui, China
2. State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-Products,Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China
3. School of Clinical Medicine, Ningxia Medical University, Yinchuan 750004, China

Received:2021-10-19 Online:2022-07-25 Published:2022-07-26
Contact: WANG Xiaofu,CAI Jian

Abstract

Abstract:

Bacterial fruit of blotch (BFB) is a quarantine disease in China, which spreads very rapidly and no commercial resistant varieties have been found at present. In order to establish rapid fluorescent recombinase polymerase amplification (RPA) detection method of BFB, the best combination of RPA primers and probe were designed and screened out according to the specific sequence of BFB, then RPA results was analyzed using two modes and compared with quantitative real-time PCR(qRT-PCR). One was real time RPA (RT-RPA), and the other was end point RPA (EP-RPA). The results showed that the established dual-mode fluorescence RPA method for detection of BFB had strong specificity, and its sensitivity for detection of pathogens was equivalent to that of qRT-PCR. The results of RT-RPA, EP-RPA and qRT-PCR were identical. The average detection time of EP-RPA, RT-RPA and qRT-PCR were about 20, 5 and 44 min,respectively. The detection speed of RPA was much faster, and the detection process did not rely on large instruments, which was convenient and fast. As a result, it was suitable for on-site detection. The establishment of this constant temperature rapid detection method provided a new technical support for the on-site detection of plant diseases.

Key words: bacterial fruit of blotch, real time RPA, end point RPA, visualization

CLC Number:

WANG Chenwei, WANG Xiaofu, WEI Wei, CHEN Xiaoyun, SHEN Jie, XU Junfeng, CAI Jian. Establishment of on-site rapid dual-mode fluorescence RPA detection method for bacterialfruit of blotch[J]. Acta Agriculturae Zhejiangensis, 2022, 34(7): 1519-1528.

Figures/Tables 9

Table 1 Pathogenic bacteria of Cucurbitaceae

编号 No.	致病菌 Pathogenic bacteria	保藏编号 Deposit number
Ac	西瓜噬酸菌Acidovorax citrulli	ATCC 29625
1	黄瓜黑星病菌Cladosporiumcu cumerinum	ATCC 38728
2	核盘菌Sclerotinia sclerotiorum	ACCC 36961
3	丁香假单胞杆菌Pseudomonas syringae pv. Lachrymans	NBRC 14078
4	嗜管欧文氏菌Erwinia tracheiphila	NCPPB 2452
5	野油菜黄单胞菌Xanthomonas campestris pv. cucurbita	ATCC 11672
6	胡萝卜软腐果胶杆菌Pectobacterium carotovorum subsp. brasiliense	ATCC 38728

Table 2 Probe and primer

目的 Purpose	名称 Name	序列 Sequence(5'→3')
RT-RPA	Ac-F1	GTCATTACTGAATTTCAACAAGCTTTCGCT
	Ac-F2	CAAGCTTTCGCTCAATTGAATATTCGTTTT
	Ac-F3	GTTTTGACGCAATCAAATTTTTGTCACCGG
	Ac-F4	TGTCACCGGCGGCACGGTGCAGTTTCCTGC
	Ac-R1	TGTTGTTGGTCCGGTATAGACCGGATCAAT
	Ac-R2	CGGCTTCGCGAGAGGCCTCTTTGTTGTTGG
	Ac-R3	CGCTCTGCGGTAGGGCGAAGAAACCAACAC
	Ac-R4	CCTCCACCAACCAATACGCTCTGCGGTAGG
	Ac-P	TTCCGCCGGCAACGCTGATTCGACTCTA/i6FAMdT/G/idSp/A/iBHQ1dT/TTTTAAAGAACAG
PCR	SEQID4	GTCATTACTGAATTTCAACA
	SEQID5	CCTCCACCAACCAATACGCT
qRT-PCR	Ac-qF	CTGATAATCCTCGGCTCAACAA
	Ac-qR	TGAGCGCATTTCTGACGAG
	Ac-qP	(FAM)-AAGAAATACGCCCTCGCCAATCTCC-(BHQ)

Fig.1 Sequence information and principle of RPA primers and probes A, Sequence information of primers and probes;B, Principle of RPA primer amplification; C,RPA probe principle.

Fig.2 On-site inspection process A,Watermelon seeds were treated in the lysate for 2-5 min, and 2 μL leaching solution was used as template for RPA amplification; B,Put the eight tubes into the hand-held fluorescent thermostatic amplifier to set the program, the total running time was 25 min, and it could be detected within 10 min; C,Visual operation.

Fig.3 Screening results of primer A,Amplification results of upstream primer F1 and all downstream primer combinations; B.Amplification results of downstream primer R2 and all upstream primer combinations.

Fig.4 Specificity of RPA and visualization results A,DNA amplification results of Ac and common pathogens of cucurbitaceae plants;B,End point visualization results of Ac and common pathogens of cucurbitaceae plants.Ac, Acidovorax citrulli; 1-6,Other bacteria in Table 1; 7, H2O.

Fig.5 Results of sensitivity test A,RT-RPA results of different concentrations of DNA;B,RT-PCR results of different concentrations of DNA;C,Visualization results after DNA amplification at different concentrations(The template DNA concentrations of tubes 0-7 were 60, 60×5-1, 60×5-2, 60×5-3, 60×5-4, 60×5-5, 60×5-6, 60×5-7 ng·μL-1);D,RT-RPA results of different concentrations of bacteria;E,qRT-PCR results of different concentrations of bacteria;F,Visualization results after amplification of bacterial solution with different concentrations(The concentration of bacterial solution selected for tubes 0-7 were 2.05×108, 2.05×107, 2.05×106, 2.05×105, 2.05×104, 2.05×103, 2.05×102 and 20.5 CFU·mL-1).

Table 3 Test results of actual samples

西瓜样品 Sample	检测结果Detection results			检测时间Detection time/ min
西瓜样品 Sample	RT-RPA	EP-RPA	qRT-PCR	RT-RPA	EP-RPA	qRT-PCR
S1	+	+	+	3.2±0.6	20	44.8±0.4(Ct=27.9±0.3)
S2	+	+	+	2.3±0.4	20	35.7±1(Ct=20.6±0.8)
S3	+	+	+	6.0±0.8	20	54±2(Ct=35.2±1.6)
S4	-	-	-	/	20	/
S5	-	-	-	/	20	/
S6	+	+	+	4.1±0.4	20	50.1±0.75(Ct=32.1±0.6)
S7	-	-	-	/	20	/
S8	+	+	+	2.6±0.6	20	37.6±0.4(Ct=22.1±0.3)
S9	+	+	+	3.1±0.3	20	39±0.5(Ct=23.21±0.4)
S10	-	-	-	/	20	/
CK	-	-	-	/	20	/

Fig.6 Detection curve and fluorescence results of positive samples

References 18

[1]	WALCOTT R R, GITAITIS R D, CASTRO A C. Role of blossoms in watermelon seed infestation by Acidovorax avenae subsp. citrulli[J]. Phytopathology, 2003, 93(5): 528-534. DOI URL
[2]	WILLEMS A, GOOR M, THIELEMANS S, et al. Transfer of several phytopathogenic Pseudomonas species to Acidovorax as Acidovorax avenae subsp.avenae subsp. nov., comb. nov., Acidovorax avenae subsp. citrulli, Acidovorax avenae subsp.cattleyae, and Acidovorax konjaci[J]. International Journal of Systematic Bacteriology, 1992, 42(1): 107-119. DOI URL
[3]	KOMIYA Y, SHIRAKAWA T, ABIKO K. The immunological diagnostic assay of watermelon bacterial fruit blotch by dot immuno-binding assay(DIBA) and tissue printing immunoassay(TPI)[J]. Japanese Journal of Phytopathology, 2002, 68(3): 291-296. DOI URL
[4]	GITAITIS R, WALCOTT R. The epidemiology and management of seedborne bacterial diseases[J]. Annual Review of Phytopathology, 2007, 45: 371-397. DOI URL
[5]	FENG J J, LI J Q, WALCOTT R R, et al. Advances in detection of Acidovorax citrulli, the causal agent of bacterial fruit blotch of cucurbits[J]. Seed Science and Technology, 2013, 41(1): 1-15. DOI URL
[6]	王吉明, 尚建立, 李娜, 等. 我国西瓜甜瓜种质资源工作70年回顾与展望[J]. 中国瓜菜, 2019, 32(8): 14-18.
	WANG J M, SHANG J L, LI N, et al. Advances in detection and prevention of melons fruit spot pathogen[J]. China Cucurbits and Vegetables, 2019, 32(8): 14-18. (in Chinese)
[7]	曾海娟, 吴淑燕, 邱实, 等. 瓜类果斑病菌的检测与防治进展[J]. 微生物学杂志, 2016, 36(1): 100-105.
	ZENG H J, WU S Y, QIU S, et al. Advances in detection and prevention of melons fruit spot pathogen[J]. Journal of Microbiology, 2016, 36(1): 100-105. (in Chinese with English abstract)
[8]	宋蕤, 刘箐, 刘雅莉, 等. 西瓜细菌性果斑病菌快速免疫PCR检测[J]. 植物检疫, 2009, 23(2): 4-6.
	SONG R, LIU Q, LIU Y L, et al. Rapid immuno-PCR detection of Acidovorax avenae subsp.citrulli[J]. Plant Quarantine, 2009, 23(2): 4-6. (in Chinese with English abstract)
[9]	周佩, 姜培, 罗金燕, 等. 瓜类果斑病菌胶体金试纸条的检测应用[J]. 中国植保导刊, 2021, 41(2): 96-99.
	ZHOU P, JIANG P, LUO J Y, et al. Application of gold immunochromatography assay strip for detection of Acidovorax citrulli[J]. China Plant Protection, 2021, 41(2): 96-99. (in Chinese with English abstract)
[10]	赵子婧, 芦钰, 田文, 等. 应用微滴数字PCR同时检测瓜类种子携带果斑病菌和角斑病菌[J]. 植物保护, 2021, 47(2): 156-163.
	ZHAO Z J, LU Y, TIAN W, et al. Detection of Acidovorax citrulli and Pseudomonas syringae pv. lachrymans from cucurbit seeds by multiplex droplet digital PCR[J]. Plant Protection, 2021, 47(2): 156-163. (in Chinese with English abstract)
[11]	LI J, MACDONALD J, VON STETTEN F. Review: a comprehensive summary of a decade development of the recombinase polymerase amplification[J]. The Analyst, 2018, 144(1): 31-67. DOI URL
[12]	LEE H J, CHO I S, JU H J, et al. Rapid and visual detection of tomato spotted wilt virus using recombinase polymerase amplification combined with lateral flow strips[J]. Molecular and Cellular Probes, 2021, 57: 101727.
[13]	KUMAR P V, SHARMA S K, RISHI N, et al. An isothermal based recombinase polymerase amplification assay for rapid, sensitive and robust indexing of Citrus yellow mosaic virus[J]. Acta Virologica, 2018, 62(1): 104-108. DOI URL
[14]	MIAO F M, ZHANG J Y, LI N, et al. Rapid and sensitive recombinase polymerase amplification combined with lateral flow strip for detecting African swine fever virus[J]. Frontiers in Microbiology, 2019, 10: 1004.
[15]	国家市场监督管理总局, 国家标准化管理委员会. 瓜类果斑病菌检疫鉴定方法: GB/T 36822—2018[S]. 北京: 中国标准出版社, 2018.
[16]	HA Y, FESSEHAIE A, LING K S, et al. Simultaneous detection of Acidovorax avenae subsp. citrulli and Didymellabryoniae in cucurbit seedlots using magnetic capture hybridization and real-time polymerase chain reaction[J]. Phytopathology, 2009, 99(6): 666-678. DOI URL
[17]	PANDA R, ARIYARATHNA H, AMNUAYCHEEWA P, et al. Challenges in testing genetically modified crops for potential increases in endogenous allergen expression for safety[J]. Allergy, 2013, 68(2): 142-151. DOI URL
[18]	CRANNELL Z A, ROHRMAN B, RICHARDS-KORTUM R. Equipment-free incubation of recombinase polymerase amplification reactions using body heat[J]. PLoS One, 2014, 9(11): e112146.

Establishment of on-site rapid dual-mode fluorescence RPA detection method for bacterialfruit of blotch

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