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

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

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