A Recombinase-Aided Amplification-Lateral Flow Dipstick Detection Technique for Early On-Site Diagnosis of Bacterial Blight Caused by Xanthomonas oryzae pv. oryzae in Rice

doi:10.1016/j.rsci.2025.01.005

摘要/Abstract

. [J]. Rice Science, 2025, 32(4): 575-584.

图/表 7

Table 1. Primers and probes for recombinase-aided amplification-lateral flow dipstick (RAA-LFD) detection of Xanthomonas oryzae pv. oryzae.

Primer set	Primer name	Primer and probe sequence (5ʹ‒3ʹ)	Amplicon length (bp)
P1	RAAF1	GCCGCTAGGAATGAGCAATGCACACGTGGA	162
P1	RAAR1	GCGTCCTCGTCTAAGCGATAGACGGCGCAC
P2	RAAF2	CACACGTGGAAAGGGACCTCAAAAAAATCC	158
P2	RAAR2	CTTGCAAGGGATAGAAGCGTCCTCGTCTAA
P3	RAAF3	CCGTGCTGTGTGGAGTACAAAGCCACCAAC	131
P3	RAAR3	ATAACTTGTGACGTAGCGAGCGTTTGAGAT
P4	RAAF4	GGTGCAACAAACTCCCATGGTGTGACGGG	134
P4	RAAR4	CAGTCCGGATTGGAGTCTGCAACTCGACTC
P5	RAAF5	ACCCAACATCCAGTTCGCATCGTTTAGGGC	221
P5	RAAR5	CTCAACCTGGGAATTGCAGTGGATACTGGG
/	RAAR2-biotin	Biotin-CTTGCAAGGGATAGAAGCGTCCTCGTCTAA	/
/	RAA2-nfo-probe	FAM-TAGTGCGCCAGGAAGGCCAGCGCACCGTAGTTGC-THF-GACGGCTACCACCGT (C3 space)	/

Fig. 1. Primers and probe screening for recombinase-aided amplification-lateral flow dipstick (RAA-LFD) detection of Xoo. A, Primer screening. B, Position of selected primer (RAAF2/R2) and probe (RAA2-nfo-probe) based on the Xoo locus XooORF0080. C, RAA-LFD results using primers (RAAF2/R2) and probe (RAA2-nfo-probe). M, 2000 bp DNA marker (TaKaRa, Japan); P1‒P5, The designed primer pairs RAAF1/R1, RAAF2/R2, RAAF3/R3, RAAF4/R4, and RAAF5/R5, respectively; Xoo, Xanthomonas oryzae pv. oryzae; Xoc, Xanthomonas oryzae pv. oryzicola; LA20 and Zhe173 are Xoo strains, and RS105 is a Xoc strain. ddH2O was used as a negative control.

Fig. 2. Optimization of reaction temperature and time for recombinase-aided amplification-lateral flow dipstick (RAA-LFD) detection of Xanthomonas oryzae pv. oryzae. A and B, Reaction temperature (from 31 ºC to 41 ºC for 20 min, A) and time (from 5 to 30 min at 37 ºC, B) optimization.

Fig. 3. Specificity assessment for recombinase-aided amplification lateral flow dipstick (RAA-LFD) detection of Xoo. Xoo, Xanthomonas oryzae pv. oryzae; Xoc, Xanthomonas oryzae pv. oryzicola; Pa, Pantoea ananatis; Bg, Burkholderia glumae; Dz, Dickeya zeae; Rs, Ralstonia solanacearum. ddH2O was used as a negative control.

Fig. 4. Sensitivity assessment for recombinase-aided amplification-lateral flow dipstick (RAA-LFD) detection of Xanthomonas oryzae pv. oryzae (Xoo). A, Sensitivity assessment was determined using Xoo genomic DNA as the template, with concentrations ranging from 100 ng/μL to 100 fg/μL. B, Sensitivity assessment was performed using Xoo culture solution as the template, with concentrations ranging from 1 × 108 CFU/mL to 10 CFU/mL. ddH2O was used as a negative control.

Fig. 5. On-site detection of Xanthomonas oryzae pv. oryzae (Xoo) by developed recombinase-aided amplification-lateral flow dipstick (RAA-LFD) system. A, Workflow of RAA-LFD detection: sample preparation with simplified DNA extraction, RAA reaction at 37 ºC for 20 min, and visual detection by LFD. B, Schematic diagram of LFD detection. FAM (carboxyfluorescein amidite) of special amplicons is first caputured by anti-FAM antibody conjuated colloidal gold in conjuated pad. Then, biotin of the complex binds to anti-biotin antibody in the test line, leading to appearing test line, indicating positive, otherwise indicating negative. The complex of anti-FAM antibody conjuated colloidal gold binds to anti-antibody-FAM antibody in the control line, leading to appearing the control line. C, RAA-LFD detection result for the inoculated rice leaves. Lanes 1‒7, 1‒7 d after inoculation. Rice variety Nipponbare was used. Scale bar, 5 mm. D, RAA-LFD detection result for the field samples suspected to be infected by Xoo from Wenzhou and Ningbo cities, Zhejiang Province, China. Lanes I‒IV, Leaf samples from Wenzhou with lesion lengths categorized as less than 1 cm, 1‒3 cm, 3‒6 cm, and greater than 6 cm, respectively; Lanes Iʹ‒IVʹ, Leaf samples from Ningbo with corresponding lesion lengths of less than 1 cm, 1‒3 cm, 3‒6 cm, and greater than 6 cm, respectively.

Table 2. Recombinase-aided amplification-lateral flow dipstick (RAA-LFD) detection results of field samples.

Geographic location	Number of samples based on lession length				RAA-LFD
Geographic location	< 1 cm	1‒3 cm	3‒6 cm	> 6 cm	Positive	Negative
Wenzhou	6	6	4	2	18	0
Ningbo	6	5	3	1	0	15
Total	12	11	7	3	18	15

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