Rice Science ›› 2025, Vol. 32 ›› Issue (4): 575-584.DOI: 10.1016/j.rsci.2025.01.005
• Experimental Technique • Previous Articles
Hou Yuxuan1,#(), Zhu Jie2,#, Lu Chenglong1, Fan Libo1, Liang Mengqi3, Zhang Xiaobo1, Cheng Benyi1, Xu Xia1, Gong Junyi1(
)
Received:
2024-10-14
Accepted:
2025-01-16
Online:
2025-07-28
Published:
2025-08-06
Contact:
Hou Yuxuan, Gong Junyi
About author:
First author contact:#These authors contributed equally to this work
Hou Yuxuan, Zhu Jie, Lu Chenglong, Fan Libo, Liang Mengqi, Zhang Xiaobo, Cheng Benyi, Xu Xia, Gong Junyi. 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[J]. Rice Science, 2025, 32(4): 575-584.
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Primer set | Primer name | Primer and probe sequence (5ʹ‒3ʹ) | Amplicon length (bp) |
---|---|---|---|
P1 | RAAF1 | GCCGCTAGGAATGAGCAATGCACACGTGGA | 162 |
RAAR1 | GCGTCCTCGTCTAAGCGATAGACGGCGCAC | ||
P2 | RAAF2 | CACACGTGGAAAGGGACCTCAAAAAAATCC | 158 |
RAAR2 | CTTGCAAGGGATAGAAGCGTCCTCGTCTAA | ||
P3 | RAAF3 | CCGTGCTGTGTGGAGTACAAAGCCACCAAC | 131 |
RAAR3 | ATAACTTGTGACGTAGCGAGCGTTTGAGAT | ||
P4 | RAAF4 | GGTGCAACAAACTCCCATGGTGTGACGGG | 134 |
RAAR4 | CAGTCCGGATTGGAGTCTGCAACTCGACTC | ||
P5 | RAAF5 | ACCCAACATCCAGTTCGCATCGTTTAGGGC | 221 |
RAAR5 | CTCAACCTGGGAATTGCAGTGGATACTGGG | ||
/ | RAAR2-biotin | Biotin-CTTGCAAGGGATAGAAGCGTCCTCGTCTAA | / |
/ | RAA2-nfo-probe | FAM-TAGTGCGCCAGGAAGGCCAGCGCACCGTAGTTGC-THF-GACGGCTACCACCGT (C3 space) | / |
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 |
RAAR1 | GCGTCCTCGTCTAAGCGATAGACGGCGCAC | ||
P2 | RAAF2 | CACACGTGGAAAGGGACCTCAAAAAAATCC | 158 |
RAAR2 | CTTGCAAGGGATAGAAGCGTCCTCGTCTAA | ||
P3 | RAAF3 | CCGTGCTGTGTGGAGTACAAAGCCACCAAC | 131 |
RAAR3 | ATAACTTGTGACGTAGCGAGCGTTTGAGAT | ||
P4 | RAAF4 | GGTGCAACAAACTCCCATGGTGTGACGGG | 134 |
RAAR4 | CAGTCCGGATTGGAGTCTGCAACTCGACTC | ||
P5 | RAAF5 | ACCCAACATCCAGTTCGCATCGTTTAGGGC | 221 |
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.
Geographic location | Number of samples based on lession length | RAA-LFD | |||||
---|---|---|---|---|---|---|---|
< 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 |
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 | |||||
---|---|---|---|---|---|---|---|
< 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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