[논문]Loop-Mediated Isothermal Amplification for the Detection of Xanthomonas arboricola pv. pruni in Peaches

Li, Weilan; Lee, Seung-Yeol; Back, Chang-Gi; Ten, Leonid N.; Jung, Hee-Young

doi:10.5423/ppj.oa.07.2019.0197

Loop-Mediated Isothermal Amplification for the Detection of Xanthomonas arboricola pv. pruni in Peaches 원문보기

The plant pathology journal, v.35 no.6, 2019년, pp.635 - 643

Li, Weilan (School of Applied Biosciences, Kyungpook National University) , Lee, Seung-Yeol (School of Applied Biosciences, Kyungpook National University) , Back, Chang-Gi (Horticultural and Herbal Crop Environment Division, National Institute of Horticultural and Herbal Science) , Ten, Leonid N. (School of Applied Biosciences, Kyungpook National University) , Jung, Hee-Young (School of Applied Biosciences, Kyungpook National University)

Abstract ▼ AI-Helper

To detect Xanthomonas arboricola pv. pruni, a loopmediated isothermal amplification (LAMP) detection method were developed. The LAMP assay was designed to test crude plant tissue without pre-extraction, or heating incubation, and without advanced analysis equipment. The LAMP primers were designed by targeting an ABC transporter ATP-binding protein, this primer set was tested using the genomic DNA of Xanthomonas and non-Xanthomonas strains, and a ladder product was generated from the genomic DNA of X. arboricola pv. pruni strain but not from 12 other Xanthomonas species strains and 6 strains of other genera. The LAMP conditions were checked with the healthy leaves of 31 peach varieties, and no reaction was detected using either the peach leaves or the peach DNA as a template. Furthermore, the high diagnostic accuracy of the LAMP method was confirmed with 13 X. arboricola pv. pruni strains isolated from various regions in Korea, with all samples exhibiting a positive reaction in LAMP assays. In particular, the LAMP method successfully detected the pathogen in diseased peach leaves and fruit in the field, and the LAMP conditions were proven to be a reliable diagnostic method for the specific detection and identification of X. arboricola pv. pruni in peach orchards.

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표/그림 (8)

그림 Fig. 1. Schematic representation of the primer design for the proposed loop-mediated isothermal amplification assay showing the posi-tion of the six primers spanning the target gene and the nucleotide sequences of the putative ABC transporter ATP-binding protein gene sequence used to design the primers.
그림 Fig. 2. Temperature optimization of the loop-mediated isothermal amplification (LAMP) primer set for the detection of Xanthomonas arboricola pv. pruni.
그림 Fig. 3. Electrophoresis of the loop-mediated isothermal amplification (LAMP) products and the LAMP products digested with the HaeIII restriction enzyme.
그림 Fig. 4. Electrophoresis analysis of the loop-mediated isothermal amplification sensitivity test and visual analysis using a UV illuminator.
표 Table 1. Xanthomonas spp. and other bacterial strains used for the analysis of the specificity of the LAMP assays developed in this study
표 Table 2. Peach varieties used for the validation of the LAMP assays developed in this study
표 Table 3. Xanthomonas arboricola pv. pruni strains isolated from various regions in Korea used for the LAMP specificity tests
그림 Fig. 5. Validation of the loop-mediated isothermal amplification (LAMP) assay using field samples.

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제안 방법

The LAMP assay were able to test crude plant tissue without pre-extraction, or heating incubation, and it does not need advanced analysis equipment. It was successfully applied in field samples, the results were obtained by 45 min, with visual inspection, which supporting a high effective and easy processing method for plant quarantine.
pruni (Pagani, 2005). Leaves and fruits bearing the shot hole disease can be validate by the LAMP assay, particularly, the pathogen were successfully detected by using leaves without visible symptoms, which supporting the high sensitivity and accuracy of the proposed LAMP assay.
1. Schematic representation of the primer design for the proposed loop-mediated isothermal amplification assay showing the position of the six primers spanning the target gene and the nucleotide sequences of the putative ABC transporter ATP-binding protein gene sequence used to design the primers. The right arrow indicates a sense sequence used for the primer, while the left arrow indicates the complementary sequence used for the primer.
The LAMP primers were designed using PrimerExplorer V4 (http://primerexplorer.jp/e/), four primers were used in this study (Fig. 1): outer primers F3 (5′-AGACGTCTAGGGCAAGCC-3′) and B3 (5′-TGCTAGAACTGACGCTGAGA-3′) and inner primers FIP (5′-ATGGGGGCCCAATCTCTAGCAATTTTGTC GGGCGACAACTTCAAC-3′) and BIP (5′-TCCACACCGATTTGCAGAAGGATTTTCGTGAGTCAATCGCAGTGT-3′).
The proposed LAMP method was applied for the diagnosis of field samples. Thirty-two peach varieties were used for the validation testing, 4 peach varieties were tested using diseased leaves, and 31 peach varieties were tested using healthy leaves.
pruni, we designed a LAMP primer system and optimized the LAMP reaction conditions using Xap1 as a positive control. The proposed LAMP method was successfully used in the diagnosis of the pathogen in field samples, providing simple and convenient field identification.
4, lane 8). To evaluate the species specificity of the designed primers, 12 other Xanthomonas species strains and six strains from other genera were tested using the LAMP reaction with their genomic DNA (Table 1), and specific PCR was used to compare the sensitivity of the proposed LAMP assays. All of the Xanthomonas and nonXanthomonas strains were tested with PCR using primers Y17CoF/Y17CoR.
To optimize the reaction temperature, the LAMP reaction was performed under various thermal conditions (50°C, 53°C, 55°C, 58°C, 60°C, 63°C, and 65°C).

대상 데이터

arboricola pv. pruni BJ15 and CFBP 5530 (accession number: MF362224 and HQ896469). Moreover, the 16S rRNA gene and ABC transporter ATP-binding protein gene sequences of strain Xap1 were compared with the 13 X.

성능/효과

All of the Xanthomonas and nonXanthomonas strains were tested with PCR using primers Y17CoF/Y17CoR. It was found that the LAMP assay was more accurate than the traditional PCR method, with only the strain Xap1 producing a positive reaction in the LAMP test, while two strains of X. campestris pv. hyacinthi and Acidovorax facilis had positive reactions in the PCR test using primers Y17CoF/Y17CoR.
For this purpose, healthy leaves and leaves with shot hole disease from naturally infected plants were tested. Leaf samples from 32 peach varieties were analyzed with the proposed LAMP assay (Table 2, Fig. 5A), and it was found that only the diseased leaves had a positive reaction, in particular, the LAMP assay were succeed for leaves without invisible symptoms, with the healthy leaves producing a negative reaction (Fig. 5B). Confirming the specific amplification, all of the positive LAMP products were digested by the HaeIII restriction enzyme (Fig.

참고문헌 (25)

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