A primer pair for detecting and identifying xanthomonas oryzae pv. oryzae and application thereof

Abstract

The application discloses a target for detecting and identifying Xanthomonas oryzae pv. oryzae (Xoo), which is a nucleotide sequence as shown in SEQ ID No. 1. The application also discloses a primer pair for detecting and identifying Xoo, which comprises a forward primer 5'-CCTTCTGCAATCTATGGCACC-3' and a reverse primer 5'-TAAATAACACGGTGAAGCCGAC-3'. The primer pair can be used for PCR and can only amplify a nucleic acid fragment with a length of 138 bp from a sample containing Xoo DNA, has specificity and universality for detecting and identifying Xoo, and realizes accurate detection and identification of Xoo. The primer pair is suitable for real-time fluorescent quantitative PCR, and the detection sensitivity reaches 2.7x10 2 fg / μL.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a primer pair for detecting and identifying rice bacterial blight pathogens and their applications. Background Technology

[0002] Bacterial blight and rice leaf streak are the most important bacterial diseases of rice. The pathogens causing rice bacterial blight (Xanthomonas oryzae pv. oryzae, Xoo) and rice leaf streak (Xanthomonas oryzae pv. oryzicola, Xoc) are two pathogenic species within the same species. Xoo and Xoc can be transmitted through seeds and are quarantine pests for imported plants in my country. Reliable methods for identifying Xoo and Xoc are crucial for accurate quarantine of these two pathogens and for early warning and control of these diseases.

[0003] Phylogenetic analysis has revealed that *Xanthomonas oryzae* (Xo) is divided into five phylogenetic groups: Xoo Asia, Xoo Africa, Xo USA, Xoc, and Xanthomonas oryzae pv. leersiae (Xol). This means that Xo does not only contain the two pathogenic species Xoo and Xoc, but also XoUS and Xol, which have weaker pathogenicity to rice. Furthermore, the pathogenic species Xoo include two phylogenetic groups, Xoo Asia and Xoo Africa, with genomic similarity differences between them approaching those between bacterial subspecies.

[0004] The most common method for detecting Xoo and Xoc is PCR, with various methods such as conventional PCR, duplex PCR, multiplex PCR, quantitative real-time PCR (qPCR), and digital PCR (dPCR) available for detecting and identifying Xoo and Xoc. The key to specifically detecting and identifying Xoo and Xoc using PCR is to use primers that are specific to Xoo or Xoc.

[0005] Based on the understanding that Xo strains differentiate into five phylogenetic groups, and utilizing the abundant whole genome sequences of Xanthomonas bacteria currently stored in the NCBI database, a BLAST search revealed two problems with existing specific primers for identifying Xoo and Xoc. First, the primers lack sufficient specificity; the primer sequence and the target sequence being amplified may have a highly consistent target sequence in the whole genome sequence of non-target strains, leading to non-specific amplification. For example, the commonly used primer pair Xoo80F / Xoo80R (Lang et al. Genomics-Based Diagnostic Marker Development for Xanthomonas oryzae pv.oryzae and X.oryzae pv.oryzicola[J]. Plant Disease, 2010, 94(3). 311-319) not only matches the target sequence in the genomes of XooAsia, XooAfrica, and XoUS strains, but also highly matches the sequence in the genomes of some Xanthomonas citri strains, and even Pseudomonas syringae strains, leading to nonspecific PCR amplification and false positives. Similarly, the primer pair Xoc2071F / Xoc2071R used for detecting and identifying Xoc not only matches the target sequence in the genome of Xoc strains, but also highly matches the sequence in the genome of Xol strains. Second, the primers lack universality. For example, the primer pair Xoo-Hpa1F / Xoo-Hpa1R (Feng Wenjie et al., Molecular marker screening and detection of rice bacterial blight and bacterial leaf streak [J]. Acta Phytopathologica Sinica, 2013, 43(6): 581-589) for detecting and identifying Xoo strains completely matched the target sequence in the XooAsia strain genome, but Xoo-Hpa1F differed from the target sequence in the XooAfrica strain genome by 5 bases. Detection revealed that the primer pair Xoo-Hpa1F / Xoo-Hpa1R could only amplify the target sequence of the XooAsia strain, but not the target sequence of the XooAfrica strain, resulting in false negatives. Summary of the Invention

[0006] This invention addresses the problems of low specificity or limited versatility of existing primers used for PCR detection and identification of Xoo, which can lead to false positives or false negatives. It provides a specific primer pair that can accurately detect and identify Xoo and its application.

[0007] The specific technical solution adopted is as follows:

[0008] This invention utilizes the abundant whole genome sequences of Xanthomonas bacteria stored in the NCBI database to mine pseudogenes of Xo that have not been addressed in existing techniques, discovering Xoo-specific pseudogenes. Primers were designed targeting the specific conserved regions of the Xoo pseudogenes, and a BLAST search was performed in the NCBI database to evaluate the universality and specificity of the primers. Primers with strong universality and specificity were selected, and PCR was performed on Xoo, its closely related variant Xoc, and other Xanthomonas strains to verify the universality and specificity of the primers, thus identifying universal primers specific to Xoo.

[0009] This invention provides a target for detecting and identifying rice bacterial blight pathogens, wherein the target is a nucleotide sequence as shown in SEQ ID No. 1.

[0010] The present invention also provides the application of the aforementioned target in the detection and identification of rice bacterial blight pathogen.

[0011] This invention provides a primer pair for detecting and identifying rice bacterial blight pathogens. The primers target a pseudogene in the Xoo genome sequence. For XooAfrica strain BAI3, the target is the C0L90_22090 site in the whole genome sequence (GenBank accession number: CP025610.1), which corresponds to the two-component sensor histidine kinase (SHK). The primer pair includes a forward primer XooSHK2F and a reverse primer XooSHK2R. The forward primer XooSHK2F (5'-CCTTCTGCAATCTATGGCACC-3') has 21 nucleotides and corresponds to positions 107-127 of the Xoo SHK pseudogene sequence; the reverse primer XooSHK2R (5'-TAAATAACACGGTGAAGCCGAC-3') has 22 nucleotides and corresponds to positions 223-244 of the Xoo SHK pseudogene sequence. The PCR amplification yielded a nucleic acid fragment of 138 bp in length, and only a 138 bp nucleic acid fragment could be amplified from Xoo.

[0012] The present invention also provides the application of the primer pair described herein in the detection and identification of rice bacterial blight pathogen.

[0013] The present invention also provides the application of the primer pair described herein in the preparation of a kit for detecting and identifying rice bacterial blight pathogen.

[0014] This invention provides a method for detecting and identifying rice bacterial blight pathogen, comprising the following steps: using the DNA of the sample to be tested as an amplification template, performing PCR amplification using the primer pair; if a 138bp gene fragment can be amplified, it indicates that the sample to be tested contains rice bacterial blight pathogen; otherwise, the sample to be tested does not contain rice bacterial blight pathogen.

[0015] Specifically, per 25 μL, the above-mentioned routine PCR amplification reaction system is as follows: 1 μL of DNA template with a concentration of 10–50 ng / μL, 1 μL of 10 μM forward primer, 1 μL of 10 μM reverse primer, 12.5 μL of 2×Taq PCR Mix, and sterile ultrapure water to make up the difference; the amplification reaction conditions are: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 15 s, 35 cycles; 72℃ extension for 5 min.

[0016] This invention also provides a method for detecting and identifying rice bacterial blight pathogen, comprising the following steps: using the DNA of the sample to be tested as an amplification template, performing real-time quantitative PCR using the primer pair; if the amplification is positive, it indicates that the sample to be tested contains rice bacterial blight pathogen; otherwise, the sample to be tested does not contain rice bacterial blight pathogen.

[0017] Specifically, the real-time quantitative PCR amplification reaction system per 20 μL is as follows: 1 μL of DNA template (10–50 ng / μL), 0.5 μL of 10 μM forward primer, 0.5 μL of 10 μM reverse primer, 10 μL of 2×SYBR Green Mix, and sterile ultrapure water to make up the volume; the amplification reaction conditions are: 95℃ pre-denaturation for 30 s; 95℃ denaturation for 10 s; 60℃ annealing for 30 s; 40 cycles.

[0018] In the two methods for detecting and identifying rice bacterial blight pathogens mentioned above, the amplification templates used are bacteria, extracted bacterial DNA, or crude extracts containing bacteria / bacterial DNA.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] (1) The present invention provides a target for detecting and identifying rice bacterial blight pathogen, the target being a nucleotide sequence as shown in SEQ ID No. 1.

[0021] (2) This invention preferably uses the forward primer XooSHK2F and the reverse primer XooSHK2R for PCR amplification, which can amplify a 138bp nucleic acid fragment from samples containing Xoo DNA, exhibiting specificity and universality for detecting and identifying Xoo. This primer pair can achieve accurate detection and identification of Xoo. In addition, the PCR-amplified nucleic acid fragment is 138bp in length, and no primer dimers are generated during the amplification process, making it suitable for qPCR detection. It is more sensitive than conventional PCR detection, with a detection limit of up to 2.7 × 10⁻⁶. 2 fg / μL.

[0022] (3) The method of the present invention has strong specificity, good versatility and high detection sensitivity, and is of great value for early warning of rice bacterial blight caused by Xoo. Attached Figure Description

[0023] Figure 1 This is an image showing the electrophoresis results of PCR detection using DNA from various bacterial cells as templates.

[0024] Figure 2 The image shows the electrophoresis results of PCR detection using Xoo PXO99A genomic DNA as a template, which was serially diluted 10-fold.

[0025] Figure 3 The images show amplification curves and dissolution peaks for real-time quantitative PCR using Xoo PXO99A genomic DNA as a template, serially diluted 10-fold. In the images, A represents the amplification curve, and B represents the dissolution peak. Labels 1-6 indicate DNA concentrations of 2.7 × 10⁻⁶. 6 fg / μL, 2.7×10 5 fg / μL, 2.7×10 4 fg / μL, 2.7×10 3 fg / μL, 2.7×10 2 fg / μL and 2.7×10 fg / μL, 7 indicates sterile ultrapure water. Detailed Implementation

[0026] The present invention will be further illustrated below with reference to the accompanying drawings and embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0027] Pseudogenes annotated in the bacterial whole-genome sequences in the NCBI database are marked with " / pseudo" in the gbk file of the whole-genome sequence. All pseudogenes and their nucleic acid sequences in the complete whole-genome sequence of XooAfrica strain BAI3 (GenBank accession number: CP025610.1) were compiled into a table, and the pseudogene names were assigned to the corresponding true gene coding products. All pseudogene sequences were then searched one by one in the NCBI database using BLASTN to select Xoo-specific pseudogenes and determine candidate targets.

[0028] Example 1

[0029] Design universal specific primers for the Xoo pseudogene.

[0030] Specific primers for Xoo were designed using NCBI's primer-BLAST database, with amplification product sizes ranging from 100 to 300 bp. These primers can be used simultaneously for conventional PCR, qPCR, and dPCR detection. The specificity of the primers for Xoo was evaluated using BLASTN. A BLASTN search was performed in the NCBI database using the target sequence amplified by the primers as the query. The retrieved homologous sequences and primer sequences were then arrayed using the MUSCLE program in MEGA5 software, and the identity between the primer and target sequences was compared using BioEdit software.

[0031] The method for evaluating the universality and specificity of primers for Xoo was as follows: the designed primers were BLASTed in the NCBI nucleic acid sequence database. The universal and specific primers for Xoo showed 100% coverage and 100% identity with the Xoo SHK pseudogene sequence, while the E value was at least one order of magnitude lower than that of Xoc and other strains of Xanthomonas.

[0032] Following the above method, the optimal forward primer XooSHK2F (5'-CCTTCTGCAATCTATGGCACC-3') and reverse primer XooSHK2R (5'-TAAATAACACGGTGAAGCCGAC-3') were selected, and the target nucleotide sequence is shown in SEQ ID No. 1.

[0033] Example 2

[0034] Establish a molecular detection method for rice bacterial blight pathogen

[0035] (1) Primer preparation: Primer nucleotide sequences were synthesized using an automated DNA synthesizer, purified by ULTRAPAGE to obtain white crystals, diluted to 10 μM with Tris-EDTA buffer (pH 8.0) or ultrapure water, aliquoted into 0.5-mL centrifuge tubes, and stored at -20℃.

[0036] (2) Template preparation: Eight XooAsia strains, one XooAfrica strain BAI3, eleven Xoc strains, seven other species of Xanthomonas strains, and four other genera of rice pathogenic bacteria strains (Table 1) were cultured on solid enriched medium. Single colonies of the test strains were picked, and the bacterial cells were suspended in 10 μL of sterile pure water. One μL of the bacterial suspension was used as a template for PCR reaction. Alternatively, bacteria were cultured in liquid enriched medium, and bacterial genomic DNA was extracted from the bacterial cells. The absorbance of the DNA at 260 nm and 280 nm was detected using a UV spectrophotometer to evaluate the quality of the extracted DNA, calculate the DNA concentration, and store it at -20℃ for later use.

[0037] Table 1. Bacterial strains used for PCR detection

[0038] strain Country where the separation occurred Xanthomonas oryzae pv.oryzae PXO99A the Philippines Xanthomonas oryzae pv.oryzae PXO86 the Philippines Xanthomonas oryzae pv.oryzae ScYc-b China Xanthomonas oryzae pv.oryzae YN11 China Xanthomonas oryzae pv.oryzae FuJ China Xanthomonas oryzae pv.oryzae OS198 China Xanthomonas oryzae pv.oryzae GD414 China Xanthomonas oryzae pv.oryzae HEN11 China Xanthomonas oryzae pv.oryzae BAI3 Burkina Faso Xanthomonas oryzae pv.oryzicola BLS256 the Philippines Xanthomonas oryzae pv.oryzicola RS105 China Xanthomonas oryzae pv.oryzicola FZ05 China Xanthomonas oryzae pv.oryzicola ACCC 05509 China Xanthomonas oryzae pv.oryzicola oxy02 China Xanthomonas oryzae pv.oryzicola oxy04 China Xanthomonas oryzae pv.oryzicola oxy05 China Xanthomonas oryzae pv.oryzicola JS China Xanthomonas oryzae pv.oryzicola AH China Xanthomonas oryzae pv.oryzicola GX China Xanthomonas oryzae pv.oryzicola YN China Xanthomonas campestris CGMCC 1.3408 U.K. Xanthomonas citri Xac29-1 China Xanthomonas hortorum ATCC 19865 Yugoslavia Xanthomonas albilineans FJ1 China Xanthomonas axonopodis CCTCC AB 2018263 China Xanthomonas sacchari ACCC 10416 China Xanthomonas sontii XQ1 China Burkholderia glumae Os48 China Acidovorax oryzae CGMCC 1.1728 Japan Dickeya oryzae ACCC 61554 China Pantoea ananatis F163 China

[0039] (3) Conventional PCR amplification: Taking a total reaction volume of 25 μL as an example, the amplification reaction system consisted of 1 μL DNA template (10–50 ng / μL), 1 μL forward primer (10 μM), 1 μL reverse primer (10 μM), 12.5 μL 2×Taq PCR Mix, and 9.5 μL sterile ultrapure water. The amplification program was as follows: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 15 s, 35 cycles; 72℃ extension for 5 min. 5 μL of the amplified reaction solution was electrophoresed in 1.5% (w / v) agarose gel and 0.5×TBE buffer (pH 8.3), and the PCR products were observed using a gel imaging system.

[0040] (4) qPCR amplification: Taking a total reaction volume of 20 μL as an example, the amplification reaction system consists of 1 μL DNA template (10–50 ng / μL), 0.5 μL forward primer (10 μM), 0.5 μL reverse primer (10 μM), 10 μL 2×SYBR Green Mix, and 8.0 μL sterile ultrapure water. The amplification program is as follows: 95℃ pre-denaturation for 30 s; 95℃ denaturation for 10 s, 60℃ annealing for 30 s, for 40 cycles.

[0041] Example 3

[0042] Primer pairs for PCR detection of rice bacterial blight pathogen

[0043] Genomic DNA was extracted from the above-mentioned 9 Xoo strains, 11 Xoc strains, 7 other species of Xanthomonas strains, and 4 other genera of rice pathogenic bacteria strains. Using 1 μL of DNA solution from each strain as a template, PCR was performed in a 25 μL reaction system using primers XooSHK2F and XooSHK2R, following the conventional PCR amplification system and procedure described in Example 2. The PCR products were detected by agarose gel electrophoresis. The results are as follows: Figure 1 As shown.

[0044] The results showed that the 138bp target fragment was amplified from the genomic DNA of all XooAsia and XooAfrica strains, while no 138bp nucleic acid fragment was amplified from the genomic DNA of other bacteria, indicating that primers XooSHK2F and XooSHK2R have the specificity for detecting and identifying Xoo.

[0045] Example 4

[0046] The sensitivity of primer pairs for PCR detection of rice bacterial blight pathogen

[0047] Xoo PXO99A genomic DNA at a concentration of 27 ng / μL was serially diluted 10-fold eight times. Using 1 μL of each diluted solution as a template, PCR was performed in a 25 μL reaction system using primers XooSHK2F and XooSHK2R and 2×Hieff PCR Master Mix (Shanghai Yisheng Biotechnology Co., Ltd.). The amplification system and procedure were the same as in Example 2. PCR products were detected by agarose gel electrophoresis. The results are shown below. Figure 2 As shown, the lowest detectable concentration of DNA is 2.7 × 10⁻⁶. 4 fg / μL.

[0048] Example 5

[0049] Sensitivity of primer pairs for qPCR detection of rice bacterial blight pathogen

[0050] Xoo PXO99A genomic DNA at a concentration of 27 ng / μL was serially diluted 10-fold eight times. Using 1 μL of each diluted solution as a template, primers XooSHK2F and XooSHK2R were used. PCR was performed in a 20 μL reaction system using 2×ChamQ SYBR Master Mix (Nanjing Novizan Biotechnology Co., Ltd.), following the qPCR amplification system and procedure described in Example 2. The results are shown in the figure. Figure 3 As shown in A, the minimum DNA concentration indicating a positive amplification is 2.7 × 10⁻⁶. 2fg / μL, compared to the sensitivity of conventional PCR for DNA detection (2.7×10⁻⁶ fg / μL). 4 The concentration (fg / μL) was 100 times higher. Furthermore, the melting curve of the amplification results showed exactly one peak. Figure 3 B) indicates that the amplification product is single and no non-specific products appear, indicating that the qPCR amplification has high specificity.

[0051] The embodiments described above provide a detailed explanation of the technical solutions of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, additions, or similar substitutions made within the scope of the principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of primer pairs in the detection and identification of rice bacterial blight pathogen, characterized in that, The primer pair is used to amplify the target, the nucleotide sequence of which is shown in SEQ ID No. 1; The primer pair includes the forward primer XooSHK2F and the reverse primer XooSHK2R, whose nucleotide sequences are as follows: Forward primer XooSHK2F: 5'-CCTTCTGCAATCTATGGCACC-3'; Reverse primer XooSHK2R: 5'-TAAATAACACGGTGAAGCCGAC-3'; The bacterial blight pathogens of rice are Asian strains and African strains of the bacterial blight pathogen.

2. The application of primer pairs in the preparation of a kit for detecting and identifying rice bacterial blight pathogen, characterized in that, The primer pair is used to amplify the target, the nucleotide sequence of which is shown in SEQ ID No. 1; The primer pair includes the forward primer XooSHK2F and the reverse primer XooSHK2R, whose nucleotide sequences are as follows: Forward primer XooSHK2F: 5'-CCTTCTGCAATCTATGGCACC-3'; Reverse primer XooSHK2R: 5'-TAAATAACACGGTGAAGCCGAC-3'; The bacterial blight pathogens of rice are Asian strains and African strains of the bacterial blight pathogen.

3. A method for detecting and identifying rice bacterial blight pathogen, characterized in that, Includes the following steps: Using the DNA of the sample to be tested as an amplification template, PCR amplification was performed using primer pairs; If a 138 bp gene fragment can be amplified, it indicates that the sample contains rice bacterial blight pathogen; otherwise, the sample does not contain rice bacterial blight pathogen. The primer pair includes the forward primer XooSHK2F and the reverse primer XooSHK2R, whose nucleotide sequences are as follows: Forward primer XooSHK2F: 5'-CCTTCTGCAATCTATGGCACC-3'; Reverse primer XooSHK2R: 5'-TAAATAACACGGTGAAGCCGAC-3'; The bacterial blight pathogens of rice are Asian strains and African strains of the bacterial blight pathogen.

4. The method for detecting and identifying rice bacterial blight pathogen as described in claim 3, characterized in that, The PCR amplification reaction system, per 25 μL, consisted of: 1 μL of DNA template (10–50 ng / μL), 1 μL of 10 μM forward primer, 1 μL of 10 μM reverse primer, 12.5 μL of 2×Taq PCR Mix, and sterile ultrapure water to make up the volume. The amplification reaction conditions were: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 15 s, for 35 cycles; and 72℃ extension for 5 min.

5. A method for detecting and identifying rice bacterial blight pathogen, characterized in that, Includes the following steps: Using the DNA of the sample to be tested as an amplification template, real-time quantitative PCR was performed using primer pairs; If the amplification is positive, it means that the sample contains rice bacterial blight pathogen; otherwise, the sample does not contain rice bacterial blight pathogen. The primer pair includes the forward primer XooSHK2F and the reverse primer XooSHK2R, whose nucleotide sequences are as follows: Forward primer XooSHK2F: 5'-CCTTCTGCAATCTATGGCACC-3'; Reverse primer XooSHK2R: 5'-TAAATAACACGGTGAAGCCGAC-3'; The bacterial blight pathogens of rice are Asian strains and African strains of the bacterial blight pathogen.

6. The method for detecting and identifying rice bacterial blight pathogen as described in claim 5, characterized in that, The real-time quantitative PCR amplification reaction system, per 20 μL, consisted of: 1 μL of DNA template (10–50 ng / μL), 0.5 μL of 10 μM forward primer, 0.5 μL of 10 μM reverse primer, 10 μL of 2×SYBR Green Mix, and sterile ultrapure water to make up the volume. The amplification reaction conditions were: 95℃ pre-denaturation for 30 s; 95℃ denaturation for 10 s; 60℃ annealing for 30 s; 40 cycles.

7. The method for detecting and identifying rice bacterial blight pathogen as described in claim 3 or 5, characterized in that, The amplification template is bacteria, extracted bacterial DNA, or a crude extract containing bacteria / bacterial DNA.

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