MNP marker site for identifying pseudomonas syringae, primer composition, reagent, kit and application

Abstract

This invention relates to the field of biotechnology and discloses an MNP marker site, primer composition, reagent, kit, and application for the identification of *Pseudomonas syringae*. The MNP marker composition includes 157 marker sites from MNP-1 to MNP-157. The MNP marker sites are genomic regions screened from the *Pseudomonas syringae* genome that are specific to *P. syringae* and exhibit multiple nucleotide polymorphisms in different pathogenic species of *P. syringae*. Based on the discovered MNP marker sites, multiplex primer compositions can be designed for multiplex PCR amplification, followed by sequencing of the amplified products using a next-generation sequencing platform. This allows for high-throughput, high-efficiency, and high-accuracy identification and differentiation of pathogenic species of *P. syringae* and pathogenic species in diseased plants, providing technical support for the investigation, identification, and control of pathogenic species of *P. syringae*.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to an MNP marker site, primer composition, reagent, kit, and application for the identification of Pseudomonas syringae. Background Technology

[0002] Pseudomonas syringae ( Pseudomonas syringae, P.syringae Bacterial pathogens (BMPs) are prevalent plant pathogens, encompassing a variety of pathogenic species that can cause numerous plant diseases, including kiwifruit canker, cucurbitaceous bacterial angular leaf spot, cruciferous bacterial black spot, and legume blight. These diseases, characterized by their insidious nature, sudden onset, and destructiveness, have become among the most devastating diseases in agricultural production, causing significant losses to the global economy.

[0003] Traditional detection methods, such as field observation of disease symptoms and biological assays of bacteria (including bacterial isolation and purification, morphological observation, and other bacteriological and physiological biochemical assays), while not requiring expensive equipment, are time-consuming, labor-intensive, and have limited specificity. Although DNA sequence-based molecular detection technologies have been applied... P.syringae While current molecular detection techniques utilize a limited number of sites and have limited discriminative power, they struggle to accurately identify pathogenic species. Therefore, there is an urgent need for a highly efficient, sensitive, and accurate detection technology to achieve [the desired outcome]. P.syringae Early and accurate detection of diseases and monitoring of seedling health.

[0004] Therefore, how to provide multiple novel molecular markers with high polymorphism to achieve detection in a single assay is a key challenge. P.syringae Multiple markers can be detected efficiently, accurately, and sensitively, thereby achieving... P.syringae Accurate identification of pathogenic species is a technical problem that urgently needs to be solved. Summary of the Invention

[0005] To address the problems in related technologies, this invention proposes an MNP marker site, primer composition, reagent, kit, and application for the identification of *Pseudomonas syringae*, which can be used for... P.syringae It can perform qualitative identification and quantitative analysis, and has the effects of multiple targets, high throughput and high sensitivity, so as to overcome the above-mentioned technical problems existing in existing related technologies.

[0006] Therefore, the specific technical solution adopted by the present invention is as follows:

[0007] According to a first aspect of the present invention, an MNP marker combination for identifying *Pseudomonas syringae* is provided, the MNP marker combination comprising 157 marker sites from MNP-1 to MNP-157, wherein the MNP marker site is a genomic region screened on the *Pseudomonas syringae* genome that is *Pseudomonas syringae*-specific and has multiple nucleotide polymorphisms in different pathogenic species of *Pseudomonas syringae*, and the MNP marker site is at least one of the 157 marker sites from MNP-1 to MNP-157.

[0008] According to a second aspect of the present invention, a multiplex PCR primer composition for detecting the MNP marker site is provided, the multiplex PCR primer composition comprising at least one of 157 primer pairs, and the nucleotide sequences of the 157 primer pairs are shown in SEQ ID NO.1-SEQ ID NO.314.

[0009] According to a third aspect of the present invention, a reagent for detecting the MNP marker site is provided, the reagent comprising the multiplex PCR primer composition described above.

[0010] According to a fourth aspect of the present invention, a kit for detecting the MNP marker site is provided, the kit comprising the multiplex PCR primer composition or the reagents described herein.

[0011] Furthermore, the kit also includes a multiplex PCR premix.

[0012] According to a fifth aspect of the present invention, the use of the MNP marker site, the multiplex PCR primer composition, or the reagent is provided in the preparation of a detection reagent for identifying and distinguishing *Pseudomonas syringae*.

[0013] According to a sixth aspect of the present invention, there is provided the use of the MNP marker site, the multiplex PCR primer composition, the reagent, or the kit in identifying *Pseudomonas syringae* and distinguishing pathogenic species of *Pseudomonas syringae* and plant lines or samples infected with *Pseudomonas syringae*.

[0014] Compared with the prior art, the present invention provides MNP marker sites, primer compositions, reagents, kits and applications for the identification of Pseudomonas syringae, and has the following beneficial effects:

[0015] The present invention provides a P.syringae The MNP marker sites used for identification were analyzed from 43 sites. P.syringaeThe genomic sequences of pathogenic species (a total of 378 sequences) were analyzed, and 157 MNP marker sites were screened. Multiplex primer combinations were designed based on the sequence information of these 157 MNP marker sites. Multiplex PCR amplification was performed using the designed multiplex primer combinations, and the amplified products were sequenced using a next-generation sequencing platform, which can meet the requirements for one-time sequencing. P.syringae This allows for high-throughput, high-efficiency, and high-accuracy detection and differentiation of plants infected with this fungus, enabling the identification and differentiation of... P.syringae The identification and differentiation of genetic diversity provide technical support for identification. Attached Figure Description

[0016] The accompanying drawings, which are included to provide a further understanding of this application and form part of this application, illustrate exemplary embodiments and are used to explain this application, but do not constitute an undue limitation of this application. In the drawings:

[0017] Figure 1 This is a schematic diagram illustrating the principle of MNP marker polymorphism according to an embodiment of the present invention;

[0018] Figure 2 It is provided according to the embodiments of the present invention. P.syringae Flowchart of MNP marker site screening and primer design;

[0019] Figure 3 This is a flowchart of the detection process for MNP marker sites provided in an embodiment of the present invention. Detailed Implementation

[0020] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] According to embodiments of the present invention, an MNP marker site, primer composition, reagent, kit, and application for the identification of Pseudomonas syringae are provided.

[0022] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0023] According to a first aspect of the invention, a *Pseudomonas syringae* strain is provided. Pseudomonas syringae , P.syringae The MNP tag combination used for identification, wherein the MNP tag combination is in P.syringae Genome screening P.syringaeSpecificity, and in P.syringae The genome contains multiple nucleotide polymorphisms; the MNP marker sites include at least one of 157 marker sites from MNP-1 to MNP-157.

[0024] According to a second aspect of the invention, based on a general inventive concept, a multiplex PCR primer composition for detecting the MNP marker site is also provided, the multiplex PCR primer composition comprising at least one of primer pairs 1 to 157; and the nucleotide sequences of the 157 primer pairs are shown in SEQ ID NO.1-SEQ ID NO.314.

[0025] In this embodiment of the invention, the composition of the specific multiplex amplification primer composition for detecting MNP marker sites is refined, enabling targeted detection. P. syringae The detection, thereby achieving the detection of P. syringae High-throughput, high-efficiency, and high-accuracy detection of pathogenic bacteria.

[0026] The multiplex PCR primer composition is designed based on the above-mentioned MNP marker site. The specific composition and region of the MNP marker site can be referred to the above embodiments. Since the multiplex PCR primer composition adopts some or all of the technical solutions of the above embodiments, it has at least all the beneficial effects brought about by the technical solutions of the above embodiments, which will not be elaborated here.

[0027] According to a third aspect of the invention, based on a general inventive concept, a reagent for detecting the MNP marker site is also provided, the reagent comprising the multiplex PCR primer composition.

[0028] This reagent is based on the above-described multiplex PCR primer composition. The specific sequence information of the multiplex PCR primer composition can be found in the above embodiments. Since this reagent adopts some or all of the technical solutions of the above embodiments, it has at least all the beneficial effects brought about by the technical solutions of the above embodiments, which will not be elaborated here.

[0029] According to a fourth aspect of the invention, based on a general inventive concept, a kit for detecting the MNP marker site is also provided, the kit comprising the multiplex PCR primer composition or the reagent, the kit further comprising a multiplex PCR premix.

[0030] This kit is based on the above-described multiplex PCR primer composition. The specific sequence information of the multiplex PCR primer composition can be found in the above embodiments. Since this kit adopts some or all of the technical solutions of the above embodiments, it has at least all the beneficial effects brought about by the technical solutions of the above embodiments, which will not be elaborated here.

[0031] According to a fifth aspect of the invention, based on a general inventive concept, a method for identifying and distinguishing the MNP marker site, the primer composition, or the reagent is also provided. P.syringae Applications in detection reagents.

[0032] This application is based on the above-mentioned MNP marker sites. The specific composition and region of the MNP marker sites can be referred to in the above embodiments. Since this application adopts some or all of the technical solutions of the above embodiments, it has at least all the beneficial effects brought about by the technical solutions of the above embodiments, which will not be elaborated here.

[0033] According to a sixth aspect of the invention, based on a general inventive concept, a method is also provided for identifying the MNP marker site, the primer composition, the reagent, or the kit in the context of identification. P.syringae Pathogenic species and infections P.syringae Application in plant strains or samples.

[0034] Specifically, when used P.syringae and infection P.syringae When identifying plant samples, the detection of [specific substances] in the test sample and the blank control is based on [specific substances]. P.syringae The number of sequencing sequences, the number of detected MNP markers, and MNP marker sequence information are used for quality control to determine whether the sample contains [MNP markers]. P.syringae The nucleic acid; wherein the quality control scheme and judgment method are based on known... P.syringae The DNA was used as the test sample to evaluate the detection capabilities of the kit. P.syringae To ensure high efficiency and accuracy, the kit was developed for detection. P.syringae The quality control plan and judgment method.

[0035] This application is based on the above-mentioned MNP marker sites. The specific composition and region of the MNP marker sites can be referred to in the above embodiments. Since this application adopts some or all of the technical solutions of the above embodiments, it has at least all the beneficial effects brought about by the technical solutions of the above embodiments, which will not be elaborated here.

[0036] The technical solution of the present invention is further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. If there is no corresponding national standard, then general international standards, conventional conditions, or conditions recommended by the manufacturer are followed.

[0037] The creative thinking behind this invention is:

[0038] The invention developed P.syringaeThe novel molecular marker is the MNP marker site. An MNP marker site is a polymorphic marker caused by multiple nucleotides within a region of the genome. Compared with SSR and SNP markers, MNP markers have the following advantages:

[0039] (1) Abundant alleles, with 2 alleles on a single MNP marker. n Species alleles, higher than SSR and SNP, are suitable for the identification and differentiation of organisms;

[0040] (2) It has strong species differentiation ability and only requires a small number of MNP markers to achieve species identification, thus reducing the detection error rate.

[0041] The method described in this invention is based on ultramultiplex PCR combined with next-generation high-throughput sequencing technology, enabling simultaneous detection. P.syringae The 157 MNP markers have the following advantages:

[0042] (1) High efficiency, using multiplex PCR for single detection P.syringae The 157 MNP markers, while existing fluorescent PCR techniques can detect only one at a time. P.syringae With just one marker, detection efficiency was improved by 157 times.

[0043] (2) High accuracy, using multiplex PCR to detect multiple [samples] at once. P.syringae Multiple markers are used to avoid high false negatives caused by amplification failure of a single marker; the amplified products are sequenced hundreds of times using a second-generation high-throughput sequencer, and the detection results are accurate and reliable.

[0044] (3) Detection and separation of mixtures, one-time detection P.syringae The 157 MNP markers were used to separate the mixture based on the detected MNP markers. P.syringae Existing methods based on fluorescence PCR technology are limited by the number of fluorescence channels and background fluorescence signals, making it difficult to detect simultaneously. P.syringae Multiple tags.

[0045] Given the above advantages and characteristics, MNP labeling and its detection technology: MNP labeling can achieve the identification and differentiation of mixtures across species. P.syringae It has application potential in the detection of pathogenic species and their early warning mechanisms. Currently in P.syringae There are no reports on MNP markers in the detection of pathogenic species, and there is also a lack of corresponding technologies.

[0046] Based on this, the present invention has developed P.syringae The MNP marker site, wherein the MNP marker site is located at... P.syringae The genome was screened for genomic regions with multiple nucleotide polymorphisms within the species, and the reference genome for each MNP marker site is shown in Table 1.

[0047] The present invention developed the aforementioned P.syringae The multiplex PCR primer composition for MNP marker sites comprises 157 primer pairs, the nucleotide sequences of which are as shown in SEQ ID NO.1 to SEQ ID NO.314. The primers do not conflict with each other and can be efficiently amplified by multiplex PCR.

[0048] The multiplex PCR primer composition can be used as the... P.syringae This invention provides a kit for detecting MNP marker sites, and the kit can accurately identify... P.syringae And to distinguish different genetic backgrounds.

[0049] In the reproducibility test of this invention, the logarithm of the difference in major MNP marker genotype between different libraries and between different library construction batches for each sample was 0, the reproducibility r = 100%, and the accuracy a = 100%.

[0050] The MNP marker of the present invention and the detection of the kit described herein P.syringae It has high specificity. Example

[0051] P.syringae Screening of MNP marker sites and design of primers for multiplex PCR amplification:

[0052] P.syringae Screening of MNP marker sites:

[0053] like Figure 1 As shown, based on 235 publicly available online resources... P.syringae The genome sequences of Ps and 143 other pathogenic species were compared and matched. A total of 157 MNP marker sites were screened, which are shown in Table 1.

[0054]

[0055]

[0056]

[0057]

[0058]

[0059]

[0060]

[0061] The specific steps for the above screening are as follows:

[0062] Select the above P.syringae The genome sequence of a representative strain (NZ_CP032631.1) was used as a reference genome. Other genome sequences were aligned with the reference genome to obtain the genome sequence. P.syringae Single nucleic acid polymorphism sites in pathogenic species;

[0063] On the reference genome, a window of 80bp to 300bp is used for translation with a step size of 30kb to screen for regions with multiple candidate MNP marker sites. The candidate MNP marker site regions are required to contain ≥1 of the single nucleotide variant sites and the single nucleotide polymorphism sites are not present on the sequences at both ends of the 50bp.

[0064] Sites with high DP (discrimination) values ​​in the candidate polynucleotide polymorphism (PNP) site region were selected; the selection criteria included:

[0065] DP = d / t;

[0066] In the formula, t is the number of comparisons when all genomes are compared pairwise in the candidate polynucleotide polymorphism site region, and d is the number of genome pairs with at least two single nucleotide polymorphism differences in the candidate polynucleotide polymorphism site region.

[0067] Other step sizes can also be used during the window translation stage, not limited to 30kb, but the use of 30kb in this invention is advantageous for... P.syringae A comprehensive screening.

[0068] Design of primers for multiplex PCR amplification:

[0069] like Figure 2 As shown, multiplex PCR amplification primers for the candidate MNP marker were designed using primer design software. The primer design followed the principle that primers do not interfere with each other, and all primers can be combined into a primer pool for multiplex PCR amplification, that is, all designed primers can be amplified normally in one amplification reaction.

[0070] Evaluation of the detection efficiency of primer combinations:

[0071] Preparation of domestically produced P.syringae DNA samples were analyzed with genome copy numbers of 1 copy / reaction, 10 copies / reaction, 100 copies / reaction, and 1000 copies / reaction, with an equal volume of sterile water as a blank control. Three replicate libraries were tested for each sample over three consecutive days, resulting in nine sequencing data sets per sample. Based on the data analysis results for each sample shown in Table 2, the reproducibility and accuracy of the detection method were evaluated, and a quality control system for contamination and target sequencing was established. P.syringaeThe detection threshold, and the detection process for MNP marker sites are as follows: Figure 3 As shown.

[0072] Sensitivity and stability: As shown in Table 2, an average of 22 sites were detected at 1 copy / reaction; an average of 64 sites were detected at 10 copies / reaction; and in the detection of positive samples at 100 copies / reaction and 1000 copies / reaction, P.syringae All 157 MNP marker sites were detected, and sequence-specific alignments were performed at the sites specified in the table. P.syringae The reference sequence indicates that the primers and detection method have technical stability, high specificity, and sensitivity as low as 1 copy / reaction in detecting the bacteria.

[0073] Example 2

[0074] Comparing Example 2 with Example 1, the difference between Example 2 and Example 1 is as follows:

[0075] MNP marker and primer identification P.syringae Performance evaluation and threshold setting:

[0076] Detection P.syringae It exhibits technical stability, high specificity, and sensitivity as low as 1 copy / reaction.

[0077] Preparation of domestically produced P.syringae DNA samples were analyzed with genome copy numbers of 1 copy / reaction, 10 copies / reaction, 100 copies / reaction, and 1000 copies / reaction, with an equal volume of sterile water as a blank control. Three replicate libraries were tested for each sample over three consecutive days, resulting in nine sequencing data sets per sample. Based on the data analysis results for each sample shown in Table 2, the reproducibility and accuracy of the detection method were evaluated, and a quality control system for contamination and target sequencing was established. P.syringae The detection threshold, and the detection process for MNP marker sites are as follows: Figure 3 As shown.

[0078]

[0079] Table 2 shows that, at 1 copy / reaction rate, an average of 22 sites were detected; at 10 copies / reaction rate, an average of 64 sites were detected; and in the detection of positive samples at 100 copies / reaction rate and 1000 copies / reaction rate, P.syringae All 157 MNP marker sites were detected, and sequence-specific alignments were performed at the sites specified in the table. P.syringae The reference sequence indicates that the primers and detection method have technical stability, high specificity, and sensitivity as low as 1 copy / reaction in detecting the bacteria.

[0080] MNP marker detection kit detects P.syringaeReproducibility and accuracy assessment:

[0081] Based on whether the genotypes of common detection sites can be reproduced in the three replicates, the MNP marker detection method is evaluated. P.syringae To assess the reproducibility and accuracy, specifically, the genotypes of each MNP locus were compared from nine sets of data generated from 1000 copies / reaction positive samples, and the results are shown in Table 3.

[0082]

[0083] Table 3 shows that the number of MNP markers with different major genotypes is 0. Based on the principle that reproducible genotypes between two replicates are considered accurate, the accuracy is calculated as a = 1 - (1 - r) / 2 = 0.5 + 0.5r, where r represents the reproducibility rate, i.e., the ratio of reproducible loci to the number of shared loci. In the reproducibility test of this invention, the logarithm of the difference in major genotypes of MNP markers between different libraries and between different library preparation batches for each sample is 0, the reproducibility rate r = 100%, and the accuracy a = 100%.

[0084] It can be stably detected in positive samples with 100 copies / reaction and 1000 copies / reaction. P.syringae Of the 157 MNP markers detected, the blank control showed a maximum of 4 MNP markers; therefore, this invention... P.syringae The criterion for a positive result is: when the sample contains... P.syringae No fewer than 22 MNP marker sites, and sequence clustering alignment to P.syringae When the reference genome was examined, the sample was determined to contain the aforementioned [subject / factor]. P.syringae Nucleic acid.

[0085] Example 3

[0086] MNP marker sites, kits, and methods in the identification of P.syringae Applications in

[0087] Five nucleic acid samples provided by the Guangxi Zhuang Autonomous Region Institute of Special Crops were analyzed using an MNP marker site detection kit. The samples were named S1-S5 as *Actinidia kiwifruit*. Pseudomonas syringae pv. actinidiae, Psa Diseased kiwi fruit samples were tested, and the results are shown in Table 4.

[0088]

[0089] As shown in Table 4, the kit and method detected the MNP marker site of Actinidia kiwifruit ulcerans in kiwifruit leaf samples infected with kiwifruit ulcer disease.

[0090] As shown in Table 4, the kit and method described herein accurately separated multiple MNP markers in a single reaction, while methods based on fluorescent PCR that detect only one marker in a single reaction required 157 tests to achieve the desired detection effect. This indicates that the kit and method described herein are superior in their ability to accurately separate multiple MNP markers in a single reaction. P.syringae Efficiency and accuracy in identification.

[0091] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0092] (1) The 157 highly polymorphic MNP marker sites provided in this embodiment of the invention are used for identification P.syringae It has high specificity.

[0093] (2) The high polymorphism multiplex PCR amplification primer composition provided in the embodiments of the present invention can amplify 157 MNP marker sites in one reaction and integrate with a second-generation sequencing platform for sequencing of amplification products, thereby achieving the differentiation of 157 MNP marker sites in one reaction.

[0094] The joint detection and differentiation of 157 MNP marker sites provides... P.syringae It provides technical support for efficient and accurate identification and differentiation.

[0095] (3) The kit for high polymorphic MNP marker sites provided in this embodiment of the invention can detect all 157 MNP marker sites in a single reaction. In the reproducibility test, all 157 marker sites were stably detected, demonstrating the high stability of the kit. The logarithm of the difference in major MNP marker genotypes between different libraries and different batches of libraries detected by the kit was 0, with a reproducibility rate r = 100% and an accuracy a = 100%, indicating the high accuracy and stability of the kit in complex templates.

[0096] (4) The primer composition provided in the embodiments of the present invention can be used to detect samples by high-throughput sequencing. By adding a unique tag to each sample, it is possible to detect hundreds or thousands of samples at once, and the detection efficiency can be further improved.

[0097] (5) The application provided in this embodiment of the invention, compared to the traditional fluorescent PCR method which relies on parallel experiments with standard samples to detect fluorescence signals, only requires high-throughput sequencing to detect the samples, and then the detected MNP-labeled base sequences are analyzed. P.syringae The identification process eliminates the need for parallel testing of standard samples, is simple to operate and data processing is straightforward, thus improving testing efficiency.

[0098] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A method for detecting *Pseudomonas syringae* (… Pseudomonas syringae A multiplex PCR primer composition for identifying MNP marker combinations, characterized in that, The multiplex PCR primer composition comprises 157 primer pairs, and the nucleotide sequences of the 157 primer pairs are shown in SEQ ID NO.1-SEQ ID NO.

314.

2. A reagent for detecting Pseudomonas syringae (Pto DC3000) Pseudomonas syringae characterized in that, The reagent includes the multiplex PCR primer composition as described in claim 1.

3. A method for detecting *Pseudomonas syringae* (… Pseudomonas syringae The reagent kit is characterized by, The kit includes the multiplex PCR primer composition as described in claim 1.

4. The reagent kit according to claim 3, characterized in that, The kit also includes a multiplex PCR premix.

5. A reagent as described in claim 2 for the preparation and identification of *Pseudomonas syringae* (… Pseudomonas syringae Application in testing reagents.

6. A reagent as described in claim 2 for the identification of *Pseudomonas syringae* (… Pseudomonas syringae ) or infection with Pseudomonas syringae ( Pseudomonas syringae Application in plant samples.

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