Novel molecular targets specific for burkholderia glumae and its pathovar phomopsis sojae and rapid detection methods thereof

Abstract

The application discloses a new molecular target specific to Burkholderia gladioli and a coconut poison pathogenic variety thereof and a rapid detection method thereof, and belongs to the technical field of molecular biology and microbial detection. A nucleotide sequence combination as a detected target in the application for identifying Burkholderia gladioli and a coconut poison pathogenic variety thereof is shown as SEQ ID NO. 1-3. The application can detect Burkholderia gladioli without physiological and biochemical identification, can make up for the defects that biochemical tests cannot identify the coconut poison pathogenic variety of high-toxicity Burkholderia gladioli, has short detection time, low cost, simple operation and strong specificity; the application can make up for the defects that existing molecular detection and molecular typing of Burkholderia gladioli have poor specificity and lack of methods for the coconut poison pathogenic variety of high-toxicity Burkholderia gladioli, the detection result is more accurate, the result determination is simpler, and the practicability is stronger.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biology and microbial testing technology, and specifically relates to a novel molecular target specific for Burkholderia gladioli and its pathogenic variants of cocos nucifera and a rapid detection method thereof. Background Art

[0002] Burkholderia gladioli is a Gram-negative bacterium widely found in air and soil. This species exhibits complex strain diversity, including toxigenic foodborne pathogens (B. gladioli. pv. cocovenenans), plant pathogens (B. gladioli. pv. agaricicola, B. gladioli. pv. alliicola, and B. gladioli. pv. gladioli), and environmental isolates. Recent studies have linked Burkholderia gladioli to nosocomial infections, cystic fibrosis, keratitis, osteomyelitis, lung infections, neonatal sepsis, and nosocomial sepsis in young children, particularly in immunocompromised / suppressed populations. Therefore, rapid and accurate identification of Burkholderia gladioli is crucial.

[0003] Burkholderia gladioli pv. cocovenenans (B. gladioli. pv. cocovenenans), also known in China as Pseudomonas cocovenenans or Pseudomonas cocovenenans, is the pathogenic form of Burkholderia gladioli that can cause severe food poisoning and is the foodborne pathogen with the highest morbidity and mortality rates discovered in my country to date. Fumaric acid, produced by B. gladioli pv. cocovenenans, is the primary toxic metabolite causing food poisoning and death. It is primarily found in black fungus, white fungus, fermented cornmeal products, and other starch products. Previously, food poisoning caused by this bacterium primarily occurred in impoverished, remote, and remote areas with a high incidence of poverty. However, in recent years, food poisoning incidents caused by B. gladioli pv. cocovenenans have occurred repeatedly in economically developed regions, leading to a continued increase in public awareness of the issue. In Guangdong Province, incidents of fumonisin poisoning, caused by the consumption of contaminated and spoiled rice noodles and long-soaked wood ear mushrooms, occurred for three consecutive years in 2018, 2019, and 2020. These incidents resulted in multiple severe illnesses and deaths, posing a serious threat to human health and food safety. Therefore, identifying and controlling food contamination with Burkholderia gladioli pv. cocovenenans is crucial for reducing fumonisin poisoning.

[0004] Currently, the detection of Burkholderia gladioli and B. gladioli pv. cocovenensis primarily relies on traditional culture methods specified in the national standard (GB 4789.29-2020). This method requires bacterial enrichment, selective isolation and purification, and biochemical identification of monoclonal colonies. This biochemical identification can only detect B. gladioli. Subsequent toxin culture for 5 days and toxin detection using high-performance liquid chromatography (HPLC) or ultra-high-performance liquid chromatography-mass spectrometry (UPLC-MS / MS) are required to identify B. gladioli pv. cocovenensis. This entire process is time-consuming, susceptible to environmental and human influences, and lacks high sensitivity. In the event of an outbreak, traditional detection methods are difficult to meet. Molecular biological detection methods, primarily based on PCR, are rapidly becoming one of the most promising technologies to replace traditional methods due to their rapidity, accuracy, and simplicity.

[0005] The key to molecular biological detection methods is whether the target gene or sequence is specific for the bacteria being tested. Currently, the target genes or sequences for detecting Burkholderia gladioli are very limited, consisting of only 16S rRNA, 23S rRNA, and 16S-23S rRNA. The four pathogenic variants of Burkholderia gladioli share very little sequence variation in these genes, and even some closely related Burkholderia species are difficult to distinguish. Therefore, molecular detection methods developed using these genes have poor specificity and cannot identify the pathogenic variant of Burkholderia gladioli. Even if they can identify Burkholderia gladioli, they are prone to false negatives or false positives. In addition, the housekeeping genes recA, atpD, gltB, gyrB, lepA, phaC, and trpB have also been used for molecular typing of Burkholderia gladioli. These methods utilize several housekeeping genes for PCR amplification, sequencing, and phylogenetic tree construction to obtain classification and identification results. However, these methods can only identify Burkholderia gladioli, but not Burkholderia calamus pv. cocovenensis. Furthermore, these methods are complex, costly, and require high professional skills. Therefore, it is extremely necessary to discover new molecular target genes or sequences specific for Burkholderia gladioli and highly virulent Burkholderia gladioli pv. cocovenensis, and to establish corresponding rapid molecular detection methods based on these genes. Summary of the Invention

[0006] In view of the above problems, the purpose of the present invention is to overcome the shortcomings of the existing technology and provide a specific new molecular target and corresponding detection method for identifying Burkholderia gladioli (B.gladioli) and its cocovenenans pathovar (B.gladioli.pv.cocovenenans).

[0007] To achieve the above-mentioned objectives, the technical solution adopted by the present invention is: the application of a nucleotide sequence combination as a detected target in the identification of Burkholderia gladioli and its pathogenic variants of Cocos nucifera, the nucleotide sequence combination is shown in SEQ ID NO.1 to SEQ ID NO.3; the application is for non-disease diagnosis and treatment purposes.

[0008] Comparative genomic analysis revealed specific sequence fragments from the genomes of Burkholderia gladioli and the highly virulent Burkholderia gladioli pv. cocovenenum. These fragments can serve as new, specific molecular target sequences for distinguishing Burkholderia gladioli and the highly virulent Burkholderia gladioli pv. cocovenenum. The nucleotide sequences are shown in SEQ ID NOs. 1 to 3. By testing the sample for the presence of the corresponding sequence, it can be determined whether the sample contains strains belonging to Burkholderia gladioli and / or the highly virulent Burkholderia gladioli pv. cocovenenum.

[0009] As a preferred embodiment of the application of the present invention, the sequences SEQ ID NO.1-2 are used to identify Burkholderia gladioli; the sequence SEQ ID NO.3 is used to identify Burkholderia gladioli pv. cocovenenans.

[0010] The present invention also provides a primer set for identifying Burkholderia gladioli and its pv. cocosvenorii. The primer set is designed based on the nucleotide sequence combination.

[0011] By designing corresponding primer sets for PCR and qPCR based on the corresponding nucleotide sequences, each primer set includes a forward primer and a reverse primer, corresponding to the detection of a nucleotide sequence. The amplification products of the primer sets correspond to all or part of the nucleotide sequences shown in SEQ ID NOs: 1 to 3.

[0012] As a preferred embodiment of the primer set of the present invention, the sequences of the primer set from 5' to 3' are shown as SEQ ID NOs. 4 to 9; wherein:

[0013] SEQ ID NO.4 and SEQ ID NO.5 are primer sets corresponding to the sequence of SEQ ID NO.1;

[0014] SEQ ID NO.6 and SEQ ID NO.7 are primer sets corresponding to the sequence of SEQ ID NO.2;

[0015] SEQ ID NO.8 and SEQ ID NO.9 are primer sets corresponding to the sequence of SEQ ID NO.3.

[0016] The present invention also provides a kit for identifying Burkholderia gladioli and its p. cocovetoxin pathogenic variants, and the kit comprises the primer set.

[0017] The present invention also provides a method for identifying Burkholderia gladioli and its pathogenic variants for non-disease diagnosis and treatment purposes, wherein the method comprises a PCR method and a quantitative qPCR method.

[0018] As a preferred embodiment of the method for identifying Burkholderia gladioli and its pv. cocovesum, the PCR method comprises the following steps:

[0019] S1: using the primer set to perform PCR amplification on the DNA of the sample to be tested;

[0020] S2: Perform gel electrophoresis to detect the amplified product;

[0021] S3: Observe whether there is a single amplification band at the position of the product size corresponding to each primer pair in the primer set. If so, it indicates that the sample to be tested contains the corresponding target bacteria; if no corresponding single amplification band appears, the sample to be tested does not contain the corresponding target bacteria; the target bacteria is Burkholderia gladioli or Burkholderia gladioli pv. cocos venom; the PCR amplification system in S1 includes PCR reaction buffer, template DNA, PCR polymerase, dNTP, MgCl2, primer set and sterile double-distilled water.

[0022] Generally, a PCR system contains only one set of primers; by setting up multiple PCR systems, the DNA of a single bacterium or two bacteria can be amplified using different primers to improve detection efficiency.

[0023] As a preferred embodiment of the method for identifying Burkholderia gladiolus and its pathogenic variants of B. cocovenenum, the PCR amplification system is: 2.5 μL of 10×PCR reaction buffer, 2.0 μL of 25 mM MgCl2, 1.0 μL of 2.5 mM dNTPs, 1-2 μL of template DNA, 1 μL each of 5 μM upstream and downstream primers, 1 U of Taq enzyme, and sterile double-distilled water to make up the volume to 25 μL; the PCR amplification program in S1 is: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s; annealing at 60-64°C for 30 s; extension at 72°C for 30 s; denaturation, annealing, and extension for a total of 30-35 cycles; and finally extension at 72°C for 10 min.

[0024] As a preferred embodiment of the method for identifying Burkholderia gladioli and its pv. cocosvenorii of the present invention, the qPCR method comprises the following steps:

[0025] S1: using one of the primers in the set to perform PCR amplification on the sample DNA on a fluorescence quantitative amplification instrument;

[0026] S2: Use corresponding fluorescence quantitative software to analyze whether the amplification result is consistent with expectations. If a fluorescent signal is generated, it indicates that the test sample contains the target bacteria; if no fluorescent signal is generated, it indicates that the test sample does not contain the corresponding target bacteria; the target bacteria is Burkholderia gladioli or Burkholderia gladioli pv. cocosvenorensis; the qPCR amplification system in S1 includes 2×TB Green Premix, template DNA, primer set and sterile double-distilled water.

[0027] Generally, a qPCR system contains only one set of primers; by setting up multiple qPCR systems, the DNA of a single bacterium or two bacteria can be amplified using different primers to improve detection efficiency.

[0028] As a preferred embodiment of the method for identifying Burkholderia gladioli and its pathogenic variants of B. cocovenensis described in the present invention, the qPCR amplification system is: 10 μL of 2×TB Green Premix reaction solution, 100 ng of template DNA, 1 μL each of 10 μmol / L upstream and downstream primers, and sterile double-distilled water to make up the volume to 20 μL; the qPCR amplification program in S1 is: pre-denaturation at 95°C for 10 min; denaturation at 95°C for 5 s, annealing at 62°C for 60 s, and 45 cycles of denaturation and annealing.

[0029] Beneficial effects of the present invention: The present invention discloses specific nucleotide sequences, related primer sets, and corresponding PCR and quantitative qPCR detection methods for identifying Burkholderia gladioli and its p. cocovenenum. Compared with the existing technology, the present invention can detect Burkholderia gladioli without physiological and biochemical identification, and can also make up for the defect that biochemical tests cannot identify highly virulent Burkholderia gladioli p. cocovenenum. It has the advantages of short detection time, low cost, simple operation, and high specificity. At the same time, the detection method of the present invention can make up for the defects of existing molecular detection and molecular typing methods for Burkholderia gladioli, which have poor specificity and lack of highly virulent Burkholderia gladioli p. cocovenenum. The detection results are more accurate, the result judgment is simpler, and the practicality is stronger. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The electrophoresis results for specific evaluation of the PCR detection method established by the primer pair SEQ ID NO. 4-5 designed based on the conserved sequence SEQ ID NO. 1 in Example 3.

[0031] Figure 2The electrophoresis results for the specific evaluation of the PCR detection method established by the primer pair SEQ ID NO. 6-7 designed based on the conserved sequence SEQ ID NO. 2 in Example 3.

[0032] Figure 3 These are the electrophoresis results for the specificity evaluation of the PCR detection method for the highly toxic gladiolus Burkholderia cereus pv. cocovenenans in Example 4.

[0033] Figure 4 This is the sensitivity evaluation result of the qPCR detection method for Burkholderia gladiolus in Example 5.

[0034] Figure 5 These are the sensitivity evaluation results of the qPCR detection method for the highly toxic gladiolus Burkholderia cereus pv. cocovenenans in Example 6. DETAILED DESCRIPTION

[0035] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0036] Example 1: Mining of new molecular targets specific to Burkholderia gladioli and highly virulent Burkholderia gladioli pathogenic variants of Cocos nucifera

[0037] Bioinformatics analysis was performed based on the GenBank database and the whole genome DNA sequence of Burkholderia gladioli tested by our team; specific gene fragments of Burkholderia gladioli and highly toxic Burkholderia gladioli pathovar cocovesum were screened and obtained. The nucleotide sequences of the gene fragments are shown in SEQ ID NO.1-SEQ ID NO.3.

[0038] Among them, the sequences SEQ ID NO.1 to 2 are used to identify Burkholderia gladioli; the sequence SEQ ID NO.3 is used to identify the highly toxic Burkholderia gladioli pathovar cocovenom.

[0039] Example 2 Rapid Detection Method for Burkholderia gladioli and Highly Toxic Burkholderia gladioli Pv. cocovenenum

[0040] 1) Primer design

[0041] According to the sequences SEQ ID NO. 1 to 3 in Example 1, a specific PCR amplification primer set (including a forward primer and a reverse primer) was designed. The primer set sequence is shown in Table 1 below.

[0042] Table 1 Specific PCR detection primer sets

[0043]

[0044] 2) PCR method for distinguishing Burkholderia gladioli and highly virulent Burkholderia gladioli pv. cocovenenum, the steps are as follows:

[0045] Preparation of S1 DNA template: Burkholderia gladioli and highly virulent Burkholderia gladioli pv. cocovenenum were cultured in BHI liquid medium, and their bacterial genomic DNA was extracted using a commercial bacterial genomic DNA extraction kit to serve as the test template.

[0046] S2 PCR amplification: using one of the primer sets 1 to 3 to perform PCR amplification on the DNA of the sample to be tested;

[0047] ①PCR detection system:

[0048]

[0049] in:

[0050] When the template DNA is used to identify the presence of Burkholderia gladioli, the primers are any of primer sets 1 to 2;

[0051] When the template DNA is used to identify the presence of highly virulent gladiolus Burkholderia spp. pv. cocovenenum, the primers are those of primer set 3;

[0052] ②PCR amplification procedure:

[0053]

[0054] S3: Run the PCR amplification product on gel electrophoresis to observe whether amplification bands are present at positions corresponding to the product size of each primer set. If amplification bands are present, the sample contains the target bacteria. If no amplification bands are present, the sample does not contain the target bacteria.

[0055] 3) Quantitative qPCR method for distinguishing Burkholderia gladioli and highly virulent Burkholderia gladioli pv. cocovenenum, as follows:

[0056] Preparation of S1 DNA template: Burkholderia gladioli and highly virulent Burkholderia gladioli pv. cocovenenum were cultured in BHI liquid medium, and their bacterial genomic DNA was extracted using a commercial bacterial genomic DNA extraction kit to serve as the test template.

[0057] S2 qPCR amplification: using one of the primer sets 1 to 3 to perform PCR amplification on the test sample DNA on a fluorescence quantitative amplification instrument;

[0058] ③qPCR detection system:

[0059]

[0060] in:

[0061] When the template DNA is used to identify the presence of Burkholderia gladioli, the primers are any of primer sets 1 to 2;

[0062] When the template DNA is used to identify the presence of highly virulent gladiolus Burkholderia spp. pv. cocovenenum, the primers are those of primer set 3;

[0063] ④qPCR amplification procedure:

[0064]

[0065] S3: Use appropriate fluorescence quantitative software to analyze the amplification results to see if they meet expectations. If a fluorescent signal is generated, provided the blank control is absent, then the sample contains the target bacteria. If no fluorescent signal is generated, then the sample does not contain the target bacteria. The concentration of the target bacteria in the system is quantitatively determined by determining the intensity of the fluorescent signal.

[0066] Example 3 Specificity Evaluation of PCR Detection Method for Burkholderia Gladiolus

[0067] 127 strains of Burkholderia gladioli (including 56 strains of Burkholderia gladioli pv. cocovenensis), 24 strains of Burkholderia, and 59 strains of non-Burkholderia (including Pseudomonas aeruginosa, Bacillus cereus, Bacillus subtilis, Enterococcus faecalis, Cronobacter sakazakii, Staphylococcus aureus, and Salmonella) were tested by PCR according to the method of Example 2. The S1 DNA template was prepared by extracting genomic DNA from each bacterium; and the primers used in the S2 PCR amplification were any of primer sets 1 and 2.

[0068] The gel results of PCR amplification products are as follows Figure 1 , M is the DL2000 DNA standard marker, 1-127 are gladiolus Burkholderia strains (including 56 gladiolus Burkholderia pv. cocovetoxin), 128-211 are non-gladiolus Burkholderia strains, and C is the negative control.

[0069] The strains and test results are shown in Table 2 below; in the table, "+" in the test result column indicates positive, and "-" indicates negative.

[0070] Table 2 Specificity evaluation test results of gladiolus Burkholderia detection of the present invention

[0071]

[0072]

[0073]

[0074]

[0075]

[0076] Depend on Figure 1 、 Figure 2 As shown in Table 2, the primer set showed specific amplification bands for all gladiolus Burkholderia strains, while no specific bands were found for non-gladiolus Burkholderia strains, indicating that the method of the present invention has high specificity.

[0077] Example 4 Evaluation of the specificity of the PCR detection method for highly toxic gladiolus Burkholderia cereus pv. cocovenenum

[0078] 127 strains of gladiolus Burkholderia, including 56 strains of gladiolus Burkholderia pv. cocovetoxin, 24 strains of Burkholderia and 59 strains of non-Burkholderia (including Pseudomonas aeruginosa, Bacillus cereus, Bacillus subtilis, Enterococcus faecalis, Cronobacter sakazakii, Staphylococcus aureus, Salmonella, etc.) were taken and PCR detection was performed according to the method of Example 2. Among them, S1 DNA template preparation was to extract genomic DNA of each bacteria respectively; during S2 PCR amplification, the primers used were primers of primer set 3. A control group was set, and the template of the control group was an aqueous solution without genome.

[0079] The gel results of PCR amplification products are as follows Figure 3 As shown; M is the DL2000 DNA standard marker, 1-127 are gladiolus Burkholderia strains (including 56 gladiolus Burkholderia pv. cocovetoxin), 128-211 are non-gladiolus Burkholderia strains, and C is the negative control.

[0080] The strains used and the test results are shown in Table 3 below; in the table, "+" in the test result column indicates positive, and "-" indicates negative.

[0081] Table 3 Specificity evaluation test results of the gladiolus Burkholderia cocos parasite detection of the present invention

[0082]

[0083]

[0084]

[0085]

[0086]

[0087] Depend on Figure 3 As shown in Table 3, the detection results of the primer set 3 showed specific amplification bands only for Burkholderia gladioli pv. cocovetoxin, while the non-toxigenic Burkholderia gladioli and non-Gladioli Burkholderia strains showed no specific bands, indicating that the method of the present invention has high specificity.

[0088] Example 5 Sensitivity Evaluation of qPCR Detection Method for Burkholderia Gladiolus

[0089] Cultivate to a concentration of 10 8 CFU / mL of Burkholderia gladioli standard strain CICC 10574 was diluted 10-fold with deionized water to obtain a concentration of 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 CFU / mL of the pure culture of the strain was used, and the DNA template was extracted according to Example 2, which was the detection standard for Burkholderia gladioli qPCR. The qPCR reaction was performed according to Example 2, and three parallel experiments were performed for each template.

[0090] Draw a standard curve: use the logarithm of the concentration of the pure culture of the standard strain as the horizontal axis and the corresponding real-time Ct value of qPCR as the vertical axis. The fitted curve is the standard curve of Burkholderia gladioli.

[0091] Wet rice noodles were sterilized by ultraviolet light to prepare sterile samples. The results of culture on NA nutrient agar plates showed that no microorganisms were present in the treated wet rice noodles samples, ensuring that the microorganisms in the wet rice noodles samples in subsequent experiments were all from artificial contamination.

[0092] The NA counting plate results showed that the initial concentration of Burkholderia gladioli in the artificially contaminated sample was 4.6×10 8 CFU / mL. The homogenate of the artificially contaminated sample was diluted 10-fold using 0.85% sterile saline to prepare a 10-fold dilution of the Burkholderia gladioli concentration. 1 CFU / mL~10 8 The artificial contaminated simulated samples were CFU / mL. qPCR reactions were performed according to Example 2 using bacterial genomic DNA extracted from each gradient homogenate as template and sterile distilled water as blank control. Three parallel experiments were performed for each template.

[0093] Depend on Figure 4As shown, the concentration of the gladiolus Burkholderia bacteria solution is 10 2 -10 8 There is a linear relationship between CFU / mL and Ct value, and the detection limit of the primer set is 10 2 CFU / ml. The detection limit of the primer set in the spiked sample was 10 3 CFU / ml.

[0094] Example 6 Evaluation of the sensitivity of the qPCR detection method for Burkholderia cerebroventricularia parasitica

[0095] Cultivate to a concentration of 10 8 CFU / mL of Burkholderia gladioli pv. cocovenenum standard strain ATCC33664 was diluted 10-fold with deionized water to obtain a concentration of 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 The pure culture of the strain with a CFU / mL was used to extract the DNA template according to Example 2, which was the detection standard for qPCR of Burkholderia gladioli pv. cocovenenans. The qPCR reaction was performed according to Example 2, and three parallel experiments were performed for each template.

[0096] Draw a standard curve: use the logarithm of the concentration of the pure culture of the standard strain as the horizontal axis and the corresponding real-time Ct value of qPCR as the vertical axis. The fitting curve is the standard curve of Burkholderia gladioli pv. cocovetoxin.

[0097] Wet rice noodles were sterilized by ultraviolet light to prepare sterile samples. The results of culture on NA nutrient agar plates showed that no microorganisms were present in the treated wet rice noodles samples, ensuring that the microorganisms in the wet rice noodles samples in subsequent experiments were all from artificial contamination.

[0098] The NA counting plate results showed that the initial concentration of Burkholderia gladioli in the artificially contaminated sample was 3.9×10 8 CFU / mL. The homogenate of the artificially contaminated sample was diluted 10-fold using 0.85% sterile saline to prepare a 10-fold dilution containing 10 CFU / mL of Burkholderia cerebroventriculariae. 1 CFU / mL~10 8 The artificial contaminated simulated samples were CFU / mL. qPCR reactions were performed according to Example 2 using bacterial genomic DNA extracted from each gradient homogenate as template and sterile distilled water as blank control. Three parallel experiments were performed for each template.

[0099] Depend on Figure 5 As shown, the concentration of the gladiolus Burkholderia bacteria solution is 10 2 -10 8 There is a linear relationship between CFU / mL and Ct value, and the detection limit of the primer set is 10 2 CFU / ml. The detection limit of the primer set in the spiked sample was 10 3 CFU / ml.

[0100] Example 7 Detection of actual samples

[0101] 100 food samples (including rice, flour, starch products, black fungus, and white fungus) were collected from local supermarkets or farmers' markets in Guangzhou to verify the practical application of the PCR detection method in Example 2. All samples were cultured in GVC enrichment broth for 18-24 hours, and bacterial genomic DNA was extracted using a kit method for detection of Burkholderia gladioli and Burkholderia gladioli pv. cocovesum. At the same time, the target bacteria in the same sample were tested as a control experiment according to the national standard GB4789.29-2020 method: All samples were enriched with GVC enrichment broth for 20-24 hours; a loopful of the enrichment broth was streaked onto mPDA and PCFA plates and selectively isolated and cultured for 24-48 hours; a single colony was picked and inoculated onto egg yolk agar plates for 24-48 hours. A single colony was selected for Gram staining and oxidase testing. Single colonies with iris rings that were Gram-negative, oxidase-negative, and lecithinase-positive were selected and inoculated and purified on PDA for 24 hours; a bacterial lawn was selected for biochemical identification (this step identified Burkholderia gladioli); the strain identified as Burkholderia gladioli was subjected to toxin culture for 5 days, and the toxin was measured by liquid chromatography-mass spectrometry. Based on the toxin results, it was identified as Burkholderia gladioli pathovar cocovetoxin. The test results are shown in Table 4.

[0102] Table 4 Detection of Burkholderia gladioli and Burkholderia gladioli pv. cocovesum in food samples

[0103]

[0104] As shown in Table 4, the present method detected 4 positive samples of Burkholderia gladioli from 100 samples, with a positive rate of 4%. Analysis by food type showed that the detection rates for wood ear mushrooms, white fungus, and starch samples were 20%, 14.29%, and 1.33%, respectively, while no samples of flour were detected. Using the national standard method, 12 strains of Burkholderia gladioli were isolated from the 4 positive samples. Toxigenic culture and toxin assays revealed that one strain (isolated from wood ear mushrooms) had a fumonisin content of 14.9 mg / L, while the levels of the other strains were all <0.015 mg / L. The detection results of the method of the present invention on the gladiolus Burkholderia and gladiolus Burkholderia spp. cocovenenans are consistent with the national standard method, indicating that the method of the present invention is highly reliable. Compared with the national standard method, the method of the present invention can identify gladiolus Burkholderia and / or gladiolus Burkholderia spp. cocovenenans at one time, and has short detection time, low cost, simple operation and strong practicality.

[0105] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present invention. SEQUENCE LISTING <110> Institute of Microbiology, Guangdong Academy of Sciences (Guangdong Microbiological Analysis and Testing Center), Guangdong Huankai Biotechnology Co., Ltd. <120> New molecular targets and rapid detection methods for Burkholderia gladioli and its pathogenic variants <130> 20220530 <160> 9 <170> PatentIn version 3.3 <210> 1 <211> 915 <212> DNA <213> artificial synthesis <400> 1 atgaattggg atgaggtacc gcgtgcgctg cgggatcgtt acaaagcgat ctcgggcgac 60 cgtctcggcg gcatgacgct gcacatactg gaatcgatga acaccggcaa gctgccgact 120 cgacccggta tcgacagcga atcctacgcc ttgttcgccg aacaattcaa ctcgaccctg 180 ctggccgcgc acttcttcga gaacctgatg cacggcgagg accgccgcct ggaaacgacc 240 ggctacgaag cgttccagat cacgatcccg gaacgttatt tccggcaccc gggcctgacc 300 gatgccgcgc cgatgagcaa ggaggaaacc cgcgagatca ggcaggccgt ggacgagacg 360 aaagcacggc tgaatttctc caaggacatg tccttcgtgg cgggccagct ctacaagctc 420 gaattcatct cggtgttctc ttatctcgaa gcctatgtcg acagtctcct cacagaattc 480 gtcgggatga gcaagctgga agccttcagg atggtccgcg acaagggctt gccggaggtc 540 ctccgcttcg cgctggacga gatcgatccg cgcatcctgc gatgtttcgc gctattcgag 600 gaggatgcgc tgaagttcat cgacttctgc catatcgtca ggaaccagca cgtccaccgg 660 ctcggcatta ccacggcgcg cgcctacaag aactacgagg aaggcggctt tctctgccac 720 gaccacttcg cggacagcgg cgaacccgac acgagcttcg cccgcaccaa tttccacttc 780 tgcagctaca ttatccgcgt cggccagccg atcaatctct cggcgatctc ccggcctttc 840 aggttgttcg tgcgcgaact ggccaccatc accgagcact tctgccaatc gcgccgcgcc 900 tcggcggcgg cttga 915 <210> 2 <211> 276 <212> DNA <213> Synthetic <400> 2 atgattcccg gcacgactcc cgatatcgca cccaagacct atcgtttccg cttcccccgc 60 cgttccacgc cctacgtgtt cgcgctctac atggcgacca tcatggcctt cctgatgtgc 120 ctggtgatca ccttcgccga attcggcctg gacgcgcatt acatggaaaa cgtgaagaat 180 gcctatcggg tggcgatgcc ttcggccttc gtctgcgtgc tgatcgtgag gccgttggtg 240 gcgcggctgg tggcctggac cgtgcatccg cattga 276 <210> 3 <211> 1014 <212> DNA <213> Synthetic <400> 3 atgccgccaa tatcatccgc cgatgcgccg acaacgatat tcatgttctc ggggcaaggt 60 tcgcactatt accagatggg acgcgacctg ttcgagcagc gcggtgcgtt ttaccgccac 120 gcgctcgaac tgaacgaact gatccgccag gatctcggcg tctcgatcct cgccgagatc 180 tacgatccgc aacgcaaggc ctcggatcgt ttcgacgcga cgccgccgac ccactgcgcg 240 atcttcctgg tcgagtacgc gctcggtcgc gcgctgatcg aggagggcat ccagcccgat 300 ttcctgctga cagccagcat gggcagcttc gcggcgctcg cgctggcacg ttgcctgacg 360 gtggaggagg cggtaagcgc ggtggtctcg caggcgcgcc tgctggacga attcggtgcg 420 ccgggcggga tcgtcgcggt gccgaacctg ccctgggagc aggtgcgcgc gctggccgag 480 cgcttcgatt gcgatctcgg cgcgatcaac aacctgtccg cgtgcacgct ttcggccgag 540 cgccagcgtc tcggagccgt cgtggcggcc ttgcgcgaat cgaacgcgat ctaccagcag 600 ctcgccgtct cgcgcgcatt ccattcgcgg tggatcgacg cggctcgcgc gccattcctg 660 aacagcatga ccttcacgcg ctggcatgcg ccgtcgatcc cgatcgtctg ctgcgccgat 720 gtcgacgtgc tgcacgagat tcgcagcagt tatctgtggg acgtggtgcg ccagccgatc 780 cgcttcgacg ataccgtgcg catgctggcc ggccggcagc gccgcgcgct gcgtttcatc 840 gacgtggggc cgtccggcac gctcgccacc agcctcaaat tcagcgagac gcgattgccg 900 gtgccttgcg aggttcacgg cgtgctgtcg ccgttcggcg gcaacgtgct ccgctatgcg 960 cgtactgccg ggctgttcgg caaggccgcg cgaggcgtgt cgccggctgt ctga 1014 <210> 4 <211> 20 <212> DNA <213> artificial synthesis <400> 4 accggctacg aagcgttcca 20 <210> 5 <211> twenty one <212> DNA <213> artificial synthesis <400> 5 ccagcttgct catcccgacg a 21 <210> 6 <211> twenty one <212> DNA <213> artificial synthesis <400> 6 atgattcccg gcacgactcc c 21 <210> 7 <211> twenty three <212> DNA <213> artificial synthesis <400> 7 acccgatagg cattcttcac gtt 23 <210> 8 <211> twenty one <212> DNA <213> artificial synthesis <400> 8 cggcgcgatc aacaacctgt c 21 <210> 9 <211> twenty three <212> DNA <213> artificial synthesis <400> 9 accacgtccc acagataact gct 23

Claims

1. The use of a nucleotide sequence combination as a detection target in identifying Burkholderia gladioli and its p. cocovetoxin pathogenic variants, characterized in that: The nucleotide sequence combination is shown in SEQ ID NO.1 to SEQ ID NO.3; the application is for non-disease diagnosis and treatment purposes; The primer set shown in SEQ ID NO.4 and SEQ ID NO.5 for detecting the sequence SEQ ID NO.1 is used to identify Burkholderia gladiolus; The primer set shown in SEQ ID NO.6 and SEQ ID NO.7 for detecting the sequence SEQ ID NO.2 is used to identify Burkholderia gladiolus; The primer set shown in SEQ ID NO.8 and SEQ ID NO.9 for detecting the sequence SEQ ID NO.3 is used to identify Burkholderia gladioli pv. cocovenenans.

2. A primer set for identifying Burkholderia gladioli and its pathogenic variants, characterized in that: The primer set is designed based on the nucleotide sequence combination according to claim 1; the sequence of the primer set from 5' to 3' is shown in SEQ ID NOs. 4 to 9; wherein: SEQ ID NO.4 and SEQ ID NO.5 are primer sets corresponding to the sequence of SEQ ID NO.1, used to identify Burkholderia gladiolus; SEQ ID NO.6 and SEQ ID NO.7 are primer sets corresponding to the sequence of SEQ ID NO.2, used to identify Burkholderia gladiolus; SEQ ID NO.8 and SEQ ID NO.9 are primer sets corresponding to the sequence of SEQ ID NO.3, which are used to identify Burkholderia gladioli pv. cocovenenans.

3. A kit for identifying Burkholderia gladioli and its pathogenic variants, characterized in that: The kit comprises the primer set according to claim 2.

4. A method for identifying Burkholderia gladioli and its pathogenic variants for purposes other than disease diagnosis and treatment, characterized in that: The method includes a PCR method and a quantitative qPCR method; the PCR method includes the following steps: S1: performing PCR amplification on the sample DNA using the primer set as described in claim 2; S2: Perform gel electrophoresis to detect the amplified product; S3: Observe whether a single amplified band exists at the position of the product size corresponding to each primer pair in the primer set. If so, it indicates that the test sample contains the corresponding target bacteria; if no corresponding single amplified band appears, the test sample does not contain the corresponding target bacteria; the target bacteria is Burkholderia gladioli or Burkholderia gladioli pv. cocovetoxin; The PCR amplification system in S1 includes PCR reaction buffer, template DNA, PCR polymerase, dNTP, MgCl2, primer set and sterile double-distilled water.

5. The method according to claim 4, characterized in that The PCR amplification system is as follows: 2.5 μL of 10× PCR reaction buffer, 2.0 μL of 25 mM MgCl2, 1.0 μL of 2.5 mM dNTP, 1-2 μL of template DNA, 1 μL each of 5 μM upstream and downstream primers, 1 U of Taq enzyme, and sterile double-distilled water to make up the volume to 25 μL; the PCR amplification program in S1 is as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s; annealing at 60-64°C for 30 s; extension at 72°C for 30 s; denaturation, annealing, and extension for a total of 30-35 cycles; and finally extension at 72°C for 10 min.

6. The method according to claim 4, characterized in that The qPCR method includes the following steps: S1: using one of the primers in the primer set as described in claim 2 to perform PCR amplification on the sample DNA to be tested on a fluorescence quantitative amplification instrument; S2: using corresponding fluorescence quantitative software to analyze whether the amplification results are consistent with expectations; if a fluorescent signal is generated, it indicates that the test sample contains the target bacteria; if no fluorescent signal is generated, it indicates that the test sample does not contain the corresponding target bacteria; the target bacteria is Burkholderia gladioli or Burkholderia gladioli pv. cocovetoxin; The qPCR amplification system in S1 includes 2× TB Green Premix, template DNA, primer set and sterile double-distilled water.

7. The method according to claim 6, characterized in that The qPCR amplification system was as follows: 10 μL of 2× TB Green Premix reaction solution, 100 ng of template DNA, 1 μL each of 10 μmol / L upstream and downstream primers, and sterile double-distilled water to make up the volume to 20 μL. The qPCR amplification program in S1 was as follows: pre-denaturation at 95°C for 10 min; denaturation at 95°C for 5 s, annealing at 62°C for 60 s, and 45 cycles of denaturation and annealing.

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