Molecular markers for the identification of brucella and use thereof

Abstract

This invention relates to the field of biotechnology, specifically to a molecular marker for identifying Brucella and its application. Based on the Brucella-specific LysR gene sequence, this invention designs a pair of PCR amplification primers: an upstream primer 5'-CTGGACCTCCGCCAAGTA-3' and a downstream primer 5'-CATCGCCAGCCATGTTTC-3'. These primers exhibit high specificity, yielding a specific 124bp band via PCR amplification, with a detection limit of 1.563 ng / μL using conventional PCR methods. This specific detection primer and related detection method can be applied to the rapid detection of Brucella, providing early diagnosis for Brucella infection and showing great promise for future applications.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically to a molecular marker for identifying Brucella and its application. Background Technology

[0002] Brucella is an α-2 Proteus species, rod-shaped, aerobic, Gram-negative bacterium that can produce acids from several carbohydrates and degrade nitrates and nitrites. It moves using abundant flagella around its head. Brucella was first discovered in 1988 when it was isolated from human clinical specimens (usually blood). Currently, Brucella is found in various environments, including water, soil, plants, and animals. In recent years, literature has reported that human Brucella can cause illness in immunocompetent individuals, thus classifying it as a zoonotic pathogen. Some strains are highly virulent, causing local abscesses and even septic shock. Research on detection methods for human Brucella is of great significance for human production and living environments, public health, and the sustainable development of animal husbandry.

[0003] Polymerase chain reaction (PCR) is a commonly used laboratory diagnostic method known for its speed. While real-time quantitative PCR, loop-mediated isothermal amplification, and droplet digital PCR offer high sensitivity, the reagents and consumables used are generally more expensive than those for conventional PCR, resulting in higher testing costs. Furthermore, these methods require specialized (and expensive) instruments, hindering widespread adoption and causing significant inconvenience. Therefore, this invention provides a conventional PCR detection method that offers high specificity and sensitivity, lower testing costs, and ease of implementation. Summary of the Invention

[0004] The first aspect of the present invention aims to provide a protein biomarker for detecting Brucella.

[0005] A second aspect of the present invention aims to provide genetic markers for detecting Brucella.

[0006] The third aspect of this invention aims to provide the application of the protein markers of the first aspect of this invention and the gene markers of the second aspect of this invention.

[0007] The fourth aspect of this invention is to provide a product.

[0008] The fifth aspect of this invention aims to provide a reagent or kit.

[0009] The sixth aspect of this invention aims to provide the application of the product of the fourth aspect of this invention and the reagent or kit of the fifth aspect of this invention.

[0010] The seventh aspect of this invention aims to provide a method.

[0011] To achieve the above-mentioned objectives of this invention, the technical solution adopted by this invention is as follows:

[0012] In a first aspect, the invention provides a protein biomarker for detecting Brucella, said protein biomarker comprising an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% homology to SEQ ID NO:1.

[0013] A second aspect of the invention provides a genetic marker for detecting Brucella, said genetic marker comprising a gene sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% homology to SEQ ID NO:2.

[0014] Preferably, the Brucella includes Brucella sp. WY7; more preferably, the Brucella is Brucella. Brucella sp. WY7 (CP049796–CP049798).

[0015] A third aspect of the present invention provides the use of a substance for detecting the protein marker of the first aspect of the present invention and / or the gene marker of the second aspect of the present invention in at least one of (a1) to (a6):

[0016] (a1) Identification of Brucella for non-diagnostic purposes;

[0017] (a2) Preparation of products for identifying Brucella;

[0018] (a3) Screening for Brucella as a non-diagnostic purpose;

[0019] (a4) Prepare products for screening Brucella;

[0020] (a5) Detection of Brucella for non-diagnostic purposes;

[0021] (a6) Prepare products for detecting Brucella.

[0022] Preferably, the substance comprises a substance selected from one or more detection techniques or methods from the group consisting of: Northern blotting, PCR, gene chip method, nucleic acid sequencing, protein chip method, and immune cross-linking method.

[0023] Preferably, the product comprises at least one of reagents, reagent kits, test strips, chips, and systems.

[0024] Preferably, the Brucella includes Brucella bacteria. Brucellasp. WY7; More preferably, the Brucella is Brucella. Brucella sp. WY7 (CP049796–CP049798).

[0025] A fourth aspect of the present invention provides a product comprising a substance for detecting protein markers of the first aspect of the present invention and / or gene markers of the second aspect of the present invention.

[0026] Preferably, the substance comprises a substance for use in one or more detection techniques or methods selected from the group consisting of: Northern blotting, PCR, gene chip method, nucleic acid sequencing, protein chip method, and immune cross-linking method.

[0027] Preferably, the product comprises at least one of reagents, reagent kits, test strips, chips, and systems.

[0028] Preferably, the product comprises primer pairs for amplifying the gene markers described in the second aspect of the present invention.

[0029] Preferably, the primer pair used to amplify the gene marker of the second aspect of the present invention has the sequences shown in SEQ ID NO: 5, 6.

[0030] A fifth aspect of the present invention provides a reagent or kit comprising the primer pairs described in the fourth aspect of the present invention.

[0031] Preferably, the kit further comprises DNA polymerase, dNTPs, PCR buffer, and Mg. 2+ At least one of them.

[0032] A sixth aspect of the invention provides the application of any one of (1) to (2) in any one of (b1) to (b3);

[0033] (1) The product described in the fourth aspect of the present invention;

[0034] (2) The reagent or kit described in the fifth aspect of the present invention;

[0035] (b1) Identification of Brucella for non-diagnostic purposes;

[0036] (b2) Screening for Brucella for non-diagnostic purposes;

[0037] (b3) Detection of Brucella for non-diagnostic purposes.

[0038] A seventh aspect of the present invention provides a method according to any one of (c1) to (c3):

[0039] (c1) A method for identifying Brucella, comprising detecting whether the proteome and / or genome of a test sample contains the protein markers described in the first aspect of the present invention and / or the gene markers described in the second aspect of the present invention; if the genome of the test sample contains the protein markers described in the first aspect of the present invention and / or the gene markers described in the second aspect of the present invention, then the test sample is Brucella; otherwise, it is not.

[0040] (c2) A method for screening Brucella, wherein the proteome and / or genome of the sample to be tested contain the protein markers described in the first aspect of the present invention and / or the gene markers described in the second aspect of the present invention, and the sample to be tested contains the protein markers described in the first aspect of the present invention and / or the gene markers described in the second aspect of the present invention in its genome;

[0041] (c3) A method for detecting Brucella, wherein the test sample proteome and / or genome contains the protein markers described in the first aspect of the present invention and / or the gene markers described in the second aspect of the present invention, wherein if the genome of the test sample contains the protein markers described in the first aspect of the present invention and / or the gene markers described in the second aspect of the present invention, then the test sample contains Brucella; otherwise, it does not contain it.

[0042] Preferably, the method is used for non-diagnostic and non-therapeutic purposes.

[0043] Preferably, the method for detecting whether the genome of the sample to be tested contains the gene marker described in the second aspect of the present invention is as follows: the DNA of the sample to be tested is mixed with the primer pair in the reagent or kit described in the fifth aspect of the present invention, and PCR detection is performed.

[0044] In some embodiments of the present invention, the PCR detection includes the following steps:

[0045] ① Extract DNA from the sample;

[0046] ② Using the DNA extracted in step ① as a template, PCR was performed using a specific primer set;

[0047] ③ Detect the PCR amplification products.

[0048] In some embodiments of the present invention, step ① uses the EZNA® Bacterial DNA Kit (Omega) to extract genomic DNA from the sample.

[0049] In some embodiments of the present invention, the PCR in step ② uses 2×T5 Super PCR Mix (Colony) reagent (TSINGKE), and the 20 μL reaction system includes: 10 μL of 2×T5 Super PCR premix (containing dye), 1 μL each of upstream and downstream primers (concentration 10 μM), 1 μL of template DNA (10-30 ng / μL), and 7 μL of ddH2O. The sequences of the primer pairs used are shown in SEQ ID NO: 5 and 6.

[0050] In some embodiments of the present invention, the PCR amplification program in step ② is as follows: 98℃ pre-denaturation for 4 min 30 s; 98℃ denaturation for 10 s, 60℃ annealing for 13 s, 72℃ extension for 10 s, for a total of 30 cycles; 72℃ extension for 2 min.

[0051] In some embodiments of the present invention, the detection step in step ③ is: detecting the PCR amplification product by 1% agarose gel electrophoresis.

[0052] In some embodiments of the present invention, if the PCR in step ③ can amplify a specific band of the correct length of 124 bp, it indicates that the Brucella is present in the test sample.

[0053] The beneficial effects of this invention are:

[0054] This invention designs a pair of PCR amplification primers based on the Brucella WY7 specific fragment LysR gene sequence: upstream primer 5'-CTGGACCTCCGCCAAGTA-3' and downstream primer 5'-CATCGCCAGCCATGTTTC-3'. These primers exhibit high specificity, yielding a specific 124 bp band via PCR amplification, with a detection limit of 1.563 ng / μL using conventional PCR methods. These specific detection primers and related detection methods can be applied to the rapid detection of Brucella, providing early diagnosis for Brucella infections and demonstrating excellent application prospects. Attached Figure Description

[0055] Figure 1 for Brucella streak diagram of sp. WY7 on LB medium plate.

[0056] Figure 2 For 16S rRNA gene sequence pair Brucella GBDP phylogenetic tree constructed using sp. WY7.

[0057] Figure 3 for Brucella sp. WY7 comparative genome Venn diagram.

[0058] Figure 4 For the specific detection of Brucella LysR gene by PCR, M: DS2000 Marker; 1: Brucella WY7-DNA1; 2: Brucella WY7-DNA2; 3: Human Brucella ATCC49188; 4: Human Brucella MCCC 1K05807; 5: Brucella wheat; 6: Acinetobacter johnsonii; 7: Rhodococcus rubrum; 8: Acinetobacter baumannii; 9: Water (negative).

[0059] Figure 5 For PCR sensitivity testing of the Brucella LysR gene, M: DS2000 Marker; DNA concentrations of 1-8 were: 50 ng / μL, 25 ng / μL, 12.5 ng / μL, 6.25 ng / μL, 3.125 ng / μL, 1.563 ng / μL, 0.781 ng / μL, and 0.391 ng / μL, respectively.

[0060] Figure 6 PCR detection using primers specific to the Brucella 2066 gene. M: DS2000 Marker; 1: Brucella WY7-DNA1; 2: Brucella WY7-DNA2; 3: Human Brucella ATCC49188; 4: Human Brucella MCCC 1K05807; 5: Brucella wheat; 6: Brucella sparrowii; 7: Escherichia coli; 8: Staphylococcus aureus; 9: Haloxylon ammodendron; 10: Acinetobacter johnsonii; 11: Bacillus; 12: Water (negative). Detailed Implementation

[0061] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0062] Example 1: Isolation and morphological characteristics of Brucella strains

[0063] Brucella of the present invention Brucella sp. WY7 (CP049796–CP049798) was isolated from Antarctic krill samples (58–60°W, 62–64°S) obtained from the East China Sea Fisheries Research Institute in November 2016.

[0064] This invention targets a strain of Brucella closely related to humans, isolated from Antarctic krill in 2016. BrucellaSpecific detection was performed on sp. WY7, and the genome information of this Brucella has been uploaded to NCBI with accession numbers: CP049796–CP049798.

[0065] Its separation operation is shown in Table 1, and its morphological structure is as follows: Figure 1 As shown.

[0066] Table 1. Procedures for strain isolation

[0067]

[0068] Example 2 Molecular identification of Brucella strains

[0069] Using the extracted total DNA from the bacterial strain as a template, PCR was performed using the universal 16S rDNA primers 27F and 1492R to amplify the bacterial 16S rDNA sequence. The amplified fragment was approximately 1500 bp in length.

[0070] The primer sequences are:

[0071] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO: 3).

[0072] 1492R: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO: 4).

[0073] The obtained PCR products were detected by 1% agarose gel electrophoresis and sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. Sequencing results were compared using NCBI BLASTn. A 16S rDNA phylogenetic tree was constructed using the Type (Strain) Genome Server with the GBDP method. Figure 2 This indicates that WY7 belongs to the Brucella genus.

[0074] Example 3 Screening of Brucella molecular markers

[0075] Brucella WY7 was cultured, and DNA was extracted and sent to Shanghai Ouyi Biotechnology Co., Ltd. for whole-genome sequencing. Based on third-generation sequencing data, the Brucella WY7 genome was reassembled from scratch. Comparative pan-genome analysis was used. Figure 3 Genes present only in Brucella WY7 were selected, and gene1983, which has low similarity to Brucella, was screened. The protein sequences of the selected genes to be tested were compared using NCBI BLASTp to obtain the specific protein sequence: a LysR family transcriptional regulatory factor, recorded as LysR. The amino acid sequence is shown in SEQ ID NO: 1, and the corresponding nucleotide sequence is shown in SEQ ID NO: 2.

[0076] The amino acid sequence of LysR is (SEQ ID NO: 1):

[0077] LDIEDLRTFVEVADTGGVSPASRRLGISKSMVSRRLIRLEKELGVQLLARTTRGAALTEAGITFREHAAKVCAEIDIAKETISPAGDLRGRLRVAAPLTFGPTHFAPVLASLAQRHPELQVQASYTDRFVDLIAEGFDCAIRVGHLADSNMLARRVGWTSAKYVATPDYIKKYGSPETPEEFLMHEVVMQGTETWLAMDGDKIITLKPQGRFKADNAVALVAAALAGIGLAGLPEELIREHLASGALVPVMTRYPPPRLGIYVIRPPGQHPARKIRVLTEILIECFEDFSADREN。

[0078] The nucleotide sequence of LysR is (SEQ ID NO: 2):

[0079] TTGGACATCGAAGATTTACGAACCTTCGTGGAGGTTGCTGATACTGGCGGGGTTTCTCCCGCCTCGCGGCGGCTCGGCATCTCGAAGTCGATGGTCAGTCGGCGGCTTATTCGGCTGGAAAAGGAATTGGGCGTGCAGTTGCTCGCTCGCACGACGCGCGGCGCCGCGCTCACGGAGGCTGGAATCACCTTTCGGGAGCACGCCGCAAAAGTCTGCGCCGAGATCGACATTGCCAAGGAGACGATATCGCCCGCCGGCGATCTTCGCGGTCGGCTAAGAGTAGCAGCTCCGCTTACGTTCGGTCCGACGCACTTCGCGCCGGTGCTCGCGTCCCTGGCACAGCGCCATCCGGAGTTGCAGGTTCAGGCCAGCTATACTGATCGGTTCGTTGACCTGATCGCAGAAGGGTTCGACTGCGCGATAAGGGTCGGTCATCTTGCCGACTCGAATATGCTTGCAAGACGGGTCGGCTGGACCTCCGCCAAGTACGTTGCAACACCGGACTACATCAAAAAGTACGGCTCCCCCGAGACGCCAGAGGAATTCCTTATGCACGAAGTCGTCATGCAGGGAACTGAAACATGGCTGGCGATGGATGGGGATAAGATCATCACTTTGAAGCCGCAGGGCCGCTTCAAAGCCGACAATGCCGTGGCTCTTGTGGCCGCAGCACTTGCGGGGATTGGCCTTGCCGGTCTTCCTGAAGAATTGATCAGGGAGCATCTGGCTTCCGGCGCGTTGGTCCCGGTCATGACACGTTATCCGCCGCCTAGACTTGGCATCTATGTGATCCGGCCGCCGGGGCAGCACCCTGCACGCAAGATACGGGTGCTCACCGAGATTCTAATCGAGTGTTTTGAAGACTTTTCGGCGGATCGTGAAAACTAG。

[0080] Example 4 Design of Brucella PCR Primers

[0081] Based on the Brucella WY7 specific fragment LysR gene sequence and gene2066, conventional PCR specific primer sets were designed, and the primer sequences are shown in Table 2.

[0082] Table 2. Brucella PCR primer design

[0083]

[0084] Brucella WY7 was cultured, and DNA was extracted as a template for detection. The PCR reaction used a 20 μL reaction system: 10 μL of 2×T5Super PCR premix (containing dye), 1 μL each of forward and reverse primers (10 μM concentration), 1 μL of template DNA (10-30 ng / μL), and 7 μL of ddH2O. The PCR amplification program was: 98℃ pre-denaturation for 4 min 30 s; 98℃ denaturation for 10 s, 60℃ annealing for 13 s, 72℃ extension for 10 s, for a total of 30 cycles; 72℃ extension for 2 min.

[0085] Both primer pairs were used for bacterial strain detection and identification, and the results were as follows: Figure 4 , 6 The results showed that all primers could amplify the correct bands in Brucella WY7. However, only primers Bru-LysR-F and Bru-LysR-R showed the specificity of the detection primers and were detected only in Brucella WY7. No similar bands were found in other bacterial species, indicating that they had better detection performance.

[0086] Example 5: Specific detection of Brucella PCR primers

[0087] The template DNAs used for specific detection were: Brucella WY7-DNA1, Brucella WY7-DNA2 (DNA1 and 2 are purified DNA extracted from different Brucella WY7 colonies), Brucella humanis (ATCC49188), Brucella humanis (MCCC 1K05807), Brucella wheat germ (MCCC 1A17435), Acinetobacter johnsonii (MCCC 1A01872), Rhodococcus rubrum (MCCC 1A18432), and Acinetobacter baumannii (JCM 6841).

[0088] PCR amplification was performed using the upstream primer 5'-CTGGACCTCCGCCAAGTA-3' (Bru-LysR-F, SEQ ID NO: 5) and the downstream primer 5'-CATCGCCAGCCATGTTTC-3' (Bru-LysR-R, SEQ ID NO: 6).

[0089] The PCR reaction used a 20 μL reaction system: 10 μL of 2×T5 Super PCR premix (containing dye), 1 μL each of forward and reverse primers (10 μM concentration), 1 μL of template DNA (10-30 ng / μL), and 7 μL of ddH2O. The PCR amplification program was as follows: 98℃ pre-denaturation for 4 min 30 s; 98℃ denaturation for 10 s, 60℃ annealing for 13 s, 72℃ extension for 10 s, for a total of 30 cycles; 72℃ extension for 2 min.

[0090] The amplification products were detected by nucleic acid gel electrophoresis, and the results are as follows: Figure 4 As shown, only lanes with Brucella WY7 as the template yielded a bright band of 124 bp in length.

[0091] Example 6 Sensitivity detection of Brucella PCR primers

[0092] Brucella WY7-DNA2 prepared in Example 5 was serially diluted to 50 ng / μL, 25 ng / μL, 12.5 ng / μL, 6.25 ng / μL, 3.125 ng / μL, 1.563 ng / μL, 0.781 ng / μL, and 0.391 ng / μL as detection templates.

[0093] PCR amplification was performed using the upstream primer 5'-CTGGACCTCCGCCAAGTA-3' (SEQ ID NO: 5) and the downstream primer 5'-CATCGCCAGCCATGTTTC-3' (SEQ ID NO: 6). The PCR reaction used a 20 μL reaction system: 10 μL of 2×T5Super PCR premix (containing dye), 1 μL each of the upstream and downstream primers (10 μM concentration), 1 μL of template DNA (10-30 ng / μL), and 7 μL of ddH2O. The PCR amplification program was as follows: 98℃ pre-denaturation for 4 min 30 s; 98℃ denaturation for 10 s, 60℃ annealing for 13 s, 72℃ extension for 10 s, for a total of 30 cycles; 72℃ extension for 2 min.

[0094] The amplification products were detected by nucleic acid gel electrophoresis, and the results are as follows: Figure 5 As shown, the sensitivity of this conventional PCR detection method is 1.563 ng / μL.

[0095] In summary, this invention proposes a method for identifying Brucella bacteria from Antarctic krill. Using conventional PCR to detect the strain's DNA, this method can accurately and rapidly detect Brucella bacteria with high sensitivity.

[0096] This invention uses conventional PCR methods to test the application of this molecular marker in the detection of Brucella. Those skilled in the art can expect that other primers can be designed based on this molecular marker, or other molecular amplification methods, including but not limited to nested PCR, RT-qPCR, RAP, LAMP, etc., can also achieve the detection effect of Brucella.

Claims

1. A primer pair for identifying Brucella, characterized in that: The primer pair consists of upstream primer 5'-CTGGACCTCCGCCAAGTA-3' and downstream primer 5'-CATCGCCAGCCATGTTTC-3'. The Brucella is Brucella. Brucella sp. WY7.

2. A reagent or kit, characterized in that: It includes the primer pair described in claim 1.

3. The reagent or kit according to claim 2, characterized in that: The kit also contains DNA polymerase, dNTPs, PCR buffer, and Mg. 2+ At least one of them.

4. The application of the primer pair of claim 1, the reagent or kit of claim 2 or 3 in the identification and screening of Brucella for non-diagnostic purposes; The Brucella is Brucella. Brucella sp. WY7.

5. A non-diagnostic destination for identifying Brucella. Brucella The method described in sp. WY7 involves PCR detection of the DNA in the sample to be tested using the reagents or kits described in claim 2 or 3; if the result is positive, the sample to be tested is Brucella. Brucella sp. WY7; otherwise, it is not Brucella. Brucella sp. WY7.

6. A non-diagnostic destination screening method for Brucella Brucella The method described in sp. WY7 involves PCR detection of the DNA in the sample to be tested using the reagents or kits described in claim 2 or 3; if the result is positive, the sample to be tested contains Brucella. Brucella sp. WY7; conversely, it does not contain Brucella. Brucella sp. WY7.

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