A method for screening specific molecular markers for microbial tracing
By employing whole-genome alignment screening and homologous recombination technology, a specific molecular marker screening method was established, which solved the problems of specificity and stability in existing microbial traceability technologies. This method achieves high specificity and high sensitivity in microbial traceability and is applicable to the detection needs of multiple fields.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- WUHAN MIAOLING BIOTECHNOLOGY CO LTD
- Filing Date
- 2026-04-13
- Publication Date
- 2026-05-29
AI Technical Summary
In existing microbial traceability technologies, molecular marker screening methods lack specificity, have low sensitivity and poor stability, and are easily affected by closely related microorganisms, leading to inaccurate traceability results.
Unique gene fragments of target microorganisms were screened by whole-genome alignment. Homologous recombination technology and multiple validation systems were combined to design specific primers for PCR amplification, construct recombinant plasmids, establish a positive control system, and ensure the specificity, sensitivity and stability of molecular markers through multiple validations.
It improves the specificity and stability of molecular markers, can accurately distinguish target microorganisms from closely related microorganisms, and is suitable for food safety production, environmental monitoring and clinical diagnosis. The test results are highly accurate and reproducible.
Abstract
Description
Technical Field
[0001] This invention belongs to the field of microbial detection and traceability technology, specifically a specific molecular marker screening method for microbial traceability. Background Technology
[0002] Microbial traceability technology is of great significance in fields such as food safety production, environmental monitoring, and clinical diagnosis. Its core is to identify the source and transmission route of target microorganisms through specific molecular markers. Currently, commonly used molecular marker screening methods mainly include random amplified polymorphic DNA (RAPD), restriction fragment length polymorphism (RFLP), and simple sequence repeats (SSR). However, these methods have problems such as insufficient specificity, low sensitivity, and poor stability, and are easily affected by closely related microorganisms, leading to inaccurate traceability results.
[0003] Homologous recombination, as a highly efficient gene cloning technique, has been widely applied in molecular biology experiments such as plasmid construction. However, its application in the screening of microbial-specific molecular markers has not been reported. Current molecular marker screening techniques often lack systematic validation frameworks, relying solely on simple specificity detection to determine markers, resulting in poor applicability of the markers in practical applications. Therefore, there is an urgent need to develop a specific molecular marker screening method that combines homologous recombination technology with multiple validation systems to improve the specificity, sensitivity, and stability of molecular markers and meet the practical needs of microbial traceability.
[0004] Based on this, a specific molecular marker screening method for microbial tracing was designed. Summary of the Invention
[0005] In view of the above situation and to overcome the shortcomings of the prior art, the present invention provides a specific molecular marker screening method for microbial tracing, which effectively solves the problems mentioned in the background.
[0006] To achieve the above objectives, the present invention provides the following technical solution: a method for screening specific molecular markers for microbial tracing, comprising the following steps:
[0007] Step 1: Microbial genome feature analysis. Genomic DNA of the target microorganism and closely related non-target microorganisms is extracted. The complete genome sequence is obtained using whole-genome sequencing technology. Bioinformatics analysis tools are used to compare and analyze the genomic differences to identify the unique gene fragment regions of the target microorganism.
[0008] Step 2: Specific candidate gene screening. Based on the unique gene fragment regions identified in Step 1, gene fragments with a length of 500-1500 bp are screened as candidate genes. The candidate genes are single copies in the target microbial genome and have no homologous sequences in the genomes of closely related non-target microorganisms.
[0009] Step 3: Homologous arm design and target fragment amplification. Specific primers are designed based on the candidate gene sequence. The 5' end of the primers has a 15-20 bp homologous arm, which is completely complementary to the flanking sequence of the restriction enzyme site of the vector plasmid. The target fragment is amplified using a high-fidelity enzyme PCR system. The PCR reaction system is 20 μl, including 7 μl of sterile water, 10 μl of 2× high-fidelity enzyme, 1 μl of upper primer, 1 μl of lower primer, and 1 μl of template DNA. The PCR program is as follows: denaturation at 94℃ for 5 min, followed by denaturation at 94℃ for 30 s, annealing at 55-65℃ for 1 min, extension at 72℃ for 30-90 s, for a total of 30 cycles, and finally extension at 72℃ for 7 min and cooling at 4℃.
[0010] Step 4: Construction of recombinant plasmid. The vector plasmid was double-digested with EcoRI and BamHI in a 50 μl digestion system, including 50-V sterile water (μl), Cut Buffer (5 μl), vector plasmid (1 μg), EcoRI (1.5 μl), and BamHI (1.5 μl). The reaction was carried out at 37℃ for 30 min. The digested vector was then subjected to homologous recombination with the target fragment amplified in Step 3 in a 10 μl recombination system, including 2× recombinase (5 μl), digested vector (1 μl), and target fragment (4 μl). The ligation was carried out at 50℃ for 30 min to obtain the recombinant plasmid.
[0011] Step 5: Establishment of the positive control system. The recombinant plasmid constructed in Step 4 was extracted using the alkaline lysis method. The specific steps are as follows: E. coli bacterial culture containing the recombinant plasmid was centrifuged at 10,000 rpm for 2 min to collect the bacterial cells. 250 μl of RNase A-containing Buffer P1 was added to suspend the bacterial cells. 250 μl of Buffer P2 was added and gently inverted 4-6 times to lyse the bacterial cells. 350 μl of Buffer N3 was added and inverted, then centrifuged at 10,000 rpm for 15 min. The supernatant was transferred to an adsorption column and washed sequentially with 150 μl of Buffer PB and 400 μl of Buffer PW. After 2 min of free space to remove ethanol, 90 μl of Buffer EB preheated at 65-70℃ was added to elute, and the purified recombinant plasmid was obtained and used as the positive control.
[0012] Step Six: Specificity Verification. The positive control obtained in Step Five, the genomic DNA of the target microorganism, and the genomic DNA of closely related non-target microorganisms are used as templates. PCR amplification is performed using the specific primers designed in Step Three. The amplification results are detected by 1.5% agarose gel electrophoresis. Candidate genes that can amplify specific bands only in the genomic DNA of the target microorganism and the positive control, and that do not amplify bands in closely related non-target microorganisms, are qualified candidate genes with specificity.
[0013] Step 7: Sensitivity detection. The genomic DNA of the target microorganism is serially diluted to obtain template solutions with concentrations of 100 ng / μl, 10 ng / μl, 1 ng / μl, 0.1 ng / μl, and 0.01 ng / μl. PCR amplification is performed using template solutions of each concentration as templates. The lowest detection limit of the specific qualified candidate gene is detected, and candidate genes with a lowest detection limit ≤ 0.1 ng / μl are screened.
[0014] Step 8: Stability assessment. The target microorganism is passaged 10 times under different culture conditions. Genomic DNA is extracted after each passage and PCR amplification is performed using specific primers. The stability of the amplified bands is detected. Candidate genes that can stably amplify specific bands after 10 consecutive passages are qualified candidate genes in terms of stability.
[0015] Step 9: Verify practicality by applying candidate genes with good stability to actual sample testing. The actual samples include food samples, environmental samples, or clinical samples contaminated with the target microorganism. The detection rate of the candidate genes in the actual samples is verified by PCR amplification combined with electrophoresis. Candidate genes with a detection rate ≥95% are considered practically qualified candidate genes.
[0016] Step 10: Standardization and finalization: Identify the practical and qualified candidate genes as specific molecular markers for microbial traceability, clarify their primer sequences, PCR amplification conditions and detection standards, and form a standardized screening scheme.
[0017] Preferably, the bioinformatics analysis tools mentioned in step one include BLAST, ClustalX, and MEGA, which determine the unique gene fragment regions of the target microorganism through multiple sequence alignment.
[0018] Preferably, the annealing temperature of the primers in step three is adjusted according to the primer Tm value, with an adjustment range of 55-65℃. The primer specificity is verified by BLAST to ensure that there are no non-specific binding sites.
[0019] Preferably, the vector plasmid in step four is a pET series plasmid. After enzyme digestion, the digestion efficiency is verified by 1.5% agarose gel electrophoresis to ensure complete digestion.
[0020] Preferably, the purity of the recombinant plasmid extracted by the alkaline lysis method in step five is detected by Nanodrop, and the A260 / A280 ratio is between 1.8 and 2.0 to ensure that there is no protein or RNA contamination.
[0021] Preferably, the specific bands amplified by PCR in step six are verified by sequencing, and the sequencing results show ≥99% homology with the candidate gene sequence.
[0022] Preferably, the gradient dilution in step seven is performed using sterile water, and the dilution process is carried out in a laminar flow hood to avoid cross-contamination.
[0023] Preferably, the different culture conditions in step eight include temperatures of 25°C, 37°C, and 42°C, pH values of 5.0, 7.0, and 9.0, and the culture media are LB medium, nutrient broth medium, and MRS medium.
[0024] Preferably, the pretreatment method for the actual samples in step nine is as follows: food samples are homogenized using a homogenizer and then centrifuged to obtain the supernatant; environmental samples are filtered using a filter membrane and then eluted; and genomic DNA is directly extracted from clinical samples.
[0025] Preferably, the detection criteria in step ten include the specific band size range, electrophoresis detection conditions, and result judgment criteria. The electrophoresis detection conditions are 120V voltage electrophoresis for 25 minutes.
[0026] Compared with the prior art, the beneficial effects of the present invention are:
[0027] 1. This invention screens unique gene fragments of target microorganisms through whole-genome alignment, and combines multiple verifications of specificity, sensitivity, stability and practicality to ensure that the screened molecular markers have high specificity and can effectively distinguish target microorganisms from closely related non-target microorganisms, avoiding cross-reactions;
[0028] 2. The innovative application of homologous recombination technology to the construction of positive control systems has improved the preparation efficiency and stability of control standards, providing a reliable standard for the verification of molecular markers;
[0029] 3. A systematic screening process has been established, with clear operating parameters and judgment criteria for each step, which avoids the influence of uncertain factors and improves the accuracy and repeatability of screening results;
[0030] 4. The selected molecular markers are suitable for the detection of various real samples, with a high detection rate, and can meet the microbial traceability needs of multiple fields such as food safety production, environmental monitoring, and clinical diagnosis, and have broad application prospects. Detailed Implementation
[0031] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments of the present invention. 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.
[0032] This invention provides a specific molecular marker screening method for microbial tracing, comprising the following steps:
[0033] Step 1: Microbial Genome Characterization
[0034] Genomic DNA was extracted from the target microorganism and closely related non-target microorganisms, and the complete genome sequences were obtained using whole-genome sequencing technology. Bioinformatics analysis tools such as BLAST, ClustalX, and MEGA were used to perform multiple alignments of the genome sequences of the target microorganism and closely related non-target microorganisms to analyze genomic differences and identify gene fragment regions unique to the target microorganism. These regions are exclusive to the target microorganism and have no homologous sequences in the genomes of closely related non-target microorganisms.
[0035] Step 2: Specific candidate gene screening
[0036] Based on the unique gene fragment regions identified in Step 1, gene fragments with a length of 500-1500 bp were screened as candidate genes. The screening criteria were: the candidate gene was a single copy in the target microbial genome, and the copy number was verified by real-time quantitative PCR; there were no homologous sequences in the genomes of closely related non-target microorganisms, and this was confirmed by BLAST alignment, ensuring the specificity of the candidate genes.
[0037] Step 3: Homologous arm design and target fragment amplification
[0038] Specific primers were designed based on candidate gene sequences. The 5' end of each primer contained a 15-20 bp homologous arm, which was perfectly complementary to the flanking sequences of the restriction enzyme sites on the vector plasmid, ensuring the efficiency of subsequent homologous recombination. After primer design, BLAST was used to verify primer specificity and avoid non-specific binding sites.
[0039] The target fragment was amplified using a high-fidelity enzyme PCR system. The PCR reaction volume was 20 μl, specifically consisting of: 7 μl sterile water, 10 μl 2× high-fidelity enzyme, 1 μl upper primer, 1 μl lower primer, and 1 μl template DNA. The PCR program was set as follows: denaturation at 94℃ for 5 min, followed by 30 cycles. Each cycle included denaturation at 94℃ for 30 s, annealing at 55-65℃ for 1 min (annealing temperature adjusted according to primer Tm value), extension at 72℃ for 30-90 s (extension time adjusted according to target fragment length, 1 min extension per 1000 bp), and a final extension at 72℃ for 7 min. The product was then stored at 4℃. The PCR product was confirmed by 1.5% agarose gel electrophoresis, indicating successful amplification of the target fragment.
[0040] Step 4: Construction of Recombinant Plasmids
[0041] pET series plasmids were selected as vectors, and double digestion with EcoRI and BamHI was performed. The digestion system consisted of 50 μl of sterile water (50-V), 5 μl of Cut Buffer, 1 μg of vector plasmid, 1.5 μl of EcoRI, and 1.5 μl of BamHI. The digestion system was incubated in a 37°C water bath for 30 min. After digestion, the digestion efficiency was verified by 1.5% agarose gel electrophoresis to ensure complete digestion of the vector plasmid.
[0042] The digested vector was subjected to homologous recombination with the target fragment amplified in step three. The recombination system consisted of 10 μl of 2× recombinase, 1 μl of digested vector, and 4 μl of the target fragment. The recombination system was ligated in a 50°C water bath for 30 min to obtain the recombinant plasmid, which contained the complete sequence of the candidate gene.
[0043] Step 5: Establishment of a positive control system
[0044] The recombinant plasmid constructed in step four was extracted using an alkaline lysis method. The specific steps are as follows:
[0045] 1. Add 2 ml of E. coli bacterial culture containing recombinant plasmid to a centrifuge tube, centrifuge at 10,000 rpm for 2 min at room temperature, and discard the supernatant;
[0046] 2. Add 250 μl of Buffer P1 containing RNase A to the centrifuge tube, mix thoroughly with a pipette, and suspend the bacterial cells.
[0047] 3. Add 250 μl of Buffer P2 and gently invert the container 4-6 times to obtain a clear lysis buffer. Avoid vigorous mixing to prevent chromosomal DNA shearing.
[0048] 4. Add 350 μl of Buffer N3, and immediately and gently invert the container 4-6 times to mix until a white flocculent precipitate forms. Centrifuge at 10,000 rpm for 15 min at room temperature.
[0049] 5. Carefully transfer the supernatant to a clean adsorption column, centrifuge at 10,000 rpm for 1 min at room temperature, and discard the waste liquid in the collection tube.
[0050] 6. Add 150 μl of Buffer PB to the adsorption column, centrifuge at 10,000 rpm for 1 min, and discard the waste liquid;
[0051] 7. Add 400 μl Buffer PW to the adsorption column (make sure anhydrous ethanol has been added), centrifuge at 10000 rpm for 1 min, discard the waste liquid, repeat this step once, and then centrifuge for 2 min to remove residual ethanol.
[0052] 8. Place the adsorption column in a new centrifuge tube, open the cap and blow air for 4 minutes to ensure complete removal of ethanol;
[0053] 9. Add 90 μl of preheated Buffer EB (65-70℃) to the middle of the adsorption membrane of the adsorption column, let it stand at room temperature for 2 min, centrifuge at 10000 rpm for 1 min, pour the liquid back into the adsorption column and centrifuge again for 1 min to obtain the purified recombinant plasmid.
[0054] 10. Detect the purity of the recombinant plasmid using Nanodrop. An A260 / A280 ratio between 1.8 and 2.0 is considered acceptable. Use the acceptable recombinant plasmid as a positive control and store it at -20℃ for later use.
[0055] Step Six: Specificity Verification
[0056] The positive control obtained in step five, the genomic DNA of the target microorganism, and the genomic DNA of closely related non-target microorganisms were used as templates for PCR amplification using the specific primers designed in step three. The PCR reaction system and procedure were the same as in step three. After amplification, the results were detected by 1.5% agarose gel electrophoresis.
[0057] Judgment criteria: Only the genomic DNA of the target microorganism and the positive control can amplify a specific band of the same size as the target fragment; closely related non-target microorganisms do not amplify bands. Candidate genes that meet this condition are qualified specific candidate genes. The specific bands are sequenced for verification; the sequencing results must show ≥99% homology with the candidate gene sequence to ensure the accuracy of amplification.
[0058] Step 7: Sensitivity Testing
[0059] The genomic DNA of the target microorganism was serially diluted with sterile water to obtain template solutions with concentrations of 100 ng / μl, 10 ng / μl, 1 ng / μl, 0.1 ng / μl, and 0.01 ng / μl. The dilution process was performed in a clean bench to avoid cross-contamination. Using these template solutions as templates, PCR amplification was performed according to the PCR system and procedure described in step three. The amplification results were detected by 1.5% agarose gel electrophoresis.
[0060] Candidate genes with a detection limit ≤ 0.1 ng / μl were screened to ensure that the selected molecular markers had high sensitivity and could detect low concentrations of target microorganisms.
[0061] Step 8: Stability Assessment
[0062] The target microorganism was passaged 10 times consecutively under different culture conditions, including temperature (25℃, 37℃, 42℃), pH (5.0, 7.0, 9.0), and culture medium (LB medium, nutrient broth medium, MRS medium). After each passage, genomic DNA was extracted according to the method in step one, and PCR amplification was performed using specific primers to detect the stability of the amplified bands.
[0063] Judgment criteria: After 10 consecutive passages, clear and specific bands can be stably amplified without band loss or blurring. Candidate genes that meet this condition are qualified candidates for stability, ensuring the applicability of molecular markers under different environmental conditions.
[0064] Step 9: Mark the utility verification
[0065] The candidate genes with good stability were applied to the detection of actual samples, including food samples (such as meat, vegetables, and dairy products), environmental samples (such as soil, water, and air) contaminated with the target microorganism, and clinical samples (such as blood, sputum, and feces).
[0066] The actual sample pretreatment methods are as follows: food samples are homogenized using a homogenizer, centrifuged at 8000 rpm for 10 min, and the supernatant is collected for DNA extraction; environmental samples are filtered through a 0.22 μm filter membrane, and the microorganisms on the filter membrane are washed off with sterile water to extract DNA; clinical samples are directly extracted for DNA according to the instructions of the genomic DNA extraction kit.
[0067] Using processed actual sample DNA as a template, PCR amplification and electrophoresis were performed, and the detection rate of candidate genes was calculated. Candidate genes with a detection rate ≥95% were selected as qualified candidate genes for practical application, ensuring the reliability of molecular markers in real-world applications.
[0068] Step 10: Standardization and Finalization
[0069] Practical and qualified candidate genes were identified as specific molecular markers for microbial traceability. Their primer sequences, PCR amplification conditions (including reaction system, annealing temperature, and extension time), and detection standards were defined. Detection standards included the range of specific band size, electrophoresis detection conditions (120V electrophoresis for 25 minutes), and result interpretation criteria (the presence of a specific band indicates a positive result, and the absence of a band indicates a negative result). This standardized screening protocol facilitates widespread application.
[0070] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.
[0071] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.
Claims
1. A specific molecular marker screening method for microbial traceability, characterized in that, Includes the following steps: Step 1: Microbial genome feature analysis. Genomic DNA of the target microorganism and closely related non-target microorganisms is extracted. The complete genome sequence is obtained using whole-genome sequencing technology. Bioinformatics analysis tools are used to compare and analyze the genomic differences to identify the unique gene fragment regions of the target microorganism. Step 2: Specific candidate gene screening. Based on the unique gene fragment regions identified in Step 1, gene fragments with a length of 500-1500 bp are screened as candidate genes. The candidate genes are single copies in the target microbial genome and have no homologous sequences in the genomes of closely related non-target microorganisms. Step 3: Homologous arm design and target fragment amplification. Specific primers are designed based on the candidate gene sequence. The 5' end of the primers has a 15-20 bp homologous arm, which is completely complementary to the flanking sequence of the restriction enzyme site of the vector plasmid. The target fragment is amplified using a high-fidelity enzyme PCR system. The PCR reaction system is 20 μl, including 7 μl of sterile water, 10 μl of 2× high-fidelity enzyme, 1 μl of upper primer, 1 μl of lower primer, and 1 μl of template DNA. The PCR program is as follows: denaturation at 94℃ for 5 min, followed by denaturation at 94℃ for 30 s, annealing at 55-65℃ for 1 min, extension at 72℃ for 30-90 s, for a total of 30 cycles, and finally extension at 72℃ for 7 min and cooling at 4℃. Step 4: Construction of recombinant plasmid. The vector plasmid was double-digested with EcoRI and BamHI in a 50 μl digestion system, including 50-V sterile water (μl), Cut Buffer (5 μl), vector plasmid (1 μg), EcoRI (1.5 μl), and BamHI (1.5 μl). The reaction was carried out at 37℃ for 30 min. The digested vector was then subjected to homologous recombination with the target fragment amplified in Step 3 in a 10 μl recombination system, including 2× recombinase (5 μl), digested vector (1 μl), and target fragment (4 μl). The ligation was carried out at 50℃ for 30 min to obtain the recombinant plasmid. Step 5: Establishment of the positive control system. The recombinant plasmid constructed in Step 4 was extracted using the alkaline lysis method. The specific steps are as follows: E. coli bacterial culture containing the recombinant plasmid was centrifuged at 10,000 rpm for 2 min to collect the bacterial cells. 250 μl of RNase A-containing Buffer P1 was added to suspend the bacterial cells. 250 μl of Buffer P2 was added and gently inverted 4-6 times to lyse the bacterial cells. 350 μl of Buffer N3 was added and inverted, then centrifuged at 10,000 rpm for 15 min. The supernatant was transferred to an adsorption column and washed sequentially with 150 μl of Buffer PB and 400 μl of Buffer PW. After 2 min of free space to remove ethanol, 90 μl of Buffer EB preheated at 65-70℃ was added to elute, and the purified recombinant plasmid was obtained and used as the positive control. Step Six: Specificity Verification. The positive control obtained in Step Five, the genomic DNA of the target microorganism, and the genomic DNA of closely related non-target microorganisms are used as templates. PCR amplification is performed using the specific primers designed in Step Three. The amplification results are detected by 1.5% agarose gel electrophoresis. Candidate genes that can amplify specific bands only in the genomic DNA of the target microorganism and the positive control, and that do not amplify bands in closely related non-target microorganisms, are qualified candidate genes with specificity. Step 7: Sensitivity detection. The genomic DNA of the target microorganism is serially diluted to obtain template solutions with concentrations of 100 ng / μl, 10 ng / μl, 1 ng / μl, 0.1 ng / μl, and 0.01 ng / μl. PCR amplification is performed using template solutions of each concentration as templates. The lowest detection limit of the specific qualified candidate gene is detected, and candidate genes with a lowest detection limit ≤ 0.1 ng / μl are screened. Step 8: Stability assessment. The target microorganism is passaged 10 times under different culture conditions. Genomic DNA is extracted after each passage and PCR amplification is performed using specific primers. The stability of the amplified bands is detected. Candidate genes that can stably amplify specific bands after 10 consecutive passages are qualified candidate genes in terms of stability. Step 9: Verify practicality by applying candidate genes with good stability to actual sample testing. The actual samples include food samples, environmental samples, or clinical samples contaminated with the target microorganism. The detection rate of the candidate genes in the actual samples is verified by PCR amplification combined with electrophoresis. Candidate genes with a detection rate ≥95% are considered practically qualified candidate genes. Step 10: Standardization and finalization: Identify the practical and qualified candidate genes as specific molecular markers for microbial traceability, clarify their primer sequences, PCR amplification conditions and detection standards, and form a standardized screening scheme.
2. The specific molecular marker screening method for microbial traceability according to claim 1, characterized in that: The bioinformatics analysis tools mentioned in step one include BLAST, ClustalX, and MEGA, which identify unique gene fragment regions of the target microorganism through multiple sequence alignment.
3. The specific molecular marker screening method for microbial traceability according to claim 1, characterized in that: The annealing temperature of the primers in step three is adjusted according to the primer Tm value, with an adjustment range of 55-65℃. The primer specificity is verified by BLAST to ensure that there are no non-specific binding sites.
4. The specific molecular marker screening method for microbial traceability according to claim 1, characterized in that: The vector plasmid mentioned in step four is a pET series plasmid. After enzyme digestion, the digestion efficiency is verified by 1.5% agarose gel electrophoresis to ensure complete digestion.
5. The specific molecular marker screening method for microbial traceability according to claim 1, characterized in that: The purity of the recombinant plasmid extracted by the alkaline lysis method described in step five was detected by Nanodrop. The A260 / A280 ratio was between 1.8 and 2.0, ensuring that there was no protein or RNA contamination.
6. The method for screening specific molecular markers for microbial tracing according to claim 1, characterized in that: The specific bands amplified by PCR in step six were verified by sequencing, and the sequencing results showed ≥99% homology with the candidate gene sequence.
7. The method for screening specific molecular markers for microbial tracing according to claim 1, characterized in that: The gradient dilution described in step seven is performed using sterile water, and the dilution process is carried out in a laminar flow hood to avoid cross-contamination.
8. The specific molecular marker screening method for microbial traceability according to claim 1, characterized in that: The different culture conditions mentioned in step eight include temperatures of 25℃, 37℃, and 42℃, pH values of 5.0, 7.0, and 9.0, and the culture media are LB medium, nutrient broth medium, and MRS medium.
9. The specific molecular marker screening method for microbial traceability according to claim 1, characterized in that: The pretreatment method for actual samples described in step nine is as follows: food samples are homogenized using a homogenizer and then centrifuged to obtain the supernatant; environmental samples are filtered using a filter membrane and then eluted; and genomic DNA is directly extracted from clinical samples.
10. The method for screening specific molecular markers for microbial tracing according to claim 1, characterized in that: The detection criteria in step ten include the specific band size range, electrophoresis detection conditions, and result judgment criteria. The electrophoresis detection conditions are 120V voltage electrophoresis for 25 minutes.