A method for extracting mycobacterium genome

Abstract

The present invention provides a method for extracting a mycobacterium genome, and belongs to the field of biological detection technology. The method for extracting the mycobacterium genome provided by the present invention uses lysase LysA and LysB for wall breaking treatment, which solves the problem of poor mycobacterium genome extraction effect in the prior art. The total genome amount of Mycobacterium tuberculosis extracted by the present invention can reach about 5-10 times that of the traditional classical CTAB genome extraction method, and the integrity of the obtained genome fragments is significantly improved. Compared with the use of lysozyme overnight treatment in the traditional CTAB genome extraction method, the LysA and LysB treatment in the present invention for 2 hours can achieve several times the extraction efficiency, greatly shortening the extraction time, being superior to the traditional CTAB genome extraction method and the genome extraction kit on the market, and being conducive to the rapid and efficient extraction of complete mycobacterium genomes in clinical and laboratory settings.

Description

Technical Field

[0001] The invention relates to the technical field of biological detection, and in particular to a method for extracting a mycobacterium genome. Background Art

[0002] Tuberculosis (TB) is a chronic wasting disease caused by infection with Mycobacterium tuberculosis. It is one of the top ten deadly diseases in the world and the single infectious disease with the highest number of deaths. Timely and accurate diagnosis of tuberculosis is one of the important and effective means to solve the problem of tuberculosis prevention and control. Due to the slow growth of Mycobacterium tuberculosis (it takes 3-4 weeks to grow visible clones on the culture plate), pathogen diagnosis and drug sensitivity detection technology based on bacterial culture have serious lags in clinical guidance; therefore, molecular diagnostic technology based on DNA detection has been increasingly valued and adopted. However, molecular detection technology depends on the efficient and complete extraction of bacterial genomic DNA. In addition, due to the short read length of second-generation sequencing technology, the detection of genomic mutation sites of clinical strains of Mycobacterium tuberculosis based on it can only detect point mutations in about 90% of the regions, and cannot detect mutations in some genomic structural variations and regions with more repetitive sequences (about 10%). To detect these mutations, third-generation sequencing technology (Nanopore or Pacbio sequencing) is required, and third-generation sequencing technology has higher requirements for the integrity of genomic DNA samples.

[0003] Mycobacterium tuberculosis has a cell wall structure and composition that is different from that of ordinary Gram-positive and Gram-negative bacteria. The cell wall of Mycobacterium tuberculosis is composed of "peptidoglycan-arabinogalactan-mycolic acid" covalently linked to form the core structure of the cell wall. Its peptidoglycan layer is thick and is covalently linked and modified by arabinogalactan polysaccharides, which is similar to the cell wall structure characteristics of Gram-positive bacteria. Unlike ordinary Gram-positive bacteria, the cell wall of Mycobacterium tuberculosis also has an outer membrane structure (the outer membrane structure of the cell wall is a characteristic of Gram-negative bacteria). Although Mycobacterium tuberculosis has an outer membrane structure, the composition of its outer membrane structure is completely different from that of the outer membrane of ordinary Gram-negative bacteria. The outer membrane of Gram-negative bacteria is composed of phospholipids and lipopolysaccharides, respectively, and the outer membrane and the inner membrane are connected by protein bridges. However, the inner leaflet of the outer membrane of Mycobacterium tuberculosis is composed of mycolic acid, which is covalently linked to the peptidoglycan through arabinogalactan. The outer leaflet of the outer membrane of Mycobacterium tuberculosis is composed of some extractable lipids unique to mycobacteria, such as Phthiocerol dimycocerosates (PDIMs), sulfatides, polyacyltrehalose, diacyltrehalose, phenol glycolipids, triacylglycerols, and trehalose mono / dimycolate, etc. Therefore, the outer membrane of Mycobacterium tuberculosis is more hydrophobic and less permeable than that of common Gram-negative bacteria.

[0004] At present, the classic method for extracting the genome of Mycobacterium tuberculosis is the CTAB method. Some commercial genome extraction kits also use the extraction principle of the CTAB method, but the silica gel column adsorption method is used in the DNA separation and purification step instead of the isopropanol / ethanol precipitation in the original CTAB method. In the process of extracting the genome of Mycobacterium tuberculosis by the CTAB method, lysozyme is first used to lyse the mycobacterial cell wall, sodium dodecyl sulfate (SDS) / proteinase K is used to destroy the bacterial cell membrane and degrade proteins, and then CTAB is used to further destroy the cell membrane and precipitate polysaccharides. Finally, phenol chloroform extraction and isopropanol / ethanol precipitation of genomic DNA are performed. Mycobacterium tuberculosis has an outer membrane structure with low permeability. Due to the steric effect, lysozyme often finds it difficult to approach its target-peptidoglycan, resulting in insufficient cell wall lysis. In addition, the cell wall acid subunits of the peptidoglycan of Mycobacterium tuberculosis also have N-glycosylation modifications, which further make the peptidoglycan of Mycobacterium tuberculosis insensitive to lysozyme degradation. Due to the network structure of cell wall polysaccharides, insufficient lysis of the cell wall by lysozyme often hinders the subsequent steps of sufficient lysis of the cell membrane and effective release of genomic DNA. Summary of the invention

[0005] The object of the present invention is to provide a method for extracting the mycobacterium genome, which can achieve a better bacterial cell lysis effect.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a method for extracting a mycobacterium genome, comprising the following steps:

[0008] S1. Mix the mycobacteria with the alcohol reagent, and retain the bacterial pellet after centrifugation for 1;

[0009] S2, mixing the bacterial precipitate 1 with an inactivator, and incubating to obtain inactivated bacterial cells; or, subjecting the bacterial precipitate 1 to a heat inactivation treatment to obtain inactivated bacterial cells;

[0010] S3, treating the inactivated bacteria with gradient concentration alcohol reagents, and retaining the bacterial precipitate 2 after centrifugation;

[0011] S4, mixing the bacterial precipitate 2 with an enzyme reagent, performing enzymatic hydrolysis treatment, and obtaining an enzymatic hydrolysis product;

[0012] S5, extracting the genome from the enzymatic hydrolysate using the CTAB method;

[0013] The enzyme reagent contains LysA protein and / or LysB protein.

[0014] Preferably, the enzyme reagent also contains lysozyme.

[0015] Preferably, the enzymatic treatment time is 2 to 12 hours;

[0016] And / or, the temperature of the enzymatic hydrolysis treatment is 35-38°C.

[0017] Preferably, when the enzyme reagent contains LysA protein and LysB protein, the weight ratio of LysA protein to LysB protein is 1:0.8-1.2.

[0018] Preferably, the inactivator contains chloroform and methanol.

[0019] Preferably, the volume ratio of chloroform to methanol in the inactivator is 1.5-2.5:1.

[0020] Preferably, the centrifugal speed is 10000-15000 r / min;

[0021] The centrifugation time is 1 to 5 minutes;

[0022] And / or, in S2, the incubation temperature is 50-70°C;

[0023] And / or, the incubation time is 1.5 to 2.5 hours.

[0024] Preferably, in S3, the gradient concentration alcohol reagent treatment comprises the following steps:

[0025] The inactivated bacteria are centrifuged, mixed with anhydrous ethanol, and the precipitate 1 is retained after centrifugation;

[0026] The precipitate 1 is mixed with an ethanol aqueous solution, and the precipitate 2 is retained after centrifugation;

[0027] The precipitate 2 is mixed with water, and centrifuged to obtain the bacterial precipitate 2;

[0028] The concentration of the ethanol aqueous solution is 40-60%.

[0029] Preferably, when in S2, the bacterial precipitate 1 is subjected to heat inactivation treatment to obtain inactivated bacteria, in S4, the enzyme reagent further contains a detergent.

[0030] Preferably, the mycobacterium includes Mycobacterium tuberculosis, Mycobacterium marinum or Mycobacterium smegmatis.

[0031] Beneficial effects of the present invention:

[0032] The method for extracting the mycobacterium genome provided by the present invention uses lysases LysA and LysB for wall-breaking treatment, which solves the problem of poor mycobacterium genome extraction effect in the prior art. The total amount of the Mycobacterium tuberculosis genome extracted by the present invention can reach about 3 to 8.2 times that of the traditional classical CTAB genome extraction method, and the integrity of the obtained genome fragments is significantly improved. Compared with the use of lysozyme overnight treatment in the traditional CTAB genome extraction method, the LysA and LysB treatment in the present invention for 2 hours can achieve several times the extraction efficiency, greatly shortening the extraction time. In general, the present invention is superior to the traditional CTAB genome extraction method and the genome extraction kit on the market in terms of genome concentration, integrity and genome extraction time, which is conducive to the rapid and efficient extraction of a complete Mycobacterium tuberculosis genome in the clinic and laboratory, and has an important role in promoting clinical testing and scientific research experiments.

[0033] The present invention provides a method for inactivating Mycobacterium tuberculosis by chloroform / methanol treatment, which can effectively remove lipids on the surface of Mycobacterium tuberculosis compared with the traditional heat inactivation method, is conducive to the destruction of bacterial cell walls by enzymatic hydrolysis, and greatly improves the integrity of the extracted genome. For scenarios where chloroform / methanol inactivation cannot be used, the present invention provides a solution of adding a detergent in the enzyme treatment step after heat inactivation, which can achieve effective inactivation and is conducive to subsequent genome extraction. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 are the test results of different inactivation methods, wherein A is the genome extraction result and agarose gel electrophoresis diagram of Mycobacterium tuberculosis after being treated with different inactivation methods; B is the genome extraction result and agarose gel electrophoresis diagram of Mycobacterium marinum after being treated with different inactivation methods; C is the genome extraction result and agarose gel electrophoresis diagram of Mycobacterium smegmatis after being treated with different inactivation methods;

[0035] Figure 2 The genome extraction results and agarose gel electrophoresis images of different treatment times, wherein A is the genome extraction results and agarose gel electrophoresis images of Mycobacterium tuberculosis after being treated with different inactivation methods; B is the genome extraction results and agarose gel electrophoresis images of Mycobacterium marinum after being treated with different inactivation methods; C is the genome extraction results and agarose gel electrophoresis images of Mycobacterium smegmatis after being treated with different inactivation methods;

[0036] Figure 3are the genome extraction results and agarose gel electrophoresis images after adding different detergents, wherein A is the genome extraction result; B is the agarose gel electrophoresis image (wherein, the left image is the gel electrophoresis image of the genome extracted from Mycobacterium tuberculosis after adding different detergents, the middle image is the gel electrophoresis image of the genome extracted from Mycobacterium marinum after adding different detergents, and the right image is the gel electrophoresis image of the genome extracted from Mycobacterium smegmatis after adding different detergents);

[0037] Figure 4 : are protein purification diagrams, wherein: A is the protein purification diagram of LysA; B is the protein purification diagram of LysB. DETAILED DESCRIPTION

[0038] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0039] The preparation of the reagents LysA and LysB of the present invention comprises the following steps:

[0040] Primer sequences:

[0041] Bxz1ysinAFw-PciI: ATATAACATGTCCatgcccgcatacacacaggacggca, as shown in SEQ ID NO.1;

[0042] Bxz1LysAHisRv-AflII:AAGCTCTTAAGCTAGTGGTGATGATGGTGATGtcggttcttctcctgccaggc, as shown in SEQ ID NO.2;

[0043] HisD29LysBFw-NcoI: ACATGCCATGGgtCATCACCATCATCACCACatgagcaagccctggctgttcacc, as shown in SEQ ID NO.3;

[0044] D29LysBRv-HindIII:AACCCAAGCTTtcagatctgtcgtaggaactcgaccgcc, as shown in SEQ IDNO.4;

[0045] 1. Using the genomic DNA of Mycobacterium tuberculosis Bxz1 phage as a template and Bxz1ysinAFw-PciI & Bxz1LysAHisRv-AflII as primers for PCR amplification (using NEB Q5 Master mix enzyme reaction complex) to obtain the PciI-LysAhis-AflII sequence fragment, double-digest the fragment with PciI-AflII, and then clone it into the pRSFDute-1 plasmid vector double-digested with NcoI-AflII to obtain the pRSF-LysAhis plasmid. Using the genomic DNA of Mycobacterium tuberculosis D29 phage as a template and HisD29LysBFw-NcoI&D29LysBRv-HindIII as primers for PCR amplification (using NEB Q5 Master mix enzyme reaction complex) to obtain the NcoI-HisD9LysB-HindIII sequence fragment, the fragment was double-digested with NcoI-HindIII and then cloned into the pRSFDute-1 plasmid vector double-digested with NcoI-HindIII to obtain the pRSF-hisLsB plasmid.

[0046] 2. Transform the pRSF-LysAhis and pRSF-hisLysB plasmids into chemically competent E. coli BL21 (DE3) cells, respectively, and screen positive transformant clones on LB solid plates containing 20 µg / ml kanamycin.

[0047] 3. Pick positive clones and culture them in 10 ml LB + 20 µg / ml kanamycin liquid medium at 37 degrees / 200 rpm overnight.

[0048] 4. Dilute the above bacterial solution 1:100 with 1 liter LB + 20 µg / ml kanamycin liquid medium and continue to culture at 37 degrees / 200rpm until OD 600 =0.4-0.6.

[0049] 5. Add sterile IPTG to the bacterial solution to a final concentration of 0.5 mM, and then continue to induce expression at 16 degrees / 200 rpm for 16 hours.

[0050] Collect the cells by centrifugation at 6.4°C.

[0051] 7. Resuspend the cells in 40 ml of 4°C pre-cooled TWEB buffer (50 mM Tris buffer + 300 mM NaCl, pH 8.0), centrifuge at 4000 rpm for 10 min, discard the supernatant, and resuspend the cells in 40 ml of 4°C pre-cooled TWEB buffer.

[0052] 8. Ultrasonicate on ice to disrupt bacteria (5×10 s pulses separated by 10 s rests or according to the instrument instructions).

[0053] 9. Centrifuge the lysate at 10,000 g for 5 min at 4°C. Collect the supernatant and place on ice (take 500 µl into an EP tube and freeze at -80°C for subsequent verification).

[0054] 10. Equilibrate Ni-NTA resin: Take 6 ml Ni-NTA agarose resin in a 50 ml centrifuge tube, centrifuge at 800g for 1 min, discard the supernatant, resuspend the resins in 20 ml TWEB buffer, centrifuge at 800g for 1 min, and discard the supernatant.

[0055] 11. Repeat step 10 twice.

[0056] 12. Pour the supernatant of the lysate in step 7 into the balanced Ni-NTA resin, tighten the tube cap, and rotate slowly at 4 degrees to mix for 60 min.

[0057] 13. Transfer the mixed solution in step 10 to a 50 ml or 30 ml column (twice). After the resins settle to the bottom, open the valve and collect the filtrate. This step and all subsequent steps should be carried out at 4 degrees.

[0058] 14. Store the filtrate at 4 degrees, take another 500 μl into an EP tube and freeze at -80 degrees for subsequent analysis.

[0059] 15.30ml pre-cooled TWEB buffer for washing once (flow-through stored at 4 degrees)

[0060] Wash twice with 16.30 ml pre-cooled TWEB buffer + 20 mM imidazole (flow-through stored at 4 degrees).

[0061] 17.20 ml pre-cooled TWEB buffer + 500 mM imidazole was used to elute the target protein, and the protein purity was detected by SDS-page electrophoresis.

[0062] 18. Use Slide-A-Lyzer™ G3 dialysis cassette, 10K MWCO (thermo, A52973) for dialysis and desalting; use protein concentrator tube (thermo 88528) to concentrate protein sample; after determining the concentration, add 100% glycerol (final concentration is 50%) in equal volume, mix well and store in aliquots at -80 degrees (purification of lysase LysA and LysB is shown in the figure) Figure 4 as shown).

[0063] In the present invention, the LysA and LysB can be prepared by other methods as long as their lysis activity is not affected; the LysA is the Mycobacterium Bxz1 phage N-acetylmuramoyl-L-alanine amidase, and its amino acid sequence information is: NP_818286.1; LysB is the Mycobacterium D29 phage mycolic acid-arabinogalactan esterase, and its amino acid sequence information is: NP_046827.1.

[0064] Example 1

[0065] The method for extracting the Mycobacterium tuberculosis genome provided in this embodiment comprises the following steps:

[0066] Collect the bacteria in a threaded tube, add 1 mL of methanol, mix thoroughly, centrifuge at 12000 r / min for 2 min, and discard the supernatant.

[0067] Add 1 mL of chloroform:methanol (volume ratio 2:1) solution to resuspend the bacterial pellet, mix thoroughly and transfer to a glass bottle, and incubate in a 60°C water bath for 2 h.

[0068] Transfer the sample in the glass bottle to a 2ml threaded tube, centrifuge at 12000r / min for 2min, and discard the supernatant; add 1mL of anhydrous ethanol, mix thoroughly, and centrifuge at 12000r / min for 2min, and discard the supernatant; add 1mL of 50% ethanol, mix thoroughly, and centrifuge at 12000r / min for 2min, and discard the supernatant; add 1mL of H2O, mix thoroughly, and centrifuge at 12000r / min for 2min, and discard the supernatant.

[0069] Add 450 μL buffer to resuspend the cells, mix thoroughly, add LysA+LysB complex lysing enzyme, 240 μg of each protein (the amount added here is based on the amount of enzyme protein. In actual applications, in order to facilitate the storage or use of reagents, the buffer is used to prepare the protease solution for use. In this embodiment, the concentration of LysA mother solution is 20 mg / mL, and the concentration of LysB mother solution is 13.6 mg / mL), and incubate at 37°C 100 r / min overnight. The buffer containing LysA+LysB is TE buffer + 0.05% tween80.

[0070] Add 87.5 μL 10% SDS and 7 μL 100 mg / mL proteinase K, mix well and incubate in a 65°C metal bath for 10 min.

[0071] After instant centrifugation, add 125 μL 5M NaCl and 125 μl CTAB solution (10% CTAB solution prepared with 0.7M NaCl solution as solvent, which needs to be preheated at 65°C in advance), vortex to mix evenly, and incubate in a metal bath at 65°C for 10 min.

[0072] After instant centrifugation, add 937.5 μL of chloroform:isoamyl alcohol (volume ratio of 24:1), vortex thoroughly to mix, and centrifuge at 11000g for 8 min. Take the supernatant to a new centrifuge tube, add about 0.6 times the volume of supernatant pre-cooled isopropanol at -20℃, mix by inversion, and freeze at -80℃ for more than 30 min.

[0073] After the sample melted, centrifuge at 11000g for 15 min at room temperature, discard the supernatant, add 1 mL of -20℃ precooled 70% ethanol, and invert several times.

[0074] Centrifuge at 11000g for 5 min, discard the supernatant with a 1mL pipette tip, then centrifuge at 11000g for 1 min, discard the residual liquid with a 20µL pipette tip, and dry in a fume hood for several minutes.

[0075] Add 50 μL TE buffer (preheated at 65°C) to dissolve the precipitate and obtain genomic DNA. Use NanoDrop, Qubit and agarose gel electrophoresis to evaluate the purity and concentration of the DNA sample, and store the extracted DNA sample at -20°C.

[0076] This genome extraction step is also applicable to NTM (non-tuberculous mycobacteria), and the subsequent H37 ), Mycobacterium marinum ( Mm ) and Mycobacterium smegmatis ( Ms ) was verified in the genome extraction example.

[0077] Example 2

[0078] The only difference from Example 1 is that after adding 450 μL of buffer to resuspend the bacteria, 240 μg of LysA, LysB and lysozyme Lyso are added after sufficient mixing (based on the amount of enzyme protein, in actual application, for the convenience of addition, the solution is prepared using buffer and then used), and incubated at 37°C 100 r / min for 2 h.

[0079] Experimental Example 1

[0080] Mycobacterium tuberculosis, Mycobacterium marinum, and Mycobacterium smegmatis were used for genome extraction. The sample pretreatment was as shown in Table 1. The remaining steps were the same as the extraction method steps of Example 1. The specific operation of the heat inactivation method was 100°C for 5 minutes. The chloroform inactivation method was the same as the extraction method steps of the Mycobacterium tuberculosis genome. After genome extraction, nanoDrop and Qubit were used to detect the concentration and calculate the total amount of DNA. At the same time, 1% agarose gel electrophoresis was used for detection. The results are shown in Figure 1 As shown (the amount of gel electrophoresis samples loaded is 1 / 20 of the total amount of genome extraction, and the subsequent results also use this loading amount. Note: The heat inactivation LysA+LysB treatment experimental group in Example 1 is a modified heat inactivation method (for details of the comparison of the results of the specific modified method, see Experimental Example 3).

[0081] Table 1 Materials, inactivation methods, buffer solutions and enzyme treatment methods used in experiments 1 to 18

[0082]

[0083] Depend on Figure 1 It can be seen that for the same amount of Mycobacterium tuberculosis, after chloroform / methanol treatment and inactivation, the total amount of genomic DNA extracted by LysA and LysB enzymes is 6.5~8.5 times that of lysozyme treatment; after heat inactivation, the total amount of genomic DNA extracted by LysA and LysB enzymes is 2.5 times that of lysozyme treatment ( Figure 1 -A). For the same amount of Mycobacterium marinum, after inactivation by chloroform / methanol treatment, the total amount of genomic DNA extracted by LysA and LysB enzymes was 13-17 times that of that extracted by lysozyme treatment; after heat inactivation, the total amount of genomic DNA extracted by LysA and LysB enzymes was 3-8 times that of that extracted by lysozyme treatment ( Figure 1 -B). For the same amount of Mycobacterium smegmatis, after inactivation by chloroform / methanol treatment, the total amount of genomic DNA extracted by LysA and LysB enzymes was 3 to 5 times that of that extracted by lysozyme treatment; after heat inactivation, the total amount of genomic DNA extracted by LysA and LysB enzymes was 3 to 7 times that of that extracted by lysozyme treatment ( Figure 1 -C).

[0084] Agarose gel electrophoresis showed that after chloroform / methanol inactivation or heat inactivation, the concentration of genome extraction treated with LysA and LysB enzymes was higher than that treated with lysozyme, and the genome integrity was better. Under the same enzyme treatment conditions, chloroform / methanol inactivation can effectively increase the genome extraction efficiency by 2 to 10 times in Mycobacterium tuberculosis, Mycobacterium marinum, and Mycobacterium smegmatis.

[0085] Experimental Example 2

[0086] Mycobacterium tuberculosis, Mycobacterium marinum, and Mycobacterium smegmatis were used for genome extraction. The sample pretreatment was as shown in Table 2. The remaining steps were the same as the above-mentioned Mycobacterium tuberculosis genome extraction method. The chloroform inactivation method was the same as the above-mentioned Mycobacterium tuberculosis genome extraction method. After genome extraction, nanoDrop and Qubit were used to detect the concentration and calculate the total amount of DNA. At the same time, 1% agarose gel electrophoresis was used for detection. The results are shown in Figure 2 shown.

[0087] Table 2 Materials, inactivation methods, buffers used, and enzyme treatment methods for experiments in groups 19 to 39

[0088]

[0089] Depend on Figure 2 It can be seen that for Mycobacterium tuberculosis, the genome extraction efficiency of the LysA and LysB enzymes treated with lytic enzymes overnight was about 7 times that of the lysozyme treatment; the genome extraction efficiency of the LysA and LysB enzymes treated with lytic enzymes for 2 hours was about 3 times that of the lysozyme treatment. For Mycobacterium marinum, the genome extraction efficiency of the LysA and LysB enzymes treated with lytic enzymes overnight was about 15-19 times that of the lysozyme treatment; the genome extraction efficiency of the LysA and LysB enzymes treated with lytic enzymes for 2 hours was about 5 times that of the lysozyme treatment. For Mycobacterium smegmatis, the genome extraction efficiency of the LysA and LysB enzymes treated with lytic enzymes overnight was about 5 times that of the lysozyme treatment; the genome extraction efficiency of the LysA and LysB enzymes treated with lytic enzymes for 2 hours was about 2.5-6 times that of the lysozyme treatment.

[0090] Agarose gel electrophoresis showed that after overnight and 2 hours of lysase treatment, the concentration of genome extraction treated with LysA and LysB enzymes was higher than that treated with lysozyme, and the genome integrity was better. Extending the enzyme treatment time from 2 hours to 24 hours can effectively increase the genome extraction efficiency by 2 to 9 times in Mycobacterium tuberculosis, Mycobacterium marinum, and Mycobacterium smegmatis.

[0091] Experimental Example 3

[0092] The sources of detergents used in the examples are as follows:

[0093] Reagent name: Tween 80 (polysorbate 80)

[0094] Manufacturer: Sigma-Aldrich

[0095] Reagent name: Triton X-100

[0096] Manufacturer: Sigma-Aldrich

[0097] Reagent name: Sarcosyl (N-Lauroylsarcosine)

[0098] Manufacturer: Sigma-Aldrich

[0099] Reagent name: Genapol X-100

[0100] Manufacturer: MACKLIN

[0101] Reagent name: N-Octyl-β-D-glucopyranoside

[0102] Manufacturer: Aladdin

[0103] Mycobacterium tuberculosis, Mycobacterium marinum, and Mycobacterium smegmatis were used for genome extraction. The sample pretreatment was shown in Table 3. The remaining steps were the same as the above-mentioned Mycobacterium tuberculosis genome extraction method. The specific operation of the heat inactivation method was 100°C treatment for 5 minutes. After genome extraction, the concentration was detected by nanoDrop and Qubit and the total amount of DNA was calculated. At the same time, 1% agarose gel electrophoresis was used for detection. The results are shown in Figure 3 shown.

[0104] Table 3 Materials, inactivation methods, buffers used, and enzyme treatment methods for experiments in groups 40 to 54

[0105]

[0106] Depend on Figure 3 It can be seen that in the scenario where chloroform / methanol inactivation (heat inactivation) cannot be used, for Mycobacterium tuberculosis, adding 1% TritonX-100 can increase the genome extraction efficiency by 1.5 times; for Mycobacterium marinum, adding 1% TritonX-100 can increase the genome extraction efficiency by 1.4 times; for Mycobacterium smegmatis, adding 1% TritonX-100 can increase the genome extraction efficiency by 2.4 times. It is suggested that 1% TritonX-100 treatment can help improve the efficiency of enzyme lysis.

[0107] Agarose gel electrophoresis showed that after adding 1% TritonX-100, the concentration of genomes extracted from Mycobacterium tuberculosis, Mycobacterium marinum and Mycobacterium smegmatis was higher. Especially in Mycobacterium smegmatis, the addition of TritonX-100 can significantly improve the efficiency of genome extraction.

[0108] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for extracting a mycobacterium genome, characterized in that: The following steps are involved: S1. Mix the mycobacteria with the alcohol reagent, and retain the bacterial pellet after centrifugation for 1; S2, mixing the bacterial precipitate 1 with an inactivator, and incubating to obtain inactivated bacterial cells; S3, treating the inactivated bacteria with gradient concentration alcohol reagents, and retaining the bacterial precipitate 2 after centrifugation; S4, mixing the bacterial precipitate 2 with an enzyme reagent, performing enzymatic hydrolysis treatment, and obtaining an enzymatic hydrolysis product; S5, extracting the genome from the enzymatic hydrolysate using the CTAB method; The enzyme reagent contains LysA protein and LysB protein; The inactivator contains chloroform and methanol; The volume ratio of chloroform to methanol in the inactivator is 1.5-2.5:1; The weight ratio of the LysA protein to the LysB protein is 1:0.8-1.

2.

2. The extraction method according to claim 1, characterized in that The enzyme reagent also contains lysozyme.

3. The extraction method according to claim 1, characterized in that The enzymatic treatment time is 2 to 12 hours; And / or, the temperature of the enzymatic hydrolysis treatment is 35-38°C.

4. The extraction method according to claim 1, characterized in that The centrifugal speed is 10000-15000 r / min; The centrifugation time is 1 to 5 minutes; And / or, in S2, the incubation temperature is 50-70°C; And / or, the incubation time is 1.5 to 2.5 hours.

5. The extraction method according to claim 1, characterized in that In S3, the gradient concentration alcohol reagent treatment comprises the following steps: The inactivated bacteria are centrifuged, mixed with anhydrous ethanol, and the precipitate 1 is retained after centrifugation; The precipitate 1 is mixed with an ethanol aqueous solution, and the precipitate 2 is retained after centrifugation; Mixing the precipitate 2 with water, and centrifuging to obtain the bacterial precipitate 2; The concentration of the ethanol aqueous solution is 40-60%.

6. The extraction method according to claim 1, characterized in that The mycobacterium includes Mycobacterium tuberculosis, Mycobacterium marinum or Mycobacterium smegmatis.

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