CN116426518A - Method for efficiently extracting mycobacterium tuberculosis nucleic acid and application thereof - Google Patents
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Abstract
The invention discloses a method for efficiently extracting mycobacterium tuberculosis nucleic acid and application thereof, which comprises the following steps: (1) preparing a liquid culture medium; (2) liquefying sputum; (3) culturing the bacterial colony; (4) mixing with the lysate; (5) centrifuging the mixed solution; (6) eluting the nucleic acid; (7) nanopore sequencing technology to detect nucleic acids. The invention firstly prepares a liquid culture medium from bovine colostrum, glucose, middlebrook7H9 broth base, coconut juice, folic acid, glycerol and tween-80, rapidly cultures a large amount of mycobacterium tuberculosis, then forms a lysate from urea, potassium chloride, tris (hydroxymethyl) aminomethane hydrochloride, polyethylene glycol octyl phenyl ether, cetyltrimethylammonium bromide and potassium phosphate buffer solution, efficiently extracts mycobacterium tuberculosis nucleic acid, improves the extraction quality of the mycobacterium tuberculosis nucleic acid, finally detects the mycobacterium tuberculosis nucleic acid by using a nanopore sequencing technology, shortens the detection time to within 1 day, has high detection rate, and achieves the aim of rapidly identifying the mycobacterium tuberculosis.
Description
Technical Field
The invention belongs to the technical field of mycobacterium tuberculosis, and particularly relates to a method for efficiently extracting mycobacterium tuberculosis nucleic acid and application thereof.
Background
Mycobacterium tuberculosis, commonly known as Mycobacterium tuberculosis, is a bacterium that causes tuberculosis. Tuberculosis is a chronic infectious disease, mainly transmitted through respiratory tract, and the infectious source is mainly sputum of tuberculosis patients who expel bacteria. Although tuberculosis is a curable disease, tuberculosis is still one of the ten causes of death worldwide, and the incidence and mortality of tuberculosis worldwide is slow to decrease. According to the world health organization report, 1000 tens of thousands of people are estimated to suffer from tuberculosis and 140 tens of thousands of people die from tuberculosis worldwide in 2019.
The existing tuberculosis diagnosis method comprises a sputum smear microscopic examination method, a mycobacterium species identification method, a sputum tuberculosis conventional culture method and the like. Wherein, the sputum smear microscopic examination method is quick and cheap, but can not distinguish dead bacteria and live bacteria and has poor specificity; the mycobacterium species identification method can accurately identify different species of mycobacterium, but has complex operation and certain danger of partially used medicines; the conventional culture method of the tubercle bacillus phlegm can identify the live bacteria of the dead bacteria, but has long time consumption and low sensitivity. In addition, since the cell wall of Mycobacterium tuberculosis contains a large amount of lipids, accounting for 23.77% of the dry weight of the bacteria, 40% of which is waxy, and Mycobacterium tuberculosis has a layer of capsule outside the cell wall, the cell wall of Mycobacterium tuberculosis is stronger than that of common bacteria, resulting in difficult extraction of Mycobacterium tuberculosis nucleic acid.
The world health organization proposed "strategy for terminal tuberculosis" in 2015, that is, the mortality and morbidity of tuberculosis are reduced by 90% and 80% respectively by 2030 and by 95% and 90% respectively by 2035. In order to achieve the aim, the invention provides a method for efficiently extracting mycobacterium tuberculosis nucleic acid and application thereof, improves the diagnosis capability of a tuberculosis laboratory, shortens the diagnosis time, carries out early and rapid diagnosis of tuberculosis and drug resistance detection thereof, and achieves the effectiveness and pertinency of therapeutic drug administration.
Disclosure of Invention
The invention aims to solve the defects in the prior art, and provides a method for efficiently extracting mycobacterium tuberculosis nucleic acid and application thereof, and the method specifically adopts the following technical scheme:
a method for efficiently extracting mycobacterium tuberculosis nucleic acid, which comprises the following steps:
(1) Preparing a liquid culture medium, wherein the liquid culture medium comprises bovine coloctrum, glucose, middlebrook7H9 broth base, coconut juice, folic acid, glycerol and tween-80, and the mass ratio of the bovine coloctrum, the glucose, the Middlebrook7H9 broth base, the coconut juice, the folic acid, the glycerol and the tween-80 is 1.5-2.5:2.0-3.0:1.0-1.8:1.2-2.4:0.05-0.15:0.5-0.7:0.6-1.0;
(2) Collecting sputum of a patient, adding NaOH solution with the mass percentage of 4% in an amount which is 1-2 times of the volume of the sputum of the patient, vibrating the sputum, and fully liquefying the sputum of the patient;
(3) Culturing bacterial colony, inoculating the sputum fully liquefied in the step (2) into the liquid culture medium prepared in the step (1), regulating the pH to 6.5-6.8, culturing at 37 ℃, growing bacterial colony for 10-14 days, and reserving supernatant to obtain high-concentration mycobacterium tuberculosis group;
(4) Mixing with a lysate, adding 1.0-1.2 times of the lysate into the high-concentration mycobacterium tuberculosis group obtained in the step (3), mixing to obtain a mixed solution, treating the mixed solution at 100 ℃ for 20min, wherein the lysate consists of 2-4mol/L urea, 2-4mol/L potassium chloride, 1.5-2.5mol/L tris hydrochloride, 1% polyethylene glycol octyl phenyl ether, 20-60g/L cetyltrimethylammonium bromide and 0.2mol/L potassium phosphate buffer solution, and the pH value of the lysate is=6.0;
(5) Centrifuging the mixed solution, mixing the supernatant with a magnetic bead suspension, and standing, wherein the magnetic bead suspension comprises polyethyleneimine modified magnetic beads, and the pH=6.0 of the magnetic bead suspension;
(6) Eluting nucleic acid, adsorbing magnetic beads through a magnetic rack, discarding supernatant, washing impurities adsorbed on the surfaces of the magnetic beads with a washing liquid, eluting the nucleic acid adsorbed on the surfaces of the magnetic beads with an eluting liquid, and separating to obtain mycobacterium tuberculosis nucleic acid, wherein the pH of the washing liquid is=4.0, and the pH of the eluting liquid is=9.0;
(7) Detecting the mycobacterium tuberculosis nucleic acid obtained in the step (6) by using a nanopore sequencing technology, and constructing a nanopore sequencing library.
Preferably, the specific steps of the step (1) are as follows: weighing 1.0-1.8 parts by mass of Middlebrook7H9 broth base, adding into 200-350 parts by mass of distilled water, sterilizing at 121 ℃ for 15min under high pressure, cooling to 45 ℃ to obtain Middlebrook7H9 broth base solution, adding 1.5-2.5 parts by mass of bovine coloctrum, 2.0-3.0 parts by mass of glucose, 1.2-2.4 parts by mass of coconut juice, 0.05-0.15 parts by mass of folic acid, 0.5-0.7 parts by mass of glycerol and 0.6-1.0 part by mass of tween-80 to Middlebrook7H9 broth base solution, and uniformly mixing to obtain the liquid culture medium.
Preferably, in the step (2), the vibration is performed for 25-30 min.
Preferably, in step (3), the colonies are grown for 12 days.
Preferably, in step (6), the cleaning solution is hydrochloric acid solution, and the eluent is sodium hydroxide solution.
Preferably, the specific steps of the step (7) are as follows:
(1) Designing primers of specific parting genes IS6100, hsp65, rpoB and gyrA of the mycobacterium tuberculosis;
(2) Adding TTTCTGTTGGTGCTGATATTG base filling sequences to the 5 'end of each forward primer-F and ACTTGCCTGTCGCTCTATCTTC base filling sequences to the 5' end of each reverse primer-R to obtain amplification primers containing a second round PCR primer binding site;
(3) Diluting the amplification primers of the binding sites of the PCR primers of the second round, and mixing forward and reverse primers of each gene; secondly, forming a primer pool by the mixed primers;
(4) Synthesizing a Barcode label primer applicable to the second round of PCR, diluting all Barcode dry powder primers, and mixing forward and reverse primers of the same Barcode to form a Barcode primer pool;
(5) Performing first round amplification and purification on the mycobacterium tuberculosis nucleic acid by using a primer pool;
(6) Performing a second round of amplification and purification with the Barcode primer;
(7) A nanopore sequencing library is constructed and purified.
Preferably, the primer sequence of IS6100 comprises a nucleotide sequence set forth in SEQ01 and comprises a nucleotide sequence set forth in SEQ 02; the primer set of hsp65 comprises the nucleotide sequence set forth in SEQ03 and comprises the nucleotide sequence set forth in SEQ 04; the primer set of rpoB comprises a nucleotide sequence shown as SEQ05 and comprises a nucleotide sequence shown as SEQ 06; the primer set for gyrA comprises the nucleotide sequence shown as SEQ07 and comprises the nucleotide sequence shown as SEQ 08.
The invention also provides an application of the method for efficiently extracting the mycobacterium tuberculosis nucleic acid in rapid identification of the mycobacterium tuberculosis.
The beneficial effects of the invention are as follows: because the mycobacterium tuberculosis has a capsule outside the cell wall, the cell wall of the mycobacterium tuberculosis is stronger than that of common bacteria, so that the extraction of the mycobacterium tuberculosis nucleic acid is difficult. According to the invention, bovine colostrum, glucose, middlebrook7H9 broth base, coconut juice, folic acid, glycerol and Tween-80 are used as raw materials to prepare a liquid culture medium, a large amount of mycobacterium tuberculosis is rapidly cultured, urea, potassium chloride, tris (hydroxymethyl) aminomethane hydrochloride, polyethylene glycol octyl phenyl ether, cetyltrimethylammonium bromide and potassium phosphate buffer solution are used to form a lysate, so that mycobacterium tuberculosis nucleic acid is efficiently extracted, the extraction quality of the mycobacterium tuberculosis nucleic acid is improved, finally, the nano-pore sequencing technology is used for detecting the mycobacterium tuberculosis nucleic acid, the detection time of the mycobacterium tuberculosis nucleic acid is shortened to within 1 day, the detection rate is high, the aim of rapidly identifying the mycobacterium tuberculosis is fulfilled, and the method is simple to operate and short in flow.
Detailed Description
The conception and technical effects of the present invention will be clearly and completely described in conjunction with examples below to fully understand the objects, aspects and effects of the present invention. It should be noted that, in the case of no conflict, the embodiments and features in the embodiments may be combined with each other.
Example 1
Rapid culture of large numbers of mycobacterium tuberculosis:
(1) Weighing 3g Middlebrook7H9 broth base in parts by mass, adding into 600g distilled water, sterilizing at 121 ℃ for 15min under high pressure, cooling to 45 ℃ to obtain Middlebrook7H9 broth base solution, adding 4g bovine coloctrum, 5g glucose, 3.6g coconut juice, 0.2g folic acid, 1.2g glycerol and 1.6g tween-80 to Middlebrook7H9 broth base solution, and uniformly mixing to obtain a liquid culture medium;
(2) Collecting patient sputum (the patient is a tuberculosis patient in a high-new hospital of a first affiliated hospital of Nanchang university in 2022), adding NaOH solution with the mass percent of 4% in 1.5 times of volume into the patient sputum, vibrating for 25min, and fully liquefying the patient sputum;
(3) Culturing bacterial colony, inoculating the sputum fully liquefied in the step (2) into the liquid culture medium prepared in the step (1), regulating the pH value to 6.5, culturing at 37 ℃, growing bacterial colony for 12 days, and reserving supernatant to obtain high-concentration mycobacterium tuberculosis group.
Example 2
Rapid culture of large numbers of mycobacterium tuberculosis:
(1) Weighing 2g Middlebrook7H9 broth base in parts by mass, adding into 400g of distilled water, sterilizing at 121 ℃ for 15min under high pressure, cooling to 45 ℃ to obtain Middlebrook7H9 broth base solution, adding 3g of bovine coloctrum, 4g of glucose, 2.4g of coconut juice, 0.1g of folic acid, 1.0g of glycerol and 1.2g of tween-80 into the Middlebrook7H9 broth base solution, and uniformly mixing to obtain a liquid culture medium;
(2) Collecting patient sputum (the patient is a tuberculosis patient in a high and new hospital of a first affiliated hospital of Nanchang university in 2022), adding an equal volume of NaOH solution with the mass percent of 4% into the patient sputum, vibrating for 30min, and fully liquefying the patient sputum;
(3) Culturing bacterial colony, inoculating the sputum fully liquefied in the step (2) into the liquid culture medium prepared in the step (1), regulating the pH to 6.8, culturing at 37 ℃, growing bacterial colony for 10 days, and reserving supernatant to obtain high-concentration mycobacterium tuberculosis group.
Example 3
Rapid culture of large numbers of mycobacterium tuberculosis:
(1) Weighing 3.6g Middlebrook7H9 broth base in parts by mass, adding into 600g distilled water, sterilizing at 121 ℃ for 15min under high pressure, cooling to 45 ℃ to obtain Middlebrook7H9 broth base solution, adding 5g bovine coloctrum, 6g glucose, 4.8g coconut juice, 0.3g folic acid, 1.4g glycerol and 2.0g tween-80 to Middlebrook7H9 broth base solution, and uniformly mixing to obtain a liquid culture medium;
(2) Collecting patient sputum (the patient is a tuberculosis patient in a high and new hospital of a first affiliated hospital of Nanchang university in 2022), adding 2 times of NaOH solution with the mass percent of 4% into the patient sputum, vibrating for 30min, and fully liquefying the patient sputum;
(3) Culturing bacterial colony, inoculating the sputum fully liquefied in the step (2) into the liquid culture medium prepared in the step (1), regulating the pH value to 6.5, culturing at 37 ℃, growing bacterial colony for 14 days, and reserving supernatant to obtain high-concentration mycobacterium tuberculosis group.
Example 4
High-efficiency extraction of tubercle bacillus nucleic acid:
(1) Adding 1.2 times of lysate into the high-concentration mycobacterium tuberculosis group prepared in the example 1, mixing to obtain a mixed solution, and treating the mixed solution at 100 ℃ for 20min to inactivate mycobacterium tuberculosis and release nucleic acid; the lysate consisted of 3mol/L urea, 3.5mol/L potassium chloride, 2mol/L tris hydrochloride, 1% polyethylene glycol octylphenyl ether, 40g/L cetyltrimethylammonium bromide and 0.2mol/L potassium phosphate buffer, adjusted to ph=6.0 with phosphoric acid;
(2) Centrifuging the mixed solution at 12000rpm for 5min, sucking 1000 mu L of supernatant into a centrifuge tube, adding 600 mu L of resuspended magnetic bead solution containing polyethyleneimine modification, uniformly mixing, and standing for 5min;
(3) Eluting nucleic acid, adsorbing magnetic beads by a magnetic rack, discarding supernatant, washing impurities adsorbed on the surfaces of the magnetic beads by using a washing liquid, eluting the nucleic acid adsorbed on the surfaces of the magnetic beads by using an eluting liquid, and separating to obtain the mycobacterium tuberculosis nucleic acid, wherein the pH of the washing liquid is=4.0, and the pH of the eluting liquid is=9.0. Placing the centrifuge tube on a magnetic rack for standing for 1min, removing liquid in the centrifuge tube, taking down the centrifuge tube, adding 500 mu L of hydrochloric acid solution with pH=4.0, and shaking and uniformly mixing for 30s; placing the centrifuge tube on a magnetic rack for standing for 1min, and removing liquid in the centrifuge tube; taking down the centrifuge tube, adding 100 mu L of sodium hydroxide solution eluent with pH value of 9.0, blowing and mixing uniformly, and standing for 3min; placing the centrifuge tube on a magnetic rack for standing; transferring the supernatant to a new centrifuge tube to obtain the mycobacterium tuberculosis nucleic acid.
Example 5
High-efficiency extraction of tubercle bacillus nucleic acid:
(1) Adding 1-fold lysate to the high-concentration mycobacterium tuberculosis group prepared in the example 2, mixing to obtain a mixed solution, and treating the mixed solution at 100 ℃ for 20min to inactivate mycobacterium tuberculosis and release nucleic acid; the lysate consisted of 4mol/L urea, 4mol/L potassium chloride, 2.5mol/L tris hydrochloride, 1% polyethylene glycol octylphenyl ether, 60g/L cetyltrimethylammonium bromide and 0.2mol/L potassium phosphate buffer, adjusted ph=6.0 with phosphoric acid;
(2) Centrifuging the mixed solution at 12000rpm for 5min, sucking 1000 mu L of supernatant into a centrifuge tube, adding 600 mu L of resuspended magnetic bead solution containing polyethyleneimine modification, uniformly mixing, and standing for 5min;
(3) Eluting nucleic acid, adsorbing magnetic beads by a magnetic rack, discarding supernatant, washing impurities adsorbed on the surfaces of the magnetic beads by using a washing liquid, eluting the nucleic acid adsorbed on the surfaces of the magnetic beads by using an eluting liquid, and separating to obtain the mycobacterium tuberculosis nucleic acid, wherein the pH of the washing liquid is=4.0, and the pH of the eluting liquid is=9.0. Placing the centrifuge tube on a magnetic rack for standing for 1min, removing liquid in the centrifuge tube, taking down the centrifuge tube, adding 500 mu L of hydrochloric acid solution with pH=4.0, and shaking and uniformly mixing for 30s; placing the centrifuge tube on a magnetic rack for standing for 1min, and removing liquid in the centrifuge tube; taking down the centrifuge tube, adding 100 mu L of sodium hydroxide solution eluent with pH value of 9.0, blowing and mixing uniformly, and standing for 3min; placing the centrifuge tube on a magnetic rack for standing; transferring the supernatant to a new centrifuge tube to obtain the mycobacterium tuberculosis nucleic acid.
Comparative example 1
Culturing mycobacterium tuberculosis:
(1) Weighing 3g Middlebrook7H9 broth base in parts by mass, adding into 600g distilled water, sterilizing at 121 ℃ for 15min under high pressure, cooling to 45 ℃ to obtain Middlebrook7H9 broth base solution, adding 4g bovine coloctrum, 5g glucose, 1.2g glycerol and 1.6g tween-80 into the Middlebrook7H9 broth base solution, and uniformly mixing to obtain a liquid culture medium;
(2) Collecting patient sputum (the patient is a tuberculosis patient in a high-new hospital of a first affiliated hospital of Nanchang university in 2022), adding NaOH solution with the mass percent of 4% in 1.5 times of volume into the patient sputum, vibrating for 25min, and fully liquefying the patient sputum;
(3) Culturing bacterial colony, inoculating the sputum fully liquefied in the step (2) into the liquid culture medium prepared in the step (1), regulating the pH value to 6.5, culturing at 37 ℃, growing bacterial colony for 12 days, and reserving supernatant to obtain high-concentration mycobacterium tuberculosis group.
Comparative example 2
Extraction of tubercle bacillus nucleic acid:
(1) Adding 1.2 times of lysate into the high-concentration mycobacterium tuberculosis group prepared in the example 1, mixing to obtain a mixed solution, and treating the mixed solution at 100 ℃ for 20min to inactivate mycobacterium tuberculosis and release nucleic acid; the lysate consisted of 3mol/L urea, 4mol/L potassium chloride, 1% polyethylene glycol octylphenyl ether and 0.2mol/L potassium phosphate buffer, adjusted to ph=6.0 with phosphoric acid;
(2) Centrifuging the mixed solution at 12000rpm for 5min, sucking 1000 mu L of supernatant into a centrifuge tube, adding 600 mu L of resuspended magnetic bead solution containing polyethyleneimine modification, uniformly mixing, and standing for 5min;
(3) Eluting nucleic acid, adsorbing magnetic beads by a magnetic rack, discarding supernatant, washing impurities adsorbed on the surfaces of the magnetic beads by using a washing liquid, eluting the nucleic acid adsorbed on the surfaces of the magnetic beads by using an eluting liquid, and separating to obtain the mycobacterium tuberculosis nucleic acid, wherein the pH of the washing liquid is=4.0, and the pH of the eluting liquid is=9.0. Placing the centrifuge tube on a magnetic rack for standing for 1min, removing liquid in the centrifuge tube, taking down the centrifuge tube, adding 500 mu L of hydrochloric acid solution with pH=4.0, and shaking and uniformly mixing for 30s; placing the centrifuge tube on a magnetic rack for standing for 1min, and removing liquid in the centrifuge tube; taking down the centrifuge tube, adding 100 mu L of sodium hydroxide solution eluent with pH value of 9.0, blowing and mixing uniformly, and standing for 3min; placing the centrifuge tube on a magnetic rack for standing; transferring the supernatant to a new centrifuge tube to obtain the mycobacterium tuberculosis nucleic acid.
Comparative example 3
Extraction of tubercle bacillus nucleic acid:
(1) Adding 1.2 times of lysate into the high-concentration mycobacterium tuberculosis group prepared in comparative example 1, mixing to obtain a mixed solution, and treating the mixed solution at 100 ℃ for 20min to inactivate mycobacterium tuberculosis and release nucleic acid; the lysate consisted of 3mol/L urea, 3.5mol/L potassium chloride, 2mol/L tris hydrochloride, 1% polyethylene glycol octylphenyl ether, 40g/L cetyltrimethylammonium bromide and 0.2mol/L potassium phosphate buffer, adjusted to ph=6.0 with phosphoric acid;
(2) Centrifuging the mixed solution at 12000rpm for 5min, sucking 1000 mu L of supernatant into a centrifuge tube, adding 600 mu L of resuspended magnetic bead solution containing polyethyleneimine modification, uniformly mixing, and standing for 5min;
(3) Eluting nucleic acid, adsorbing magnetic beads by a magnetic rack, discarding supernatant, washing impurities adsorbed on the surfaces of the magnetic beads by using a washing liquid, eluting the nucleic acid adsorbed on the surfaces of the magnetic beads by using an eluting liquid, and separating to obtain the mycobacterium tuberculosis nucleic acid, wherein the pH of the washing liquid is=4.0, and the pH of the eluting liquid is=9.0. Placing the centrifuge tube on a magnetic rack for standing for 1min, removing liquid in the centrifuge tube, taking down the centrifuge tube, adding 500 mu L of hydrochloric acid solution with pH=4.0, and shaking and uniformly mixing for 30s; placing the centrifuge tube on a magnetic rack for standing for 1min, and removing liquid in the centrifuge tube; taking down the centrifuge tube, adding 100 mu L of sodium hydroxide solution eluent with pH value of 9.0, blowing and mixing uniformly, and standing for 3min; placing the centrifuge tube on a magnetic rack for standing; transferring the supernatant to a new centrifuge tube to obtain the mycobacterium tuberculosis nucleic acid.
Example 6
Detecting mycobacterium tuberculosis nucleic acid by using a nanopore sequencing technology, and constructing a nanopore sequencing library:
(1) Designing primers of specific parting genes IS6100, hsp65, rpoB and gyrA of the mycobacterium tuberculosis, and the sequence information of the specific primers IS shown in table 1;
(2) Adding TTTCTGTTGGTGCTGATATTG base filling sequences to the 5 'end of each forward primer-F and ACTTGCCTGTCGCTCTATCTTC base filling sequences to the 5' end of each reverse primer-R to obtain amplification primers containing a second round PCR primer binding site, wherein the specific primer sequences are shown in Table 2;
(3) All primers in Table 2 were diluted to 100. Mu.M, and the forward and reverse primers of each gene were mixed in equal proportions; secondly, forming a primer pool by the mixed primers, wherein the concentration ratio of the primers corresponding to IS6100, hsp65, rpoB and gyrA in the primer pool IS 0.5:1:1.2:1. primer mixing in different proportions, which is preferred, may also achieve the results of the present invention.
(4) And synthesizing the Barcode tag primers suitable for the second round of PCR, wherein each Barcode primer corresponds to one sample, and each sample is provided with a different Barcode tag. All barcode dry powder primers were diluted to 10. Mu.M, and the same forward and reverse primers of the same barcode were mixed in equal proportions to form a pool of barcode primers, the specific primer sequences are shown in Table 3.
(5) The Mycobacterium tuberculosis nucleic acids obtained in example 4, example 5, comparative example 2 and comparative example 3 were PCR amplified (plus 1 PCR negative control) using the mixed primer pools: the total reaction was 50. Mu.l, of which 2X PhusionHFBu fferPCRMastermix (NEB) was 25. Mu.l, the optimized primer pool was 5. Mu.l, and 50ng of nucleic acid, ddH2O was added to 50. Mu.l. The reaction procedure is: pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 15s, annealing at 65℃for 60s, 1℃decrease per cycle, extension at 72℃for 15s,10 cycles; denaturation at 95℃for 15s, annealing at 62℃for 60s, extension at 72℃for 15s,20 cycles; after the cycle was completed, the sample was extended at 72℃for 5 minutes and stored at 4 ℃.
Adding 30 μl of AMpureXPBeads into 50 μl of multiplex PCR product, blowing, mixing, standing at room temperature for 5min to allow magnetic beads to be fully combined with PCR product; placing the purification system on a magnetic frame, waiting for 5min until the magnetic beads are completely adsorbed on one side of the magnetic frame, and removing the supernatant; adding 200 μl of freshly prepared 80% ethanol, standing on a magnetic rack for 30s, discarding supernatant, and washing again to remove impurities thoroughly; removing residual ethanol by using a pipette, uncovering, and air-drying at room temperature for 5min until the magnetic beads are dried; taking the purification tube off the magnetic rack, adding 25 μl ddH2O to elute nucleic acid, blowing and mixing uniformly to make the magnetic beads in a completely suspended state, standing at room temperature for 3min, and eluting nucleic acid sufficiently; the purification tube was again placed on a magnetic rack for 3min, and 23 μl of eluent was extracted for the second round of amplification after the beads were completely adsorbed on one side of the magnetic rack.
(6) Two rounds of PCR amplification were performed using Barcode tag primers (BC 01-BC 05) with different tags on different samples to facilitate mixing together for on-machine sequencing. The reaction system was 50. Mu.l, 2X PhusionHFBufferPCRMastermix (NEB). Mu.l of the reaction system, 2. Mu.l of the Barcode primer, and 23. Mu.l of the purified product after the first round of amplification. The reaction procedure is: pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 15s, annealing at 62℃for 15s, elongation at 72℃for 30s,6 cycles; after the cycle was completed, the sample was extended at 72℃for 5 minutes and stored at 4 ℃.
Adding 30 μl of AMpureXPBeads into 50 μl of multiplex PCR product, blowing, mixing, standing at room temperature for 5min to allow magnetic beads to be fully combined with PCR product; placing the purification system on a magnetic frame, waiting for 5min until the magnetic beads are completely adsorbed on one side of the magnetic frame, and removing the supernatant; adding 200 μl of freshly prepared 80% ethanol, standing on a magnetic rack for 30s, discarding supernatant, and washing again to remove impurities thoroughly; removing residual ethanol by using a pipette, uncovering, and air-drying at room temperature for 5min until the magnetic beads are dried; taking the purification tube off the magnetic rack, adding 30 μl ddH2O to elute nucleic acid, blowing and mixing uniformly to make the magnetic beads in a completely suspended state, standing at room temperature for 3min, and eluting nucleic acid sufficiently; the purification tube was again placed on a magnetic rack for 3min, and after the beads were completely adsorbed on one side of the magnetic rack, 27 μl was aspirated for library construction.
(7) Constructing a nanopore sequencing library and purifying:
(1) library mixing (pooling): 5 parts of purified product after two rounds of amplification were subjected to poolin g (less than 100ng plus 10. Mu.l) per sample, and then the total volume was made up to 50. Mu.l with ddH 2O.
(2) End repair and 3' end addition of tail a: the total reaction system was 65. Mu.l, 50. Mu.l of the DNA solution after mixing, and 15. Mu.l of end-repair mix (Vazyme). The reaction procedure is: the reaction was stopped at 30℃for 15min,65℃for 15min and stored at 4 ℃. (8) (9) herba Pogostemonis
(3) Purifying a tail end repair product: after adding 39. Mu.l of AMpureXPBeads to 65. Mu.l of PCR product, the purification was performed in the same manner as the magnetic bead purification method in step (5), and finally eluting DNA with 30. Mu.lddH 2O, 25. Mu.l was used for the ligation reaction.
(4) Nanopore sequencing linker ligation: the total reaction system was 50. Mu.l, 25. Mu.l of the end repair purification product, 10. Mu.l of 5×LiationnBuffer (NEB), T4RapidDNALigase (NEB). Mu.l, adapterMix (Nanopore). Mu.l, and ddH2O 6. Mu.l. The reaction procedure is: the reaction was carried out at 20℃for 15min and stored at 4 ℃.
(5) Purifying a joint connection product: adding 30 mu.l of AMpureXPBeads into 50 mu.l of the joint connection product, blowing and mixing uniformly, and standing at room temperature for 5min to fully combine the magnetic beads with the PCR product; placing the purification system on a magnetic frame, waiting for 5min until the magnetic beads are completely adsorbed on one side of the magnetic frame, and removing the supernatant by using a pipettor; 200. Mu.l SFB (supplied by NanoporeLSK109 kit) was added and the supernatant was discarded after standing on a magnetic rack for 30s and repeated once; sucking residual SFB, uncovering, airing for 5min at room temperature, and adding 15 mu lddH2O to elute DNA after the magnetic beads are dried; and (3) standing for 3min at room temperature after blowing and mixing uniformly, and sucking 13 μl to obtain the final library.
(6) Nanopore sequencing: using a Nanopore company MinION sequencer (sequencing kit: SQK-LSK109, sequencing chip: R9) R9 chip, and performing chip activity induction for 5min before adding the sample into the chip, so that the preservation solution in the chip is completely replaced by the sequencing buffer solution; library loading is carried out according to oxford nanopore official instructions; the sequencing kit selects SQK-LSK109, trimbarcode and Highaccuracy mode for nanopore sequencing.
(7) And (3) database analysis and comparison: after the sequencing data is taken off the machine, the nanopore sequencer automatically converts the sequencing result to generate a sequencing file in fastq format for subsequent analysis. Performing quality control analysis on the off-machine data, wherein the quality control flow selects Qscore to be more than or equal to 9, and removes low-quality reads and connector sequences; filtering the sequences with the sequence length smaller than 450bp or larger than 750bp to obtain a target sequence which accords with the expected design; comparing the target sequence with the genome sequence of the mycobacterium tuberculosis in the database; the detected species and sequence numbers were generated as shown in table 4.
TABLE 1 Mycobacterium tuberculosis specific primer sequences
| Primer name | Primer sequence (5 '-3') | Sequence numbering |
| my-IS6100-F | GTGGCCAACTCGACATCC | SEQ01 |
| my-IS6100-R | AGTGTGGCTAACCCTGAACC | SEQ02 |
| my-hsp65-F | AACGTCGTCCTGGARAAGAA | SEQ03 |
| my-hsp65-R | GTCGAGACCTTGGAGCTGAC | SEQ04 |
| my-rpoB-F | CGGTCGCTATAAGGTCAACAA | SEQ05 |
| my-rpoB-R | CGGYCGCTAYAAGGTCAACAA | SEQ06 |
| my-gyrA-F | GGATCGAACCGGTTGACATC | SEQ07 |
| my-gyrA-R | GATTCTCCAGCGCCCAGAA | SEQ08 |
TABLE 2 Mycobacterium tuberculosis related gene amplification primers
| Primer name | Primer sequence (5 '-3') | Sequence numbering |
| tNGS-my-IS6100-F | TTTCTGTTGGTGCTGATATTGCGTGGCCAACTCGACATCC | SEQ09 |
| tNGS-my-IS6100-R | ACTTGCCTGTCGCTCTATCTTCAGTGTGGCTAACCCTGAACC | SEQ10 |
| tNGS-my-hsp65-F | TTTCTGTTGGTGCTGATATTGCAACGTCGTCCTGGARAAGAA | SEQ11 |
| tNGS-my-hsp65-R | ACTTGCCTGTCGCTCTATCTTCGTCGAGACCTTGGAGCTGAC | SEQ12 |
| tNGS-my-rpoB-F | TTTCTGTTGGTGCTGATATTGCCGGTCGCTATAAGGTCAACAA | SEQ13 |
| tNGS-my-rpoB-R | ACTTGCCTGTCGCTCTATCTTCCGGYCGCTAYAAGGTCAACAA | SEQ14 |
| tNGS-my-gyrA-F | TTTCTGTTGGTGCTGATATTGCGGATCGAACCGGTTGACATC | SEQ15 |
| tNGS-my-gyrA-R | ACTTGCCTGTCGCTCTATCTTCGATTCTCCAGCGCCCAGAA | SEQ16 |
TABLE 3 barcode primers
Table 4 4 number of detection sequences of samples and negative control group
| Example 4 | Example 5 | Comparative example 2 | Comparative example 3 | Negative control group | |
| IS6100 | 1864 | 1642 | 935 | 896 | 0 |
| hsp65 | 1935 | 1757 | 1024 | 922 | 0 |
| rpoB | 1572 | 1494 | 878 | 630 | 0 |
| gyrA | 1320 | 1263 | 796 | 594 | 0 |
In summary, (1) the invention prepares the liquid culture medium by taking bovine coloctrum, glucose, middlebrook7H9 broth base, coconut juice, folic acid, glycerol and tween-80 as raw materials, wherein the coconut juice, the folic acid and the like have synergistic effect, and the culture rate of tubercle bacillus is accelerated; (2) Because the mycobacterium tuberculosis has a layer of capsule outside the cell wall, the cell wall of the mycobacterium tuberculosis is stronger than that of common bacteria, so that the extraction of the mycobacterium tuberculosis nucleic acid is more difficult; (3) According to the invention, the detection time IS shortened to within 1 day by using a nanopore sequencing technology, primers of specific parting genes IS6100, hsp65, rpoB and gyrA of the mycobacterium tuberculosis are designed, and the target genes of the pathogenic bacteria to be detected are amplified in one reaction by using a multiplex PCR technology, so that a plurality of mycobacterium tuberculosis can be detected simultaneously.
The present invention is not limited to the above embodiments, but is merely preferred embodiments of the present invention, and the present invention should be construed as being limited to the above embodiments as long as the technical effects of the present invention are achieved by the same means. Various modifications and variations are possible in the technical solution and/or in the embodiments within the scope of the invention.
Claims (8)
1. A method for efficiently extracting mycobacterium tuberculosis nucleic acid, which is characterized by comprising the following steps:
(1) Preparing a liquid culture medium, wherein the liquid culture medium comprises bovine coloctrum, glucose, middlebrook7H9 broth base, coconut juice, folic acid, glycerol and tween-80, and the mass ratio of the bovine coloctrum, the glucose, the Middlebrook7H9 broth base, the coconut juice, the folic acid, the glycerol and the tween-80 is 1.5-2.5:2.0-3.0:1.0-1.8:1.2-2.4:0.05-0.15:0.5-0.7:0.6-1.0;
(2) Collecting sputum of a patient, adding NaOH solution with the mass percentage of 4% in an amount which is 1-2 times of the volume of the sputum of the patient, vibrating the sputum, and fully liquefying the sputum of the patient;
(3) Culturing bacterial colony, inoculating the sputum fully liquefied in the step (2) into the liquid culture medium prepared in the step (1), regulating the pH to 6.5-6.8, culturing at 37 ℃, growing bacterial colony for 10-14 days, and reserving supernatant to obtain high-concentration mycobacterium tuberculosis group;
(4) Mixing with a lysate, adding 1.0-1.2 times of the lysate into the high-concentration mycobacterium tuberculosis group obtained in the step (3), mixing to obtain a mixed solution, treating the mixed solution at 100 ℃ for 20min, wherein the lysate consists of 2-4mol/L urea, 2-4mol/L potassium chloride, 1.5-2.5mol/L tris hydrochloride, 1% polyethylene glycol octyl phenyl ether, 20-60g/L cetyltrimethylammonium bromide and 0.2mol/L potassium phosphate buffer solution, and the pH value of the lysate is=6.0;
(5) Centrifuging the mixed solution, mixing the supernatant with a magnetic bead suspension, and standing, wherein the magnetic bead suspension comprises polyethyleneimine modified magnetic beads, and the pH=6.0 of the magnetic bead suspension;
(6) Eluting nucleic acid, adsorbing magnetic beads through a magnetic rack, discarding supernatant, washing impurities adsorbed on the surfaces of the magnetic beads with a washing liquid, eluting the nucleic acid adsorbed on the surfaces of the magnetic beads with an eluting liquid, and separating to obtain mycobacterium tuberculosis nucleic acid, wherein the pH of the washing liquid is=4.0, and the pH of the eluting liquid is=9.0;
(7) Detecting the mycobacterium tuberculosis nucleic acid obtained in the step (6) by using a nanopore sequencing technology, and constructing a nanopore sequencing library.
2. The method according to claim 1, wherein the step (1) comprises the specific steps of: weighing 1.0-1.8 parts by mass of Middlebrook7H9 broth base, adding into 200-350 parts by mass of distilled water, sterilizing at 121 ℃ for 15min under high pressure, cooling to 45 ℃ to obtain Middlebrook7H9 broth base solution, adding 1.5-2.5 parts by mass of bovine coloctrum, 2.0-3.0 parts by mass of glucose, 1.2-2.4 parts by mass of coconut juice, 0.05-0.15 parts by mass of folic acid, 0.5-0.7 parts by mass of glycerol and 0.6-1.0 part by mass of tween-80 to Middlebrook7H9 broth base solution, and uniformly mixing to obtain the liquid culture medium.
3. The method of claim 1, wherein in step (2), the shaking is performed for 25min to 30min.
4. The method of claim 1, wherein in step (3), the colonies are grown for 12 days.
5. The method of claim 1, wherein in step (6), the cleaning solution is hydrochloric acid solution and the eluent is sodium hydroxide solution.
6. The method according to claim 1, wherein the step (7) comprises the specific steps of:
(1) Designing primers of specific parting genes IS6100, hsp65, rpoB and gyrA of the mycobacterium tuberculosis;
(2) Adding TTTCTGTTGGTGCTGATATTG base filling sequences to the 5 'end of each forward primer-F and ACTTGCCTGTCGCTCTATCTTC base filling sequences to the 5' end of each reverse primer-R to obtain amplification primers containing a second round PCR primer binding site;
(3) Diluting the amplification primers of the binding sites of the PCR primers of the second round, and mixing forward and reverse primers of each gene; secondly, forming a primer pool by the mixed primers;
(4) Synthesizing a Barcode label primer applicable to the second round of PCR, diluting all Barcode dry powder primers, and mixing forward and reverse primers of the same Barcode to form a Barcode primer pool;
(5) Performing first round amplification and purification on the mycobacterium tuberculosis nucleic acid by using a primer pool;
(6) Performing a second round of amplification and purification with the Barcode primer;
(7) A nanopore sequencing library is constructed and purified.
7. The method of claim 6, wherein in step (1), the primer sequence of IS6100 comprises a nucleotide sequence set forth in SEQ01 and comprises a nucleotide sequence set forth in SEQ 02; the primer set of hsp65 comprises the nucleotide sequence set forth in SEQ03 and comprises the nucleotide sequence set forth in SEQ 04; the primer set of rpoB comprises a nucleotide sequence shown as SEQ05 and comprises a nucleotide sequence shown as SEQ 06; the primer set for gyrA comprises the nucleotide sequence shown as SEQ07 and comprises the nucleotide sequence shown as SEQ 08.
8. A method for efficiently extracting mycobacterium tuberculosis nucleic acid is applied to rapidly identifying mycobacterium tuberculosis.
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| CN117025587B (en) * | 2023-10-10 | 2024-03-01 | 北京博晖创新生物技术集团股份有限公司 | Lysate of sputum liquefaction and mycobacteria nucleic acid extraction, kit, extraction method and application |
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