WO2011158815A1 - Method for simultaneous achievement of bacteriolysis of acid-fast bacterium and separation of nucleic acid from the bacterium - Google Patents

Abstract

Disclosed is a method for achieving the bacteriolysis of an acid-fast bacterium and the separation of a nucleic acid from the bacterium simultaneously, with high efficiency and in a simple manner. The method is characterized by comprising preparing a mixed solution containing a chaotropic salt and the acid-fast bacterium and repeating the suction of the mixed solution and the ejection of the mixed solution using a chip having, melt-adhered to the inside thereof, a continuous body having a pore structure, wherein the pore structure has a pore diameter of 10 to 100 µm. In the method, it is preferred to repeat the suction and the ejection at least three times.

Description

Method for simultaneous lysis of mycobacteria and separation of its nucleic acids

The present invention relates to a method for simultaneously performing lysis of acid-fast bacteria typified by Mycobacterium tuberculosis and separation of its nucleic acids by simple operations.

Tuberculosis is a bacterial disease that is showing signs of rebirth in Japan, and improvement of its diagnostic method is extremely important. As a method for diagnosing tuberculosis, in recent years, a genetic test that can obtain highly accurate results in a short time has been carried out instead of a culture test that takes several weeks.

Tuberculosis genetic testing is performed by identifying the presence of M. tuberculosis in sputum samples by PCR using primers specific to the M. tuberculosis gene. In order to perform a genetic test, as a pretreatment, it is necessary to lyse (destroy) the bacterial cells to be tested in the sample and then to separate the nucleic acid.

However, acid-fast bacteria typified by Mycobacterium tuberculosis have a strong cell wall including a lipid layer of mycolic acid, and are therefore difficult to lyse compared with general bacteria such as Escherichia coli.

As a general lysis method of acid-fast bacteria, a method of physically destroying bacterial cells using ultrasonic waves (see Patent Document 1), a method of destroying bacterial cells using phenol as a lysis agent, 60 A method of lysing acid-fast bacteria by heating to ˜100 ° C. (see Patent Document 2).

These conventionally known mycobacterial lysis methods are problematic in that they are expensive, complicated to operate, lack of safety, and have low lysis efficiency. It was. In addition, these methods require a separate operation for separating the nucleic acid after lysis of the acid-fast bacterium, so that the entire operation is complicated.

WO 2007/094506 JP-A-6-319527

The present invention was devised in view of the current state of the prior art, and an object of the present invention is to provide a method capable of efficiently simultaneously lysing mycobacterial lysis and its nucleic acid with a simple operation. It is in.

As a result of intensive studies to achieve the above-mentioned object, the present inventor first brought an acid-fast bacterium into contact with a chaotropic salt to soften the cell wall of the bacterium, and this was then passed through a pore structure continuum having a specific pore size. By repeatedly passing the cells, the cell wall was efficiently destroyed, and it was found that lysis of acid-fast bacteria and separation of nucleic acids could be performed simultaneously, and the present invention was completed.

The present invention has the following configurations (1) to (6).
(1) Prepare a mixed solution containing a chaotropic salt and acid-fast bacteria, and then repeat the suction and discharge of the mixed solution using a chip in which a pore structure continuous body having a pore size of 10 to 100 μm is welded. A method comprising simultaneously lysing acid-fast bacilli and separating nucleic acids thereof.
(2) The method according to (1), wherein the concentration of the chaotropic salt in the mixed solution is 2 to 6M.
(3) The method according to (1) or (2), wherein the pH of the mixed solution is 4.5 to 6.5.
(4) The method according to any one of (1) to (3), wherein the temperature of the mixed solution is set to 35 to 80 ° C., and the suction and discharge of the mixed solution are repeated.
(5) The method according to any one of (1) to (4), wherein the suction and discharge are repeated three or more times.
(6) The method according to any one of (1) to (5), wherein the mixed solution further contains acetate having a concentration of 0.1 to 1.0M.
(7) The mixed solution further includes a Good buffer solution having a concentration of 0.05 to 0.5 M, and the buffer solution has a buffer pH range of pH 4.5 to 6.5. The method according to any one of (5).

In the method of the present invention, the acid-fast bacterium having a cell wall softened with a chaotropic salt is repeatedly passed through a chip in which a continuous pore structure having a specific pore size is welded. Lysis and its nucleic acid can be separated easily and efficiently at the same time.

It is the schematic of an example of the structure of the chip | tip used by the method of this invention.

Hereinafter, the method of the present invention will be described in detail.
The method of the present invention uses acid-resistant bacteria lysis by repeating suction and discharge of a mixed solution containing a chaotropic salt and acid-fast bacteria using a chip in which a continuous pore structure having a specific pore size is welded. The nucleic acids are separated at the same time.

Examples of acid-fast bacteria to be lysed by the method of the present invention include Mycobacterium avium, M. intracellulare, M. gordonae, and M. tuberculosis. (M. tuberculosis), M. kansasii, M. fortuitum, M. chelonae, M. bovis, M. scroflace. ), M. paratuberculosis, M. phlei, M. marinum, M. simiae, M. scraceum (M. scr). fulaceum, M. szulgai, M. leprae, M. xenopi, M. ulcerans, M. lepraemurium, M. flavescens (M. flavescens), M. terrae, M. nonchromogenicum, M. malmoense, M. asiaticum, M. vaquee (M) Vaccae), M. gastri, M. triviale, M. haemophilum, M. africanum, e. - it can be mentioned thermo register table (M.thermoresistable) and M. smegmatis (M. smegmatis).

The chaotropic salt used in the method of the present invention is a chemical substance that has the property of destabilizing molecular structures such as proteins, and is used as a lysing agent for lysing cells during nucleic acid extraction in the field of biochemistry. It is a substance. Any conventionally known chaotropic salt can be used, such as guanidine salt, sodium isothiocyanate, sodium iodide, potassium iodide, urea, sodium bromide, potassium bromide, calcium bromide, bromide. It can be ammonium, sodium perchlorate, sodium thiocyanate, potassium thiocyanate, ammonium isothiocyanate, sodium chloride, potassium chloride, ammonium chloride. Among these, a guanidine salt is preferable in terms of cell solubility and nucleic acid recovery efficiency. Examples of the guanidine salt include guanidine hydrochloride, guanidine thiocyanate (guanidine thiocyanate), guanidine sulfate, and guanidine isothiocyanate. Among these, guanidine hydrochloride or guanidine thiocyanate is preferable from the viewpoint of lysis efficiency. These salts may be used alone or in combination.

The acid-fast bacterium and the chaotropic salt are used in the form of a mixed solution containing both, and can be easily prepared into a mixed solution by dissolving the chaotropic salt and the acid-fast bacterium in an appropriate solvent such as water or a buffer solution. For example, a mixed solution can be prepared by dissolving a chaotropic salt and a buffering agent in deionized water and adding a bacterial solution in which acid-fast bacteria are prepared at a constant concentration.

The concentration of the chaotropic salt in the mixed solution is preferably 2 to 6 M (mol / L), more preferably 3 to 5 M (mol / L). If the concentration of the chaotropic salt in the mixed solution is less than the lower limit, the lysis efficiency of the acid-fast bacterium cell wall may be reduced, and if it exceeds the upper limit, the chaotropic salt may be precipitated.

The pH of the mixed solution is preferably 4.5 to 6.5, more preferably 5.0 to 6.0. If the pH of the mixed solution is less than the above lower limit or exceeds the above upper limit, the nucleic acid separation efficiency may be lowered.

In order to maintain the pH of the mixed solution in the above range, it is preferable to add an appropriate buffer to the solution. An example of such a buffering agent is acetate. Among the acetates, monovalent metal acetates such as potassium acetate and sodium acetate are particularly effective. The concentration of these acetates in the mixed solution is preferably 0.1 to 1.0 M (mol / L) from the viewpoint of the buffering effect. As another example of such a buffer, MES (2-morpholinoethanesulfonic acid), ACES (N- (2-acetamido) -2-aminoethanesulfonic acid), PIPES (piperazine-1,4-bis (2) -Good buffer solutions having a buffer capacity on the acidic side, such as ethanesulfonic acid)). The concentration of these Good buffers in the mixed solution is preferably 0.05 to 0.5 M (mol / L) from the viewpoint of the buffering effect.

In the method of the present invention, a mixed solution containing a chaotropic salt and an acid-fast bacterium can be used as it is at room temperature, but it is preferable to use it after heating to a specific temperature. The heating temperature is 35 to 80 ° C., preferably 40 to 75 ° C. When heated, the dissolution effect of the chaotropic salt is likely to be improved, but if the heating temperature is too high, the evaporation of the liquid increases and the concentration of the chaotropic salt may change. In addition, the nucleic acid adsorption ability to the pore structure continuum may be reduced.

The heating method is not particularly limited, and examples thereof include a method in which the mixed solution is put in an appropriate tube and heated by a known heating means such as a heat block, a water bath, a microwave oven, or an air bath. The acid-fast bacterium may be added to the mixed solution from the beginning, or may be added after the mixed solution is heated to a predetermined temperature.

The method of the present invention uses the tip having a pore structure continuous body having a pore diameter of 10 to 100 μm welded therein to repeatedly suck and discharge the above mixed solution, whereby the acid-fast bacteria and the pore structure continuous body are The pores are in contact with each other, and not only the acid-fast bacteriolysis but also the nucleic acid separation is performed at the same time.

FIG. 1 shows a schematic diagram of an example of the structure of a chip used in the method of the present invention. The chip 1 used in the present invention basically has a conventionally known configuration (for example, an elongated inverted conical hollow body shown in FIG. 1), and the inside thereof has a pore structure continuous body over a certain length from the tip. 2 is welded. The pore structure continuum has a continuous pore structure, and the pore diameter is 10 to 100 μm, preferably 20 to 70 μm. If the pore diameter deviates from the above range, acid-fast bacteria lysis and nucleic acid separation cannot be performed effectively. The material of the pore structure is not particularly limited, but silica is preferable. Such structures are also referred to as “monolithic silica” or “silica monolith”. Monolithic silica has a structure that integrates a three-dimensional network-like framework and pore channels, and can control micro-sized through-holes and nano-sized pores on the framework surface during synthesis. Have

The pore structure continuum can generally be produced by a sol-gel method. For example, a metal alkoxide is partially hydrolyzed to produce a reactive monomer, and this monomer is polycondensed to produce a colloidal oligomer. Can be produced by forming a sol, further hydrolyzing to promote polymerization and crosslinking, and forming a three-dimensional structure to form a gel. The pore structure continuous body is shaped into a size accommodated in the chip, and then welded to the chip using, for example, ultrasonic waves.

The chip with the pore structure continuous body welded inside is attached to a syringe and the like, and repeatedly sucks and discharges the liquid mixture containing the chaotropic salt and acid-fast bacteria. The number of repetitions of suction and discharge is preferably 3 times or more. If it is less than 3 times, there is a possibility that acid-fast lysis and nucleic acid cannot be sufficiently separated. There is no upper limit to the number of repetitions, but a maximum of about 20 is sufficient.

Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

Example 1
(1) Preparation of acid-fast bacterium As the acid-fast bacterium, the Mycobacterium bovis BCG strain (hereinafter abbreviated as BCG strain) was used. The BCG strain was cultured in 3% Ogawa medium (Nissui Pharmaceutical Co., Ltd.) at 35 ° C. for 2 weeks, then inoculated into MycoBroth (manufactured by Kyokuto Pharmaceutical Co., Ltd.), a liquid medium for acid-fast bacteria culture, and at 37 ° C. for 6 days. Further culture was performed. In order to obtain a highly dispersible bacterial solution, the cultured liquid medium was filtered with a hydrophilic filter having a pore size of 5 μm, and then the concentration of McFarland 1 was measured according to the McFarland turbidimetric method while measuring OD600 with a turbidimeter. The bacterial solution was adjusted. Next, in order to remove nucleic acid that has already been released in the liquid medium, 1.0 mL of this bacterial solution is added to a 1.5 mL tube, and only bacterial cells are precipitated by centrifugation, and the supernatant is removed to remove 1 The cells were resuspended with 0 mL of phosphate buffer.

(2) Preparation of chaotropic salt solution Guanidine thiocyanic acid as a chaotropic salt and potassium acetate as a buffering agent were dissolved in deionized water to prepare a solution of a chaotropic salt containing a buffering agent.

(3) Preparation of Mixed Solution To 500 μL of the chaotropic salt solution prepared in (2), a solution obtained by diluting the McFarland 1 bacterial solution prepared in (1) 1000 times with a phosphate buffer was mixed. The concentration of guanidine thiocyanic acid in the mixed solution was 5M, the concentration of potassium acetate was 0.8M, and the pH of the solution was 5.5.

(4) Lysis of acid-fast bacteria and separation and extraction of nucleic acid Next, using a chip having a pore structure continuous body as shown in FIG. Nucleic acid was separated and extracted. Specifically, a porous structure continuous body (monolith silica, pore diameter 30 μm, cutting area 3.14 square mm, thickness 1 mm) was welded to a Terumo 1 mL syringe and mixed in a commercially available 250 μL chip. The solution was sucked and discharged 10 times at a rate of 100 μL / second, the acid-fast bacteria in the mixed solution were lysed, and the nucleic acid was adsorbed to the pore structure continuum in the chip. After the mixed solution was completely discharged, the tip was moved to a tube containing 500 μL of the cleaning solution, and the cleaning solution was sucked and discharged three times at a rate of 100 μL / second for cleaning. This washing process was repeated once more. Finally, the tip is moved to a tube containing 100 μL of eluate, and the eluate is aspirated and discharged 5 times at a rate of 100 μL / sec to elute the acid-fast bacilli nucleic acids adsorbed on the continuous pore structure in the chip. did. In order to suppress a decrease in elution efficiency, care was taken so that air did not contact the continuous pore structure in the chip during suction and discharge. Note that a 0.8 M potassium acetate solution in 70% ethanol was used as the washing solution, and a 10 mM potassium hydroxide aqueous solution was used as the eluent.

(5) Detection and quantification of acid-fast bacterium genes by real-time PCR Next, the acid-fast bacterium genes were detected and quantified by real-time PCR for the nucleic acid eluate obtained in (4). The PCR reagent composition and PCR conditions were as shown below.

Reagent composition:
Oligo 1 250 nM,
Oligo 2 1500 nM,
Oligo 3 (5 ′ end labeled with BODIPY-FL) 250 nM,
× 10 buffer 1 μL,
dNTP 0.2 mM,
MgSO ₄ 4 mM,
KOD plus DNA polymerase 0.2U,
Eluate 1μL
(Adjust the total volume to 10 μL with Milli-Q water)
(The sequences of Oligo 1 to Oligo 3 are as shown in SEQ ID Nos. 1 to 3 in the sequence listing.)

PCR conditions:
Thermal denaturation: 94 ° C for 2 minutes, 98 ° C for 0 seconds, annealing: 60 ° C for 5 seconds (fluorescence detection), 50 cycles

The above reagent composition is a combination of a primer and a probe that can specifically detect the BCG strain. As a positive control, genomic DNA extracted from the BCG strain by the phenol / chloroform method was diluted with 10 mM Tris buffer so as to contain 1000 copies in 1 μL. Real-time PCR detects a specific nucleic acid sequence in a sample based on the fact that the fluorescence of a fluorescent dye labeled on the probe is quenched when the amplification product and the probe are hybridized. A light cycler (registered trademark) manufactured by Roche Diagnostics was used for amplification and detection of nucleic acids. As the measurement mode, 530 nm was used, and the obtained QProbe extinction ratio was calculated using the obtained real-time detection data. Furthermore, the number of cycles that reached a quenching rate of 2% was determined and analyzed in the same manner as the real-time quantitative PCR method using SYBR Green I. The nucleic acid recovery efficiency of mycobacteria was expressed as a percentage relative to 100% positive control. In addition, it is considered that the nucleic acid recovery efficiency of acid-fast bacteria is good if it is 30% or more. The results are shown in Table 1.

Example 2
The mixture obtained in (3) was put into a 1.5 mL tube with a screw cap and heated and heated at 65 ° C. for 5 minutes on a heat block, except that suction and discharge were performed in (4). In the same manner as in Example 1, the nucleic acid recovery efficiency of acid-fast bacteria was displayed. The results are shown in Table 1.

Example 3
The recovery efficiency of acid-fast bacilli nucleic acids was displayed in the same manner as in Example 2 except that the pore diameter of the pore structure continuum of (4) was changed to 10 μm. The results are shown in Table 1.

Example 4
The recovery efficiency of acid-fast bacterium nucleic acids was displayed in the same manner as in Example 2 except that the pore diameter of the pore structure continuum in (4) was changed to 20 μm. The results are shown in Table 1.

Example 5
The recovery efficiency of acid-fast bacterium nucleic acids was displayed in the same manner as in Example 2 except that the pore diameter of the pore structure continuum in (4) was changed to 40 μm. The results are shown in Table 1.

Example 6
The nucleic acid recovery efficiency of acid-fast bacteria was displayed in the same manner as in Example 2 except that the pore diameter of the pore structure continuous body of (4) was changed to 50 μm. The results are shown in Table 1.

Example 7
The recovery efficiency of acid-fast bacilli nucleic acids was displayed in the same manner as in Example 2 except that the pore diameter of the pore structure continuum in (4) was changed to 70 μm. The results are shown in Table 1.

Example 8
The efficiency of acid-fast bacilli nucleic acid recovery was displayed in the same manner as in Example 2, except that guanidine hydrochloride was used in place of guanidine thiocyanate as the chaotropic salt. The results are shown in Table 1.

Example 9
The recovery efficiency of the acid-fast bacterium's nucleic acid was displayed in the same manner as in Example 2 except that the number of suction / discharge in the process of lysis of the acid-fast bacterium and the separation of the nucleic acid was changed to 5. The results are shown in Table 1.

Example 10
The recovery efficiency of acid-fast bacilli nucleic acids was displayed in the same manner as in Example 2 except that the number of suction / discharge in the acid-fast bacilli lysis and nucleic acid separation steps was changed to 20. The results are shown in Table 1.

Example 11
The recovery efficiency of the acid-fast bacterium nucleic acid was displayed in the same manner as in Example 2 except that the heating temperature was changed to 40 ° C. The results are shown in Table 1.

Example 12
The recovery efficiency of acid-fast bacilli nucleic acids was displayed in the same manner as in Example 2 except that the heating temperature was changed to 75 ° C. The results are shown in Table 1.

Example 13
The recovery efficiency of acid-fast bacterium nucleic acids was displayed in the same manner as in Example 2 except that the heating temperature was changed to 95 ° C. The results are shown in Table 1.

Comparative Example 1
The nucleic acid recovery efficiency of acid-fast bacteria was displayed in the same manner as in Example 2 except that the pore diameter of the pore structure continuous body of (4) was changed to 120 μm. The results are shown in Table 1.

Comparative Example 2
The recovery efficiency of the acid-fast bacterium's nucleic acid was displayed in the same manner as in Example 2 except that the pore diameter of the continuous pore structure (4) was changed to 5 μm. The results are shown in Table 1.

Comparative Example 3
The recovery efficiency of acid-fast bacterium nucleic acids was displayed in the same manner as in Example 2 except that milliQ water was used instead of the chaotropic salt solution of (2). The results are shown in Table 1.

Comparative Example 4
The recovery efficiency of the acid-fast bacterium's nucleic acid was displayed in the same manner as in Example 2 except that the number of suction / discharge in the acid-fast bacterium lysis and nucleic acid separation step was changed to 1. The results are shown in Table 1.

From Table 1, in Examples 1 to 13 in which suction and discharge were repeated using a chip in which a pore structure continuous body having a specific pore diameter was welded, pore structure continuous outside the pore diameter range of the present invention. Compared with Comparative Examples 1 and 2, which were treated with the body, Comparative Example 3 in which the solution of chaotropic salt was not used, and Comparative Example 4 in which the suction and discharge were not repeated, the nucleic acid recovery efficiency was remarkably high. Understandable.

Since the method of the present invention can efficiently perform lysis of mycobacteria and its nucleic acid, which were difficult to lyse due to a strong cell wall, simultaneously and at low cost and safely by a simple operation, tuberculosis This is extremely useful for pretreatment of genetic testing of bacteria.

SEQ ID NO: 1 is the designed polynucleotide sequence described as Oligo 1 in the Examples.
SEQ ID NO: 2 is the designed polynucleotide sequence described as Oligo 2 in the Examples.
SEQ ID NO: 3 is the sequence of the designed polynucleotide described as Oligo 3 in the Examples.

Claims (7)

A mixed solution containing chaotropic salt and acid-fast bacterium is prepared, and then the mixture is repeatedly sucked and discharged by using a chip in which a pore structure continuous body having a pore size of 10 to 100 μm is welded. A method comprising simultaneously lysing bacteria and separating nucleic acids thereof. The method according to claim 1, wherein the concentration of the chaotropic salt in the mixed solution is 2 to 6M. The method according to claim 1 or 2, wherein the pH of the mixed solution is 4.5 to 6.5. 4. The method according to claim 1, wherein the temperature of the mixed liquid is set to 35 to 80 ° C., and the suction and discharge of the mixed liquid are repeated. 5. The method according to claim 1, wherein the suction and discharge are repeated three or more times. The method according to any one of claims 1 to 5, wherein the mixed solution further contains acetate having a concentration of 0.1 to 1.0M. The mixed solution further comprises a Good's buffer having a concentration of 0.05 to 0.5M, and the buffer has a buffered pH range of pH 4.5 to 6.5. The method according to claim 1.

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