JP5773280B2 - Method for simultaneous lysis of mycobacteria and separation of its nucleic acids - Google Patents

Description

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

  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 for 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 containing a lipid layer of mycolic acid, and are therefore difficult to lyse compared to 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 The method (refer patent document 2) of lysing acid-fast bacteria by heating to -100 degreeC is mentioned.

  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, bring the temperature of the mixed solution to 35 to 80 ° C., and then chip with a pore structure continuous body having a pore diameter of 30 to 70 μm welded inside A method characterized in that lysis of acid-fast bacteria and separation of nucleic acids thereof are simultaneously carried out by repeating suction and discharge of the mixed solution.
(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 suction and discharge are repeated three or more times.
(5) The method according to any one of (1) to (4) , wherein the mixed solution further contains acetate having a concentration of 0.1 to 1.0M.
(6) 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 (4) .

  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 pore structure continuous body having a specific pore size is welded. The nucleic acids are separated at the same time.

  Examples of mycobacteria to be lysed by the method of the present invention include Mycobacterium avium, M. intracellularellae, 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. scroflacem (M. scro) ulaceum, M. szulgai, M. leprae, M. xenopi, M. ulcerans, M. lepraemurium, M. flavesens (M. flavescens), M. terrae, M. nonchromogenicum, M. malmoense, M. asiaticum, M. vaquee (M) Vaccae), M. gastri, M. triviale, M. haemophilum, M. africanum, M. Mention may be made of M. thermoresistable and 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 the mixture can be easily prepared 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.

  It is preferable that pH of a liquid mixture is 4.5-6.5, More preferably, it is 5.0-6.0. 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 buffer 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 solution 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 acid-fast bacteria 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.

  Although it does not specifically limit as a heating method, The method of putting a liquid mixture in a suitable tube and heating with well-known heating means, such as a heat block, a water bath, a microwave oven, an air bath, is mentioned. 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.

In the method of the present invention, the acid-fast bacterium and the pore structure continuous body are obtained by repeatedly sucking and discharging the above mixed solution using a chip having a pore structure continuous body having a pore diameter of 30 to 70 μm welded therein. In addition to the lysis of acid-fast bacteria, the nucleic acids are separated at the same time.

A schematic diagram of an example of the structure of a chip used in the method of the present invention is shown in FIG. The chip 1 used in the present invention basically has a conventionally known structure (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. Pore structure continuum are those having a continuous pore structure, the pore size is 30 ~ 70 μ m. 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 skeleton and pore channels, and can control micro-sized through-holes and nano-sized pores on the skeleton 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 in which the pore structure continuous body is welded is attached to a syringe or the like and repeatedly sucks and discharges the mixed liquid 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.

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

Reference 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 incubated 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 0.0 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 (monolithic silica, pore diameter 30 μm, cut area 3.14 square mm, thickness 1 mm) welded to a commercially available 250 μL chip is attached to a Terumo 1 mL syringe and mixed. 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 sucked and discharged five times at a rate of 100 μL / sec to elute nucleic acid of acid-fast bacteria adsorbed on the pore structure continuum 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 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 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 the 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 acid. 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 acid-fast bacteria 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 Reference Example 1, the recovery efficiency of acid-fast bacterium nucleic acids was displayed. The results are shown in Table 1.

Reference 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.

Reference 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.

Reference 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, Examples 2 and 5 to 12 in which suction and discharge were repeated at a specific mixed liquid temperature using a chip in which a pore structure continuous body having a specific pore diameter was welded, were used in the present invention. Compared with Comparative Examples 1 and 2 treated with a pore structure continuum outside the range of pore diameter, Comparative Example 3 in which no chaotropic salt solution was used, and Comparative Example 4 in which suction and discharge were not repeated, It can be understood that the nucleic acid recovery efficiency is remarkably high.

  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 (6)

A mixture containing a chaotropic salt and acid-fast bacilli was prepared, the temperature of the mixture was 35 to 80 ° C., and then a chip having a pore structure continuous body having a pore diameter of 30 to 70 μm was used. A method comprising simultaneously lysing acid-fast bacteria and separating nucleic acids by repeating suction and discharge of the mixed solution.   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. The method according to any one of claims 1 to 3, wherein the repetition of the suction and discharge is not less than 3 times. The method according to any one of claims 1 to 4 , wherein the mixed solution further contains acetate having a concentration of 0.1 to 1.0M. Any mixture further comprises a Good's buffer at a concentration of 0.05-0.5 M, according to claim 1-4 in which the buffer is characterized by having a buffered pH range pH4.5~6.5 The method according to claim 1.

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