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WO2011158815A1 - Procédé pour la réalisation simultanée d'une bactériolyse d'une bactérie résistante aux acides et d'une séparation d'acides nucléiques provenant de la bactérie - Google Patents

Procédé pour la réalisation simultanée d'une bactériolyse d'une bactérie résistante aux acides et d'une séparation d'acides nucléiques provenant de la bactérie Download PDF

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WO2011158815A1
WO2011158815A1 PCT/JP2011/063555 JP2011063555W WO2011158815A1 WO 2011158815 A1 WO2011158815 A1 WO 2011158815A1 JP 2011063555 W JP2011063555 W JP 2011063555W WO 2011158815 A1 WO2011158815 A1 WO 2011158815A1
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acid
mixed solution
fast
bacterium
nucleic acid
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Japanese (ja)
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圭一 氏内
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Toyobo Co Ltd
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Toyobo Co Ltd
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Priority to JP2012520447A priority Critical patent/JP5773280B2/ja
Priority to CN2011800301817A priority patent/CN102947449A/zh
Publication of WO2011158815A1 publication Critical patent/WO2011158815A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor

Definitions

  • 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.
  • 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.
  • PCR primers specific to the M. tuberculosis gene.
  • 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.
  • Patent Document 1 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).
  • 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.
  • 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 ⁇ m 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.
  • 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.
  • 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.
  • thermo register table M.thermoresistable
  • 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.
  • guanidine salt is preferable in terms of cell solubility and nucleic acid recovery efficiency.
  • the guanidine salt include guanidine hydrochloride, guanidine thiocyanate (guanidine thiocyanate), guanidine sulfate, and guanidine isothiocyanate.
  • guanidine hydrochloride or guanidine thiocyanate is preferable from the viewpoint of lysis efficiency.
  • 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.
  • 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.
  • an appropriate buffer to the solution.
  • An example of such a buffering agent is acetate.
  • 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.
  • MES 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ m 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 ⁇ m, preferably 20 to 70 ⁇ 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.
  • 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.
  • the pore structure continuum can generally be produced by a sol-gel method.
  • a metal alkoxide is partially hydrolyzed to produce a reactive monomer, and this monomer is polycondensed to produce a colloidal oligomer.
  • 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.
  • Example 1 Preparation of 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.
  • the cultured liquid medium was filtered with a hydrophilic filter having a pore size of 5 ⁇ m, 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.
  • 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.
  • the tip was moved to a tube containing 500 ⁇ L of the cleaning solution, and the cleaning solution was sucked and discharged three times at a rate of 100 ⁇ L / second for cleaning. This washing process was repeated once more. Finally, the tip is moved to a tube containing 100 ⁇ L of eluate, and the eluate is aspirated and discharged 5 times at a rate of 100 ⁇ L / 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.
  • Reagent composition Oligo 1 250 nM, Oligo 2 1500 nM, Oligo 3 (5 ′ end labeled with BODIPY-FL) 250 nM, ⁇ 10 buffer 1 ⁇ L, dNTP 0.2 mM, MgSO 4 4 mM, KOD plus DNA polymerase 0.2U, Eluate 1 ⁇ L (Adjust the total volume to 10 ⁇ L 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.
  • 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 ⁇ L.
  • 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.
  • 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 ⁇ m. 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 ⁇ m. 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 ⁇ m. 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 ⁇ m. 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 ⁇ m. 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 ⁇ m. 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 ⁇ m. 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.
  • 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.

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Abstract

L'invention concerne un procédé pour la réalisation simultanée de la bactériolyse d'une bactérie résistante aux acides et de la séparation d'un acide nucléique provenant de la bactérie avec une efficacité élevée et d'une manière simple. Le procédé est caractérisé en ce qu'il comprend la préparation d'une solution mélangée contenant un sel chaotropique et la bactérie résistante aux acides et la répétition de l'aspiration de la solution mélangée et de l'éjection de la solution mélangée à l'aide d'une puce ayant, adhérant par fusion à l'intérieur de celle-ci, un corps continu ayant une structure poreuse, la structure poreuse ayant un diamètre de pore de 10 à 100 µm. Dans le procédé, il est préféré de répéter l'aspiration et l'éjection au moins trois fois.
PCT/JP2011/063555 2010-06-18 2011-06-14 Procédé pour la réalisation simultanée d'une bactériolyse d'une bactérie résistante aux acides et d'une séparation d'acides nucléiques provenant de la bactérie Ceased WO2011158815A1 (fr)

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JP2012520447A JP5773280B2 (ja) 2010-06-18 2011-06-14 抗酸菌の溶菌とその核酸の分離を同時に行う方法
CN2011800301817A CN102947449A (zh) 2010-06-18 2011-06-14 同时进行抗酸菌的溶菌和抗酸菌的核酸的分离的方法

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Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH06319527A (ja) * 1991-12-18 1994-11-22 Becton Dickinson & Co ミコバクテリアの溶菌方法
JPH11266864A (ja) * 1998-03-19 1999-10-05 Hitachi Ltd 核酸の精製方法および精製用装置
WO2002078846A1 (fr) * 2001-03-28 2002-10-10 Hitachi, Ltd. Instrument et procede de recolte d'acides nucleiques
WO2005078088A1 (fr) * 2004-02-12 2005-08-25 Gl Sciences Incorporated Mécanisme de séparation et de purification d'adn et ainsi de suite
JP2006296220A (ja) * 2005-04-15 2006-11-02 Gl Sciences Inc Ｄｎａなどの分離精製方法及び分離精製機構
JP2007306867A (ja) * 2006-05-19 2007-11-29 Hitachi High-Technologies Corp 核酸抽出装置及び核酸抽出方法
WO2009058432A1 (fr) * 2007-10-31 2009-05-07 Akonni Biosystems Dispositif de préparation d'échantillon
JP2011110025A (ja) * 2009-11-30 2011-06-09 Toyobo Co Ltd 抗酸菌の溶菌方法

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP4090443B2 (ja) * 2004-02-24 2008-05-28 株式会社日立ハイテクノロジーズ 核酸回収器具、その部品、及び核酸回収器具の生産方法

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH06319527A (ja) * 1991-12-18 1994-11-22 Becton Dickinson & Co ミコバクテリアの溶菌方法
JPH11266864A (ja) * 1998-03-19 1999-10-05 Hitachi Ltd 核酸の精製方法および精製用装置
WO2002078846A1 (fr) * 2001-03-28 2002-10-10 Hitachi, Ltd. Instrument et procede de recolte d'acides nucleiques
WO2005078088A1 (fr) * 2004-02-12 2005-08-25 Gl Sciences Incorporated Mécanisme de séparation et de purification d'adn et ainsi de suite
JP2006296220A (ja) * 2005-04-15 2006-11-02 Gl Sciences Inc Ｄｎａなどの分離精製方法及び分離精製機構
JP2007306867A (ja) * 2006-05-19 2007-11-29 Hitachi High-Technologies Corp 核酸抽出装置及び核酸抽出方法
WO2009058432A1 (fr) * 2007-10-31 2009-05-07 Akonni Biosystems Dispositif de préparation d'échantillon
JP2011110025A (ja) * 2009-11-30 2011-06-09 Toyobo Co Ltd 抗酸菌の溶菌方法

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BOOM R ET AL.: "Rapid and simple method for purification of nucleic acids", J. CLIN. MICROBIOL., vol. 28, no. 3, 1990, pages 495 - 503 *
O'MAHONY J ET AL.: "Rapid real-time PCR assay for detection and quantitation of Mycobacterium avium subsp. paratuberculosis DNA in artificially contaminated milk", APPL. ENVIRON. MICROBIOL., vol. 70, no. 8, 2004, pages 4561 - 4568 *
WILSON SM ET AL.: "Progress toward a simplified polymerase chain reaction and its application to diagnosis of tuberculosis", J. CLIN. MICROBIOL., vol. 31, no. 4, 1993, pages 776 - 782 *

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JP5773280B2 (ja) 2015-09-02
JPWO2011158815A1 (ja) 2013-08-19

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