JP2007143495A - Method for crushing material to be crushed in sample - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for crushing a sample which is difficult to crush and efficiently extracting a nucleic acid in a short time. <P>SOLUTION: In the method for crushing a material to be crushed in a sample by adding a powdery solid material which is amorphous to a sample containing a material to be crushed which is a microorganism or an organic tissue, and stirring the sample, major axis of the powdery solid material is ≤32 μm and the specific gravity is ≥3.5 and the hardness (Hv10) is ≥600. A method for extracting a nucleic acid from the microorganism and the organic tissue are also provided. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for efficiently crushing microorganisms or biological tissues in a sample using a powdery solid.

  In order to make a definitive diagnosis of the cause of infection or other diseases caused by bacteria or viruses, it is necessary to detect and identify the bacteria or viruses that cause the disease.

  In the field of clinical diagnosis and the like, a definite diagnosis of a disease is performed by extracting a nucleic acid from a sample and amplifying and detecting a target bacterial or viral gene (Japanese Patent Laid-Open No. 2000-14400, “Quantification of target nucleic acid”). Method (Patent Document 1); Anal. Biochem., 314, 77-86 (2003) (Non-Patent Document 1)). Furthermore, nucleic acids extracted from cells in human tissues can be used as diagnostic markers for cancer and other diseases (Ann. Surg. Oncol., 11, 778-785 (2004) (Non-patent Document 2)). ), Early diagnosis of disease, treatment monitoring, prognosis determination, and index of treatment policy determination (J. Clin. Microbiol., 42, 4284-4292 (2004) (Non-patent Document 3); J. Clin. Microbiol., 43, 2489-2491 (2005) (non-patent document 4)). In these genetic tests, extraction of nucleic acids from a sample is an important operation. Cell disruption methods for nucleic acid extraction include mechanical methods using homogenizers, chemical methods using denaturants, etc. (Molecular Cloning: A laboratory manual Appendix 7.23-7.25 (New York: Cold Spring Harbor). (Laboratory, 1989), and biological methods using enzymes, etc. (Molecular Cloning: A laboratory manual Appendix E3-E4 (New York: Cold Spring Harbor Laboratory, 1989). There is a method of crushing cells by mixing them and stirring the sample.

  In genetic testing in clinical diagnosis, these conventional nucleic acid extraction methods have problems that some microorganisms have a hard cell wall that cannot sufficiently crush bacteria, making it difficult to efficiently extract bacterial genes. There was a point. In addition, it takes time to crush the cell wall for nucleic acid extraction, and as a result, it takes time to extract nucleic acid. In addition, in the case where tissues and cells are disrupted by adding and mixing particulate solids to a frozen sample and stirring the sample, it is necessary to freeze the sample in advance, which takes time for freezing, There was a problem that the sample was dissolved while the sample was being processed, and the cells could not be efficiently disrupted. In that case, a special apparatus for crushing the sample while it was frozen was necessary.

  As described above, if extraction of nucleic acids from target microorganisms or tissues contained in a sample is difficult or takes a long time, the microorganisms causing the disease are detected and identified with high sensitivity or rapidity by genetic testing. There were problems that could not be used, and that genes in tissues could not be used as genetic markers for diseases.

JP 2000-14400 A

Anal. Biochem. , 314, 77-86 (2003) Ann. Surg. Oncol. , 11, 778-785 (2004) J. et al. Clin. Microbiol. , 42, 4284-4292 (2004) J. et al. Clin. Microbiol. , 43, 2489-2491 (2005)

  An object of the present invention is to provide a method for crushing a sample that is difficult to crush and efficiently extract nucleic acid in a short time. Thereby, a nucleic acid is efficiently extracted from a microorganism in a short time, and a gene is detected and identified, so that a definitive diagnosis of an infection caused by bacteria or viruses can be performed. Further, by extracting nucleic acid from a tissue in a short time and detecting a marker gene for a disease, it can be used as an index for determination of treatment policy during surgery and monitoring of treatment.

As a result of repeated studies, the present inventors have found a method for efficiently disrupting microorganisms and tissues in a sample.
The present invention relates to a method for crushing a material to be crushed in a sample by adding the powdered solid material to a sample containing the material to be crushed and stirring the sample, and the major axis of the powdered solid material is 32 μm or less. There is provided a method for crushing an object to be crushed, having a specific gravity of 3.5 or more and a hardness (Hv10) of 600 or more.
In a preferred embodiment, the powdered solid is amorphous. Furthermore, the material to be crushed can be a microorganism or a biological tissue. In a more preferred embodiment, the powdered solid is zirconia.
In another aspect, the present invention also provides a method for extracting nucleic acid, characterized in that nucleic acid is extracted from the material to be crushed by the above method.

  According to the present invention, microorganisms or biological tissues in a sample can be efficiently disrupted even if they have a hard cell wall, and their nucleic acids can be extracted to perform high-sensitivity and rapid testing of genes. Is possible. In addition, since it is not necessary to freeze the sample, it is possible to reduce the cost and size of the apparatus. Thereby, a nucleic acid is efficiently extracted from a microorganism in a short time, and a gene is detected and identified, so that a definitive diagnosis of an infection caused by bacteria or viruses can be performed. Further, by extracting nucleic acid from a tissue in a short time and detecting a marker gene for a disease, it can be used as an index for determination of treatment policy during surgery and monitoring of treatment.

Hereinafter, the present invention will be described in detail.
The powdered solid substance in the present invention may be any inorganic substance as long as it satisfies the condition that the major axis is 32 μm or less, the specific gravity is 3.5 or more, and the hardness (Hv10) is 600 or more. However, it may be an organic material. Non-limiting examples of the material of the powdered solid substance include inorganic materials such as zirconia, diamond, iron, alloys such as tungsten carbide-cobalt alloy (WC-Co alloy).

  The powdered solid in the present invention is preferably a non-uniform and amorphous powder, but may be of a uniform shape. The powdered solid material preferably has an angular shape with sharp corners, not a shape with smooth corners such as a sphere or an ellipse. Specific examples of such a shape include, for example, a hemisphere, a cone, a triangular pyramid, a quadrangular pyramid, a cylinder, a cube, a triangular to a hexagonal prism, a plate, For example, a polygonal plate including a triangle, a quadrangle, a pentagon, and the like, a shape in which these shapes are combined, a star shape having a sharp protrusion, and the like can be given. The shape of the powdered solid can be confirmed by observation with, for example, a phase-contrast optical microscope, a scanning type, or a transmission type electron microscope.

  As for the size of the particles, the longest part of the diameter of the powder may be 32 μm or less, preferably 20 μm or less. The specific gravity is preferably larger, because the tissue is crushed using the kinetic energy of stirring, and may be 3.5 or more. The upper limit of the specific gravity of the particles is not particularly limited, but in consideration of the production and availability of the particles, for example, it will be about 20 at the maximum, more preferably about 10 at the maximum. The sample and the powdered solid may be separated by centrifugation. When the specific gravity is larger than the sample solution, the powdered solid can settle and be separated from the sample by standing. Moreover, separation by magnetic force is possible by incorporating a magnetic substance in the powder solid. The hardness of the powdery solid is preferably as hard as possible, and is preferably 600 or more, preferably 1000 or more in terms of Vickers hardness (Hv10). For the hardness, the amount of powdered solid added to the sample is appropriately determined according to the type of sample, the stirring method, and the stirring time. The hardness can be measured, for example, according to JIS Handbook: Ceramics, Japanese Standards Association, p315-317.

  The powdery solid in the present invention may be commercially available or prepared. For example, a material that is a raw material of the powdered solid material in the present invention, for example, plate-shaped zirconia is appropriately crushed by a pulverizer, and the obtained powder has an appropriate pore size for selecting a powder having a desired size. Sieve (for example, when a powder having a major axis of particles of 32 μm or less is desired, a sieve having a diameter of 32 μm or less, and when a powder having a major axis of particles of 20 μm or less is desired, a powder having a major axis having a desired size) Can be recovered, and preferably sterilized. The size and shape of the powder can be confirmed, for example, by observation with a scanning electron microscope.

  Examples of the sample in the present invention include sample suspensions of biological samples such as sputum, pus, colonies and swabs, sample suspensions such as homogenization of food samples, and sample solutions such as blood, urine and blood culture fluid. It is done. When the sample is sputum, a more preferable result can be obtained by performing a pretreatment such as NALC (N-acetyl-L-cysteine) treatment to reduce the viscosity of the sample. In addition, environmental water and wastewater in environmental analysis and the like, soil suspension, and the like can be mentioned.

  Microorganisms in the sample are gram-positive bacteria such as Escherichia coli, Salmonella, Klebsiella pneumoniae, Serratia bacteria, Vibrio parahaemolyticus, H. influenzae, etc., Staphylococcus aureus, Streptococcus, Tetanus, Clostridium botulinum, Bacillus anthracis, etc. , And Mycobacterium tuberculosis. Examples of viruses include norovirus, hepatitis A virus, hepatitis B virus, hepatitis C virus, AIDS virus, SARS virus, avian influenza virus and the like. Examples of biological tissues in the sample include human pathological tissues, such as breast cancer tissues and liver cancer tissues, human organ tissues, animal organs, and plant tissues.

  The sample can be stirred by reciprocating the container containing the sample by hand, and the direction of the amplitude may be vertical or horizontal, but is preferably about 2 to 10 reciprocations per second in the vertical direction. It is better to reciprocate. Or you may stir with a vortex mixer. The agitation time may be until the cell membranes of microorganisms and tissues are destroyed, specifically, 2 minutes or more. If the tissue has a strong cell wall and is hard like a plant tissue, it may be longer.

  By disrupting the target bacteria and tissues according to the present invention, various physiologically active substances such as nucleic acids, proteins, carbohydrates, lipids, hormones and the like are easily extracted from them. Preferably, disrupted bacteria or tissue debris is removed by well-known means such as centrifugation or filtration. The method of the present invention is particularly useful for nucleic acid extraction. The nucleic acid extracted through the disruption method of the present invention can be subjected to various treatments, and a typical example is a nucleic acid amplification test. Nucleic acid amplification tests include PCR (Polymerase Chain Reaction), RT-PCR (Reverse-Transscript PCR), LCR (Ligase Chain Reaction), LAMP (Loop-Mediated Amplification of DNA Amplification DNA) Sequence Based Amplification), TMA (Transscription-Mediated Amplification), or TRC (Transcribation Reverse Transaction Reaction, Ayumi of Medicine, Vol. 206, No. 200, No. 200, etc.). RNA amplification methods.

  Nucleic acid extraction is performed by destroying cell membranes, cell walls, or coat proteins with enzymes and surfactants, decomposing complex proteins to release nucleic acids, and then adding phenol / chloroform to extract the released nucleic acids. The so-called phenol / chloroform method (Molecular Cloning: A laboratory manual Appendix E3-E4 (New York: Cold Spring Harbor Laboratory, 1989)), disrupting cell membranes or protein walls by treating guanidine hydrochloride or cell wall, Then, the protein forming a complex with the nucleic acid is denatured to release the nucleic acid, and then the so-called guanidine method (Molecular Cloning) in which the nucleic acid is extracted by adding ethanol or the like. A laboratory manual Appendix 7.23~7.25 (New York: Cold Spring Harbor Laboratory, 1989 years)), and the like. However, these methods have many processing steps, are complicated, and require time. In addition, phenol as an acidic solution, chloroform as an organic solvent, and guanidine hydrochloride or guanidine thiocyanate as strong modifiers are irritating and toxic. Therefore, it is harmful to the human body and needs to be handled with care, and the waste liquid has a great negative effect on the natural environment. In the present invention, bacterial cells and tissues can be crushed and their nucleic acids can be extracted in a short time without using these acidic solutions, organic solvents, and denaturing agents.

  When nucleic acid is extracted for nucleic acid amplification test, depending on the sample, the bacterial cell wall is hard or the tissue is hard, and it may be difficult to extract the nucleic acid. Eosinophils and the like in the cocoon have a hard cell wall, and it is difficult to extract nucleic acids by efficiently crushing all the eosinophilic bacteria with a normal nucleic acid extraction method. However, according to the present invention, it is possible to efficiently disrupt bacteria and tissues having hard cell walls and extract nucleic acids.

  Until now, there was a method of crushing cells and tissues by adding beads etc. to the sample and stirring, but the sample must be frozen or frozen to prevent dissolution of the sample during stirring The state had to be stirred. For this reason, it takes a long time to freeze the sample, and it was impossible to perform a rapid inspection. Moreover, the apparatus must have a refrigeration function, is expensive in price, and it is difficult to downsize the apparatus. In addition, when the sample is not frozen, the crushing efficiency is low, so it must be stirred for a long time, the temperature of the sample rises, and the protein in the cell may be denatured. In the present invention, since it is not necessary to freeze the sample, the time required for freezing can be omitted, and the stirring time can be shortened. Therefore, it is possible to perform a rapid test using the extracted nucleic acid. Further, since no refrigeration function is required, the apparatus can be reduced in price and size.

  Examples are shown below to describe the invention in more detail, but the invention is not limited to these Examples.

1. Preparation of amorphous zirconia powder Plate-shaped zirconia TZ-8YS (manufactured by Tosoh Corporation, 110 mm x 110 mm, thickness 0.3 mm, specific gravity 6.05) was crushed at 120 rpm for 5 minutes with a knit-electric stamp mill ANS143 (manufactured by Aichi Electric Co., Ltd.) did. The obtained powdery zirconia was passed through a sieve having a diameter of 32 μm, and a zirconia powder having a major particle diameter of 32 μm or less was collected and sterilized at 121 ° C. for 20 minutes to obtain an amorphous zirconia powder (φ32 μm or less). A scanning electron micrograph of the amorphous zirconia powder (φ32 μm or less) is shown in FIG. It is shown that the powder is a non-uniform shaped amorphous powder having a long axis of 32 μm or less and a sharp angle. The remaining zirconia powder applied to a sieve of φ32 μm is similarly applied to a sieve of φ75 μm, and a powder of φ32 μm or more and φ75 μm or less and a powder of φ75 μm or more are respectively collected and sterilized at 121 ° C. for 20 minutes. φ75 μm or less) and amorphous zirconia powder (φ75 μm or more).

2. Preparation of samples containing bacteria Transformation was performed with pUC19 incorporating the mecA gene (FEBS Lett. 221 (1), 167-171, 1987) encoding PBP-2 ′ of MRSA (mesitillin-resistant Staphylococcus aureus). JM109 was cultured overnight at 37 ° C. in LB medium supplemented with 100 μg / ml ampicillin. The culture solution was diluted with PBS so that the absorbance at a wavelength of 600 nm was 0.5 to prepare a sample.

3. Nucleic acid extraction by disruption of bacteria in the sample 1 ml of the sample prepared by the above procedure is put into a 2 ml Eppendorf tube (manufactured by Eppendorf). 3.0 g of amorphous zirconia powder (φ32 μm or less), amorphous zirconia powder (φ32 μm or more and φ75 μm or less) or amorphous zirconia powder (φ75 μm or more) prepared by the above was added. The tube containing the sample and amorphous zirconia powder was vigorously stirred at room temperature for 2 minutes. Thereafter, the supernatant was collected by centrifugation. On the other hand, spherical zirconia (φ30 μm) or alumina powder (specific gravity 3.5, φ50 μm or less) having the same volume as the amorphous zirconia powder is added to 1 ml of the sample placed in a 2 ml Eppendorf tube, and the sample is subjected to the same procedure as above. The supernatant was recovered.

4). 2. Gene detection The supernatant prepared in (2) was diluted 2-fold with sterilized water, 2 μl of which was used as a primer (MecP4: 5′-TGCTATCCACCCTCAAACAGG-3 ′ (SEQ ID NO: 1) and primer (MecP2: 5′-AACGTTGTATAACCACCCCCAAGA-3 ′ (sequence The mecA gene in the supernatant was quantified by quantitative PCR using a light cycler (manufactured by Roche Diagnostics) using No. 2) The PCR reaction was performed at 94 ° C. for 20 seconds and at 43 ° C. for 30 seconds. And 40 cycles of 2 minutes at 72 ° C. As a control, a plasmid was extracted from the precipitate prepared by centrifuging at 13,000 rpm for 10 minutes using QIA prep Spin Miniprep Kit (manufactured by QIAGEN). Quantitative PCR was performed.

The results are shown in FIG. The graph shown in FIG. 2 shows the nucleic acid extraction rate when various kinds of zirconia powder or alumina powder are used to crush E. coli from a sample containing E. coli and extract the nucleic acid. The amount of plasmid extracted from 1 ml of an E. coli solution having an absorbance at a wavelength of 600 nm of 0.5 was defined as 100%, and the ratio of the plasmid extracted from each sample relative thereto was defined as the nucleic acid extraction rate.
When (i) crushes E. coli with amorphous zirconia powder having a major axis of 32 μm or less and collects nucleic acid, (ii) crushes Escherichia coli with amorphous zirconia powder with a major axis of 32 μm or more and 75 μm or less to collect nucleic acid In the case of (iii), when the nucleic acid is recovered by crushing E. coli with amorphous zirconia powder having a major axis of 75 μm or more, and (iv) in the case of recovering the nucleic acid by crushing E. coli with spherical zirconia having a particle size of 30 μm or less (V) shows the case where the nucleic acid was recovered by crushing Escherichia coli with amorphous alumina powder having a major axis of 50 μm or less.

  As a result, assuming that the control result is 100%, 3. The recovery rate of the gene obtained by crushing and extracting bacteria with amorphous zirconia powder (φ32 μm or less) was about 100%. On the other hand, genes obtained by crushing and extracting bacteria with amorphous zirconia powder (φ32 μm or more and φ75 μm or less), amorphous zirconia powder (φ75 μm or more), spherical zirconia (φ30 μm), alumina powder (specific gravity 3.5, particle size 50 μm or less) The recovery rates of all were about 50% or less. This shows that it is possible to efficiently crush bacteria in the sample and recover nucleic acid with 100% efficiency by this method.

Example 1. 2 is a scanning electron micrograph of amorphous zirconia powder (φ32 μm or less) shown in FIG. The side line in the lower right of the photograph indicates 30 μm.

The nucleic acid extraction rate is shown when nucleic acids are extracted by crushing E. coli from a sample containing E. coli using various zirconia powders or alumina powders.

Claims (5)

  In the method of crushing the material to be crushed by adding the powdered solid material to the sample containing the material to be crushed and stirring the sample, the major axis of the powdered solid material is 32 μm or less, and its specific gravity Is a method of crushing an object to be crushed, having a hardness (Hv10) of 600 or more.   The method of claim 1, wherein the powdered solid is amorphous.   The method according to claim 1 or 2, wherein the material to be crushed is a microorganism or a biological tissue.   The method according to claim 1, wherein the powdered solid is zirconia.   A nucleic acid extraction method, wherein nucleic acids are extracted from the crushed material by crushing the crushed material by the method according to claim 1.