JP2006288257A - Reagent for stabilizing microorganism, and utilization thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a reagent for stabilizing microorganisms, enabling the microorganisms to be preserved and stabilized by a very simple composition, and exhibiting the preserving and stabilizing effects even at room temperature; to provide a method for stabilizing the microorganisms by using the reagent; to provide a pretreating liquid of a sample, capable of readily and rapidly pretreating the sample in a stable state of the microorganisms, a pretreating method of the sample by using the pretreating liquid, and a kit for the pretreatment; and to provide a method for detecting the microorganisms by which the microorganisms can readily and rapidly be detected in the stable state of the microorganisms. <P>SOLUTION: The reagent for stabilizing the microorganisms is a buffer solution containing tris and sodium chloride regulated so that the concentration of the sodium chloride may be >0.6% and <1.2%, the concentration of the tris may be ≥10 mM and <50 mM, and the pH may be ≥7.0 and <8.0. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a microorganism stabilizing reagent capable of storing microorganisms in a stable state, a microorganism stabilizing method, a sample pretreatment liquid, a sample pretreatment method, a sample pretreatment kit, and a microorganism detection. Regarding the method.

Known methods for preserving microorganisms include freeze-dried storage, and methods of suspending in sterilized distilled water or sugar solution and storing under low temperature conditions of 5 ° C. or lower. For example, Patent Document 1 proposes a method of suspending cells grown and cultured in a solution in which distilled water or a storage stabilizer is dissolved, and storing the suspension at a low temperature.
Patent Document 2 discloses a microorganism in a solution in which at least one substance selected from the group consisting of sugar, sugar alcohol, phosphate, magnesium salt, sodium chloride, ammonium salt, amino acid and polyethylene glycol is dissolved. And a method of storing the suspension at a low temperature is proposed.

However, in any case, it is necessary to store the microorganisms at a low temperature, and when the temperature exceeds 10 ° C., the storage stabilization effect of the microorganisms is reduced (for example, refer to paragraph 0016 of Patent Document 1). Therefore, when these preservation solutions are used as solutions when processing specimens containing microorganisms with enzymes, etc., the enzyme activity decreases significantly at low temperatures, so that processing takes time and the enzyme activity is maintained. When kept at room temperature to about 37 ° C., there is a problem that microorganisms are affected.
JP 2002-95464 A Japanese Patent Laid-Open No. 5-292934

  An object of the present invention is to provide a microorganism stabilizing reagent that can preserve and stabilize microorganisms with a very simple composition and that exhibits a storage stability effect even at room temperature, and a microorganism stabilizing method using the reagent. And The present invention also provides a sample pretreatment liquid that can pretreat a sample easily and quickly in a stable state of microorganisms, a sample pretreatment method using the pretreatment liquid, and a pretreatment kit. . Furthermore, an object of the present invention is to provide a microorganism detection method capable of detecting microorganisms simply and quickly in a stable state.

  In order to solve the above-mentioned problems, the microorganism stabilizing reagent of the present invention is a buffer solution containing Tris and sodium chloride, wherein the sodium chloride concentration is more than 0.6% and less than 1.2%. Is 10 mM or more and less than 50 mM, and pH is 7.0 or more and less than 8.0. The concentration% described in the present invention is mass / mass% unless otherwise specified. In the present invention, tris (hydroxymethyl) aminomethane is referred to as tris.

The Tris concentration is preferably 15 mM or more and 30 mM or less.
The sodium chloride concentration is preferably 0.8% or more and 1.1% or less.
It is preferable that the microorganism is a virus, rickettsia, bacterium or fungus.

  The microorganism stabilization method of the present invention is characterized in that the microorganism is suspended in the stabilization reagent of the present invention.

  The sample pretreatment liquid of the present invention is a sample pretreatment liquid for detecting microorganisms present in the sample, and the pretreatment liquid contains a proteolytic enzyme as an active ingredient. It consists of a stabilizing reagent.

The proteolytic enzyme is preferably at least one selected from the group consisting of semi-alkaline protease and cysteine protease.
It is preferable that the cysteine protease is at least one selected from the group consisting of bromelain, papain, chymopapain and ficin.
The sample is whole blood, plasma, serum, urine, spinal fluid, semen, saliva, breast milk, sweat, mucus, body fluid, feces, lesion tissue and its extract, pus, sputum, nasal discharge, nasal wash, nasal rinse, It is preferably a throat swab or a microorganism culture sample.

  The sample pretreatment method of the present invention is characterized by using the pretreatment liquid of the present invention.

  A first aspect of the microorganism detection method of the present invention is characterized by using the stabilizing reagent of the present invention.

  A second aspect of the microorganism detection method of the present invention is characterized in that a sample is suspended in the stabilizing reagent of the present invention and the microorganism is detected.

  A third aspect of the microorganism detection method of the present invention is characterized in that the microorganism is detected after the sample is pretreated with the pretreatment liquid of the present invention.

  In the method for detecting a microorganism of the present invention, the sample is whole blood, plasma, serum, urine, spinal fluid, semen, saliva, breast milk, sweat, mucus, body fluid, stool, lesion tissue and its extract, pus, sputum Nasal discharge, nasal wash, nasal rinse, throat swab, or microbial culture sample.

The sample pretreatment kit of the present invention comprises the stabilizing reagent of the present invention and a sample pretreatment agent. The pretreatment agent is preferably a proteolytic enzyme.
It is preferable that the proteolytic enzyme is at least one selected from the group consisting of semi-alkaline protease and cysteine protease.
The cysteine protease is preferably at least one selected from the group consisting of bromelain, papain, chymopapain and ficin.

  According to the microorganism stabilization reagent and stabilization method of the present invention, microorganisms can be stabilized with a very simple composition, and the number of microorganisms can be suppressed to a constant without growing, and further stored at room temperature. Since the stabilizing effect is exhibited, the influence on the enzyme reaction is extremely small, and it is extremely effective for the detection of microorganisms, particularly for the pretreatment of a sample for detecting microorganisms. In addition, the microorganism stabilization reagent of the present invention can be used as a common stabilization reagent for a plurality of general bacteria. Furthermore, the microorganism stabilizing reagent of the present invention is effective for temporary storage of samples such as sputum for transportation and inspection purposes.

  According to the sample pretreatment liquid, the pretreatment method and the pretreatment kit of the present invention, the sample can be pretreated easily and quickly in a stable state without affecting microorganisms while maintaining the enzyme activity. it can. According to the microorganism detection method of the present invention, microorganisms can be easily and rapidly detected in a stable state with little influence on microorganisms.

  Embodiments of the present invention will be described below, but these are exemplarily shown, and it goes without saying that various modifications are possible without departing from the technical idea of the present invention.

The microorganism stabilizing reagent of the present invention is a buffer containing Tris and sodium chloride.
The concentration of sodium chloride in the stabilizing reagent is more than 0.6% and less than 1.2%, preferably 0.8% or more and 1.1% or less, more preferably about 9%.
The Tris concentration in the stabilizing reagent is 10 mM or more and less than 50 mM, preferably 15 mM or more and 30 mM or less. The pH of the buffer solution is 7.0 or more and less than 8.0. The acid for titrating the pH is not particularly limited, but hydrochloric acid is preferred.

  The stabilizing reagent of the present invention may contain a known microorganism storage stabilizer, for example, a sugar such as glucose, trephalose, sucrose and the like.

  The microorganism can be stabilized by suspending the microorganism in the stabilizing reagent of the present invention. By suspending the sample in the stabilizing reagent and then detecting the microorganisms, the microorganisms present in the sample can be detected in a stable state. The detection method of microorganisms is not specifically limited, A well-known method can be used widely. In the detection of microorganisms, the microorganisms may be detected directly, or the microorganisms may be detected by detecting a biological substance.

  In the present invention, the microorganism is not particularly limited. For example, herpes virus (HSV, CMV, ZVZ, EBV, HHV, etc.), influenza virus, human immunodeficiency virus (HIV), human adult T cell leukemia virus (HTLV) , Viruses such as hepatitis viruses (HBV, HCV, HDV, HGV), other diseases such as Kaze syndrome, gastrointestinal diseases, central nervous system diseases, respiratory diseases, hemorrhagic fever, etc., rickettsia, Staphylococcus aureus And bacteria such as streptococci, coliforms, Pseudomonas aeruginosa, Legionella, Moraxella, influenza bacteria, Klebsiella, Chlamydia, mycoplasma, and the like.

  In the present invention, examples of the biological substance include nucleic acids (DNA, RNA), proteins, peptides and the like. Preparation of DNA or RNA from living cells can be performed by a known method, for example, the method of Blin et al. (Blin et al., Nucleic Acids Res. 3: 2303 (1976)) for DNA extraction, Extraction can be performed by the method of Favaloro et al. (Favaloro et al., Methods Enzymol. 65: 718 (1980)) and the like. For rRNA, cells may be lysed with an aqueous sodium hydroxide solution and then neutralized with hydrochloric acid or the like. As detection methods thereof, PCR (Polymerase chain reaction), hybridization, PALSAR reaction (see, for example, Japanese Patent No. 3267576), DNA chip, protein chip, antigen-antibody reaction, etc. can be used. .

  In the present invention, the sample is not particularly limited, but a biological sample or a biological sample is preferable, and specifically, whole blood, plasma, serum, urine, spinal fluid, semen, saliva, breast milk, sweat, mucus, etc. Body fluids and feces, lesioned tissue and extracts thereof, pus, sputum, nasal discharge, nasal wash, nasal rinse, throat swab, and microbial culture samples such as viruses.

The stabilizing reagent of the present invention is also suitably used as a lysing solution when processing a sample in the method for detecting microorganisms present in the sample. In particular, it is very effective as a solution for pretreatment of a sample with a proteolytic enzyme.
The sample pretreatment solution of the present invention is a sample pretreatment solution for detecting microorganisms present in the sample, and is a solution in which a proteolytic enzyme is contained as an active ingredient in the stabilizing reagent. The proteolytic enzyme is not particularly limited and may be appropriately selected depending on the sample and the treatment method. These proteolytic enzymes can be used alone or in combination of two or more.

For example, in the case of homogenization treatment using sputum as a sample, it is preferable to use at least one selected from the group consisting of a semi-alkaline protease (for example, sputazyme (manufactured by Kyokuto Pharmaceutical Co., Ltd.)) and cysteine protease. .
The cysteine protease is not particularly limited, but is preferably a plant-derived cysteine protease, more preferably one or more selected from the group consisting of bromelain, papain, chymopapain and ficin, and bromelain is particularly preferable. These cysteine proteases may be commercially available or those obtained by known methods.
As the bromelain, a plant-derived cysteine protease belonging to Bromeliaceae is used, but one derived from pineapple (Ananas comosus M.) is more preferable.

  In the present invention, after pretreatment of a test sample with the pretreatment liquid, microorganisms present in the test sample can be detected without culturing or culturing the microorganism.

  The present invention will be described more specifically with reference to the following examples. However, it is needless to say that these examples are shown by way of illustration and should not be construed in a limited manner.

(Experimental example 1)
A 10 mM Tris-HCl buffer solution (pH 7.0, 7.5, or 8.0) containing 0.9% (150 mM) sodium chloride was prepared as a suspension. Haemophilus influenzae was added to this suspension at 10 ³ CFU / mL, and the number of viable cells in the bacterial suspension was measured at room temperature (26 ° C.) at 0 and 2 hours. . In addition, the method for measuring the number of viable cells is as follows. Three bacterial suspensions of the above-mentioned bacterial suspension are plated on each plate of chocolate agar EX (manufactured by Nissui Pharmaceutical Co., Ltd.) and cultured at 37 ° C. for 18 hours. The number of colonies was measured for each sheet, and the average value of the three sheets was defined as the number of viable bacteria.
As a control, the number of viable bacteria was similarly measured using a 0.9% sodium chloride aqueous solution as a suspension. The results are shown in Table 1.

  As shown in Table 1, it was clarified that the decrease in the number of viable bacteria can be suppressed by setting the pH of the Tris buffer containing sodium chloride to 7.0 or more and less than 8.0.

(Experimental example 2)
Tris-HCl buffer solutions (pH 7.0) having various concentrations shown in Table 2 containing 0.9% sodium chloride were prepared and used as suspensions. Haemophilus influenzae was added to this suspension so as to be 10 ³ CFU / mL, and the viable cell counts were compared at 0 hours and 2 hours at room temperature (26 ° C.). The method for measuring the number of viable bacteria is the same as in Experimental Example 1. As a control, the number of viable bacteria was similarly measured using a 0.9% sodium chloride aqueous solution as a suspension. The results are shown in Table 2.

  As shown in Table 2, by reducing the Tris concentration of the Tris buffer containing sodium chloride to 10 mM or more and less than 50 mM, the decrease in the number of viable bacteria can be suppressed. It became clear that the decrease in the number of bacteria was suppressed.

(Experimental example 3)
A 25 mM Tris-HCl buffer (pH 7.0) containing sodium chloride solutions having respective concentrations shown in Table 3 was prepared and used as a suspension. Haemophilus influenzae was added to this suspension so that it might become 10 < ³ > CFU / mL, and the viable cell count was compared in 0 hour and 2 hours at room temperature (26 degreeC). The method for measuring the number of viable bacteria is the same as in Experimental Example 1. The results are shown in Table 3.

  As shown in Table 3, the use of Tris buffer solution containing sodium chloride exceeding 0.6% and less than 1.2 can suppress the decrease in the number of viable bacteria. By doing so, it was clarified that the decrease in the number of viable bacteria was significantly suppressed.

(Example 1 and Comparative Example 1)
A 25 mM Tris-HCl buffer (pH 7.0) containing 0.9% sodium chloride and 0.1 mM EDTA was prepared and used as a suspension. In this suspension, Haemophilus influenzae, Streptococcus pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, Legionella pneumonia Lella pneumonia (Klebsiella pneumoniae) was added to 10 ³ CFU / mL, and the viable cell counts were compared at room temperature (26 ° C.) at 0 hour and 2 hours. The method for measuring the number of viable bacteria is the same as in Experimental Example 1.
Further, as a control (Comparative Example 1), a viable cell count was similarly measured using a 0.9% aqueous sodium chloride solution as a suspension. The results are shown in Table 4.

  As shown in Table 4, according to the present invention, it was clarified that the decrease in the number of viable bacteria can be significantly suppressed against various bacteria as compared with conventional physiological saline.

(Example 2)
0.25% (2000 U / mL) Bromelain F (Bromelain F, Amano Enzyme) was dissolved in a solution [25 mM Tris buffer (pH 7.0), 0.9% sodium chloride, 0.1 mM sodium ethylenediaminetetraacetate ( EDTA)] to prepare a pretreatment solution. This pretreatment solution was added in a 20-fold amount to purulent sputum (P2 and P3, Miller & Jones classification) and allowed to react at room temperature (26 ° C.). At that time, the suspension was vortexed every 10 minutes and the dissolution time was measured. The soot dissolution time was the reaction time required for the soot to be almost completely in solution. The results are shown in Table 5.

(Example 3)
In the pretreatment solution prepared in the same manner as in Example 2, Haemophilus influenzae, Streptococcus pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus aureus, (Legionella pneumophila) or Klebsiella pneumoniae was added at 10 ³ CFU / mL, and the viable cell counts were compared at room temperature (26 ° C.) at 0 and 2 hours. The method for measuring the number of viable bacteria is the same as in Experimental Example 1. The results are shown in Table 6.

  As shown in Tables 5 and 6, by using the pretreatment liquid of the present invention, the decrease in the number of viable bacteria is greatly suppressed against various bacteria without impairing the activity of bromelain F in sputum lysis. It became clear that

Example 4
Sputazyme (manufactured by Kyokuto Pharmaceutical Co., Ltd., semi-alkaline protease) is dissolved in a solution [25 mM Tris buffer (pH 7.0), 0.9% sodium chloride, 0.1 mM sodium ethylenediaminetetraacetate (EDTA)]. A pretreatment liquid was used. This pretreatment solution was added in a 20-fold amount to purulent sputum (P3, Miller & Jones classification) and allowed to react at room temperature (26 ° C.). At that time, the suspension was vortexed every 10 minutes and the dissolution time was measured. The soot dissolution time was the reaction time required for the soot to be almost completely in solution. The results are shown in Table 7.

(Comparative Example 2)
Sputazyme (manufactured by Kyokuto Pharmaceutical Co., Ltd., semi-alkaline protease) was dissolved in a phosphate buffer which is a dissolution reagent of the kit to prepare a pretreatment solution. The experiment was performed in the same manner as in Example 4 except that this pretreatment liquid was used. The results are shown in Table 7.

  As shown in Table 7, it was revealed that even if the stabilizing reagent of the present invention was used for semi-alkaline protease, the dissolution effect on sputum was equal or better.

Claims (18)

  A buffer containing Tris and sodium chloride, wherein the sodium chloride concentration is more than 0.6% and less than 1.2%, the Tris concentration is 10 mM or more and less than 50 mM, and the pH is 7.0 or more and less than 8.0. A microorganism-stabilizing reagent.   The microorganism stabilization reagent according to claim 1, wherein the Tris concentration is 15 mM or more and 30 mM or less.   The microorganism stabilizing agent according to claim 1 or 2, wherein the sodium chloride concentration is 0.8% or more and 1.1% or less.   4. The microorganism stabilizing reagent according to any one of 1 to 3, wherein the microorganism is a virus, a rickettsia, a bacterium, or a fungus.   A method for stabilizing a microorganism, comprising suspending a microorganism in the stabilizing reagent according to any one of claims 1 to 4.   A stabilization reagent according to any one of claims 1 to 4, which is a sample pretreatment liquid for detecting microorganisms present in the sample, wherein the pretreatment liquid contains a proteolytic enzyme as an active ingredient. A sample pretreatment liquid comprising:   The sample pretreatment solution according to claim 6, wherein the proteolytic enzyme is at least one selected from the group consisting of semi-alkaline protease and cysteine protease.   The sample pretreatment solution according to claim 7, wherein the cysteine protease is at least one selected from the group consisting of bromelain, papain, chymopapain and ficin.   The sample is whole blood, plasma, serum, urine, spinal fluid, semen, saliva, breast milk, sweat, mucus, body fluid, feces, lesion tissue and its extract, pus, sputum, nasal discharge, nasal wash, nasal rinse, The sample pretreatment liquid according to any one of claims 6 to 8, which is a pharyngeal wiping liquid or a microorganism culture system sample.   A sample pretreatment method using the pretreatment liquid according to any one of claims 6 to 9.   A method for detecting microorganisms, comprising using the stabilizing reagent according to any one of claims 1 to 4.   A method for detecting microorganisms, comprising suspending a sample in the stabilizing reagent according to any one of claims 1 to 4 and detecting microorganisms.   A microorganism detection method, comprising: pretreating a sample with the pretreatment liquid according to any one of claims 6 to 9, and then detecting the microorganism.   The sample is whole blood, plasma, serum, urine, spinal fluid, semen, saliva, breast milk, sweat, mucus, body fluid, feces, lesioned tissue and its extract, pus, sputum, nasal discharge, nasal wash, nasal swab The method for detecting microorganisms according to claim 12 or 13, wherein the microorganism is a throat swab or a microorganism culture sample.   A sample pretreatment kit comprising the stabilizing reagent according to any one of claims 1 to 4 and a sample pretreatment agent.   The pretreatment kit according to claim 15, wherein the pretreatment agent is a proteolytic enzyme.   The sample pretreatment kit according to claim 16, wherein the proteolytic enzyme is at least one selected from the group consisting of semi-alkaline protease and cysteine protease.   The sample pretreatment kit according to claim 17, wherein the cysteine protease is at least one selected from the group consisting of bromelain, papain, chymopapain and ficin.