JP2011177045A - Virus detection method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for simultaneously, simply, and specifically isolating and detecting a virus nucleic acid from a biological sample possibly containing at least one kind of virus. <P>SOLUTION: The virus detection method includes (a) a step of preparing a mixture by mixing the biological sample and a composition for dissociating the virus nucleic acid and other steps (b) to (e), wherein the composition for dissociating the virus nucleic acid contains at least one kind of a chaotropic agent selected from guanidine thiocyanate, guanidine hydrochloride and sodium iodide, an anionic surfactant and a reducing agent, and the concentration of the anionic surfactant in the mixture in the step (a) is not less than 2.5 mass% and not more than 30 mass%. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a method for specifically isolating and detecting viral nucleic acid from a biological sample potentially containing virus in the same solution, in particular hepatitis B virus, hepatitis C virus, and human immunity. The present invention relates to a method for specifically isolating and detecting viral nucleic acid in a same solution from a biological sample possibly containing at least one virus selected from defective viruses.

  Conventionally, the method for isolating nucleic acids to detect viruses has been different for each virus (see, for example, Patent Document 1). In order to simultaneously detect a plurality of viruses such as hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus (HIV), an operation of releasing nucleic acids from each virus in the same solution is performed. Necessary.

  However, since the cell wall and cell membrane of HBV (hereinafter also simply referred to as “membrane”) are stronger than other viruses, in order to lyse the HBV membrane and isolate nucleic acid, for example, a high concentration Severe conditions such as addition of denaturants, high temperature or alkaline environment are required.

  On the other hand, since membranes of hepatitis C virus and human immunodeficiency virus are physicochemically weak, the conditions for dissolving the HBV membrane described above are too harsh. In addition, since the nucleic acid isolated from hepatitis C virus or human immunodeficiency virus is ribonucleic acid (RNA), the released RNA molecules are decomposed into short fragments under the above HBV membrane lysis conditions, thereby detecting the gene. It will interfere.

  Research has been conducted on a technique for making the dissolution conditions of the HBV film, the HCV film, and the HIV film the same, and an enzyme method using a proteolytic enzyme is generally studied. However, in the enzymatic method using a proteolytic enzyme, when the released viral nucleic acid is detected in the subsequent gene amplification reaction, the proteolytic enzyme degrades the enzyme that acts on the gene amplification reaction, so that nucleic acid detection is not possible. There is a risk of disappearing. That is, there is a fatal wound that transforms into an inhibitory substance in a subsequent nucleic acid detection reaction by a proteolytic enzyme necessary for dissolving a viral membrane.

  On the other hand, after elution of the virus, a method of inactivating the proteolytic enzyme with heat and alkali and bringing it into the subsequent reaction has been devised. However, when these proteolytic enzymes are inactivated by physicochemical methods such as heat or alkali, the RNAs of hepatitis C virus and human immunodeficiency virus that have already been released are also degraded at the same time. There is a problem that can not be done.

  As described above, the viral nucleic acid is released by dissolving the hepatitis B virus membrane, and at the same time, the viral nucleic acid is released by dissolving the hepatitis C virus or human immunodeficiency virus membrane. The technology for performing genetic testing using sapphire has not been established yet.

JP 2005-505289 A

  An object of the present invention is to provide a method for specifically isolating and detecting a viral nucleic acid simultaneously and conveniently from a biological sample possibly containing at least one virus.

  SUMMARY An advantage of some aspects of the invention is to solve at least a part of the problems described above, and the invention can be implemented as the following aspects or application examples.

[Application Example 1]
One aspect of the virus detection method according to the present invention is:
A method of specifically isolating viral nucleic acid from a biological sample potentially containing at least one virus in the same solution and then detecting it,
(A) mixing the biological sample with a composition for releasing viral nucleic acid;
(B) adding to the mixture of step (a) an oligonucleotide that is hybridizable with at least a partial sequence of the viral nucleic acid and biotinylated at least at one end;
(C) adding a solid phase carrier having avidin or streptavidin immobilized on the surface thereof to the mixture of step (b);
(D) separating the solid support from the mixture of step (c);
(E) detecting the viral nucleic acid by a nucleic acid amplification method using the solid phase carrier;
Including
The composition contains at least one chaotropic agent selected from guanidinium thiocyanate, guanidinium hydrochloride, and sodium iodide, an anionic surfactant, and a reducing agent, and the mixture of the step (a) The concentration of the anionic surfactant inside is 2.5% by mass or more and 30% by mass or less.

[Application Example 2]
In application example 1,
The virus to be detected can be at least one selected from hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus (HIV).

[Application Example 3]
In application example 1 or application example 2,
Furthermore, the step (e) can include a step of eluting the viral nucleic acid by adding an eluate to the solid phase carrier obtained in the step (d).

[Application Example 4]
In application example 1 or application example 2,
The step (e) can include a step of detecting the whole solid phase carrier by a nucleic acid amplification method without isolating the viral nucleic acid using the solid phase carrier obtained in the step (d).

[Application Example 5]
In any one of Application Examples 1 to 4,
The solid phase carrier may be particles having an average particle size of 0.1 μm or more and 20 μm or less and capable of being solid-liquid separated by gravity or centrifugal force.

[Application Example 6]
In any one of Application Examples 1 to 4,
The solid phase carrier may be particles having an average particle diameter of 0.1 μm or more and 20 μm or less and capable of being solid-liquid separated by electromagnetic force.

  According to the above-described virus detection method, viral nucleic acids can be simultaneously and easily identical from biological samples that may contain a plurality of viruses such as hepatitis B virus, hepatitis C virus, and human immunodeficiency virus. Viruses can be detected by specific isolation in solution.

  Hereinafter, preferred embodiments of the present invention will be described in detail.

1. Virus detection method Virus detection method according to an embodiment of the present invention,
A method of specifically isolating viral nucleic acid from a biological sample potentially containing at least one virus in the same solution and then detecting it,
(A) mixing the biological sample with a composition for releasing viral nucleic acid;
(B) adding to the mixture of step (a) an oligonucleotide that is hybridizable with at least a partial sequence of the viral nucleic acid and biotinylated at least at one end;
(C) adding a solid phase carrier having avidin or streptavidin immobilized on the surface thereof to the mixture of step (b);
(D) separating the solid support from the mixture of step (c);
(E) detecting the viral nucleic acid by a nucleic acid amplification method using the solid phase carrier;
Including
The composition contains at least one chaotropic agent selected from guanidinium thiocyanate, guanidinium hydrochloride, and sodium iodide, an anionic surfactant, and a reducing agent, and the mixture of the step (a) The concentration of the anionic surfactant inside is 2.5% by mass or more and 30% by mass or less.

  According to the virus detection method of the present invention, for example, from a biological sample that may contain at least one virus selected from hepatitis B virus, hepatitis C virus, and human immunodeficiency virus. Viral nucleic acids can be specifically isolated in the same solution to detect viruses.

  In the present invention, the “biological sample” refers to animal and plant tissues, cell lysates, blood, plasma, serum, urine, saliva, and other body fluids, cultured cell lysates, and the like that may contain viruses. There is a thing with sex. Such a sample may be as collected or diluted with a buffer or the like.

  Hereinafter, each step will be described in detail.

1.1. Step (a)
This step is a step of mixing the biological sample and a composition for releasing viral nucleic acid to prepare a mixture and releasing the nucleic acid from the virus contained in the biological sample.

  Hereinafter, a composition for releasing viral nucleic acid used in this step (hereinafter also simply referred to as “composition”) will be described.

  The composition used in this step is mixed with a biological sample that may contain at least one virus selected from hepatitis B virus, hepatitis C virus, and human immunodeficiency virus. The viral nucleic acid can be released into the solution by dissolving the membrane.

  The composition is an aqueous solution containing at least a chaotropic agent, an anionic surfactant, and a reducing agent.

  The composition contains at least one selected from guanidinium thiocyanate, guanidinium hydrochloride, and sodium iodide as the chaotropic agent. When guanidinium thiocyanate is used, the content of the chaotropic agent is preferably 0.5M to 6.0M, more preferably 0.8M to 5.5M as the concentration after mixing with the biological sample. Especially preferably, it is 1.0M-5.0M. When guanidinium hydrochloride is used, the content after mixing with the biological sample is preferably 0.5M to 8.0M, more preferably 1.0M to 7.5M. Preferably it is 1.5M-7.0M. When sodium iodide is used, the content after mixing with the biological sample is preferably 3.0M to 8.0M, more preferably 4.0M to 7.5M. Most preferably, it is 5.0M-7.0M. When the content of the chaotropic agent is within the above range, HBV, HCV, and HIV membranes can be surely dissolved, and viral RNA released from HCV and HIV is not degraded. If the content of the chaotropic agent is less than the lower limit, the HBV membrane may not be completely dissolved, or RNA released from HCV and HIV may be degraded by the enzyme RNase that degrades RNA. On the other hand, if the content of the chaotropic agent exceeds the above upper limit, it becomes difficult to prepare a composition for releasing viral nucleic acid, and even if a composition for releasing viral nucleic acid can be prepared, the viscosity becomes high and accurate. Since it is difficult to accurately weigh, it is not practical and is not preferable. Urea is well known as a chaotropic agent, but cannot be applied to the present invention because it has an action of inhibiting the hybridization of the viral nucleic acid to be isolated in the step (b) described later.

  As said anionic surfactant, what has a sulfonic acid group, a carboxyl group, a phosphoric acid group etc. as a hydrophilic group generally is preferable. Examples of those having a sulfonic acid group include sodium dodecyl sulfate and dodecylbenzene sulfonate. Examples of those having a carboxyl group include dipotassium alkenyl succinate, alkyl ether carboxylates, alkanoyl glutamates, and sodium N-lauroyl sarcosine. As what has a phosphoric acid group, polyoxyethylene alkyl ether potassium phosphate etc. are mentioned, for example. These anionic surfactants can be used singly or in combination of two or more.

  The reason why an anionic surfactant is suitable as a component in the composition is that the ability to form an emulsion with respect to the virus membrane is high and that it easily penetrates into the virus membrane. Furthermore, by using an anionic surfactant, a reaction promoting effect in the hybridization reaction between the oligonucleotide and the viral nucleic acid is expected.

  The content of the anionic surfactant is 2.5% by mass or more and 30% by mass or less as the concentration after mixing with the biological sample. When the content of the anionic surfactant is within the above range, an effect of dissolving the target three types of viruses (HBV, HCV, HIV) and assisting the release of viral nucleic acid can be obtained. If the content of the anionic surfactant is less than the above range, the virus membrane may not be emulsified and dissolved. If the content of the anionic surfactant exceeds the above range, the composition for releasing the viral nucleic acid becomes highly viscous, and accurate weighing becomes difficult, which is not practically preferable.

  Examples of the reducing agent include 2-mercaptoethanol, thioglycerol, dithiothreitol, 2-mercaptoethylamine hydrochloride, tris (2-carboxyethyl) phosphine hydrochloride, mercaptoacetic acid, and 2-mercaptopropionic acid. These can be used alone or in combination of two or more. When the reducing agent exemplified above is added, the -S-S- bond in the virus membrane or virus protein can be dissociated to form two -SH bonds. Thereby, the higher order structure of the viral protein is decomposed, and the viral protein is more easily denatured by the composition.

  The content of the reducing agent is preferably 0.05% by mass or more and 20% by mass or less, more preferably 0.01% by mass or more and 10% by mass or less, as the concentration after mixing with the biological sample. . When the content of the reducing agent is less than the above range, the above-described reducing action is not preferable. On the other hand, if the content of the reducing agent exceeds the above range, the substantial effect becomes constant, and no further effect is seen, which is not preferable.

  If necessary, a chelating agent such as ethylenediaminetetraacetic acid (EDTA) may be added to the composition. In some cases, an enzyme called RNase that degrades RNA is mixed in a biological sample, and RNA obtained by dissolving an HCV or HIV membrane is degraded by the RNase. In this case, since several types of RNase show magnesium dependence, RNA degradation can be inhibited by adding a chelating agent. The chelating agent content is preferably 0 M or more and 1 M or less. If it exceeds 1M, the substantial effect becomes constant, and no further effect is seen.

  The pH of the composition is preferably 5.0 or more and 10.0 or less, more preferably 6.0 or more and 9.0 or less. Adjustment of the pH of the composition can be easily achieved by using a good buffer which is usually used in the biochemical field. Examples of the good buffer include Tris buffer.

  The composition for releasing viral nucleic acid described above contains not only the present step but also contains at least one virus selected from hepatitis B virus, hepatitis C virus, and human immunodeficiency virus. It can be used for other applications as long as it aims to dissolve the viral membrane and release the viral nucleic acid from a potential biological sample.

1.2. Step (b)
In this step, the mixture of step (a) can be hybridized with at least a partial sequence of the viral nucleic acid and biotinylated at least at one end (hereinafter also simply referred to as “oligonucleotide”). .) Is added, and the mixture is heated and lowered.

  The addition amount of the oligonucleotide is preferably 0.5 pmol or more and 400 pmol or less, more preferably 5 pmol or more and 100 pmol or less. If the amount of oligonucleotide added is less than the above range, the amount of hybridization is insufficient, which is not preferable. On the other hand, when the amount of the oligonucleotide added exceeds the above range, it may not be captured by the avidin particle.

  After the mixture of the step (a) and the oligonucleotide are mixed, the mixture is heated and incubated to accelerate the dissolution of the virus membrane. The incubation time is preferably 5 to 60 minutes, more preferably 10 to 30 minutes. The incubation temperature is preferably 30 to 85 ° C, more preferably 50 to 80 ° C. Moreover, in order to make heating uniform, you may add slow stirring and vibration at the time of a heating as needed. When the temperature is lowered to room temperature, the viral nucleic acid and the oligonucleotide hybridize, and biotin is introduced into the viral nucleic acid.

  “Hybridize” means that the target nucleic acid and the oligonucleotide form a complex complementarily via Watson-Crick base pairing. The hybridizing sequences need not have perfect complementarity. For example, a stable complex can be formed even when less than about 10% of the base sequences do not match.

  “Capturing viral nucleic acid specifically” does not mean that the oligonucleotides are 100% identical but means that a stable complex can be formed with the probe under the capture conditions.

1.3. Step (c)
This step is a step of adding and dispersing in the mixture of step (b) a solid phase carrier having avidin or streptavidin that specifically reacts with biotin immobilized on the surface. It is one of the features of the present invention to specifically capture a viral nucleic acid via an avidin-biotin reaction. This makes it possible to efficiently amplify the target viral nucleic acid without being affected by other contaminating nucleic acids in the subsequent nucleic acid amplification method.

  Examples of the solid phase carrier on which avidin or streptavidin is immobilized on the surface include avidin magnetic particles exemplified below.

  The average particle diameter of the avidin magnetic particles is preferably from 0.1 μm to 20 μm, more preferably from 0.3 μm to 17 μm, and particularly preferably from 0.5 μm to 10 μm. If the average particle size of the avidin magnetic particles is less than the above range, it may take a long time to separate and purify the particles. On the other hand, exceeding the above range is not preferable because the surface area becomes small and the amount of viral nucleic acid captured is insufficient. In addition, in this invention, an average particle diameter means the value measured using the particle diameter distribution measuring apparatus which makes a dynamic light scattering method a measurement principle.

  Avidin magnetic particles are particles that respond to an external magnetic field, and the solid-liquid separation operation of the particles can be performed by an external magnet. Therefore, the solid-liquid separation operation can be performed not only by a manual method such as centrifugation or natural sedimentation but also by a commercially available automatic machine equipped with a magnet.

  The avidin magnetic particles are preferably superparamagnetic without residual magnetization. Superparamagnetic avidin magnetic particles move in the direction of the magnetic field when an external magnetic field is applied, but lose the residual magnetization when the external magnetic field is interrupted, and therefore agglutinate with each other without being affected by the avidin magnetic particles. Can behave independently without any problems. Therefore, superparamagnetic avidin magnetic particles have an advantage that the avidin magnetic particles after magnetic separation can be easily redispersed. By using such superparamagnetic magnetic particles in the process of capturing the viral nucleic acid of the present invention, unnecessary components and contaminants other than the target viral nucleic acid can be easily washed away and the subsequent nucleic acid amplification reaction proceeds with high efficiency. In addition, it is possible to capture, isolate and detect a very small amount of viral nucleic acid of about several molecules.

  The avidin magnetic particles that can be used in this step are useful for automating or apparatusating the capture of viral nucleic acid, but the operation of capturing viral nucleic acid may be performed by a manual method. In the manual method, separation by centrifugal separation or natural sedimentation may be performed instead of magnetic separation, or nonmagnetic particles may be used instead of magnetic particles.

1.4. Step (d)
This step is a step of separating the solid phase carrier from the mixture of the step (c).

  First, the mixture containing the solid phase carrier that has captured the viral nucleic acid in step (c) is separated into a solid phase carrier and another liquid, and only the solid phase carrier that has captured the viral nucleic acid is recovered. Thereby, components and contaminants such as serum, protein, sugar, and lipid contained in the specimen in the mixture are removed. Separation of the solid phase carrier from other liquids may be carried out by allowing the mixture to stand on a magnetic stand so that the solid phase carrier is magnetically attracted and separated into solid and liquid, or by light centrifugation at several thousand revolutions / minute. Separation may be used.

  The surface of the solid phase carrier obtained as described above contains free metal ions, proteins, and other contaminants in addition to the target viral nucleic acid. These substances may inhibit nucleic acid amplification in nucleic acid detection methods. For this reason, in this step, it is preferable to wash the solid phase carrier with a washing buffer that does not cause nucleic acid dissociation.

  The washing buffer preferably contains, for example, a salt such as NaCl or KCl at a concentration of about 0.5M to 2.0M. The addition of this amount of salt has the effect of removing the components adsorbed on the surface of the solid phase carrier by electrostatic interaction among the impurities. When the salt concentration is lower than 0.5M, the virus nucleic acid captured and bound to the surface of the solid phase carrier may be dissociated and lost, and the isolation efficiency of the virus nucleic acid may decrease. . The Watson-Crick base pairing of nucleic acid is an attractive force due to hydrogen bonding between bases acting on two single-stranded nucleic acids and electrostatic repulsive force acting between anionic phosphate groups of the phosphodiester group constituting the nucleic acid skeleton. It is formed when the attractive force due to hydrogen bonding between bases is excellent in balance. When the salt concentration of the solution is high, the salt in the solution neutralizes the electrostatic repulsion between the phosphate groups, so that the attractive force due to hydrogen bonding between the bases is dominant and the formation of Watson-Crick base pairing is achieved. Promoted. In the present invention, in the case of temperature conditions near room temperature, when the salt concentration is lower than 0.5M, electrostatic repulsion between phosphate groups is dominant, and the pairing dissociation between the oligonucleotide and the viral nucleic acid is dissociated. As a result, the capture efficiency of the viral nucleic acid using the pairing of the nucleic acid may decrease, and as a result, the isolation efficiency of the viral nucleic acid may decrease. On the other hand, when the salt concentration is higher than 2.0M, the attractive force due to hydrogen bonding between the bases is superior, and the pairing of the oligonucleotide and the viral nucleic acid is stable, but even after removing the washing buffer, A salt that remains in a trace amount and cannot be removed and is brought into the next step is not preferable because it reduces the efficiency of the elution step of the viral nucleic acid, which is an optional step in the next step (e).

  The washing buffer preferably contains a surfactant such as Triton X-100 or Tween 20 at a concentration of 0.01% by mass to 2.0% by mass. The addition of this level of surfactant has the effect of removing the components adsorbed on the surface of the magnetic particles by hydrophobic interaction among the impurities.

  The pH of the washing buffer is preferably 5.0 or more and 10.0 or less, more preferably 6.0 or more and 9.0 or less.

  In this step, after washing the solid phase carrier with a washing buffer, the separated solid phase carrier is redispersed with a washing buffer or an eluate described later to obtain an aqueous dispersion of the solid phase carrier. It is also preferable.

1.5. Step (e)
This step is a step of detecting the viral nucleic acid isolated in step (d) by a nucleic acid amplification method. Viral nucleic acid captured on the surface of the solid support is brought into a nucleic acid amplification reaction system such as PCR while being captured on the surface of the solid support (that is, an aqueous dispersion of the solid support) to amplify the nucleic acid. Can do.

  On the other hand, the viral nucleic acid captured on the surface of the solid phase carrier may be eluted from the solid phase carrier and then detected by a nucleic acid amplification method. Since the viral nucleic acid can be recovered as an aqueous solution after the elution step, it can be applied to any downstream application.

  These methods can be appropriately used depending on the type of nucleic acid amplification apparatus used and the purpose of use. In general, a method of bringing the nucleic acid amplification reaction system in a state of being captured on the surface of the solid phase carrier is simpler. However, when using a real-time fluorescence detection apparatus, in some models, the fluorescence signal may be shielded or absorbed by the solid phase carrier, which may reduce the detection efficiency of the viral nucleic acid. The method of amplifying a nucleic acid by elution of a viral nucleic acid from a solid phase carrier has a demerit of increasing the elution step of the viral nucleic acid, but has an advantage that an inhibitory factor of the PCR reaction is surely removed.

  Hereinafter, a method for eluting viral nucleic acid from a solid phase carrier will be described.

  As an eluent for elution of the viral nucleic acid from the solid phase carrier, a solution capable of dissociating the paired viral nucleic acid such as distilled water or an alkaline aqueous solution may be used. In addition, you may use what does not have a bad influence on subsequent PCR enzyme reaction, such as DEPC water and ultrapure water, instead of distilled water. In the present invention, the virus nucleic acid paired with the oligonucleotide can be dissociated by adding distilled water to reduce the salt concentration of the aqueous dispersion of the solid phase carrier. In such a case, distilled water is preferable as the eluent, but a salt such as NaCl or KCl having a concentration of 0.2 M or less may be added as long as it does not prevent dissociation of the viral nucleic acid paired with the oligonucleotide. Similarly, a salt having a buffering action such as Tris having a concentration of 0.2 M or less may be added as a range that does not prevent dissociation. In such a case, the pH of the eluate is preferably 5.0 or more and 9.0 or less, and more preferably 6.0 or more and 8.5 or less. In order to minimize aggregation of the solid phase carrier and adhesion to a container such as a tube, a trace amount of a surfactant may be added. Specifically, a surfactant such as Triton, Briji, Tween, and NP40 may be added in an amount of 0.01 to 0.5% by mass.

  It is also possible to use an alkaline aqueous solution as the eluent. The alkaline aqueous solution has a denaturing action of double-stranded nucleic acid and can dissociate the nucleic acid into a single strand. As such an alkaline aqueous solution, an aqueous solution of sodium hydroxide or potassium hydroxide can be used. The concentration of sodium hydroxide or potassium hydroxide is preferably 30 mM or less. When an alkali thicker than 30 mM is used, hydrolysis of nucleic acids, particularly viral RNA, is caused, and the isolation efficiency of viral nucleic acids is reduced. In the case of using an alkaline aqueous solution as the eluent, as in the case of using distilled water as the eluent, a salt such as NaCl or KCl having a concentration of 0.2 M or less may be added as a range that does not hinder the dissociation of nucleic acids. Is possible.

  In order to elute the viral nucleic acid, the eluate is preferably incubated for about 1 to 20 minutes after being added to the solid phase carrier, and the sample may be stirred during the incubation.

  When distilled water is used as the eluent, heating is also effective as a method for promoting the elution of viral nucleic acid. As a method of heating, either a method using an eluate that has been heated in advance (for example, 70 ° C.) or a method of heating after mixing with a solid phase carrier can be used. When an alkaline aqueous solution is used as an eluent, viral nucleic acid may be hydrolyzed and lost by heating, and caution is required for heating.

  It is also effective to repeat this elution step 2 to 3 times to ensure elution of the viral nucleic acid.

  In the case of using an alkaline aqueous solution as the eluent, a step of neutralizing pH by adding an acidic aqueous solution or an aqueous solution having a buffering action may be added as needed after the nucleic acid solution is recovered.

The viral nucleic acid solution obtained as described above can be used for nucleic acid amplification reaction. The nucleic acid amplification method is not particularly limited, but for example, Roche PCR (Polymerase chain)
reaction) method, TMP (Transcription mediated amplification-hybridization protection assay) method by Gen-Probe, LCR (Ligase chain reaction) method by Abbott, LAMP (Loop-mediated isothermal amplification of DNA) method by Eiken Chemical, Takara Shuzo The company's ICAN (Isothermal and chimeric primer-initiated amplification of nucleic acid) method can be used.

  The virus detection method according to the present invention can be used mainly for virus screening of blood transfusions or blood products. The virus detection method according to the present invention may be performed by an artificial operation, but is often used in a dedicated device to process a large number of specimens intensively. The virus detection method and the composition for releasing viral nucleic acid according to the present invention are suitable for both artificial manipulation and automatic machine manipulation.

  The method for detecting the virus has been described in detail by taking hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus (HIV) as examples, but influenza virus, pneumonia virus, rubella virus, measles It can also be applied to other viruses such as viruses.

2. Example 2.1. HBV, HCV, HIV probe The sequences of the HBV probe, HCV probe, and HIV probe used in this example are shown below.

[HBV capture probe sequence]:
5′-TCCTAGGACCCCCTGCTCGTGTTACAGGCGGGGTTTTCT-Biotin 3 ′ (hereinafter referred to as “sequence 1”)
[HCV capture probe sequence]:
5′-GTGAAGACAGTAGTTTCCTCACAGGGGAGTGATCTA-biotin 3 ′ (hereinafter referred to as “sequence 2”)
[HIV capture probe sequence]:
5′-TTATGTCCAGAAATTGCTGTAGGGCTATACATCTCTTAC-biotin 3 ′ (hereinafter referred to as “sequence 3”)

  As the HCV probe sequence, a partially modified sequence described in the prior patent document, Japanese Patent Laid-Open No. 10-1493 was used.

  The HIV probe sequence is the sequence described in the prior art document “Tano, H. et.al. Journal of Clinical Microbiology, Sept. 1995, p.2489-2491”.

2.2. Preparation of composition for releasing viral nucleic acid As a composition for releasing viral nucleic acid, the concentrations of each reagent are mixed so as to be the concentrations shown in Tables 1 and 2 below, and dissolved in distilled water. It was. The composition was filtered through a syringe filter (0.45 μm) and stored protected from light until use. The order of adding each reagent is not particularly limited. Moreover, about the component which is hard to melt | dissolve in distilled water, it heated to 50 degreeC and was made to melt | dissolve slowly.

2.3. Examples and Comparative Examples 2.3.1. Example 1
Each virus was mixed with 10 ³ IU / mL each by mixing a specific amount from plasma containing HBV (subtype C), HCV (subtype Ib) and HIV (subtype B) and diluting with virus negative plasma. Samples to be contained Samples containing 10 ² IU / mL each were prepared. In addition, in order to confirm the concentration of each virus prepared here, an experiment was conducted side by side with a WHO standard product purchased from NIBSC, and the concentration deviation was corrected. 250 μL each of these specimens and virus-negative plasma were divided into test tubes, and 250 μL of “Composition A” prepared in “2.2. Preparation of composition for releasing viral nucleic acid” was added in an equal volume (above) Step (a)).

  Next, 5 μL of a solution in which oligonucleotides of the sequence 1, the sequence 2, and the sequence 3 were prepared to 10 pmol / μL each was added, heated at 75 ° C. for 12 minutes, and then cooled to 25 ° C. b)).

Next, Magnosphere ^™ M300 / Streptavidin (manufactured by JSR Corporation) 4 mass% particle dispersion, 50 μL, was added to each test tube, and incubated at 25 ° C. for 10 minutes, whereby virus-derived nucleic acid released from the specimen was avidin- The particles were captured via a biotin reaction (step (c)).

  Subsequently, a test tube was set on the magnetic stand, the particles in the specimen were magnetically separated, and the liquid was removed. Thereafter, the particles were washed once with 250 μL of 10 mM Tris-HCl (pH 7) /0.7 M NaCl washing solution, and the washing solution was removed after separating the particles again using magnetism (step (d)). ).

  The magnetic particles were dispersed by adding 80 μL of distilled water as an eluent, heated at 75 ° C. for 2 minutes and then returned to room temperature, and the nucleic acid hybridized to the magnetic particles was eluted in the dispersion. From this dispersion, the magnetic particles are solid-liquid separated, divided into three 20 μL portions, and real-time PCR for HBV, HCV and HIV detection is performed to verify the presence or absence of recovery of each viral nucleic acid. e)).

  For detection of HBV, HCV, and HIV viruses, Cobas Taqman HBV “Auto”, Cobas Taqman HCV “Auto”, and Cobas Taqman HIV-1 “Auto” reagents manufactured by Roche Diagnostics were used for detection and determination. It was. Real-time PCR reagent formulation, PCR amplification conditions, PCR apparatus condition setting, analysis method, and criteria were in accordance with the manufacturer's manual.

  Each sample was measured five times, and the number of positive determinations is summarized in Table 3.

As shown in Table 3, by using the virus detection method according to Example 1, HBV, HCV, and HIV virus nucleic acids can be isolated in the same solution and detected by real-time PCR detection method. I was able to. The detection sensitivity of HBV, HCV and HIV is very high, and it was possible to detect three viruses with a 100% probability even in specimens containing each virus at a very low concentration of 10 ² IU / mL. It was.

A whole blood sample was used in place of the plasma sample, and virus lysis, nucleic acid capture and detection were performed in the same manner as described above. The results are shown in Table 4. When a whole blood sample was used, the detection sensitivity was slightly lower than when a plasma sample was used. In the case of a whole blood sample, considering that the blood cell volume accounts for approximately half of the whole blood, the sample that can be used for the test is substantially half that of the plasma sample. In other words, it was confirmed that even in the case of a whole blood sample, the three viruses can be detected without any problem up to 10 ³ IU / mL.

  In addition, since the result at the time of using a serum sample was the same as a plasma sample, data is omitted.

2.3.2. Example 2 to Example 7
In the step (a) of the “2.3.1. Example 1”, except that the compositions B to G described in Table 1 were used instead of the “composition A”, the “2.3. 1. The same steps as in the case of using the plasma specimen of Example 1 ”were performed. Each sample was measured five times, and the number of times determined to be positive was summarized in Tables 5 to 10.

As shown in Table 5 to Table 10, by using the virus detection methods according to Examples 2 to 7, viral nucleic acids of HBV, HCV, and HIV can be isolated in the same solution, and they are real-time. It could be detected by PCR detection method. The detection sensitivity of HBV, HCV and HIV is very high, and it was possible to detect three viruses with a 100% probability even in specimens containing each virus at a very low concentration of 10 ² IU / mL. It was.

2.3.3. Comparative Examples 1 to 5
In the step (a) of the “2.3.1. Example 1”, except that the compositions H to L shown in Table 2 were used instead of the “composition A”, the “2.3. 1. The same steps as in the case of using the plasma specimen of Example 1 ”were performed. Each sample was measured 5 times, and the number of times determined to be positive was summarized in Tables 11 to 15.

  As shown in Tables 11 to 15, the virus detection methods according to Comparative Examples 1 to 5 could not detect HBV, HCV and HIV at any concentration.

2.3.4. Example 8
The step (d) and the step (e) of the above “2.3.1. Example 1” were changed as follows.

  A test tube was set on the magnetic stand, the particles in the specimen were magnetically separated, and the liquid was removed. Thereafter, the particles were washed once with 250 μL of 10 mM Tris-HCl (pH 7) /0.7 M NaCl washing solution (step (d)).

  The step of eluting viral nucleic acid from the magnetic particles obtained in the step (d) was omitted, and the magnetic particles obtained in the step (d) were dispersed as they were in 50 μL of the PCR reaction cocktail, and a real-time PCR reaction was performed.

  Each sample was measured five times, and the number of times determined to be positive is summarized in Table 16.

  As shown in Table 16, HBV, HCV, and HIV can be detected with high sensitivity even when the magnetic particles recovered in step (e) are dispersed as they are in 50 μL of the PCR reaction cocktail and a real-time PCR reaction is performed. did it. Even in a specimen containing each virus at a very low concentration of 10 IU / mL, it was possible to detect three viruses with a probability of 80 to 100%.

2.3.5. Example 9
The step (e) of the “2.3.1. Example 1” was changed as follows.

  Disperse the magnetic particles obtained in step (d) by adding 40 μL of 20 mM sodium hydroxide aqueous solution as an eluent, heat at 75 ° C. for 2 minutes, return to room temperature, and disperse the nucleic acid hybridized to the magnetic particles. It was eluted in the liquid. The magnetic particles were separated from the dispersion by solid-liquid separation to recover the eluate. The recovered eluate was neutralized with 40 μL of 20 mM hydrochloric acid aqueous solution, then divided into three 20 μL portions and subjected to real-time PCR for detection of HBV, HCV, and HIV, respectively, to verify whether each viral nucleic acid was recovered.

  Measurements were performed 5 times for each sample, and the number of positive determinations is summarized in Table 17.

2.3.6. Example 10
Instead of the “20 mM aqueous sodium hydroxide solution” used as the eluent in “2.3.5. Example 9”, the “30 mM aqueous sodium hydroxide solution” was used. The recovered eluate was neutralized with 40 μL of a 30 mM hydrochloric acid aqueous solution.

  Measurements were performed 5 times for each sample, and the number of positive determinations is summarized in Table 18.

2.3.7. Reference example 1
Instead of the “20 mM sodium hydroxide aqueous solution” used as the eluent in “2.3.5. Example 9”, “40 mM sodium hydroxide aqueous solution” was used. The recovered eluate was neutralized with 40 μL of 40 mM hydrochloric acid aqueous solution.

  The measurement was performed 5 times for each sample, and the number of positive determinations is summarized in Table 19.

2.3.8. Reference example 2
Instead of the “20 mM aqueous sodium hydroxide solution” used as the eluent in “2.3.5. Example 9”, the “50 mM aqueous sodium hydroxide solution” was used. The collected eluate was neutralized with 40 μL of 50 mM hydrochloric acid aqueous solution.

  Measurements were performed 5 times for each sample, and the number of positive determinations is summarized in Table 20.

  As shown in Table 17 to Table 18, when 20 mM or 30 mM sodium hydroxide aqueous solution was used as an eluent, HBV, HCV and HIV could be detected with high sensitivity. Even in a specimen containing each virus at a very low concentration of 10 IU / mL, it was possible to detect three viruses with a probability of 80 to 100%.

  On the other hand, as shown in Table 19 to Table 20, when 40 mM or 50 mM sodium hydroxide aqueous solution was used, HBV could be detected with high sensitivity, but the detection rate of HCV and HIV could be significantly reduced. I understood.

Claims (6)

A method of specifically isolating viral nucleic acid from a biological sample potentially containing at least one virus in the same solution and then detecting it,
(A) mixing the biological sample with a composition for releasing viral nucleic acid;
(B) adding to the mixture of step (a) an oligonucleotide that is hybridizable with at least a partial sequence of the viral nucleic acid and biotinylated at least at one end;
(C) adding a solid phase carrier having avidin or streptavidin immobilized on the surface thereof to the mixture of step (b);
(D) separating the solid support from the mixture of step (c);
(E) detecting the viral nucleic acid by a nucleic acid amplification method using the solid phase carrier,
The composition contains at least one chaotropic agent selected from guanidinium thiocyanate, guanidinium hydrochloride, and sodium iodide, an anionic surfactant, and a reducing agent.
The virus detection method wherein the concentration of the anionic surfactant in the mixture of the step (a) is 2.5% by mass or more and 30% by mass or less. In claim 1,
The virus detection method, wherein the virus to be detected is at least one selected from hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus (HIV). In claim 1 or claim 2,
Furthermore, the step (e) includes a step of adding an eluate to the solid phase carrier obtained in the step (d) to elute the viral nucleic acid. In claim 1 or claim 2,
The virus detection method, wherein the step (e) includes a step of detecting the whole solid phase carrier by a nucleic acid amplification method without isolating the viral nucleic acid using the solid phase carrier obtained in the step (d). In any one of Claims 1 thru | or 4,
The virus detection method, wherein the solid phase carrier is a particle having an average particle diameter of 0.1 μm or more and 20 μm or less and capable of solid-liquid separation by gravity or centrifugal force. In any one of Claims 1 thru | or 4,
The virus detection method, wherein the solid-phase carrier is a particle having an average particle diameter of 0.1 μm or more and 20 μm or less and capable of being solid-liquid separated by electromagnetic force.