JP5505646B2 - Biological sample quantification method - Google Patents

Description

  The present invention relates to a biological sample quantification chip and a biological sample quantification method used for quantification of nucleic acids, for example.

  A method of performing chemical analysis, chemical synthesis, bio-related analysis, or the like using a microfluidic chip in which a fine flow path is provided on a glass substrate or the like has attracted attention. Microfluidic chips are also called Micro Total Analytical System (Micro TAS), Lab-on-a-chip, etc., and require less sample and reagents than conventional devices, and reaction time It has the advantages of being short and less waste, and is expected to be used in a wide range of fields such as medical diagnosis, on-site analysis of the environment and food, and production of pharmaceuticals and chemicals. Since the amount of the reagent may be small, it is possible to reduce the cost of the inspection, and since the necessary amount of the sample and the reagent is small, the reaction time can be greatly shortened and the inspection can be made more efficient. In particular, when used for medical diagnosis, it is possible to reduce the necessary amount of a sample such as blood as a sample, which has the advantage of reducing the burden on the patient.

As a method for amplifying a gene such as DNA or RNA used as a sample, a polymerase chain reaction (PCR) method is well known. In the PCR method, a mixture of target DNA and reagents is put in a tube, and a temperature control device called a thermal cycler is used to repeatedly react, for example, three steps of temperature changes of 55 ° C, 72 ° C, and 94 ° C with a period of several minutes. Therefore, only the target DNA can be amplified by about 2 times per temperature cycle by the action of the enzyme (DNA polymerase).

  In recent years, real-time PCR capable of quantifying DNA while performing an amplification reaction using a special fluorescent probe has been put into practical use. Real-time PCR is widely used for research and clinical testing because of its high sensitivity and reliability of measurement.

  However, in the real-time PCR method, when a substance that inhibits the amplification reaction is present in the sample, the reliability of the obtained result may be low due to the influence of the substance. In addition, a standard amount of reaction solution necessary for PCR is several tens of μl, and there is a problem that basically only one gene can be measured in one reaction system. On the other hand, since the amount of DNA extracted from a specimen is generally small, and the reagent used is generally expensive, it is usually difficult to measure many reaction systems simultaneously.

  On the other hand, International Publication No. WO2005 / 059548 describes a measurement method using a fluorescent probe that hybridizes to both a target nucleic acid and an internal standard nucleic acid. According to this measurement method, since the target nucleic acid and the internal standard nucleic acid are co-amplified, it is difficult to be influenced by the amplification inhibitor and an expensive real-time PCR dedicated device is not required. However, in this measurement method, particularly when the concentration difference between the target nucleic acid and the internal standard nucleic acid is large, the quantification accuracy is remarkably lowered and the concentration range of the standard nucleic acid that can be quantified is narrow.

International Publication Number WO2005 / 059548

  When quantifying a target nucleic acid contained in a specimen, the present invention is less susceptible to amplification inhibition factors that can be contained in the specimen, can easily perform a nucleic acid amplification reaction and target nucleic acid quantification, Provided are a biological sample quantification chip and a biological sample quantification method having a wide nucleic acid quantification range.

The biological sample quantification chip according to the first aspect of the present invention comprises:
A biological sample quantification chip used for quantification of a target nucleic acid contained in a specimen,
Including a plurality of reaction vessels,
The plurality of reaction vessels are:
A known amount of a competing nucleic acid that can be amplified with a primer common to the target nucleic acid and has a sequence different from the target nucleic acid;
A primer common to the target nucleic acid and the competing nucleic acid;
A fluorescent probe that binds to a part of the amplification product of the target nucleic acid and the competing nucleic acid, and exhibits a fluorescence change that is different between the amplification product of the target nucleic acid and the amplification product of the competing nucleic acid;
Including
The amount of the competing nucleic acid contained in each reaction vessel is different.

In the biological sample quantification chip,
The apparatus may further include a reaction vessel introduction channel connected to the plurality of reaction vessels, a reaction solution storage unit connected to the reaction solution introduction channel, and a waste solution storage unit.

  The biological sample quantification chip includes a plurality of reaction container groups each including the plurality of reaction containers, and the plurality of reaction containers constituting one reaction container group are configured to amplify the same target nucleic acid. In the plurality of reaction containers constituting another reaction container group, a target nucleic acid different from the target nucleic acid amplified in the reaction container constituting the one reaction container group is amplified. Different primers can be placed.

The biological sample quantification chip according to the second aspect of the present invention comprises:
A biological sample quantification chip used for quantification of a target nucleic acid contained in a specimen,
A first reaction vessel;
A second reaction vessel;
With
The first reaction vessel comprises:
A primer for amplifying the target nucleic acid;
A first amount of a competing nucleic acid that can be amplified with the primers and has a sequence different from the target nucleic acid;
A fluorescent probe that binds to a part of the amplification product of the target nucleic acid and the competing nucleic acid, and exhibits a fluorescence change that is different between the amplification product of the target nucleic acid and the amplification product of the competing nucleic acid;
Including
The second reaction vessel comprises:
A second amount of the competing nucleic acid different from the first amount;
The primer;
The fluorescent probe;
including.

The biological sample quantification method according to the third aspect of the present invention comprises:
A biological sample quantification method for quantifying a target nucleic acid contained in a specimen using the biological sample quantification chip according to the first aspect,
Introducing each sample into the plurality of reaction vessels;
Performing a nucleic acid amplification reaction in the plurality of reaction vessels;
Measuring the fluorescence intensity emitted by the fluorescent probe bound to a part of the amplified nucleic acid in each of the reaction vessels;
Obtaining a regression curve represented by the following formula (1) based on the fluorescence intensity measured in each reaction container and the amount of the competing nucleic acid used in each reaction container;
Estimating the amount of target nucleic acid contained in the specimen based on the regression curve;
including.

F = a / (C + b) + c (1)
(In the formula, F represents fluorescence intensity, C represents the amount of competing nucleic acid, b represents the amount of target nucleic acid contained in the sample, and a and c represent predetermined values).

In the biological sample quantification method,
In the step of obtaining the regression curve, the ratio of the fluorescence intensity measured before the nucleic acid amplification reaction to the fluorescence intensity measured after the nucleic acid amplification reaction, and the competitive nucleic acid used in each reaction vessel The regression curve can be obtained based on the relationship with the quantity.

  In this case, in the step of obtaining the regression curve, after the nucleic acid amplification reaction, the fluorescence intensity measured in the first state where the fluorescent probe is bound to a part of the amplified nucleic acid, and after the nucleic acid amplification reaction In the second state in which the fluorescent probe is dissociated from the amplified nucleic acid and the amount of the competing nucleic acid used in each of the reaction vessels. A curve can be obtained.

The biological sample quantification method according to the fourth aspect of the present invention comprises:
A biological sample quantification method for quantifying a target nucleic acid contained in a sample using a biological sample quantification chip used for quantification of a target nucleic acid contained in the sample,
The biological sample quantification chip includes a first reaction container and a second reaction container, and the first reaction container is capable of amplifying with the primer for amplifying the target nucleic acid, the primer, A first amount of a competitive nucleic acid having a sequence different from the target nucleic acid binds to the target nucleic acid and a part of the amplified product of the competitive nucleic acid, and the amplified product of the target nucleic acid is different from the amplified product of the competitive nucleic acid. A fluorescent probe that exhibits a change in fluorescence, and wherein the second reaction vessel includes a second amount of the competitive nucleic acid different from the first amount, the primer, and the fluorescent probe,
Introducing the specimen into the first and second reaction vessels;
Performing a nucleic acid amplification reaction in the first and second reaction vessels;
Measuring the fluorescence intensity emitted by the fluorescent probe bound to a part of the amplified nucleic acid in the first and second reaction vessels;
Based on the fluorescence intensity measured in the first and second reaction vessels and the amount of the competing nucleic acid contained in the first and second reaction vessels, a regression curve represented by the following formula (1) is obtained. Seeking and
Estimating the amount of target nucleic acid contained in the specimen based on the regression curve;
including.

F = a / (C + b) + c (1)
(In the formula, F represents the fluorescence intensity, C represents the amount of the competing nucleic acid, b represents the amount of the target nucleic acid contained in the sample, and a and c represent predetermined values).

  According to the biological sample quantification chip, a known amount of a competing nucleic acid having a sequence different from the target nucleic acid that can be amplified in the plurality of reaction containers with a common primer with the target nucleic acid, and the target nucleic acid A primer common to the competing nucleic acid, a fluorescent probe that binds to a part of the amplification product of the target nucleic acid and the competing nucleic acid, and exhibits a fluorescence change in which the amplification product of the target nucleic acid differs from the amplification product of the competing nucleic acid. The amount of the competing nucleic acid contained in each of the reaction containers is different, and the target nucleic acid is quantified by introducing a sample into the plurality of reaction containers by performing a nucleic acid amplification reaction using the plurality of reaction containers. Therefore, nucleic acid amplification reaction and target nucleic acid quantification can be easily performed. In addition, the target nucleic acid quantification using the above-described biological sample quantification chip is not easily affected by amplification inhibiting factors that can be included in the specimen, and the target nucleic acid quantification range is wide. Further, since the dispensing work other than the introduction of the specimen into the biological sample quantification chip is not required, the work amount is small and the necessary amount of the reagent can be reduced. Therefore, the biological sample quantification chip is used. Thus, the target nucleic acid can be quantified at low cost and with high accuracy.

  Further, according to the biological sample quantification method, the biological sample quantification method for quantifying a target nucleic acid contained in a specimen using the biological sample quantification chip, wherein the biological sample quantification method is performed in each of the reaction containers. A step of obtaining a regression curve represented by the above formula (1) based on the fluorescence intensity and the amount of the competitive nucleic acid used in each of the reaction containers, and a target nucleic acid contained in the sample based on the regression curve Including the step of estimating the amount of the target nucleic acid, and is difficult to be influenced by an amplification inhibiting factor that may be contained in the specimen, and the quantification range of the target nucleic acid is wide. In addition, since dispensing work other than introducing the sample into the biological sample quantification chip is unnecessary, the work volume is small and the necessary amount of reagent can be reduced, so that the target nucleic acid can be quantified at low cost. And it can be performed with high accuracy.

1A is a plan view illustrating a schematic configuration of a microreactor array according to an embodiment of the present invention, and FIG. 1B is a cross-sectional view taken along a line CC in FIG. FIG. 2 is a front view of the centrifuge provided with the microreactor array according to the embodiment of the present invention as viewed from the side. FIG. 3 is a plan view of the centrifugal device of FIG. 2A as viewed from above. The figure which looked at the microreactor array with which the holder of the centrifuge of FIG. 2 was mounted | worn from the top. FIG. 3 is a cross-sectional view of a microreactor array mounted on a holder of the centrifuge device of FIG. 2. The figure which shows the example of a sequence of the target nucleic acid and competitor nucleic acid which were used in Example 1 of this invention. The figure which shows the example of a sequence | arrangement of the primer used in Example 1 of this invention. The figure which shows the arrangement | sequence of Q-Probe used in Example 1 of this invention. The figure which shows the relationship (regression curve) between the quantity (logC) of the competition nucleic acid in the sample quantified in Example 1 of this invention, and the fluorescence intensity (change amount) F. FIG.

  Hereinafter, a biological sample quantification chip and a biological sample quantification method according to an embodiment of the present invention will be described in detail.

1. Embodiment 1.1. Configuration of Biological Sample Quantification Chip FIG. 1A is a plan view showing a schematic configuration of a microreactor array (biological sample quantification chip) 10 according to an embodiment of the present invention, and FIG. It is CC sectional drawing of FIG. 1 (A).

  The microreactor array 10 is a biological sample quantification chip (biochip) used for quantification of a target nucleic acid contained in a specimen. As shown in FIGS. 1 (A) and 1 (B), a plurality of reaction containers are used. The reaction container group 201, 202, 203, 204, 205, 206 including 104 is included. The microreactor array 10 further includes a reaction solution introduction channel 105 connected to each reaction vessel 104, a reaction solution storage unit 107 connected to the reaction solution introduction channel 105, and a waste solution storage unit 106. . The waste liquid storage unit 106 is connected to the reaction liquid storage unit 107. Further, the reaction liquid is supplied to the microreactor array 10 from the outside of the microreactor array 10 and the flow path 108 connecting the reaction liquid introduction flow path 105 and the waste liquid storage section 106 and the reaction liquid storage section 107. The reaction liquid supply port 109 used for the above is provided.

  As shown in FIG. 1B, the microreactor array 10 is configured by bonding transparent substrates 101, 102, and 103 together. The transparent substrate 101 is provided with a plurality of reaction vessels 104, a reaction solution introduction channel 105, a reaction solution storage unit 107, and a reaction solution supply port 109. In the transparent substrate 102, a waste liquid storage portion 106 and a flow path 108 are formed. The transparent substrates 101, 102, 103 can be, for example, resin substrates, and each part can be formed by, for example, injection molding.

  Each of the reaction vessels 104 can be amplified with a primer common to the target nucleic acid to be quantified, and has a known amount of a competitive nucleic acid having a sequence different from the target nucleic acid, a primer common to the target nucleic acid and the competitive nucleic acid, A fluorescent probe that binds to a part of the amplification product of the target nucleic acid and the competitive nucleic acid, and exhibits a fluorescence change in which the amplification product of the target nucleic acid and the amplification product of the competitive nucleic acid differ. More specifically, the competitive nucleic acid, primer, and fluorescent probe can be placed on the surface of the reaction vessel 104 by applying the competitive nucleic acid, primer, and fluorescent probe to the surface of the reaction vessel 104 and then drying. . In the microreactor array 10, the amount of competing nucleic acids contained in a plurality of reaction vessels 104 belonging to the same reaction vessel group is adjusted to be different.

  As described above, the microreactor array 10 includes a plurality of reaction vessel groups 201, 202, 203, 204, 205, 206. The same competing nucleic acid, the same primer, and the same fluorescent probe are arranged in a plurality of reaction vessels 104 included in the same reaction vessel group. Further, in the microreactor array 10, the same reaction container row included in different reaction container groups (when the arrangement direction of the reaction containers 104 included in the same reaction container group is the row direction, the reaction in the direction perpendicular to the row direction is performed. The same amount of competing nucleic acids can be placed in the reaction vessels 104 belonging to the arrangements A to F of the vessel 104.

  The same primer for amplifying the same target nucleic acid is arranged on the surface of a plurality of reaction vessels 104 constituting one reaction vessel group, and the surface of the plurality of reaction vessels 104 constituting another one reaction vessel group is arranged. Can be arranged with different primers for amplifying a target nucleic acid different from the target nucleic acid amplified in the reaction vessel 104 constituting one reaction vessel group. Thereby, different target nucleic acids can be quantified in one microreactor array 10.

  The surface of a plurality of reaction vessels 104 constituting one reaction vessel group is designed to bind to a target nucleic acid amplified in the reaction vessel 104 constituting one reaction vessel group and show a change in fluorescence amount. The same fluorescent probe is arranged. This fluorescent probe is designed to bind to a competing nucleic acid of the target nucleic acid and show a change in the amount of fluorescence. Furthermore, this fluorescent probe is designed so that the amount of change in fluorescence when bound to the target nucleic acid is different from the amount of change in fluorescence when bound to the competing nucleic acid.

  Further, a target nucleic acid different from the target nucleic acid amplified in the reaction container 104 constituting one reaction container group and its competing nucleic acid are formed on the surfaces of a plurality of reaction containers 104 constituting another one reaction container group. The same fluorescent probe that binds to and shows a change in the amount of fluorescence is disposed. Thereby, regression curves of different target nucleic acids can be obtained in one microreactor array 10.

  The reaction vessel 104 is, for example, a cylindrical shape having a circular cross section with a diameter of 500 μm and a depth of 100 μm. For example, the reaction liquid introduction flow path 105 has a cross section perpendicular to the direction in which the reaction liquid flows, having a width of 200 μm and a depth of 100 μm. The distance between the adjacent reaction vessels 104 is sufficiently secured so as to prevent mixing of the reaction liquid between the reaction vessels 104. Note that the surfaces of the reaction vessel 104 and the reaction solution introduction channel 105 are preferably subjected to surface treatment so that the inner wall surface becomes lyophilic to prevent adsorption of bubbles. The inner wall surfaces of the reaction vessel 104 and the reaction solution introduction channel 105 are preferably subjected to surface treatment that suppresses nonspecific adsorption of biomolecules such as proteins.

  The waste liquid storage unit 106 is connected to the reaction liquid introduction flow path 105 via the flow path 108. As will be described later, the reaction liquid filled in the reaction liquid introduction flow path 105 is discharged to the waste liquid storage section 106, so that the waste liquid storage section 106 has a larger volume than the reaction liquid introduction flow path 105. Good. The channel 108 is provided so as to vertically penetrate the transparent substrate 102 (for example, φ = 90 ° in FIG. 1B).

  Further, the surface of the transparent substrates 101, 102, 103 that are in contact with each other is subjected to surface treatment so as to have liquid repellency, or the contact surface is provided with a sealing property, whereby the reaction solution leaks from the reaction vessel 104, It is possible to prevent another reaction vessel 104 from entering the substrate surface. Specifically, a method of coating the contact surface with silicone rubber or fluororesin can be used.

1.2. Biological Sample Quantification Method A biological sample quantification method according to an embodiment of the present invention uses a microreactor array (biological sample quantification chip) 10 according to the present embodiment to quantitate a target nucleic acid contained in a specimen. A method for quantification, a step of introducing a sample into each of a plurality of reaction vessels 104, a step of performing a nucleic acid amplification reaction in the plurality of reaction vessels 104, and a part of the amplified nucleic acid in each reaction vessel 104 Based on the step of measuring the fluorescence intensity emitted by the fluorescent probe bound to, the fluorescence intensity measured in each reaction vessel 104 and the amount of competing nucleic acid used in each reaction vessel 104, the following equation (1) A step of obtaining a regression curve represented, and a step of estimating an amount of a target nucleic acid contained in the specimen based on the regression curve.

F = a / (C + b) + c (1)
(In the formula, F represents fluorescence intensity, C represents the amount of competing nucleic acid, b represents the amount of target nucleic acid contained in the sample, and a and c represent predetermined values).

1.2.1. Introduction of specimen into reaction vessel 104 (reaction liquid filling method)
First, the process of introducing the specimen into the reaction container 104 of the microreactor array 10 will be described. In the present embodiment, a method for filling a reaction vessel 104 with a reaction solution prepared from a specimen will be described. First, the reaction solution is supplied from the reaction solution supply port 109 to the reaction solution storage unit 107 using a pipette or the like.

  The reaction solution is prepared from a specimen, and for example, a solution containing a target nucleic acid, DNA polymerase, and nucleotide (dNTP) is contained at a predetermined concentration suitable for the reaction.

  Examples of the target nucleic acid include DNA extracted from a biological sample such as blood, urine, saliva, and cerebrospinal fluid, or cDNA reverse transcribed from the extracted RNA.

  Next, the microreactor array 10 is rotated using the centrifuge device 50 shown in FIGS. FIG. 2 is a front view of the centrifugal device 50 viewed from the side, and FIG. 3 is a plan view of the centrifugal device 50 viewed from above.

  As shown in FIG. 2 and FIG. 3, the centrifuge 50 includes a holder (rotated portion) 51 and a rotation motor (rotating means) 52 to which the microreactor array 10 can be attached. The holder 51 is inclined at an angle θ with respect to the direction from the rotation axis O toward the microreactor array 10. For this reason, the microreactor array 10 mounted on the holder 51 is also inclined at an angle θ with respect to the direction from the rotation axis O toward the microreactor array 10. Here, θ = 45 °. Note that θ may be in the range of 0 ° <θ <90 °.

  FIG. 4 is a plan view of the microreactor array 10 mounted on the holder 51 of the centrifuge 50 as viewed from above, and FIG. 5 is a cross-sectional view of the microreactor array 10 mounted on the holder 51. FIGS. 5A to 5C correspond to the DD cross sections of FIGS. 4A to 4C, respectively.

  First, as shown in FIGS. 4A and 5A, the microreactor array 10 is mounted on the holder 51 and rotated so that the transparent substrate 101 is on the outside as viewed from the rotation axis O. As a result, a centrifugal force is applied in a direction from the reaction solution storage unit 107 toward the reaction vessel 104, and the reaction solution in the reaction solution storage unit 107 advances while filling the reaction solution introduction channel 105 to fill the reaction vessel 104. . Air having a specific gravity lower than that of the reaction solution is pushed into the reaction solution introduction channel 105 and is replaced with the reaction solution, thereby filling the reaction vessel 104 with the reaction solution.

  At this time, the reaction liquid is not sent to the waste liquid container 106. As shown in FIG. 5A, this is because the direction of the flow path 108 from the reaction liquid introduction flow path 105 to the waste liquid storage section 106 is 135 with respect to the direction of centrifugal force (direction of arrow F in the figure). This is because the centrifugal force component in the direction from the reaction liquid introduction flow path 105 toward the waste liquid storage section 106 is 0 or less because the angle is at an angle.

  If the angle formed between the direction of the flow path 108 from the reaction liquid introduction flow path 105 to the waste liquid storage section 106 and the direction of the centrifugal force is 90 degrees or more and 180 degrees or less, the reaction liquid is not sent to the waste liquid storage section 106. . Therefore, in the case of θ = 45 °, if the angle φ formed by the transparent substrate 102 and the flow path 108 shown in FIG. 1B is in the range of 45 ° <φ ≦ 135 °, the reaction liquid is a waste liquid container. It is not sent to 106.

  As described above, since the reaction liquid does not flow toward the waste liquid storage unit 106, all the reaction vessels 104 can be efficiently filled with the reaction liquid, and after the rotation, the reaction liquids shown in FIGS. ), The reaction liquid is filled in all the reaction vessels 104 and the reaction liquid introduction flow path 105.

  Next, the rotation of the centrifugal device 50 is temporarily stopped, and this time, as shown in FIG. 4C and FIG. 5C, the microreactor array so that the transparent substrate 103 is on the outside as viewed from the rotation axis O. 10 is mounted on the holder 51 and rotated. As a result, the reaction solution in the reaction solution introduction channel 105 is sent out to the waste solution storage unit 106 this time. As shown in FIG. 5C, this is because the direction of the flow path 108 from the reaction liquid introduction flow path 105 to the waste liquid storage section 106 is in the direction of centrifugal force (the direction of arrow F in the figure). This is because since the angle is 45 degrees, the centrifugal force component in the direction from the reaction liquid introduction flow path 105 toward the waste liquid container 106 becomes 0 or more. If the angle between the direction of the flow path 108 from the reaction liquid introduction flow path 105 to the waste liquid storage section 106 and the direction of the centrifugal force is not less than 0 degrees and less than 90 degrees, the reaction liquid is sent to the waste liquid storage section 106. The Therefore, in the case of θ = 45 °, if the angle φ formed by the transparent substrate 102 and the flow path 108 shown in FIG. 1B is in the range of 45 ° <φ ≦ 135 °, the reaction liquid is a waste liquid container. 106. The reaction solution in the reaction solution introduction channel 105 is sent to the waste solution storage unit 106, but the reaction solution in the reaction vessel 104 remains in the reaction vessel 104.

  In this way, each reaction vessel 104 can be separated by sending the reaction solution in the reaction solution introduction channel 105 to the waste solution storage unit 106.

  When rotating in the state shown in FIG. 4C and FIG. 5C, mineral oil is previously supplied from the reaction liquid supply port 109 to the reaction liquid storage unit 107 using a pipette or the like. May be. When the microreactor array 10 is rotated in this state, the reaction liquid introduction channel 105 is filled with mineral oil. At this time, since the specific gravity of the reaction liquid is heavier than that of mineral oil, the reaction liquid in the reaction vessel 104 is not replaced with mineral oil. As a result, the individual reaction vessels 104 are separated, and contamination between the reaction vessels 104 (the reaction environment in the reaction vessel is contaminated by mixing the reaction solution in one reaction vessel with another reaction vessel). Can be prevented. It is also possible to prevent the inside of the reaction vessel 104 from being dried during the reaction process. Instead of mineral oil, a liquid that has a specific gravity lighter than that of the reaction liquid and is not miscible with the reaction liquid and is less likely to evaporate than the reaction liquid may be used. 4C and 5C, the reaction liquid in the reaction liquid introduction flow path 105 is sent to the waste liquid storage section 106 after being rotated, and then the reaction liquid storage section Mineral oil may be supplied to 107 and the centrifuge 50 may be rotated again.

  After supplying the reaction solution to the microreactor array 10 in the above procedure, PCR processing (biological sample reaction processing) can be performed. Specifically, after the opening of the microreactor array 10 is sealed, the microreactor array 10 is placed on a thermal cycler to perform PCR processing. In general, first, the step of dissociating the double-stranded DNA at 94 ° C. is performed, then the step of annealing the primer at about 55 ° C. is performed, and then the temperature is increased using a thermostable DNA polymerase. The cycle including the step of replicating the complementary strand at 72 ° C. is repeated.

  After PCR, the fluorescence intensity in each reaction vessel 104 can be measured using a fluorescence microscope, and the amount of target nucleic acid contained in the reaction solution in each reaction vessel 104 can be quantified.

  As described above, according to the microreactor array 10 according to the present embodiment, the reaction solution is supplied into the reaction vessel 104 through the reaction solution introduction channel 105 by using centrifugal force, and quantified with a pipette. This makes it possible to carry out a reaction process with a very small amount of reaction solution that is difficult to achieve. In addition, since processing can be performed in a large number of reaction vessels 104 at a time, various types of inspections and the like can be performed efficiently.

  Further, after filling the reaction liquid introduction flow path 105 and the reaction vessel 104 with the centrifugal force with the reaction liquid, the direction of the centrifugal force is changed, and the reaction liquid in the reaction liquid introduction flow path 105 is again with the centrifugal force. Since the individual reaction vessels 104 can be separated during the reaction process, contamination between the reaction vessels can be prevented. In the present embodiment, an example in which the centrifugal force is used to fill the reaction vessel 104 with the reaction solution has been described. However, instead of the centrifugal force, the reaction vessel 104 is filled with a capillary force or a pressure by a pump. Also good.

  In this embodiment, the microreactor array 10 is used as a reaction apparatus for PCR reaction, but the microreactor array 10 can be used for other DNA amplification reactions (for example, a lamp (LAMP) method).

1.2.2. Quantification of Target Nucleic Acid The biological sample quantification method according to the present embodiment further includes a step of performing a nucleic acid amplification reaction in a plurality of reaction vessels 104, and fluorescence bound to a part of the amplified nucleic acid in each reaction vessel 104. Based on the step of measuring the fluorescence intensity emitted by the probe, the fluorescence intensity measured in each reaction vessel 104 and the amount of competing nucleic acid used in each reaction vessel 104, the regression represented by the above formula (1) A step of obtaining a curve, and a step of estimating the amount of the target nucleic acid contained in the specimen based on the regression curve.

  As described above, a known amount of competing nucleic acid, primer, and fluorescent probe are arranged on the surface of the reaction vessel 104. Therefore, in the step of performing the nucleic acid amplification reaction in the reaction vessel 104, both the competing nucleic acid previously arranged in the reaction vessel 104 and the target nucleic acid contained in the reaction solution introduced into the reaction vessel 104 are amplified in the reaction vessel 104. Is done.

  In the nucleic acid amplification reaction, for example, the fluorescent probe can be bound to both the target nucleic acid and the competing nucleic acid, and furthermore, quenching of the fluorescence can be caused when the fluorescent probe is bound to either one. . In this state, the fluorescence intensity generated from each reaction vessel 104 is measured. Next, based on the fluorescence intensity measured in each reaction vessel 104 and the amount of competing nucleic acid used in each reaction vessel 104, a regression curve represented by the above formula (1) is obtained, and based on this regression curve. Thus, the amount of target nucleic acid contained in the specimen can be estimated.

  The fluorescence intensity (F) in the above formula (1) may be the fluorescence intensity itself emitted by the fluorescent probe, or (i) the fluorescence intensity measured before the nucleic acid amplification reaction and the fluorescence intensity measured after the nucleic acid amplification reaction. Ratio to fluorescence intensity (fluorescence change amount) or (ii) Heated to a temperature at which amplification product and fluorescent probe dissociate after nucleic acid amplification (fluorescent probe dissociates from amplified nucleic acid after nucleic acid amplification reaction) Fluorescence intensity measured in the first state) and the temperature at which the fluorescent probe is bound (second state in which the fluorescent probe is bound to a part of the amplified nucleic acid after the nucleic acid amplification reaction). It may be a ratio (fluorescence change amount) to the fluorescence intensity.

  Fluorescence changes when Ft is the amount of change in fluorescence when all amplification products obtained in the nucleic acid amplification reaction are amplifications of the target nucleic acid (X), and fluorescence when all amplification products are amplifications of the competitive nucleic acid (C) Let Fc be the amount of change. When both the competing nucleic acid and the target nucleic acid are present in the diffusion amplification reaction, both amplification products are produced, and the fluorescence change amount F at that time can be expressed by the following formula (2).

F = FtX / (X + C) + FcC / (X + C)
= [X (Ft−Fc) / (X + C)] + Fc (2)

  In the above formula (2), C is the amount of competing nucleic acid in the reaction vessel 104 (copy number in the reaction vessel 104), and X is the amount of target nucleic acid in the reaction vessel 104 (copy number in the reaction vessel 104). The above formula (1) can be derived from the above formula (2).

  That is, by drawing a graph with the amount of change in fluorescence (F) of each reaction vessel 104 belonging to the same reaction vessel group as the vertical axis and the amount of competitive nucleic acid (log C) contained in each reaction vessel 104 as the horizontal axis, Thus, a regression curve represented by the equation shown in the above equation (1) is obtained (for example, see FIG. 9 described later). From this regression curve, three parameters a, b and c in the above equation (1) are obtained, and the value of b corresponds to the amount of the target nucleic acid.

  The fluorescent probe is not particularly limited as long as it binds to a part of the target nucleic acid amplified by the PCR reaction and distinguishes between the target nucleic acid and the competing nucleic acid and shows a fluorescence change. For example, Taqman probe (registered trademark), Hyb probe (Registered trademark), Molecular Beacon (registered trademark), Q-Probe (registered trademark), and the like can be used. Q-Probe is a probe that detects a target gene using a “fluorescence quenching phenomenon” in which fluorescence emitted decreases when a guanine base approaches a labeled fluorescent dye.

  Q-Probe has a fluorescently labeled cytosine at the end and is designed to specifically bind to the target gene. When Q-Probe binds to the target gene, the fluorescence is affected by guanine. Decrease. When the biological sample quantification method according to this embodiment employs a competitive PCR method using Q-Probe, if the competitive nucleic acid is a guanine as a base corresponding to the fluorescently labeled terminal base of Q-Probe, the target nucleic acid Requires that the base corresponding to the fluorescently labeled terminal base of Q-Probe is a base other than guanine. Thereby, when Q-Probe co-amplifies both the target nucleic acid and the competing nucleic acid, when the competing nucleic acid and Q-Probe hybridize, the fluorescence emission of the labeled fluorescent dye decreases (quenches), while the target nucleic acid and Q-Probe When hybridized with Probe, the fluorescence emission of the labeled fluorescent dye does not decrease. In addition, about said description, even if it replaces a target nucleic acid and a competition nucleic acid, it is materialized. Therefore, it can be selected whether fluorescence quenching occurs when Q-Probe binds to a target nucleic acid or a competing nucleic acid.

  In the biological sample quantification method according to the present embodiment, a large number of test items can be quantified from one specimen using a plurality of reaction container groups of the microreactor array 10. Examples of such quantification include genetic testing of food poisoning causative bacteria (more specifically, testing for pathogenic microorganisms in foods and testing for pathogenic microorganisms in samples (feces) collected from food poisoning patients). It is done.

  In this case, the target nucleic acid used in the reaction container 104 constituting each reaction container group is a gene of a pathogenic microorganism that can cause food poisoning, and competing nucleic acids corresponding to these target nucleic acids are set. Deploy. Different causative bacteria can be quantified in each reaction container group. Examples of food poisoning causative bacteria include Campylobacter, Salmonella, Pseudomonas aeruginosa, pathogenic Escherichia coli O-157, Staphylococcus aureus, and the like. Then, a fluorescent probe (for example, Q-Probe) designed to correspond to each of the above target nucleic acids and competing nucleic acids is placed in the reaction vessel 104 constituting each reaction vessel group, and according to the above-described quantification method, Perform quantification. As a result, multi-item target nucleic acids can be easily quantified from one sample at a time.

2. Examples Hereinafter, the present invention will be described in more detail by way of examples. However, the present invention is not limited to the examples.

In this example, the case of using Q-Probe (Kurata et al., Nucleic acids Research, 2001, vol. 29, No. 6 e34) as a fluorescent probe is taken as an example, and the microreactor array 10 shown in FIG. A method for quantifying a target nucleic acid in a sample by PCR will be described.

Q-Probe interacts with guanine contained in the bound nucleic acid, and its fluorescence is significantly quenched. Therefore, Q-Probe can bind to both the target nucleic acid and the competing nucleic acid,
Furthermore, by making fluorescence quenching occur when Q-Probe binds to either one, the relative amount of the target nucleic acid relative to the known amount of competing nucleic acid can be estimated.

Target nucleic acid and competitive nucleic acid were purchased from J-Bio21 (Co). Further, as a buffer, 10 mM Tris-HCl buffer (pH: 8.3), KCl: 50 mM, Mg
A mixture of Cl ₂ : 1.5 mM was used.

FIG. 6 shows sequence examples of the target nucleic acid and the competing nucleic acid. Q-Prob in the underlined part
e binds. As shown in FIG. 6, immediately after the binding portion (underlined portion) of the fluorescent probe, the target nucleic acid is “TTTT” and the competing nucleic acid is “GGGT”. Therefore, Q-Pr
When obe binds to a competing nucleic acid, it reacts with the binding moiety guanine (G), and thus the fluorescence is quenched.

A primer having the sequence shown in FIG. 7 and a fluorescent probe (Q-Probe) having the sequence shown in FIG. 8 are applied in advance to the surface of the reaction vessel 104 of the microreactor array 10 shown in FIG. As a result, the primer and the fluorescent probe were arranged on the surface of the reaction vessel 104. Q-Probe (purchased from J-Bio21) is BODI
What was fluorescently labeled using PY FL (manufactured by Molecular probes) was used.

  In addition, different known amounts of competing nucleic acids were previously applied to the surfaces of the reaction vessels 104 in the reaction vessel rows A to F and vacuum-dried for placement. Table 1 shows the amount (copy number) of competing nucleic acids arranged in each reaction vessel 104 of the reaction vessel groups A to F.

  Subsequently, a reaction solution containing the target nucleic acid was filled in the reaction vessel 104, and PCR was performed with a thermal cycler (Master Cycler (Eppendorf), Light Cycler 480 (Roche Diagnostics)). Such a reaction solution contains a light cycler 480 genotyping master and uracil DNA glucosylase (purchased from Roche Diagnostics). Four microreactor arrays 10 were prepared, and four samples 1 to 4 having different target nucleic acid amounts (copy numbers) were prepared, and the quantitative accuracy was confirmed (Table 2). The fluorescence measurement was performed before and after the amplification reaction at room temperature, and further, at 60 ° C. and 95 ° C. after the amplification reaction.

  Since the amount of competing nucleic acid is different in reaction container rows A to F, the amount of change in fluorescence (F) of each reaction container 104 is plotted on the vertical axis and the amount of competing nucleic acid (log C) is plotted on the horizontal axis for each of samples 1 to 4. FIG. 9 is a graph obtained in this manner. In such a graph, a regression curve is obtained for each of samples 1 to 4, and based on these regression curves, three parameters a, b, and c in the above equation (1) are obtained, and the value of b is the amount of the target nucleic acid. It corresponds to. From FIG. 9 and the above formula (1), the value of b (amount of target nucleic acid) in samples 1 to 4 was calculated to be 40 copies, 850 copies, 9210 copies, and 89700 copies, respectively.

  In this example, the quantification result of one type of target nucleic acid was shown. As described above, different target nucleic acids and reagents for amplifying and quantifying the target nucleic acid (primer, fluorescence) for each reaction container group. By introducing a probe), a plurality of different target nucleic acids can be quantified in one microreactor array 10.

  As described above, according to the microreactor array 10 according to the present embodiment, a nucleic acid amplification reaction (PCR) is performed using different known amounts of competing nucleic acids in each of a plurality of reaction containers 104 included in the same reaction container group. By measuring the fluorescence intensity emitted by the fluorescent probe bound to a part of the amplified nucleic acid, based on the fluorescence intensity measured in each reaction vessel 104 and the amount of competing nucleic acid used in each reaction vessel 104 Then, the relationship between the fluorescence change amount F and the amount of competing nucleic acid (logC) in the sample is obtained as a regression curve represented by the above formula (1), and the amount of target nucleic acid contained in the sample based on the regression curve Can be estimated. Thereby, the target nucleic acid can be quantified with high accuracy and efficiency.

  This is the end of the description of the embodiment according to the present invention. The present invention includes configurations that are substantially the same as the configurations described in the embodiments (for example, configurations that have the same functions, methods, and results, or configurations that have the same purposes and results). In addition, the invention includes a configuration in which a non-essential part of the configuration described in the embodiment is replaced. In addition, the invention includes a configuration that achieves the same effect as the configuration described in the embodiment or a configuration that can achieve the same object. Further, the invention includes a configuration in which a known technique is added to the configuration described in the embodiment.

  DESCRIPTION OF SYMBOLS 10 ... Microreactor array, 50 ... Centrifugal apparatus, 51 ... Holder, 52 ... Rotation motor, 101, 102, 103 ... Transparent substrate, 104 ... Reaction container, 105 ... Reaction liquid introduction flow path, 106 ... Waste liquid storage part, 107 ... reaction liquid container, 108 ... flow path, 109 ... reaction liquid supply port, 201, 202, 203, 204, 205, 206 ... reaction container group, A, B, C, D, E, F ... reaction container row

SEQ ID NO: 1 is the sequence of the target nucleic acid.
SEQ ID NO: 2 is the sequence of a competing nucleic acid.
SEQ ID NO: 3 is the sequence of the forward primer.
SEQ ID NO: 4 is the sequence of the reverse primer.
SEQ ID NO: 5 is the sequence of Q-Probe.

Claims (4)

A biological sample quantification method for quantifying a target nucleic acid contained in a sample using a biological sample quantification chip used for quantification of a target nucleic acid contained in the sample,
The biological sample quantification chip includes a plurality of reaction containers, and the plurality of reaction containers can be amplified with a primer common to the target nucleic acid and have a known amount of a competitive nucleic acid having a sequence different from the target nucleic acid. A primer common to the target nucleic acid and the competing nucleic acid, and a part of the amplification product of the target nucleic acid and the competing nucleic acid, wherein the amplification product of the target nucleic acid is different from the amplification product of the competing nucleic acid. And the amount of the competing nucleic acid contained in each of the reaction containers is different,
The biological sample quantification method comprises:
Introducing each sample into the plurality of reaction vessels;
Performing a nucleic acid amplification reaction in the plurality of reaction vessels;
Measuring the fluorescence intensity emitted by the fluorescent probe bound to a part of the amplified nucleic acid in each of the reaction vessels;
Obtaining a regression curve represented by the following formula (1) based on the fluorescence intensity measured in each reaction container and the amount of the competing nucleic acid used in each reaction container;
Estimating the amount of target nucleic acid contained in the specimen based on the regression curve;
A biological sample quantification method comprising:
F = a / (C + b) + c (1)
(In the formula, F represents fluorescence intensity, C represents the amount of competing nucleic acid, b represents the amount of target nucleic acid contained in the sample, and a and c represent predetermined values). In claim 1,
In the step of obtaining the regression curve, the ratio of the fluorescence intensity measured before the nucleic acid amplification reaction to the fluorescence intensity measured after the nucleic acid amplification reaction, and the competitive nucleic acid used in each reaction vessel A biological sample quantification method for obtaining the regression curve based on a relationship with an amount. In claim 1,
In the step of obtaining the regression curve, after the nucleic acid amplification reaction, the fluorescence probe is bonded to a part of the amplified nucleic acid, and the fluorescence intensity is measured in the first state. The regression curve is obtained based on the relationship between the ratio of the fluorescence intensity measured in the second state in which the probe is dissociated from the amplified nucleic acid and the amount of the competing nucleic acid used in each of the reaction vessels. Quantitative method for biological sample. A biological sample quantification method for quantifying a target nucleic acid contained in a sample using a biological sample quantification chip used for quantification of a target nucleic acid contained in the sample,
The biological sample quantification chip includes a first reaction container and a second reaction container, and the first reaction container is capable of amplifying with the primer for amplifying the target nucleic acid, the primer, A first amount of a competitive nucleic acid having a sequence different from the target nucleic acid binds to the target nucleic acid and a part of the amplified product of the competitive nucleic acid, and the amplified product of the target nucleic acid is different from the amplified product of the competitive nucleic acid. A fluorescent probe that exhibits a change in fluorescence, and wherein the second reaction vessel includes a second amount of the competitive nucleic acid different from the first amount, the primer, and the fluorescent probe,
Introducing the specimen into the first and second reaction vessels;
Performing a nucleic acid amplification reaction in the first and second reaction vessels;
Measuring the fluorescence intensity emitted by the fluorescent probe bound to a part of the amplified nucleic acid in the first and second reaction vessels;
Based on the fluorescence intensity measured in the first and second reaction vessels and the amount of the competing nucleic acid contained in the first and second reaction vessels, a regression curve represented by the following formula (1) is obtained. Seeking and
Estimating the amount of target nucleic acid contained in the specimen based on the regression curve;
A biological sample quantification method comprising:
F = a / (C + b) + c (1)
(In the formula, F represents the fluorescence intensity, C represents the amount of the competing nucleic acid, b represents the amount of the target nucleic acid contained in the sample, and a and c represent predetermined values).

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