JP2021193942A - Nucleic acid determination method and reagent kit - Google Patents

Abstract

To determine nucleic acid more accurately by LAMP method.SOLUTION: A nucleic acid determination method according to an embodiment is a method for determining a target nucleic acid sequence by LAMP method. The method includes: an amplification process of amplifying a target nucleic acid sequence; a detection process of detecting a first amplification product including at least one of F3 sequence, B3 sequence, F3c sequence and B3c sequence; and a determination process of determining the concentration of the target nucleic acid sequence from the detection result.SELECTED DRAWING: Figure 1

Description

The embodiments disclosed herein and in the drawings relate to nucleic acid quantification methods and reagent kits.

The PCR (Polymerase Chain Reaction) method and the LAMP (Loop-mediated Isothermal Expression) method are used for the detection of the target sequence. However, although the PCR method can quantify nucleic acids, it has a long reaction time and requires a device such as a thermal cycler. In addition, it is difficult to accurately quantify nucleic acids in the quantification method using the rise time of turbidity by the LAMP method.

Japanese Unexamined Patent Publication No. 2002-186781.

One of the problems to be solved by the embodiments disclosed in the present specification and the drawings is to quantify nucleic acid more accurately by the LAMP method. However, the problems to be solved by the embodiments disclosed in the present specification and the drawings are not limited to the above problems. The problem corresponding to each effect by each configuration shown in the embodiment described later can be positioned as another problem.

The nucleic acid quantification method according to the embodiment is a method of quantifying a target nucleic acid sequence by the LAMP method. The method comprises an amplification step of amplifying a target nucleic acid sequence, a detection step of detecting a first amplification product containing at least one of an F3 sequence, a B3 sequence, an F3c sequence, and a B3c sequence, and a detection step of detecting the target nucleic acid. Includes a determination step to determine the concentration of the sequence. The target nucleic acid sequence is a double-stranded nucleic acid, and one strand is a F3c sequence, an F2c sequence, an F1c sequence, a B1 sequence, a B2 sequence, and a B3 sequence in this order from the 3'end side to the 5'end side. The other chain contains B3c sequence, B2c sequence, B1c sequence, F1 sequence, F2 sequence and F3 sequence from the 3'end side to the 5'end side, and contains F1 sequence and F1c sequence, F2 sequence and F2c. The sequences, F3 and F3c sequences, B1 and B1c sequences, B2 and B2c sequences, and B3 and B3c sequences are complementary to each other.

FIG. 1 is a schematic diagram showing an example of a primer set and a target nucleic acid sequence according to the first embodiment. FIG. 2 is a flowchart showing an example of the nucleic acid quantification method according to the first embodiment. FIG. 3 is a schematic diagram showing an example of a target nucleic acid sequence and an amplification product according to the first embodiment. FIG. 4 is a graph showing an example of the relationship between the amplification time and the detected value of the signal according to the first embodiment. FIG. 5 is a schematic diagram showing an example of a primer set and a target nucleic acid sequence according to the second embodiment. FIG. 6 is a schematic diagram showing an example of the amplification product according to the second embodiment. FIG. 7 is a graph showing an example of the relationship between the amplification time and the detected value of the signal according to the second embodiment. FIG. 8 is a schematic diagram showing a target nucleic acid sequence and an amplification product according to the third embodiment. FIG. 9 is a block diagram showing an example of the target nucleic acid sequence quantifying device according to the fifth embodiment.

Hereinafter, the nucleic acid quantification method and the reagent kit of the embodiment will be described with reference to the drawings.

(First Embodiment)
-Nucleic acid quantification method The nucleic acid quantification method according to the embodiment is a method of quantifying a target nucleic acid sequence by using the LAMP method. The target nucleic acid sequence is, for example, a nucleic acid sequence to be analyzed that can be contained in a sample.

Samples include body fluids obtained from animals, such as blood, serum, blood cells, plasma, interstitial fluid, urine, stool, sweat, saliva, oral mucosa, sputum, lymph, spinal fluid, tears, breast milk, and amniotic fluid. , Saliva, cell extract or plasma, etc. The sample may be cells, tissues, or the like. Alternatively, the sample may be a liquid derived from the environment such as plants, river water or seawater, a liquid expected to contain bacteria, fungi or viruses, a liquid containing artificial nucleic acid, or the like.

In the nucleic acid quantification method, a recognition sequence for binding a primer is set in the target nucleic acid sequence as in the general LAMP method.

The target nucleic acid sequence and the primers used in the nucleic acid quantification method will be described with reference to FIG. As shown in FIG. 1, the target nucleic acid sequence 11 is, for example, a double-stranded nucleic acid. One strand of the target nucleic acid sequence 11 contains the F3c sequence, the F2c sequence, the F1c sequence, the B1 sequence, the B2 sequence, and the B3 sequence in this order from the 3'end side to the 5'end side. The other strand contains the B3c sequence, the B2c sequence, the B1c sequence, the F1 sequence, the F2 sequence, and the F3 sequence in this order from the 3'end side to the 5'end side. It is preferable to leave a space of about 100 to 300 bases between the F1c sequence and the B1 sequence.

Here, the F1 to F3 sequences and the B1 to B3 sequences are recognition sequences for binding the primers. The F1 and F1c sequences, the F2 and F2c sequences, the F3 and F3c sequences, the B1 and B1c sequences, the B2 and B2c sequences, and the B3 and B3c sequences are complementary to each other.

A total of four types of primers, FIP primer, F3 primer, BIP primer, and B3 primer, are set for the target nucleic acid sequence 11 as described above. The FIP primer contains the F1c sequence and the F2 sequence from the 5'side to the 3'side in this order, and the BIP primer contains the B2 sequence and the B1c sequence from the 5'side to the 3'side in this order. include. The F3 primer comprises the F3 sequence and the B3 primer comprises the B3 sequence.

At least one of the F3 primer and the B3 primer is labeled with the first substance 14. FIG. 1 shows an example in which the F3 primer is labeled. The first substance 14 is a substance that emits a first signal 15 when the F3 primer or B3 primer labeled thereby binds to the target nucleic acid sequence 11.

As the first substance 14, it is preferable to use a substance that emits an optical first signal 15. The optical signal is, for example, fluorescence, light emission, coloration, or the like. As the first substance 14 that emits the first optical signal 15, for example, what is generally called a fluorescent dye, for example, Alexa flow, BODIPY, Cy3, Cy5, FAM, Fluorescein, HEX, JOE, Marina Blue (registered trademark). ), Fluorescein, Pacific Blue®, Rhodamine, Rhodol Green, ROX, TEMRA, TET and Texas Red®.

The optical first signal 15 may be the generation of light or an increase in light intensity when the primer binds to the target nucleic acid sequence 11, or may be the disappearance of light or a decrease in light intensity. It may be a change in the wavelength of light.

For example, one in which a dye is bound to one end of an F3 or B3 primer and a quencher is bound to the other end can be used. In such a primer, for example, in a single state, the generation of the optical signal of the dye is suppressed by the quencher, but when it binds to the target nucleic acid sequence 11, the suppression is released and the optical signal is generated. Further, a dye that generates an optical signal when the F3 or B3 primer binds to the target nucleic acid sequence 11 may be bound to one end of the F3 or B3 primer without using a quencher.

Alternatively, for example, a Quenching probe (Q probe) may be used as the first substance 14 that can obtain the disappearance of light or the reduction of light intensity. Alternatively, a substance that emits an electrical signal as the first signal 15 may be used as the first substance 14. Alternatively, the first substance 14 that presents a detectable substance such as a protein may be used as the first signal 15.

The first substance 14 is not limited to the above, and any substance that emits a signal that can be detected by a method known per se can be used.

The first substance 14 may be bound to the primer by a general method used when binding the substance to nucleic acid.

Although the example in which the F3 primer is labeled is shown in FIG. 1, the B3 primer may be labeled with the first substance 14 instead of the F3 primer, and both the F3 primer and the B3 primer are labeled. May be.

Further, in addition to these four types of primers, a loop primer (not shown) may be used in combination for amplification of the target nucleic acid sequence 11. As the loop primer, at least one of an LF primer and an LB primer can be used. The LF primer is designed to have a sequence complementary to, for example, the sequence between the F1 and F2 sequences of FIG. 1, and the LB primer is, for example, complementary to the sequence between the B1 and B2 sequences of FIG. It can be designed to have an array. By using at least one of the LF primer and the LB primer, the amplification efficiency is increased, and the time required for amplification can be shortened to 1/2 to 1/3.

As shown in FIG. 2, the target quantification method according to the first embodiment is a first amplification including at least one of the amplification steps S1, F3 sequence, B3 sequence, F3c sequence, and B3c sequence for amplifying the target nucleic acid sequence 11. It may include a detection step S2 for detecting the product 12 and a determination step S3 for determining the concentration of the target nucleic acid sequence 11 from the result of the detection.

The target nucleic acid sequence 11 may be, for example, a reverse transcriptase of the RNA to be detected. For example, the reverse transcription reaction of the RNA may be performed before the amplification step S1.

The amplification step S1 is performed using a primer set according to the above-described embodiment, that is, a primer set in which at least one of the F3 primer and the B3 primer is labeled with the first substance 14. Other materials and procedures may be performed in the same manner as the general LAMP method.

The amplification step S1 is performed, for example, by maintaining the reaction solution containing the sample and the amplification reagent under the amplification conditions. The amplification reagent includes, for example, a substrate, an amplification enzyme, and at least the above-mentioned primer set.

The substrate contains a building block of nucleic acid. The substrate is, for example, dNTP or the like.

The amplifying enzyme can be selected based on the type of target nucleic acid, primers and the like. The amplifying enzyme can be, for example, a strand-substituted DNA polymerase.

The amplification reagent may contain additional reagents necessary for the amplification reaction, and the additional reagents may be, for example, a buffer such as a pH regulator, a salt, a thickener or a buffer, or a mixture thereof. A reagent or the like necessary for detecting the first signal 15 used in the detection step S2 may be added to the reaction solution.

The amplification conditions are selected based on, for example, the type of amplification method used, the type of primer set, the type of target nucleic acid, the type of amplification enzyme, and the like. The amplification conditions are isothermal amplification reaction conditions. When maintaining the isothermal amplification condition, for example, it is more preferable to maintain the temperature of the reaction field at 55 ° C to 65 ° C.

In the amplification step S1, if the target nucleic acid sequence 11 is present in the sample, an amplification reaction may occur and an amplification product may be produced. For example, among the amplification products generated from the target nucleic acid sequence 11, an amplification product containing at least one of the F3 sequence B3 sequence, the F3c sequence, and the B3c sequence (hereinafter referred to as “first amplification product 12”) and F3. There is a dumbbell-type amplification product (hereinafter referred to as “second amplification product 13”) that does not contain a sequence, a B3 sequence, an F3c sequence, and a B3c sequence. Examples of these amplification products will be described with reference to FIG.

The first amplification product 12 can be, for example, an amplification product obtained by amplification starting from the binding of an F3 primer or a B3 primer. An example of the first amplification product 12 has the same sequence structure as the target nucleic acid sequence 11 as shown in FIG. 3, for example, and contains at least one of the F3 sequence B3 sequence, the F3c sequence, and the B3c sequence.

The first amplification product 12 can be produced by the F3 primer and the B3 primer in proportion to the reaction rate of the amplification enzyme, as in the case of amplification in the PCR method. Therefore, the amount of the first amplification product 12 produced can correlate with the abundance of the target nucleic acid sequence 11 in the sample. Further, at least one of the F3 sequence, B3 sequence, F3c sequence, and B3c sequence of the first amplification product 12 is obtained by binding the F3 primer and the B3 primer to the target nucleic acid sequence 11, and thus is the first. By producing the amplification product 12, the first signal 15 is obtained from the first substance 14. Therefore, the detected amount of the first signal 15 can correlate with the abundance of the target nucleic acid sequence 11 in the sample.

On the other hand, the second amplification product 13 can be an amplification product obtained by amplification starting from the binding of the FIP primer or the BIP primer. An example of the second amplification product 13 is, for example, one including an F1 sequence, an F2c sequence, an F1c sequence, a B1 sequence, a B2 sequence, and a B1c sequence in the direction from the 3'end to the 5'end as shown in FIG. It is a chain nucleic acid. At the 3'end, the F1 sequence binds to the F1c sequence, and at the 5'end, the B1c sequence binds to the B2 sequence to form two stem-loop structures.

The dumbbell-shaped second amplification product 13 shown in FIG. 3 is subsequently repeatedly amplified by a FIP primer and a BIP primer, and if necessary, an LF primer and an LB primer, and the F3 sequence and B3 derived from the second amplification product 13 are obtained. A large amount of sequence-free amplification product is produced. That is, the second amplification product 13 can be amplified more rapidly in a shorter time than the first amplification product 12 due to its amplification mechanism. The second amplification product 13 is not limited to that shown in FIG. 3, and includes amplification products that do not contain these F3 sequences and B3 sequences. Therefore, the amount of the second amplification product 13 produced has a low correlation with the amount of the target nucleic acid sequence 11 present in the sample. Since the second amplification product 13 does not contain a sequence complementary to the F3 sequence and the B3 sequence, the first amplification product 12 is not produced from the second amplification product 13.

Next, the first amplification product 12 is detected (detection step S2).

The detection step S2 can be performed, for example, by measuring the first signal 15 derived from the production of the first amplification product 12. For example, when the first signal 15 is fluorescent, the fluorescence intensity is detected by using, for example, a spectroscopic fluorometer or the like.

The detection is preferably performed over time, for example, in parallel with the amplification step S1. Here, "detection over time" may be continuous, or may be detected at least three times or more at regular time intervals. For example, detection is performed over time from the start of amplification in the amplification step S1 until the first signal 15 does not change.

For example, detection over time gives the graph shown in FIG. The horizontal axis of the graph shown in FIG. 4 indicates the amplification time, and the vertical axis indicates the detected value of the first signal 15. This graph shows an example in which the first signal 15 increases as the amplification product increases. The detected value (solid line) of the first signal 15 can be, for example, an S-shaped curve that starts to increase at a specific time point, increases in proportion to the amplification time, saturates at a specific time point, and does not change.

When the target nucleic acid sequence 11 in the sample is small, the production of the amplification product is delayed and the S-curve shifts to the right. That is, the detected value of the first signal 15 in a constant amplification time becomes small. On the contrary, when the target nucleic acid sequence 11 is abundant, the production of the amplification product occurs at an early stage, and the S-shaped curve shifts to the left side. That is, the detected value of the first signal 15 in a certain amplification time becomes large.

Next, the concentration of the target nucleic acid sequence 11 is determined from the result of the detection step S2 (determination step S3).

The concentration of the target nucleic acid sequence 11 is preferably determined, for example, by comparing with the detection result of a standard target nucleic acid sequence having a known concentration. The standard target nucleic acid sequence has the same sequence as the target nucleic acid sequence 11 and is labeled with the same first substance. Standard target nucleic acid sequences having different concentrations are prepared, amplified by the same method as in the amplification step S1 and the detection step S2, respectively, and the detected amount of the first signal 15 is measured over time. As a result, a plurality of standard curves having an amplification start time proportional to the concentration of the standard target nucleic acid sequence are obtained, for example, as shown by a broken line in FIG. The concentration of the target nucleic acid sequence 11 can be calculated by comparing the position of the standard curve with the position of the S-shaped curve obtained from the sample.

For example, a plurality of standard target nucleic acid sequences having different concentrations are housed in separate containers, a sample is housed in a separate container, the same amplification reagent is added to each container, and the amplification step S1 and the detection step S2 are simultaneously performed. It is preferable to perform the above comparison by recording the results in each container in one graph as shown in FIG. However, it is also possible to use the curves of the standard target nucleic acid sequences obtained in the past for comparison.

Alternatively, from the measurement result of the standard target nucleic acid sequence, a calibration curve showing the detection amount of the first signal 15 with respect to the concentration of the standard target nucleic acid sequence in a constant amplification time may be created. It is also possible to determine the concentration of the target nucleic acid sequence 11 in the sample by comparing this calibration curve with the amount of the first signal 15 detected in the sample at the same amplification time.

According to the nucleic acid quantification method according to the embodiment, the target nucleic acid sequence 11 can be accurately quantified by using the amount of the first amplification product 12 produced as an index. According to this method, the quantification can be performed in a shorter time than the quantification by the PCR method. Further, in the conventional quantitative LAMP method, the first amplification product 12 and the second amplification product 13, which is rapidly amplified as compared with the first amplification product 12, are amplified and quantified without distinction. .. Therefore, it lacked the accuracy of quantification. However, according to this method, since the first amplification product 12 is targeted for detection, more accurate quantification is possible as compared with the conventional method.

(Second Embodiment)
The method for quantifying the target nucleic acid sequence 11 according to the second embodiment further comprises detecting the second amplification product 13 in the detection step S2.

As shown in FIG. 5, in the second embodiment, at least one of the F3 primer and the B3 primer was labeled with the first substance 14, and at least one of the FIP primer and the BIP primer was labeled with the second substance 16. The amplification step S1 is performed using the primer set. FIG. 5 shows an example in which the FIP primer is labeled with the second substance 16, but the BIP primer may be labeled with the second substance 16 instead of the FIP primer, or the FIP primer and The BIP primer may be labeled with the second substance 16. Further, a primer set in which at least one of the LF primer and the LB primer, which are loop primers, is labeled with the second substance 16 may be further used.

The second substance 16 is a substance that emits a second signal 17 when the FIP primer or BIP primer to which it is bound binds to the target nucleic acid sequence 11. As the second substance 16, for example, the same substance as the first substance 14 described above can be used, but it is preferable that the first signal 15 and the second signal 17 are different from each other. The second signal 17 is preferably, for example, fluorescence or light emission having a wavelength different from that of the first signal 15 described above.

According to the amplification step S1 according to the second embodiment, as shown in FIG. 6, the first signal 15 is generated due to the production of the first amplification product 12 as in the first embodiment, and in addition, the FIP primer is generated. Alternatively, the second amplification product 13 is produced due to the binding of the BIP primer, and the second signal 17 is generated at that time.

Although not shown, when an LF primer and an LB primer are included, and at least one of them uses a primer set labeled with the second substance 16, the second due to the binding of the LF primer or the LB primer in the process of amplification. Signal 17 is generated.

The detection step S2 includes detecting both the first amplification product 12 and the second amplification product 13. For example, the detection of the first amplification product 12 may be performed by the detection of the first signal 15, and the detection of the second amplification product 13 may be performed by the detection of the second signal 17. The detection of the first amplified product 12 and the second amplified product 13 is preferably performed simultaneously over time, but it is also possible to sequentially perform either of them first.

The detection of the first signal 15 and the quantification of the target nucleic acid sequence 11 based on the detection can be performed in the same manner as in the amplification step S1, the detection step S2, and the determination step S3 of the first embodiment.

As described in the first embodiment, the second amplified product 13 can be amplified more rapidly than the first amplified product 12. Therefore, as shown in FIG. 7, the second signal 17 (broken line) can be detected earlier than the first signal 15 (solid line). The second signal 17 is independent of the concentration of the target nucleic acid sequence 11 in the sample and can increase similarly rapidly at any concentration. Therefore, although it is difficult to quantify the target nucleic acid sequence 11 from the second signal 17, the presence or absence of the target nucleic acid sequence 11 can be determined from the second signal 17.

Therefore, according to the method of the second embodiment, the target nucleic acid sequence 11 is quantified by the detection of the first amplification product 12, and at the same time, the presence or absence of the target nucleic acid sequence 11 is determined by the detection of the second amplification product 13. It can be carried out. By simultaneously performing the presence / absence determination and the quantification, it is possible to determine whether or not the first signal 15 is the result of non-specific amplification (false positive) of the first amplification product 12. For example, if the first signal 15 is detected and the second signal 17 is not detected, it is determined to be a false positive. In that case, it is preferable to change the type of reagent and / or the amplification condition and detect the sample again.

(Third Embodiment)
In the method for quantifying the target nucleic acid sequence 11 according to the third embodiment, instead of labeling at least one of the FIP primer and the BIP primer of the second embodiment with the second substance 16 to detect the target nucleic acid sequence 11. The target nucleic acid sequence 11 is detected by detecting the double-stranded amplification product (which may contain both the first amplification product 12 and the second amplification product 13) using the double-stranded recognition substance 18. .. Other than that, the configuration is the same as that of the first embodiment, and the labeling of the F3 primer and / or the B3 primer by the first substance 14 and the detection of the first signal 15 are performed in the same manner as in the first embodiment.

The double-stranded recognition substance 18 is preferably a substance that binds to the double-stranded nucleic acid and emits a signal (hereinafter referred to as "third signal 19"). The third signal 19 may be, for example, an optical signal, an electrical signal, a presentation of a substance such as a protein, or the like. The double-stranded recognition substance 18 may be, for example, a substance generally called an intercalator, and TB Green (registered trademark) or the like can be used. The third signal 19 is preferably different from the first signal 15. It is preferable that the third signal 19 can be separated and recognized even if it exists at the same time as the first signal 15.

The double-stranded recognition substance 18 can be added to the reaction solution together with other amplification reagents in the amplification step S1. Other than that, the amplification step S1 can be performed in the same manner as the nucleic acid detection method described in the first embodiment.

By performing the amplification step, as shown in FIG. 8, both the first amplification product 12 and the second amplification product 13 can be labeled with an intercalator.

In the detection step S2 according to the third embodiment, for example, the detection of the first signal 15 derived from the first amplification product 12 and the third signal derived from the intercalator labeled with the first amplification product 12 and the second amplification product 13 19 detections are performed. It is preferable that these detections are performed simultaneously over time, but it is also possible to perform either of them sequentially first. Although the first amplification product 12 and the second amplification product 13 cannot be distinguished by the third signal 19 alone, the first signal 15 generated from the first amplification product 12 is different from the third signal 19, so that the third signal 19 is the third signal. It can be detected separately from the signal 19 of 3.

The first signal 15 is detected in the same manner as in the first embodiment, and the target nucleic acid sequence 11 is quantified based on the detection.

Since the third signal 19 is derived from the first amplification product 12 and the second amplification product 13, it is faster and faster than the first signal 15 like the second signal 17 described in the second embodiment. Can be amplified to. Therefore, the third signal 19 does not depend on the concentration of the target nucleic acid sequence 11 in the sample, and the presence or absence of the target nucleic acid sequence 11 can be determined.

Therefore, according to the method of the third embodiment, the target nucleic acid sequence 11 is quantified by detecting the first amplification product 12, and at the same time, the double-stranded portion containing the first amplification product 12 and the second amplification product 13 is contained. The target nucleic acid sequence 11 can be detected by detecting the amplification product. As a result, it is possible to prevent false positives.

(Fourth Embodiment)
-Reagent Kit According to the fourth embodiment, a reagent kit for use in the nucleic acid quantification method of the first to third embodiments is provided.

For example, a reagent kit according to the method of the first embodiment contains at least a primer set containing an FIP primer, an F3 primer, a BIP primer and a B3 primer contained in a solvent, and at least one of the F3 primer and the B3 primer is as described above. It is labeled with the first substance 14. The primer set may further include at least one of an LF primer and an LB primer. These primers may be contained together in one container or may be individually contained in a container.

The reagent kit may further include an amplification reagent, a detection reagent, and the like. The reagent kit may further individually further include a standard target nucleic acid sequence of known concentration. For example, a reagent kit may contain one solution containing a standard target nucleic acid sequence of a given concentration, and the user may dilute this solution to the desired concentration to prepare multiple different concentrations of the standard target nucleic acid sequence. can.

The reagent kit may further include a container such as a tube strip for mixing each component.

The reagent kit according to the method of the second embodiment can be the same as the reagent kit of the first embodiment except that, for example, at least one of the FIP primer and the BIP primer is further labeled with the second substance 16. .. Further, at least one of the LF primer and the BF primer may be further labeled with the second substance 16.

The reagent kit according to the third embodiment may be the same as the reagent kit according to the first embodiment, except that the double-stranded recognition substance 18 is further contained.

(Fifth Embodiment)
-Target Nucleic Acid Sequence Quantitator According to the fifth embodiment, a nucleic acid quantifier for carrying out the above nucleic acid quantification method is provided.

The nucleic acid quantification apparatus is configured so that, for example, the amplification steps S1 to the determination step S3 of the nucleic acid quantification method can be automatically performed.

As shown in FIG. 9, the nucleic acid quantifying device 100 includes, for example, a measuring unit 200, a storage unit 300, a CPU 400, an input unit 500, and a display unit 600. The storage unit 300, the CPU 400, the input unit 500, and the display unit 600 are connected to each other via, for example, the bus 810.

The measuring unit 200 includes an introduction unit 210, a temperature controller 220, and a measuring instrument 230.

The introduction unit 210 includes a plurality of bottomed holes in which a container containing a reaction solution containing a sample, an amplification reagent, a detection reagent, and the like is placed. The bottomed hole has an inner diameter slightly wider than that of a container such as a tube strip, and can insert and support the container. Alternatively, the introduction unit 210 may include a mechanism for automatically weighing and mixing the sample, the amplification reagent, and the detection reagent.

The temperature controller 220 includes a heater capable of maintaining the temperature of the bottomed hole at a constant temperature. The temperature controller 220 is connected to the bus 810 via, for example, an interface (I / F) 710.

The measuring instrument 230 is configured to detect the first signal 15 from, for example, a plurality of containers installed in a bottomed hole. The measuring instrument 230 is selected according to the type of the first signal 15, and for example, an optical detector can detect the optical first signal 15. The optical detector can measure fluorescence at wavelengths in the range 220-900 nm, for example. Further, it is preferable that the configuration is such that two or more different wavelengths can be detected. In this case, the container arranged in the bottomed hole is preferably made of a light-transmitting material. The measuring instrument 230 is connected to the bus 810 via, for example, an interface (I / F) 720.

The storage unit 300 is based on the measurement data 310 including the measurement value of the first signal 15 and the measurement value of the second signal 17 or the third signal 19 obtained from the measuring device 230, and the measurement value of the first signal 15. An arithmetic expression 320, which includes an arithmetic expression for calculating the concentration of the target nucleic acid sequence 11 in the sample and an arithmetic expression for calculating the presence or absence of the target nucleic acid sequence 11 from the measured values of the second signal 17 or the third signal 19. The calculation result 330 obtained by the calculation and the program P for controlling each part are included.

The CPU 400 performs information processing such as the above calculation and control of each part according to the program P.

The input unit 500 has a configuration in which the operator inputs parameters and the like for starting or stopping each operation of the nucleic acid quantifying device 100. The input unit 500 includes a mouse, a keyboard, a button, and the like.

The display unit 600 displays information such as the calculation result 330. The display unit 600 includes, for example, a display.

Hereinafter, the operation method of the nucleic acid quantifying device 100 will be described.

First, the container is installed in the bottomed hole provided in the introduction portion 210. For example, a container containing a sample and a plurality of containers containing a standard target nucleic acid sequence of known concentration may be installed.

Next, the amplification sequence is amplified (amplification step S1). For example, the CPU 400 commands the drive of the temperature controller 220 according to the program P, and the temperature controller 220 heats the bottomed hole. For example, the temperature and heating time of the heater of the temperature controller 220 may be input from the input unit 500. By heating, the container is warmed and amplification of the target nucleic acid sequence 11 contained in the container and the standard target nucleic acid sequence is started.

Next, detection is performed by the measuring instrument 230 (detection step S2). For example, the CPU 400 sends a command to the measuring instrument 230 according to the program P, and the first signal 15 is measured from each container. The obtained measured value is sent to the storage unit 300 and stored. Further, the measured value may be displayed on the display unit 600 as a graph as shown in FIG. The detection step S2 can be performed in parallel with the amplification step S1.

When detecting the second signal 17 or the third signal 19, for example, it is input from the input unit 500 that these signals are additionally measured. As a result, the CPU 400 sends a command to the measuring instrument 230 according to the program P, and the second signal 17 or the third signal 19 is measured in the detection step S2.

Next, the concentration of the target nucleic acid sequence 11 contained in the sample is determined (determination step S3). The CPU 400 takes out the measurement data 310 and the arithmetic expression 320 stored in the storage unit 300, and calculates the concentration of the target nucleic acid sequence 11. The concentration is calculated, for example, by comparing the measured values of the sample and the measured values of the standard target nucleic acid sequence over time. Also, past measurements may be used instead of the measurements of known standard target nucleic acid sequences. In that case, it is preferable that the past measured values are stored in the storage unit 300 in advance. The calculated calculation result 330 may be output to the storage unit 300 and stored, or may be output to the display unit 600.

If necessary, the presence or absence of the target nucleic acid sequence 11 in the sample may be determined at the same time as the determination step S3. The CPU 400 retrieves the measurement data 310 and the arithmetic expression 320 for the second signal 17 or the third signal 19 stored in the storage unit 300. Further, if necessary, past measured values, measured values of the standard target nucleic acid sequence, patterns, and the like are retrieved from the storage unit 300. Any of these values is compared with the measurement data 310 of the second signal 17 or the third signal 19, and the presence or absence of the target nucleic acid sequence 11 in the sample is determined. The calculated result may be output to the storage unit 300 and stored, or may be displayed on the display unit 600, as in the above-mentioned density calculation result 330.

According to at least one embodiment described above, the target nucleic acid sequence can be accurately quantified by using the LAMP method.

Although some embodiments have been described, these embodiments are presented as examples and are not intended to limit the scope of the invention. These novel embodiments can be implemented in various other embodiments, and various omissions, replacements, and changes can be made without departing from the gist of the invention. These embodiments and modifications thereof are included in the scope and gist of the invention, and are also included in the scope of the invention described in the claims and the equivalent scope thereof.

11 ... target nucleic acid sequence, 12 ... first amplification product, 13 ... second amplification product, 14 ... first substance, 15 ... first signal, 16 ... second substance, 17 ... second signal, 18 ... Double-stranded recognition substance, 19 ... Third signal

Claims (10)

It is a method of quantifying the target nucleic acid sequence by the LAMP method.
Amplification step of amplifying the target nucleic acid sequence,
Here, the target nucleic acid sequence is a double-stranded nucleic acid, and one strand has an F3c sequence, an F2c sequence, an F1c sequence, a B1 sequence, a B2 sequence, and a B3 sequence from the 3'end side to the 5'end side. Containing in this order, the other strand contains B3c sequence, B2c sequence, B1c sequence, F1 sequence, F2 sequence and F3 sequence from the 3'end side to the 5'end side.
The F1 sequence and the F1c sequence, the F2 sequence and the F2c sequence, the F3 sequence and the F3c sequence, the B1 sequence and the B1c sequence, the B2 sequence and the B2c sequence, and the B3 sequence and the B3c sequence complement each other. Being targeted
A detection step of detecting a first amplification product containing at least one of the F3 sequence, the B3 sequence, the F3c sequence, and the B3c sequence, and a determination step of determining the concentration of the target nucleic acid sequence from the result of the detection.
Nucleic acid quantification method including. The amplification step is performed using a primer set containing at least one of an F3 primer labeled with the first substance and a B3 primer labeled with the first substance.
The F3 primer comprises the F3 sequence and the B3 primer comprises the B3 sequence.
The first substance is a substance that emits a first signal when at least one of the F3 primer to which it is bound and the B3 primer binds to the target nucleic acid sequence.
The method of claim 1, wherein the detection step comprises measuring the first signal. The amplification step further comprises individually amplifying a plurality of standard target nucleic acid sequences having different concentrations.
The standard target nucleic acid sequence comprises the same sequence as the target nucleic acid sequence.
The detection step further comprises detecting an amplification product comprising at least one of the F3 sequence and the B3 sequence obtained from the standard target nucleic acid sequence.
The determination step is further performed using the detection results in the standard target nucleic acid sequence.
The method according to claim 1 or 2. The method according to any one of claims 1 to 3, wherein the detection step further comprises detecting a second amplification product that does not contain the F3 sequence and the B3 sequence. The amplification step is performed using a primer set containing at least one of a FIP primer labeled with the second substance and a BIP primer labeled with the second substance.
Here, the FIP primer contains the F1c sequence and the F2 sequence in this order from the 5'side to the 3'side, and the BIP primer has the B1c sequence from the 5'side to the 3'side. And the B2 sequence are included in this order.
The second substance is a substance that emits a second signal when at least one of the FIP primer and the BIP primer to which it binds binds to the target nucleic acid sequence.
The method of claim 4, wherein the detection step further comprises measuring the second signal. The method according to claim 4 or 5, wherein the first substance and the second substance are different from each other. The amplification step is further carried out using a double-stranded recognition substance.
The double-stranded recognition substance is a substance that binds to a double-stranded nucleic acid and emits a third signal.
The method according to any one of claims 1 to 3, wherein the detection step further comprises measuring the third signal. The method according to any one of claims 1 to 7, comprising a primer set comprising at least one of the F3 primer labeled with the first substance and the B3 primer labeled with the first substance. Reagent kit for use in. The reagent kit according to claim 8, wherein the primer set further comprises at least one of the FIP primer labeled with the second substance and the BIP primer labeled with the second substance. The reagent kit according to claim 8, wherein the primer set further contains the double-stranded recognition substance.

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