JP2021048785A - Internal control nucleic acid, internal control kit and nucleic acid detection method - Google Patents

Abstract

To provide an internal control nucleic acid, an internal control kit and a nucleic acid detection method that can be used for a sample in any kingdom of living things and can determine whether a target nucleic acid is positive or negative with improved accuracy.SOLUTION: An internal control nucleic acid according to an embodiment is an internal control nucleic acid used for the detection of a target nucleic acid by a nucleic acid amplification reaction, comprising a non-natural base pair.SELECTED DRAWING: Figure 1

Description

Embodiments of the present invention relate to internal control nucleic acids, internal control kits and nucleic acid detection methods.

In the detection of the target nucleic acid using the nucleic acid amplification method, if the detection result is negative, it is either true negative (that is, the target nucleic acid is not present in the sample) or false negative (that is, the target nucleic acid is present but the amplification reaction is somehow. It is important to determine whether it is (inhibited by a substance).

As a method for determining the truth of negativeness, there is a method in which an internal control nucleic acid having the same base sequence as the target nucleic acid is added to the sample for amplification and detection. According to this method, if the internal control nucleic acid is positive, the amplification reaction is not inhibited, so that the target nucleic acid can be determined to be truly negative. However, this method requires labor and cost because it is necessary to synthesize an internal control nucleic acid having the same sequence for each type of target nucleic acid.

Therefore, it is desired to develop a common internal control nucleic acid that does not depend on the type of target nucleic acid. As such a common internal control nucleic acid, it is necessary to select a base sequence that is considered not to be contained in the sample. It is possible that if the primer for the internal control nucleic acid binds to other nucleic acids in the sample, the amplification efficiency of the internal control nucleic acid will decrease or non-specific amplification products will be generated, and a correct negative judgment cannot be made. Because there is.

For example, there is a method of using a plant-derived photosynthetic gene as an internal control nucleic acid in an animal sample. According to this method, only the internal control nucleic acid can be specifically and efficiently amplified in the animal sample.

Japanese Patent No. 5590438

Rosenstraus M, et al. J Clin Microbiol. 1998; 36 (1): 191-197

However, plant-derived genes are often contaminated with animal samples such as livestock. Therefore, there is a high possibility that the primer designed for the internal control nucleic acid, which is a plant-derived photosynthetic gene, will also bind to the plant-derived gene mixed in the added sample. It is difficult to judge. Further, since this internal control nucleic acid cannot be used when the sample is derived from a plant, it is necessary to prepare the internal control nucleic acid for each biological world, which is troublesome and costly.

The present invention provides an internal control nucleic acid, an internal control kit, and a nucleic acid detection method that can be used in any sample of the biological world and can more accurately determine the positive and negative authenticity of a target nucleic acid. The purpose is to do.

The internal control nucleic acid according to the embodiment is an internal control nucleic acid for use in detecting a target nucleic acid by a nucleic acid amplification reaction, and includes unnatural base pairs.

FIG. 1 is a schematic diagram showing an internal control nucleic acid of the first embodiment and a first primer set. In this figure, only unnatural base pairs are shown in alphabets X or Y, and natural base pairs are omitted. FIG. 2 is a flowchart showing the nucleic acid detection method of the first embodiment. FIG. 3 is a flowchart showing the nucleic acid detection method of the first embodiment. FIG. 4 is a block diagram showing the nucleic acid detection device of the first embodiment. FIG. 5 is a flowchart showing the nucleic acid detection method of the second embodiment. FIG. 6 is a schematic diagram showing the internal control nucleic acid of the third embodiment and the first primer set. In this figure, only unnatural base pairs are shown in alphabets X or Y, and natural base pairs are omitted.

Hereinafter, embodiments of an internal control nucleic acid, an internal control kit containing the internal control nucleic acid, and a nucleic acid detection method using the internal control nucleic acid will be described.

(First Embodiment)
First, the internal control nucleic acid of the first embodiment and a primer set for amplifying the internal control nucleic acid (hereinafter, referred to as “first primer set”) will be described.

As shown in FIG. 1, the internal control nucleic acid 1 is double-stranded DNA. The internal control nucleic acid 1 includes a forward primer binding region 2a and a reverse primer binding region 2b (hereinafter, collectively referred to as “primer binding region 2”). For example, the region consisting of the forward primer binding region 2a and its complementary strand contains unnatural base pair 3 consisting of unnatural bases X and Y. The same applies to the reverse primer binding region 2b. In this figure, natural base pairs are omitted.

Non-natural bases are artificially produced nucleotides that have a different chemical structure than natural bases, namely A (adenine), T (thymine), G (guanine) and C (cytosine), but are non-natural. Bases X and Y can form base pairs with each other. In addition, unnatural base pair 3 can form a DNA sequence in the same manner as natural base pair.

式中、Ｒは、

である。
又は、非天然塩基対は以下に化学構造式を示すｄ５ＳＩＣＳ−ｄＮＡＭ塩基対であってもよい。

又は、非天然塩基対は以下に化学構造式を示すＰＩＣＳ−ＰＩＣＳ塩基対、５ＳＣＩＳ−ＮａＭ塩基対、５ＳＣＩＳ−ＭＭＯ２塩基対、ＴＰＴ３−ＮａＭ塩基対、イソグアニン−イソシトシン塩基対、又はＰ−Ｚ塩基対などであってもよい。

The unnatural base pair 3 is, for example, a Ds-Px base pair whose chemical structural formula is shown below.

In the formula, R is

Is.
Alternatively, the unnatural base pair may be d5SICS-dNAM base pair whose chemical structural formula is shown below.

Alternatively, the unnatural base pairs are PICS-PICS base pairs, 5SSIS-NaM base pairs, 5SSIS-MMO2 base pairs, TPT3-NaM base pairs, isoguanine-isocytosine base pairs, or PZ base pairs, which have the following chemical structural formulas. And so on.

However, the types of unnatural base pair 3 are not limited to those described above. Although omitted in the above chemical structural formula, deoxyribose is bonded to the tip of the wavy line.

In the internal control nucleic acid 1, natural base pairs (not shown) may be arranged between unnatural base pairs 3, and for example, unnatural base pairs 3 and natural base pairs may be arranged alternately. , Unnatural base pairs 3 may be arranged consecutively.

FIG. 1 shows an example in which 3 pairs of unnatural base pairs 3 are contained in one primer binding region 2, but the number of unnatural base pairs 3 is not limited to this. Alternatively, all of the primer binding region 2 may be composed of unnatural base pairs 3.

Further, FIG. 1 shows an example containing one type of unnatural base pair 3 composed of unnatural bases X and Y, but even if one internal control nucleic acid 1 contains a plurality of types of unnatural base pair 3. Good.

The ratio of the number of unnatural base pairs to 3 to the total number of bases of the internal control nucleic acid 1 is preferably 25% or more, for example. By containing unnatural base pair 3 in this ratio, the internal control nucleic acid 1 can be amplified more specifically.

Internal control nucleic acid 1 such as region 5a between forward primer binding region 2a and reverse primer binding region 2b, and each region 5b from the end of internal control nucleic acid 1 to forward primer binding region 2a or reverse primer binding region 2b. The base sequence of the region other than the primer binding region 2 of the above is not particularly limited. For example, region 5 does not have to contain unnatural base pair 3, but in that case, it is a sequence to which the primers contained in the primer set (second primer set) for amplifying the target nucleic acid described later do not bind. Is preferable. Alternatively, the entire sequence of region 5 may consist of unnatural base pairs.

When a substance that inhibits the amplification of the target nucleic acid is present in the sample, the sequence of the internal control nucleic acid 1 is preferably a sequence in which the amplification is inhibited by the inhibitor, for example.

The base length of the internal control nucleic acid 1 is not particularly limited, and may be, for example, about 80 bases to about 150 bases or more.

The base length of the region 5a is preferably adjusted to, for example, about 70 to about 300 bases. Further, the region 5b does not have to be. Within this range, the internal control nucleic acid can be amplified more efficiently.

The first primer set is a primer set for amplifying the entire length or a part of the base sequence of the internal control nucleic acid 1. For example, the first primer set includes a forward primer 4a and a reverse primer 4b as shown in FIG. The forward primer 4a has a sequence complementary to the forward primer binding region 2a, and the reverse primer 4b has a sequence complementary to the reverse primer binding region 2b. Therefore, the forward primer 4a and the reverse primer 4b each contain an unnatural base X and / or Y according to the base sequence of the corresponding primer binding region 2.

The following is an example of the base sequences of the forward primer 4a, the reverse primer 4b, and the internal control nucleic acid 1.

Forward primer 4a:
5'-ATGCATGCATGNANNCNGNC-3'(SEQ ID NO: 1)
Reverse primer 4b:
5'-GCATGATCANGNANCNTNC-3'(SEQ ID NO: 2)
5'end of internal control nucleic acid 1:
5'-ATGCATGCATGNANNCNGNC-3'(SEQ ID NO: 3)
3'end of internal control nucleic acid 1:
5'-GNNANGNTCNTGATCATGC-3'(SEQ ID NO: 4)
In the sequence, the unnatural base X or Y is arranged at "N".

Next, a nucleic acid detection method using the internal control nucleic acid 1 of the first embodiment and the first primer set will be described. The nucleic acid detection method is a method of detecting a target nucleic acid in a sample by using a polymerase chain reaction (PCR) method and simultaneously performing a negative authenticity determination.

As shown in FIG. 2, the nucleic acid detection method of the embodiment includes (S1) an amplification reagent, an internal control nucleic acid containing an unnatural base pair, a first primer set for amplifying the internal control nucleic acid, and a sample. A mixing step of mixing a second primer set for amplifying the target nucleic acid of the above with a sample, (S2) an amplification step of maintaining the obtained mixture under nucleic acid amplification conditions, and (S3) an amplification product of the target nucleic acid (target). The presence or absence of the target nucleic acid in the sample is determined from the detection step for detecting the amplification product) and the amplification product (control amplification product) of the internal control nucleic acid, and (S4) the detection result of the target amplification product and the detection result of the control amplification product. Judgment Judgment step is included.

An example of the nucleic acid detection method of the first embodiment will be described in detail below.

First, prepare a sample. The sample is an analysis target that may contain a target nucleic acid, for example, a body fluid obtained from a living body. Body fluids include, for example, blood, serum, blood cells, plasma, interstitial fluid, urine, stool, sweat, saliva, oral mucosa, sputum, lymph, cerebrospinal fluid, tears, breast milk, amniotic fluid, semen, cell extracts, Cerebrospinal fluid, or cleaning fluid generated during treatment or examination of the test object. Alternatively, the sample may be cells or tissues collected from a living body, cultured cells or microorganisms, or a culture supernatant thereof.

The living body is, for example, a primate such as a human or a monkey, a rodent such as a mouse, a rat or a guinea pig, a domestic animal such as a pig, a cow or a horse, or a mammal such as a companion animal such as a dog, a cat or a rabbit. .. Alternatively, the living body may be a bird, a reptile, an amphibian, a fish, or the like.

The sample may be a liquid containing a nucleic acid consisting of a bacterium, a fungus or a virus, or an artificially synthesized natural base. Alternatively, the sample may be a sample derived from the environment such as soil, river water, seawater or water and sewage.

As a method for collecting a sample, a general method selected according to the type of sample may be used. The collected sample may be subjected to a treatment such as homogenization, dilution or concentration, drug addition or extraction, if necessary. Further, the sample may be a sample that has been stored for a certain period of time by freezing or refrigerating and then subjected to a treatment such as thawing.

Next, the sample is mixed with the amplification reagent, the internal control nucleic acid, the first primer set and the second primer set (mixing step (S1)).

The amplification reagent may be any commonly used in the PCR method, and includes, for example, an amplification enzyme such as DNA polymerase and a substrate such as deoxynucleoside triphosphate (dNTP). Further, a salt, a thickener, a cushioning material for pH adjustment and / or a surfactant and the like may be contained.

The second primer set may be a general primer set used for amplifying the target nucleic acid by the PCR method, and includes at least a forward primer and a reverse primer. The base sequences of these primers are designed to amplify a specific base sequence contained in the target nucleic acid.

The target nucleic acid is DNA that is amplified and detected in the nucleic acid detection method of the embodiment. The length of the target nucleic acid is, for example, about 50 bases to about 500 bases, more preferably about 100 bases to about 300 bases.

In the mixing step (S1), an amplification reagent, an internal control nucleic acid, a first primer set and a second primer set contained separately or in the same solvent may be prepared and mixed with the sample. Alternatively, these may be freely fixed inside a flow path such as a DNA chip and released into the sample by pouring a liquid sample into the flow path.

The mixing amount of the internal control nucleic acid, the first primer set and the second primer set is selected according to the type and amount of the sample. The internal control nucleic acid, the first primer set and the second primer set are preferably added at a concentration of, for example, 0.2 to 1.0 μM.

Next, the obtained mixture (hereinafter, also referred to as “first mixture”) is maintained under nucleic acid amplification conditions (amplification step (S2)). In the amplification step, the temperature of the first mixture may be controlled so that the target nucleic acid and the internal control nucleic acid 1 are amplified by the PCR method. For example, the first mixture is set in a thermal cycler and the temperature cycle selected based on the type of target nucleic acid, the composition and composition of the amplification reagent, the type of internal control nucleic acid and / or the type of the second primer set, etc. Just set it up and run it.

When the amplification step (S2) is executed, if the target nucleic acid is present in the sample, the target nucleic acid is amplified by the components contained in the second primer set and the amplification reagent, and the target amplification product is obtained. Further, the internal control nucleic acid 1 is amplified by the components contained in the first primer set and the amplification reagent regardless of the presence or absence of the target nucleic acid, and a control amplification product is obtained.

Next, the target amplification product and the control amplification product are detected (detection step (S3)). The method used in the detection step is not particularly limited, and it is sufficient that the presence or absence of the target amplification product and the control amplification product can be detected separately from each other.

For example, the detection step (S3) is performed by agarose gel electrophoresis after the amplification step (S2) is completely completed. In this case, if the primers are designed in advance so that the lengths of the target amplification product and the control amplification product are different from each other, the target amplification product and the control amplification product can be detected separately according to the position of the band (migrating distance). Is. Alternatively, the first primer set and the second primer set are labeled with substances that emit different signals (for example, a fluorescent substance) in advance, and are incorporated into each amplification product in the amplification step (S2) to be incorporated into each amplification product in the detection step (S2). In S3), each signal may be detected separately. Alternatively, a probe that specifically binds to the target amplification product and a probe that specifically binds to the control amplification product labeled with a substance that emits different signals (for example, a fluorescent substance) (preferably contains an unnatural base). May be used to distinguish between the target amplification product and the control amplification product. Alternatively, the detection step (S3) may be performed at the same time as the amplification step (S2). In that case, a real-time PCR method, a method using a DNA chip, or the like can be used).

Next, the presence or absence of the target nucleic acid is determined from the result of the presence or absence of the target amplification product and the control amplification product obtained in the detection step (S3) (determination step (S4)).

Hereinafter, an example of the determination step (S4) will be described with reference to FIG. Here, the fact that the detection target is detected is referred to as "positive", and the fact that it is not detected or is below the detection limit is referred to as "negative". After executing the mixing step (S1), the amplification step (S2), and the detection step (S3), it is first determined whether or not the target amplification product is positive based on the result of the detection step (S3) (S41). When the target amplification product is positive (YES), it means that the target nucleic acid is present in the sample and amplified, so that it is determined to be positive for the target nucleic acid (S42). Then, the determination step (S4) is completed.

In (S41), when the target amplification product is negative (NO), it may be because the target nucleic acid is not actually present in the sample (true negative), or the target nucleic acid is present but the amplification reaction is inhibited. It is not clear whether it is due to the absence of the first amplification product (false negative). Therefore, the next step is to determine whether or not the control amplification product is positive (S43).

If the control amplification product is positive (YES), it means that the internal control nucleic acid 1 has been amplified, indicating that the amplification reaction has not been inhibited. Since the internal control nucleic acid 1 and the target nucleic acid are amplified by the same mechanism, if the target amplification product is negative under the condition that the amplification reaction is not inhibited, it is determined that the target nucleic acid does not actually exist in the sample. can do. Therefore, in this case, it is determined that the target nucleic acid is truly negative (S44). Then, the determination step (S4) is completed.

On the other hand, when the control amplification product is negative in (S43) (NO), it means that the internal control nucleic acid 1 always present in the mixture was not amplified, so it is determined that the amplification reaction is inhibited. Can be done (S45). Then, the determination step (S4) is completed. In this case, the target amplification product may be negative (false negative) despite the presence of the target nucleic acid.

When the determination step (S4) is completed in (S45), inhibition of the amplification reaction is performed, for example, by removing the inhibitory substance in the sample, changing the conditions of the amplification reaction, or changing the component or composition of the amplification reagent. Measures can be taken to prevent it. After that, the nucleic acid detection method of the embodiment may be executed again. Alternatively, since it is possible that the amount of the internal control nucleic acid 1 is small and below the detection limit, the internal control nucleic acid 1 and the first primer set are mixed in a larger amount in the mixing step (S1), and the subsequent steps are performed. You may go again.

According to the nucleic acid detection method described above, the internal control nucleic acid 1 and the first primer set contain an unnatural base. Since a sample from any biological world does not contain an unnatural base unless it is artificially contained, the primer contained in the first primer set in the sample is an internal control nucleic acid. Internal control nucleic acid 1 can be amplified very specifically and efficiently without binding to nucleic acids other than 1. Therefore, it is possible to accurately determine the negative authenticity of the target nucleic acid using any sample of the biological world.

In addition, the internal control nucleic acid 1 of one type of embodiment and the first primer set can be commonly used in samples of all biological worlds, and the base sequence thereof is designed according to the type of sample and the biological world from which it is derived. There is no need to change it, and it is not necessary to change it when the sample is changed. As a result, it is possible to greatly reduce the labor and cost for preparing the internal control nucleic acid 1 and the first primer set.

The internal control nucleic acid 1 and the first primer set may be provided as an internal control kit separately or contained in one solvent. As the solvent, for example, a buffer solution can be used. As the buffer solution, for example, a TE buffer solution (10 mM Tris-HCl (pH 8.0), 1 mM EDTA) or the like can be used.

The internal control kit may further include an amplification reagent and / or a second primer set. In addition, the internal control kit may further include a target amplification product, a fluorescently labeled probe for detecting the control amplification product, and the like.

The nucleic acid detection method described above may be performed by a nucleic acid detection device that automatically performs all the steps. As shown in FIG. 4, the nucleic acid detection device 10 includes, for example, a measurement unit 20, a storage unit 30, an information processing unit 40, an output unit 50, and a control unit 60.

The measuring unit 20 includes a liquid feeding device 21, a nucleic acid amplification unit 22, and a detecting device 25. The liquid feeding device 21 feeds liquids such as a sample, an amplification reagent, an internal control nucleic acid, a first primer set, and a second primer set to a sample storage section 23 included in the nucleic acid amplification section 22 (mixing step (S1). )). Alternatively, any of the above liquids may be manually mixed with the sample container 23. Next, the temperature of the sample accommodating unit 23 is adjusted by the temperature control device 24 included in the nucleic acid amplification unit 22, and the amplification step (S2) is performed. The target amplification product and the control amplification product are detected by the detection device 25 after the amplification step (S2) or during the amplification step (S2) (detection step (S3)). For example, the detection device 25 includes an optical detection element, an electrical detection element, and the like.

The storage unit 30 is, for example, a program 31 for executing the determination step (S4) shown in FIG. 3, a detection result 32 obtained by the detection device 25, and a determination step (S4) obtained by the information processing unit 40 described later. ) Judgment result 33 and the like are stored. The storage unit 30 may further store a program, parameters, and the like for executing control of each unit by the control unit 90.

The information processing unit 40 takes out the detection result 32 stored in the storage unit 30 and performs the determination step (S4) according to the program 31. The determination result 33 is stored in the storage unit 30.

The output unit 50 includes, for example, a display or a printer, and outputs a determination result 33.

The control unit 60 controls the behaviors of the measurement unit 20, the storage unit 30, the information processing unit 40, and the output unit 50.

The storage unit 30, the information processing unit 40, the output unit 50, and the control unit 60 may be a computer including each of these units as a unit. All the steps of the nucleic acid detection method may be performed automatically using the nucleic acid detection device 10 as described above, or any of the steps may be performed manually if necessary.

According to the above nucleic acid detection device 10, it is possible to accurately determine whether the target nucleic acid is negative or false.

(Second embodiment)
According to the nucleic acid detection method of the second embodiment, the determination step (S4) further includes a positive authenticity determination for the target nucleic acid. The determination step (S4) of the second embodiment will be described with reference to FIG.

In the second embodiment, the amplification reagent, the internal control nucleic acid, the first primer set and the second primer set can be the same as those in the first embodiment. Further, the mixing step (S1) to the detection step (S3) can be performed in the same manner as in the first embodiment.

In the determination step (S40) of the second embodiment, whether or not the control amplification product is positive even when the target amplification product is positive (YES) in the determination of whether or not the target amplification product is positive (S41). (S46). If the control amplification product is positive (YES), it means that the target nucleic acid is present and amplified in the sample, and the internal control nucleic acid 1 is also amplified, so that the amplification reaction is occurring normally. Shown. Therefore, it is determined that the target nucleic acid is truly positive (S47). Then, the determination step (S4) is completed.

If the control amplification product is not detected (negative) in (S47), it indicates that the amplification reaction may not have occurred normally (NO). Therefore, it is determined that the amplification reaction is not normally performed (S48). Then, the determination step (S4) is completed.

When the determination step (S4) is completed in (S48), some measures are taken so that the amplification reaction can be performed more normally, for example, by changing the conditions of the amplification reaction or changing the components or compositions of the amplification reagent. Can be done. After that, the nucleic acid detection method of the embodiment may be executed again.

In (S41), when the target amplification product is negative (NO), (S43) to (S45) may be performed in the same manner as shown in FIG.

According to the nucleic acid detection method described above, since the internal control nucleic acid 1 containing an unnatural base and the first primer set are used, the authenticity of the positive can be accurately determined.

The nucleic acid detection method of the second embodiment can also be performed using the same nucleic acid detection device as that described in the second embodiment including the program for performing such a determination step (S40).

(Third Embodiment)
In the nucleic acid detection method of the third embodiment, the amplification step (S2) is performed using the LAMP method.

As shown in FIG. 6, the internal control nucleic acid 1 of the third embodiment contains, for example, six primer binding regions 2, that is, F1, F2 region, F3 region, B1 region, B2 region and B3 region, and each region. Has a region complementary to (a region with a "c" at the end).

An unnatural base pair 3 is arranged in the region where these primer binding regions 2 are provided. However, of the six primer binding regions 2, there may be regions that do not contain unnatural base pair 3, and at least one of the F3 region, F2 region, and F1 region, and the B3 region, B2 region, and B1 region. It is preferable that unnatural base pair 3 is arranged in at least one region.

The first primer set comprises a FIP primer, a BIP primer, an F3 primer and a B3 primer, each containing an unnatural base X and / or Y corresponding to the sequence of the primer binding region 2. The first primer set may further include LP primers, i.e., LF and / or LB primers, etc. (not shown).

The second primer set may be any common primer used in the LAMP method designed to amplify the target nucleic acid. For example, it may contain at least a FIP primer and a BIP primer, and may further contain an F3 primer, a B3 primer, an LP primer, that is, an LF primer and / or an LB primer and the like.

The amplification reagent may be any one generally used in the LAMP method, and includes, for example, an amplification enzyme such as DNA polymerase and a substrate such as deoxynucleoside triphosphate (dNTP). It may further contain salts, thickeners, pH adjusting cushions, surfactants and the like.

The nucleic acid detection method in the third embodiment will be described.

The type of sample and the method of collecting the sample may be the same as those in the first embodiment. In the mixing step (S1), the amplification reagent, the internal control nucleic acid 1, the first primer set and the second primer set of the third embodiment are mixed with the sample. The mixing method may be the same as in the first embodiment.

In the amplification step (S2), the temperature of the first mixture obtained in the mixing step (S1) is controlled so that the target nucleic acid and the internal control nucleic acid 1 are amplified by the LAMP method under isothermal amplification conditions. Good. For example, the first mixture is placed in an incubator set to a temperature selected based on the type of target nucleic acid, the composition and composition of the amplification reagent, the type of internal control nucleic acid and / or the type of the second primer set, etc. do it.

In the detection step (S3), a method capable of distinguishing between the target amplification product and the control amplification product produced by the LAMP method is used. For example, the first primer set and the second primer set are labeled with substances that emit different signals (for example, a fluorescent substance) in advance, and are incorporated into each amplification product in the amplification step (S2) to be incorporated into each amplification product in the detection step (S2). In S3), each signal may be detected separately. Alternatively, two types of probes that specifically bind to the target amplification product and the control amplification product may be used.

The determination step (S4) can be performed, for example, in the same manner as the method shown in the first embodiment (FIG. 3) or the second embodiment (FIG. 5).

According to the third embodiment described above, it is possible to more accurately determine the positive and negative authenticity of the target nucleic acid in any sample of the biological world.

In the first to third embodiments, an example is shown in which amplification and detection of the target nucleic acid and amplification and detection of the internal control nucleic acid 1 are performed in one mixture. However, in a further embodiment, the same sample may be added to separate containers, with target nucleic acid amplification and detection on the one hand and internal control nucleic acid mixing, amplification and detection on the other. Alternatively, the amplification step (S2) is performed in one container, the obtained mixture is distributed into two containers, the target amplification product is detected in one mixture, and the control amplification product is detected in the other mixture. You may go.

In this case, in the detection step (S3), the target amplification product and the control amplification product are selected according to i) presence / absence of cloudiness due to magnesium pyrophosphate, ii) presence / absence of fluorescence due to calcein, ii) presence / absence of fluorescence by a fluorescence intercalator such as EtBr, and the like. It may be detected.

The amplification method is not limited to the PCR method and the LAMP method, and other amplification methods accompanied by temperature changes or isothermal amplification methods may be used. For example, RT-PCR (Reverse Translation-PCR) method, RT-LAMP (Reverse Translation-LAMP) method, TMA method, NASBA method, 3SR method, RCA method, LCR method, SDA method, SmartAmp (registered trademark) or ICAN (registered trademark). The registered trademark) method, etc. can be used. In that case, the number and position of the region where the unnatural base pair 3 is provided, for example, the primer binding region, is determined according to the type of amplification method used and the type of primer set.

If the target is RNA, the second primer set may include reverse transcription primers and / or primer sets for extending RNA and the like. The amplification reagent may include reverse transcriptase. In that case, first, reverse transcription and / or elongation of the target RNA may be performed, and then the nucleic acid detection method of FIG. 2 may be performed using the obtained product as the target nucleic acid.

Further, the first primer set is specifically bound to the internal control nucleic acid 1 and replaced with a nucleic acid probe containing an unnatural base, and a signal amplification method such as an RCA method, an INVADER method, a CPT method or a PALSTER method is used. Is also possible.

1 ... Internal control nucleic acid 2 ... Primer binding region 2a ... Forward primer binding region 2b ... Reverse primer binding region 3 ... Unnatural base pair 4a ... Forward primer 4b ... Reverse primer

Claims (10)

An internal control nucleic acid for use in detecting a target nucleic acid by a nucleic acid amplification reaction.
Internal control nucleic acid containing unnatural base pairs. The internal control nucleic acid according to claim 1, wherein the internal control nucleic acid contains a primer-binding region, and the unnatural base pair is contained at least in the primer-binding region. The unnatural base pairs are Ds and Px base pairs, d5SICS and dNAM base pairs, PICS and PICS base pairs, 5SSIS and NaM base pairs, 5SSIS and MMO2 base pairs, TPT3 and NaM base pairs, isoguanine and isocitosine base pairs, or The internal control nucleic acid according to claim 1 or 2, which is a P and Z base pair. The internal control nucleic acid according to any one of claims 1 to 3 and
Used for detection of a target nucleic acid by a nucleic acid amplification reaction, which comprises a primer set containing an unnatural base for amplifying the internal control nucleic acid and / or a nucleic acid probe containing an unnatural base that specifically binds to the internal control nucleic acid. Internal control kit for. The kit according to claim 4, further comprising a primer set for amplifying a target nucleic acid and an amplification reagent. An amplification reagent, an internal control nucleic acid containing unnatural base pairs, a first primer set containing an unnatural base for amplifying the internal control nucleic acid, and a second primer for amplifying a target nucleic acid in a sample. Mixing step of mixing the set with the sample,
An amplification step of maintaining the mixture obtained in the mixing step under nucleic acid amplification conditions,
From the detection step of detecting the amplification product (target amplification product) of the target nucleic acid and the amplification product (control amplification product) of the internal control nucleic acid, the detection result of the target amplification product, and the detection result of the control amplification product. A nucleic acid detection method comprising a determination step of determining the presence or absence of the target nucleic acid in the sample. The determination step according to claim 6, wherein the determination step includes determining that the target nucleic acid is truly negative when the target amplification product is negative and the control amplification product is negative in the detection step. Method. The determination step is
i) When the target amplification product is positive and the control amplification product is positive in the detection step, it is determined that the target nucleic acid is truly positive in the determination step.
ii) When the target amplification product is positive and the control amplification product is negative in the detection step, it is determined that the amplification reaction is not normally performed in the determination step.
iii) If the target amplification product is negative in the detection step and the control amplification product is negative, it is determined in the determination step that the target nucleic acid is truly negative, or iv) the detection step. The method according to claim 6, wherein when the target amplification product is negative and the control amplification product is positive, it is determined that the amplification reaction is inhibited in the determination step. The method according to any one of claims 6 to 8, wherein the internal control nucleic acid contains a primer-binding region, and the unnatural base pair is contained at least in the primer-binding region. The unnatural base pairs are Ds and Px base pairs, d5SICS and dNAM base pairs, PICS and PICS base pairs, 5SSIS and Nam base pairs, 5SSIS and MMO2 base pairs, TPT3 and NaM base pairs, isoguanine and isocytosine base pairs, or The method according to any one of claims 6 to 9, which is a P and Z base pair.

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