Internal control nucleic acid, method for designing the nucleic acid, and kit containing the nucleic acid

Abstract

The object of the present invention is to provide an internal control nucleic acid that can be used with any biological sample and can accurately determine the positive and false negative status of a target nucleic acid, a method for designing the nucleic acid, and a kit containing the nucleic acid. [Solution] The present invention relates to an internal control nucleic acid having a unique base sequence not found in nature, a method for designing the nucleic acid, and a kit containing the nucleic acid.

Description

TECHNICAL FIELD

[0001] The present invention relates to an internal control nucleic acid used for determining whether an amplification reaction correctly proceeds in the amplification reaction of a target gene nucleic acid during genetic testing, a method for designing the nucleic acid, and a kit comprising the nucleic acid. BACKGROUND ART

[0002] In detection of a target nucleic acid using a nucleic acid amplification method, when a detection result is negative, it is important to determine whether the result is a true negative (that is, the target nucleic acid is not present in a sample) or a false negative (that is, the target nucleic acid is present but not detected for some reason).

[0003] Reasons for false negative results include sample degradation, failure in the step of extracting nucleic acid from a sample, inclusion of inhibitory substances in the extracted nucleic acid, degradation of the extracted nucleic acid, and failure of nucleic acid amplification reagents or devices. As a method for determining false negatives caused by inclusion of inhibitory substances in extracted nucleic acid, there is a method in which an internal control nucleic acid having a nucleotide sequence that does not react with primers and probes used for detection of a test object is added to a sample, and the internal control nucleic acid is amplified and detected simultaneously with the target nucleic acid. According to this method, when no internal control nucleic acid is detected, it can be determined as a false negative result caused by inhibition of the amplification reaction. However, this method has a problem that it requires labor and cost because it is necessary to design, synthesize, and confirm reactivity of an internal control nucleic acid for each type of target nucleic acid. Accordingly, development of a common internal control nucleic acid independent of the type of target nucleic acid and a method for designing the same is desired.

[0004] Patent Document 1 discloses an internal control nucleic acid molecule containing at least one forward primer binding site, at least one reverse primer binding site, and at least one amplifiable region, all randomly generated, and also discloses a method for designing it. However, while the internal control nucleic acid described in the document is edited to a desired length by concatenating 10 random sequences of approximately 50 base pairs, no specific selection criteria such as E-value for the design method, or specific criteria for acceptable sequence homology between the obtained sequence and known sequences, are disclosed. Therefore, there remains a need to develop a method for designing sequences that have sufficiently low homology to known sequences and are independent of the type of target nucleic acid, in a simple and highly reproducible manner. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] Patent No. 4805158 [Overview of the project] [Problems that the invention aims to solve]

[0006] The present invention aims to provide an internal control nucleic acid that can be used with any biological sample and can accurately determine the positive and false negative results of a target nucleic acid, a method for designing the nucleic acid, and a kit containing the nucleic acid. [Means for solving the problem]

[0007] The inventors recognized that a common internal control nucleic acid needs to have a base sequence that is not thought to be present in the sample. They discovered that the above problem could be solved by designing an internal control nucleic acid with a unique base sequence that does not exist in nature using E-value, and that by using an internal control nucleic acid with a unique base sequence that does not exist in nature in a nucleic acid amplification method for detecting the target nucleic acid, it is possible to easily determine whether it is a false negative. After repeated trial and error, they completed the present invention.

[0008] This invention relates to the following invention. [1] An internal control nucleic acid containing a sequence that is at least 90% identical to a sequence of 50 or more consecutive bases selected from the base sequences shown in Sequence ID No. 1. [2] The nucleic acid according to [1], wherein the internal control nucleic acid comprises a sequence of 50 or more consecutive bases selected from the base sequences shown in Sequence ID No. 1. [3] The nucleic acid according to [1], wherein the internal control nucleic acid consists of a sequence of 50 or more consecutive bases selected from the base sequences shown in Sequence ID No. 1. [4] The nucleic acid according to [1], wherein the internal control nucleic acid consists of the base sequence shown in Sequence ID No. 1. [5] [1] to [4] any of the internal control nucleic acids listed below; Forward primers and reverse primers for amplifying part or all of the nucleic acid; and, A composition containing a sample for the detection and / or quantification of nucleic acids. [6] The composition according to [5], further comprising an oligonucleotide probe complementary to the internal control nucleic acid in the region sandwiched between the forward primer and the reverse primer. [7] The composition according to [5], wherein the oligonucleotide of the forward primer is identical to the base sequence shown in SEQ ID NO: 2 by at least 95%. [8] The composition according to [5], wherein the oligonucleotide of the reverse primer is identical to the base sequence shown in SEQ ID NO: 3 by at least 95%. [9] The composition according to [3], wherein the oligonucleotide probe is identical to the base sequence shown in SEQ ID NO: 4 by at least 95%.

[10] A method for detecting and / or quantifying nucleic acids in a sample, a) A step of mixing an internal control nucleic acid containing at least 90% of a sequence identical to 50 or more consecutive bases selected from the base sequences shown in Sequence ID No. 1 with a forward primer, a reverse primer, and a sample for amplifying part or all of the nucleic acid. b) extending the forward primer and reverse primer to generate at least one target amplicon, and c) A step of detecting and / or quantifying the amplification of the target amplicon by electrophoresis. The method, including the method described above.

[11] A method for detecting and / or quantifying nucleic acids in a sample, a) A step of mixing an internal control nucleic acid containing at least 50 consecutive bases selected from the base sequences shown in Sequence ID No. 1 and at least 90% of the same sequence with a forward primer, reverse primer, oligonucleotide probe, and sample for amplifying part or all of the nucleic acid, b) extending the forward primer and reverse primer to generate at least one target amplicon, c) The step of binding the oligonucleotide probe to the at least one internal control nucleic acid or target amplicon, and d) detecting a signal proportional to the amount of said at least one internal control nucleic acid or target amplicon, The method as described above, comprising the above steps.

[12] The method according to

[11] , wherein the internal control consists of a contiguous sequence of 50 or more bases selected from the base sequence set forth in SEQ ID NO: 1.

[13] The method according to

[12] , wherein the internal control comprises a sequence consisting of the base sequence set forth in SEQ ID NO: 1.

[14] The method according to

[10] or

[11] , wherein the oligonucleotide of the forward primer is at least 95% identical to the base sequence set forth in SEQ ID NO: 2.

[15] The method according to

[10] or

[11] , wherein the oligonucleotide of the reverse primer is at least 95% identical to the base sequence set forth in SEQ ID NO: 3.

[16] The method according to

[10] or

[11] , wherein the oligonucleotide of the forward primer is the base sequence set forth in SEQ ID NO: 2, and the oligonucleotide of the reverse primer is the base sequence set forth in SEQ ID NO: 3.

[17] The method according to

[10] or

[11] , wherein the internal control nucleic acid comprises a contiguous sequence of 50 or more bases selected from the base sequence set forth in SEQ ID NO: 1.

[18] The method according to

[10] or

[11] , wherein the internal control nucleic acid consists of a contiguous sequence of 50 or more bases selected from the base sequence set forth in SEQ ID NO: 1.

[19] The method according to

[10] or

[11] , wherein the oligonucleotide probe is the base sequence set forth in SEQ ID NO: 4.

[20] The method according to

[10] or

[11] , further comprising a step of extracting nucleic acid from the sample before or after step a).

[21] A method for designing an internal control nucleic acid in the detection and / or quantification of nucleic acids, comprising: a) generating a random nucleotide sequence with a length of 500 to 1500 bases, and b) comparing said random nucleotide sequence with a database to select a sequence with low homology, wherein the comparison with said database is performed using BLAST, and the E-value of said BLAST selection condition is E-value > 30, the method comprising the above steps.

[22] The method according to

[21] , further comprising the step of splicing regions with high E-value from one nucleotide sequence selected in step b), or The method according to

[21] , further comprising the step of splicing regions with high E-value from two or more nucleotide sequences selected in step b).

[23] The method according to

[21] , further comprising the step of removing nucleotide sequences having five or more consecutive identical bases from the nucleotide sequence selected in step b).

[24] The method according to

[21] , further comprising the step of adjusting the GC content of the nucleotide sequence selected in step b) to 40% to 60%.

[25] The method according to any one of

[21] to

[24] , wherein the designed internal control nucleic acid has an E-value of 300 or more as determined by BLAST, and has a length of 500 to 1500 bases.

[26] A method for designing an internal control nucleic acid in the detection and / or quantification of nucleic acids, comprising: a) generating a random nucleotide sequence with a length of 500 to 1500 bases, b) selecting a sequence with low homology from said random nucleotide sequence by comparison with a database, wherein the comparison with said database is performed using BLAST, and the E-value of said BLAST selection condition is E-value > 30, Steps to remove sequences from the base sequences selected in c) and b) that contain five or more consecutive identical bases. d) In the base sequence selected in b), the step of adjusting the GC content to 40-60%, and / or e) Further includes the step of selecting a region with a high E-value from one base sequence selected in b) and joining the selected regions, or the step of selecting regions with a high E-value from two or more base sequences selected in b) and joining the selected regions. Includes, The method wherein the designed inner control nucleic acid has an E-value of 300 or more according to BLAST, and a length of 500 to 1500 bases. [Effects of the Invention]

[0009] Because the internal control nucleic acid of the present invention has low homology to known nucleic acid sequences, it can be used as an internal control in nucleic acid amplification methods for detecting various genes regardless of the sample type (animal, plant, microorganism, virus). Furthermore, while it was previously necessary to prepare internal control nucleic acids and primers / probes for detecting internal control nucleic acids for each sample type and target gene, the present invention allows for the commonization of internal control nucleic acids and primers / probes for detecting internal control nucleic acids, thereby reducing the development speed and cost of gene testing kits. The internal control nucleic acid of the present invention can be used to distinguish between true negatives and false negatives, and to confirm that the nucleic acid amplification reaction proceeded without problems. [Brief explanation of the drawing]

[0010] [Figure 1]Figure 1 shows the results of real-time PCR detection of a PCR reaction mixture prepared using DNA from Pseudomonas aeruginosa (containing the blaVIM gene), the internal control nucleic acid of SEQ ID NO: 1, the forward primer of SEQ ID NO: 2, the reverse primer of SEQ ID NO: 3, the oligonucleotide probe of SEQ ID NO: 4, the forward primer of SEQ ID NO: 5, the reverse primer of SEQ ID NO: 6, and the oligonucleotide probe of SEQ ID NO: 7 as the sample. [Figure 2] Figure 2 shows the results of real-time PCR detection of a PCR reaction mixture prepared using DNA from Escherichia coli (without the blaVIM gene), the internal control nucleic acid of SEQ ID NO: 1, the forward primer of SEQ ID NO: 2, the reverse primer of SEQ ID NO: 3, the oligonucleotide probe of SEQ ID NO: 4, the forward primer of SEQ ID NO: 5, the reverse primer of SEQ ID NO: 6, and the oligonucleotide probe of SEQ ID NO: 7 as the sample.

[0011] [Figure 3] Figure 3 shows the results of real-time PCR detection of a PCR reaction mixture prepared with TE buffer, internal control nucleic acid of SEQ ID NO: 1, forward primer of SEQ ID NO: 2, reverse primer of SEQ ID NO: 3, oligonucleotide probe of SEQ ID NO: 4, forward primer of SEQ ID NO: 5, reverse primer of SEQ ID NO: 6, and oligonucleotide probe of SEQ ID NO: 7 as the sample. [Figure 4] Figure 4 shows the results of a PCR reaction prepared using DNA from Pseudomonas aeruginosa (containing the blaVIM gene), the internal control nucleic acid of SEQ ID NO: 1, the forward primer of SEQ ID NO: 2, the reverse primer of SEQ ID NO: 3, the oligonucleotide probe of SEQ ID NO: 4, the forward primer of SEQ ID NO: 5, the reverse primer of SEQ ID NO: 6, and the oligonucleotide probe of SEQ ID NO: 7. After real-time PCR, the sample was subjected to capillary electrophoresis, and the graph shows the results with fluorescence intensity on the vertical axis and size (bp: base pairs) on the horizontal axis.

[0012] [Figure 5] Figure 5 shows the results of a PCR reaction prepared using DNA from Escherichia coli (without the blaVIM gene), an internal control nucleic acid of SEQ ID NO: 1, a forward primer of SEQ ID NO: 2, a reverse primer of SEQ ID NO: 3, an oligonucleotide probe of SEQ ID NO: 4, a forward primer of SEQ ID NO: 5, a reverse primer of SEQ ID NO: 6, and an oligonucleotide probe of SEQ ID NO: 7. After real-time PCR, the sample was subjected to capillary electrophoresis, and the graph shows the results with fluorescence intensity on the vertical axis and size (bp) on the horizontal axis. [Figure 6] Figure 6 shows the results of a PCR reaction prepared using TE buffer, internal control nucleic acid of SEQ ID NO: 1, forward primer of SEQ ID NO: 2, reverse primer of SEQ ID NO: 3, oligonucleotide probe of SEQ ID NO: 4, forward primer of SEQ ID NO: 5, reverse primer of SEQ ID NO: 6, and oligonucleotide probe of SEQ ID NO: 7 as the sample. After real-time PCR, the sample was subjected to capillary electrophoresis, and the graph shows the results with fluorescence intensity on the vertical axis and size (bp) on the horizontal axis.

[0013] [Figure 7] Figure 7 shows the results of real-time PCR detection of a PCR reaction mixture prepared using DNA from mouse hybridoma cells (containing the β-globin gene), the internal control nucleic acid of SEQ ID NO: 1, the forward primer of SEQ ID NO: 2, the reverse primer of SEQ ID NO: 3, the oligonucleotide probe of SEQ ID NO: 4, the forward primer of SEQ ID NO: 8, the reverse primer of SEQ ID NO: 9, and the oligonucleotide probe of SEQ ID NO: 10 as the sample. [Figure 8]Figure 8 shows the results of real-time PCR detection of a PCR solution prepared using TE buffer, internal control nucleic acid of SEQ ID NO: 1, forward primer of SEQ ID NO: 2, reverse primer of SEQ ID NO: 3, oligonucleotide probe of SEQ ID NO: 4, forward primer of SEQ ID NO: 8, reverse primer of SEQ ID NO: 9, and oligonucleotide probe of SEQ ID NO: 10 as the sample.

[0014] [Figure 9] Figure 9 shows the results of a PCR reaction prepared using a sample containing DNA from mouse hybridoma cells (containing the β-globin gene), the internal control nucleic acid of SEQ ID NO: 1, the forward primer of SEQ ID NO: 2, the reverse primer of SEQ ID NO: 3, the oligonucleotide probe of SEQ ID NO: 4, the forward primer of SEQ ID NO: 8, the reverse primer of SEQ ID NO: 9, and the oligonucleotide probe of SEQ ID NO: 10. After real-time PCR, the sample was subjected to capillary electrophoresis, and the graph shows the results with fluorescence intensity on the vertical axis and size (bp) on the horizontal axis. [Figure 10] Figure 10 shows the results of a PCR reaction prepared using TE buffer, internal control nucleic acid of SEQ ID NO: 1, forward primer of SEQ ID NO: 2, reverse primer of SEQ ID NO: 3, oligonucleotide probe of SEQ ID NO: 4, forward primer of SEQ ID NO: 8, reverse primer of SEQ ID NO: 9, and oligonucleotide probe of SEQ ID NO: 10. After real-time PCR, the sample was subjected to capillary electrophoresis, and the graph shows the results with fluorescence intensity on the vertical axis and size (bp) on the horizontal axis.

[0015] [Figure 11]Figure 11 shows the results of a real-time RT-PCR detection of an RT-PCR reaction mixture prepared containing the RS virus-positive control nucleic acid (SEQ ID NO: 11), the internal control nucleic acid (SEQ ID NO: 1), the forward primer (SEQ ID NO: 2), the reverse primer (SEQ ID NO: 3), the oligonucleotide probe (SEQ ID NO: 4), the forward primer (SEQ ID NO: 12), the reverse primer (SEQ ID NO: 13), and the oligonucleotide probe (SEQ ID NO: 14) as samples. [Figure 12] Figure 12 shows the results of detecting an RT-PCR solution prepared using real-time RT-PCR, containing TE buffer, internal control nucleic acid of SEQ ID NO: 1, forward primer of SEQ ID NO: 2, reverse primer of SEQ ID NO: 3, oligonucleotide probe of SEQ ID NO: 4, forward primer of SEQ ID NO: 12, reverse primer of SEQ ID NO: 13, and oligonucleotide probe of SEQ ID NO: 14 as the sample.

[0016] [Figure 13] Figure 13 shows the results of an RT-PCR reaction mixture prepared containing the RS virus-positive control nucleic acid (SEQ ID NO: 11), the internal control nucleic acid (SEQ ID NO: 1), the forward primer (SEQ ID NO: 2), the reverse primer (SEQ ID NO: 3), the oligonucleotide probe (SEQ ID NO: 4), the forward primer (SEQ ID NO: 12), the reverse primer (SEQ ID NO: 13), and the oligonucleotide probe (SEQ ID NO: 14). After real-time RT-PCR, the sample was subjected to capillary electrophoresis, and the graph shows the results with fluorescence intensity on the vertical axis and size (bp) on the horizontal axis. [Figure 14]Figure 14 shows the results of an RT-PCR reaction mixture prepared containing TE buffer, internal control nucleic acid of SEQ ID NO: 1, forward primer of SEQ ID NO: 2, reverse primer of SEQ ID NO: 3, oligonucleotide probe of SEQ ID NO: 4, forward primer of SEQ ID NO: 12, reverse primer of SEQ ID NO: 13, and oligonucleotide probe of SEQ ID NO: 14. After real-time RT-PCR, the sample was subjected to capillary electrophoresis, and the graph shows the results with fluorescence intensity on the vertical axis and size (bp) on the horizontal axis. [Modes for carrying out the invention]

[0017] The internal control nucleic acid of the present invention is used to accurately determine the positive and false negative results of a target nucleic acid in the amplification reaction of a target gene in a sample.

[0018] The sample is not particularly limited and may be any biological sample. For example, the sample may be derived from animals, plants, microorganisms, viruses, etc.

[0019] [Internal control nucleic acid] One aspect of the present invention relates to providing an internal control nucleic acid having a unique base sequence not found in nature, which is artificially constructed to have low homology to known nucleic acid sequences, and includes at least one forward primer binding site, at least one reverse primer binding site, and at least one amplified region. The internal control nucleic acid of the present invention may also include a probe binding site.

[0020] In one aspect of the present invention, an internal control nucleic acid is provided which comprises or comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to a sequence of 50 to 1000 consecutive bases selected from the base sequence shown in Sequence ID No. 1. In one embodiment, the internal control nucleic acid contains or comprises a sequence of 50 or more consecutive bases, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 150 or more, 200 or more, 250 or more, 300 or more, 350 or more, 400 or more, 450 or more, 500 or more, 550 or more, 600 or more, 650 or more, 700 or more, 750 or more, 800 or more, 850 or more, 900 or more, 950 or more, or more, selected from the base sequence shown in Sequence ID No. 1. In a preferred embodiment, the internal control nucleic acid consists of the base sequence shown in Sequence ID No. 1.

[0021] The internal control nucleic acid in this invention is not limited to the following sequences (E-value: 348 (as of November 7, 2024)): GCACATGTGC TGACCCAGAT TAGACGTGAG ATGAAGGTCT TGTAAACCAG GTCAAGTACT 60 CTTGAAGTGA CACGATCCAC GATGTCCACC GTTGGGACGT GAGTTCCAAC GGTGTAAGGT 120 GAAGATCCGT GACTTCTCAT GGAAGCACGA TGGACTTGCA CATATCTATT CTGACACGGA 180 GTCTAGAGGA GCTAGTCTCT TAGGTGCGAC CAGGATTAGT CCCAACATTA CGGGAGTCGT 240 CAATTAGACG TCTATCCATG GCAGAATCCT AATGGGTACC GAGATGGAGT CGAACCTCTC 300 TAGTCCTAAG CTCGAGAGCG ATGGTTCTAT CGGATTCTGC TACCTATGAT CGTAAGGACT 360 TAATACGCTG GGGTCAACTA GTAATGTGCC AGTGGCATAG TTTCATGCAG ATACTGCTCC 420 GCTAAGTGGC TCAAGGGTCA GTTGATATCT TGCACCGTTC AGCCATCTCC CTAGCATGCA 480 CACAAAGTTC TCTTATGTAC GGACTTCGCC ACGAACTAAC AATCTGGTGT AGCATCATCC 540 ACTGACGCGA ATGCATTCCG CTATCATCAC CATATAGTGG TGCTTGTATG AGGTCAAGTC 600 CCTGATTAAT CCGCATAAGC AATCCTTTGG GCTACACCCA GTTAGATTAG CGATCAGGGA 660 TGTATTCATT CATGAGTAGG CTAGTGGCTG GAACTTCTAT GCCCATCTAC GTCCTCTACA 720 AGACAGCTCG TCATAAGTTG AGTATACGGA CCGGACCAAG ATTGGATTGC TCACTGGTCG 780 CGATGTGCAA TCTCTACCGA GTTTCTCGTA CCGAAGGTAC GATCTTCCCA TAGACGCATA 840 CATCGAGGAT CGAACTGCCT TAAATGACGG AACGCCATTA CTAGGCTCAT CGACGCGCTA 900 CGGTATGTTC AACTCAGGAG ACCTTCTCTA CCCATCGTTA ACGTGCTGAA CTAATCAGGT 960 TCGACAATCT CGATTGATAC ACGGATCGGA ATTGGCTTAG 1000 [SEQ ID NO: 1]

[0022] [Method for designing internal control nucleic acids] Another aspect of the present invention relates to providing a method for designing internal control nucleic acids.

[0023] One aspect of the present invention provides a method for designing an internal control nucleic acid in the detection and / or quantification of nucleic acids. Such a design method is typically, below: a) A step of creating a random base sequence with a length of 500 to 1500 bases, where step a) may be performed using computer software. b) A step of selecting sequences with low homology from the random base sequences by comparing them with a database, wherein the comparison with the database is performed using BLAST, and the selection criterion for BLAST is an E-value of 30 or higher. Here, the length of the random base sequence in a) may be 500 to 1500 bases, preferably 600 to 1400 bases, 700 to 1300 bases, 800 to 1200 bases, or more preferably 900 to 1100 bases. The E-value of the BLAST selection criterion in b) may be 0.1 or higher, 1 or higher, 2 or higher, 3 or higher, 4 or higher, 5 or higher, 6 or higher, 7 or higher, 8 or higher, 9 or higher, 10 or higher, 20 or higher, 30 or higher, 40 or higher, 50 or higher, 100 or higher, or higher. The E-value of the selection criterion may be 20 or lower, 30 or lower, 40 or lower, 50 or lower, 100 or lower, 200 or lower, 300 or lower, 400 or lower, 500 or lower, 600 or lower, 700 or lower, 800 or lower, 900 or lower, or 1000 or lower. The above upper and lower limits can be appropriately selected and used within that range as the selection criterion.

[0024] The internal control nucleic acid in this invention can be artificially designed.

[0025] "Artificially" means designing a sequence randomly while taking specific design characteristics into consideration. The internal control nucleic acid can be determined by any method for determining a random base sequence of the four nucleic acid bases (A, T, G, and C). Examples of such methods, but not limited to them, include using computer software designed to generate random base sequences. Any text editing software can be used as such computer software. Examples of text editing software include Python's random module, random character generation software, and password generation software.

[0026] In one embodiment of the present invention, random nucleotide sequences are generated at least once, for example, five times, ten times, fifty times, 100 times, 500 times, 1000 times, 5000 times, or 10000 times or more. From the random nucleotide sequences, sequences with low homology are selected by comparing them with a database. Such comparison with a database may be performed using a BLAST such as nucleotide BLAST (blastn). BLAST is basically a tool that searches for sequences with low E-value (high homology), and by default, it displays E-values ​​of 10 or less. In the present invention, the lower limit of the E-value for the selection criteria of blastn is usually set to a higher value, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or higher. In the present invention, the upper limit (cutoff value) of the E-value of the selection condition for blastn is usually selected to be a number higher than the default setting (10), for example, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or 2000. In a preferred embodiment of the present invention, the E-value of the selection condition is 30. In a preferred embodiment, the E-value of the selection condition may be between 30 and 1000. In the present invention, the above lower and upper limits can be appropriately selected and used as selection conditions within that range. Since the E-value fluctuates daily, the above numbers should be considered as examples only and are not limited to those numbers.

[0027] The method for designing internal control nucleic acids in the present invention may include a step of further processing a sequence obtained using computer software designed to generate random base sequences. For example, regions with high E-values ​​in the sequence obtained using computer software may be selected and joined together, or multiple sequences with high E-values ​​may be selected from sequences obtained using computer software and combined. Furthermore, the design method may include a step of removing consecutive bases or sequences from the selected base sequence, where the consecutive bases are 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, or more. Here, a high E-value means that the E-value is 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, or more, but is not limited to these. Processing is not limited to this, but can include methods such as using computer software. Any computer software for gene analysis or text editing can be used as such computer software.

[0028] The homology of the internal control nucleic acid in this invention to known or natural sequences is determined by analysis using BLAST (Basic Local Alignment Search Tool), and the E-value is prepared to be 100 or higher, 200 or higher, preferably 300 or higher. The E-value (Expectation value) is the expected value that the sequence would happen to exist in the database. An E-value of 1 indicates that there is one instance of the sequence in the database. The higher the homology, the smaller the E-value, and the lower the homology, the larger the E-value. As a guideline, an E-value less than 0.0001 is considered to indicate homology. As the number of sequences registered in the database increases, it is expected that the E-value will decrease in the future, even for the same sequence. Generally, the E-value is thought to decrease with database updates, so the E-value is not necessarily limited to the range mentioned above, and the upper and lower limits of the E-value, as well as the values ​​in between, may decrease.

[0029] The internal control nucleic acid in the present invention is preferably designed so as not to contain repeating regions of the same base pair of 10 or more bases, 9 or more bases, 8 or more bases, 7 or more bases, 6 or more bases, 5 or more bases, or 4 or more bases within its sequence. In a preferred embodiment of the present invention, the internal control nucleic acid is prepared so as not to contain repeating regions of the same base pair of 5 or more bases. Furthermore, the method for designing the internal control nucleic acid in the present invention may include adjusting the GC content in the sequence.

[0030] The method for designing internal control nucleic acids may further include a step of removing sequences from the base sequence selected in c) and b) in which five or more identical bases are consecutive.

[0031] The method for designing the internal control nucleic acid may further include a step of adjusting the GC content in the base sequence selected in d)b). The GC content after step d) may be 20% to 80%, preferably 30% to 70%, and more preferably 40% to 60%. In one embodiment of the present invention, the GC content after step d) is adjusted to 48%.

[0032] The method for designing internal control nucleic acids may further include the step of selecting a region with a high E-value from one base sequence selected in e)b) and joining the selected regions, or the step of selecting a region with a high E-value from two or more base sequences selected in b) and joining the selected regions. Here, a high E-value means, but is not limited to, 1 or greater, 2 or greater, 3 or greater, 4 or greater, 5 or greater, 6 or greater, 7 or greater, 8 or greater, 9 or greater, 10 or greater, 20 or greater, 30 or greater, or more. Here, the minimum E-value of the designed internal control nucleic acid by BLAST may be, for example, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, or 400. The maximum E-value of the designed internal control nucleic acid by BLAST may be, for example, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 or higher. Here, in a preferred embodiment, the E-value of the designed inner control nucleic acid by BLAST is 300 or higher, and its length is 500 to 1500 bases.

[0033] [Methods for amplification, detection, and quantification of nucleic acids using internal control nucleic acids] In one aspect of the present invention, a method for detecting and / or quantifying nucleic acids in a sample is provided. In the said method, a) A step of mixing a sample with forward primers, reverse primers, and an internal control nucleic acid that contains or is identical to, or comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more of the same sequence, selected from the base sequence shown in Sequence ID No. 1, for part or all of the nucleic acid. b) extending the forward primer and reverse primer to generate at least one target amplicon, and c) The step may include detecting and / or quantifying the amplification of the target amplicon by electrophoresis. In a preferred embodiment, the internal control nucleic acid consists of the nucleotide sequence shown in SEQ ID NO: 1.

[0034] The generation and amplification of the target amplicon are not particularly limited, and known nucleic acid amplification methods can be applied. Examples include PCR, RT-PCR (Reverse Transcription-Polymerase Chain Reaction), LAMP (Loop-mediated Isothermal Amplification), RT-LAMP (Reverse Transcription-loop-mediated isothermal amplification of DNA), TMA (Transcription-Mediated Amplification), NASBA (Nucleic Acid Sequence-Based Amplification), SDA (Standard Displacement Amplification), and ICAN (Isothermal and Chimeric primer-initiated Amplification of Nucleic acids). In each nucleic acid amplification method, a person skilled in the art can appropriately select the conditions by conventional means of the art.

[0035] Detection of amplified target amplicons is not particularly limited and can be performed by measuring the presence or absence of amplified nucleic acids by known methods. Such known detection methods include, for example, agarose gel electrophoresis, methods for measuring fluorescence using an intercalator, methods for measuring fluorescence using a fluorescently labeled probe, and methods for detecting by measuring the turbidity of magnesium pyrophosphate produced during nucleic acid amplification.

[0036] The quantification of the amplified target amplicon is not particularly limited and can be performed by measuring the amount of amplified nucleic acid by known methods. Such known quantification methods include, for example, agarose gel electrophoresis, a method of measuring fluorescence using an intercalator, a method of measuring fluorescence using a fluorescently labeled probe, and a method of quantification by measuring the turbidity of magnesium pyrophosphate produced during nucleic acid amplification.

[0037] Because the internal control nucleic acid has low homology to other sequences, primers can be designed at any location on the internal control nucleic acid, allowing for modification of the amplification region and length. Furthermore, the length of the original internal control nucleic acid can be arbitrarily selected to match the amplification region. Alternatively, primers may be selected based on the desired size of the amplified region to design the internal control nucleic acid to a desired size. Primers can be used, for example, those listed in the following [Kits Containing Internal Control Nucleic Acids].

[0038] In a preferred embodiment, the method further includes a step of extracting nucleic acids from the sample before or after step a). This extraction step has the advantage of making the subsequent PCR reaction more efficient.

[0039] In a preferred embodiment of the present invention, standard two-step PCR conditions are: (i) 94°C to 98°C for 5 to 300 seconds (thermal denaturation of template double-stranded DNA) (ii) 55°C to 72°C for 5 to 180 seconds (primer annealing and DNA extension, probe degradation and fluorescence detection) This involves temperature cycling (25 to 50 cycles of (i) and (ii)). Standard 3-step PCR conditions are: (i) 94°C to 98°C for 5 to 300 seconds (thermal denaturation of template double-stranded DNA) (ii) 55°C to 65°C for 5 to 60 seconds (primer annealing) (iii) 72°C for 5 to 180 seconds (DNA elongation, fluorescence detection) This includes temperature cycles of (i), (ii), and (iii) for 25 to 50 cycles.

[0040] Another aspect of the present invention provides a method for detecting and / or quantifying nucleic acids in a sample, the method being a) A sequence of 50 or more consecutive bases selected from the base sequence shown in Sequence ID No. 1: 60 or more bases, 70 or more bases, 80 or more bases, 90 or more bases, 100 or more bases, 150 or more bases, 200 or more bases, 250 or more bases, 300 or more bases, 350 or more bases, 400 or more bases, 450 or more bases, 500 or more bases, 550 or more bases, 600 or more bases, 650 or more bases, 700 or more bases, 750 or more bases, 800 or more bases, 850 or more bases, 900 or more bases, 950 salt A step of mixing a sample with forward primers, reverse primers, oligonucleotide probes, and an internal control nucleic acid that contains or comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical sequences to a sequence of 1000 bases or more. b) extending the forward primer and reverse primer to generate and amplify at least one target amplicon, c) The step of binding the oligonucleotide probe to the at least one internal control nucleic acid or target amplicon, and d) Step of detecting a signal proportional to the amount of at least one internal control nucleic acid or target amplicon, This includes. In a preferred embodiment, the method further includes a step of extracting nucleic acid from the sample before or after step a). In a preferred embodiment, the internal control nucleic acid consists of the nucleotide sequence shown in SEQ ID NO: 1.

[0041] In c), the internal control nucleic acid binds to a probe-binding region constructed complementary to the oligonucleotide probe. In c), the target amplicon binds to the oligonucleotide probe complementarily. The internal control nucleic acid molecule may also contain at least one probe-binding region. The probe-binding region is constructed complementary to the oligonucleotide probe. The probe for the complementary probe-binding region may be any probe commonly known and used in nucleic acid (DNA and / or RNA) detection and / or quantification assays. The nucleotide sequence of the probe may be designed to bind to the complementary strand of the internal control nucleic acid.

[0042] In real-time PCR, PCR amplification products are detected by fluorescence. There are two types of fluorescence detection methods: one using an intercalator and another using a fluorescently labeled probe. Probes such as those listed in the following [kits including internal control nucleic acids] can be used.

[0043] d) Signal detection is performed by conventional means in the art, such as tracking fluorescence intensity over time. Intercalators include SYBR GreenI, TB Green, ResoLight, etc. Fluorescent dyes include, for example, FAM, HEX, VIC, ROX, Red610, Cy5, etc. Quencher dyes include, for example, TAMRA, BHQ-1, BHQ-2, BHQ-3, etc.

[0044] [Kit containing internal control nucleic acid] Another aspect of the present invention relates to a kit for determining false negatives of a target nucleic acid, comprising an internal control nucleic acid of the present invention and a primer pair for amplifying the internal control nucleic acid. The internal control nucleic acid may be one of those described above under [Internal Control Nucleic Acid]. The kit may include instructions.

[0045] Examples of forward primer sequences that can be used for amplification of internal control nucleic acids include, but are not limited to, the following sequences: TAAGTGGCTC AAGGGTCAG [Sequence No. 2]

[0046] Examples of reverse primer sequences that can be used to amplify internal control nucleic acids include, but are not limited to, the following sequences: CCTAGCATGC ACACAAAGTT [Sequence ID 3]

[0047] The above kit may, in one embodiment, include a probe. Examples of probe sequences that can be used for the detection of internal control nucleic acids include, but are not limited to, the following sequences: Cy5- TATCTTGCAC CGTTCAGCCA TCTC-BHQ-2 [Sequence ID 4]

[0048] Examples While the present invention has been broadly described above, it is believed that understanding the invention will be easier by referring to the following examples. However, the examples are provided solely for illustrative purposes and are not intended to limit the present invention.

[0049] [Manufacturing example] [Preparation of internal control nucleic acids] The method for preparing internal control nucleic acids is not limited to these, but the internal control nucleic acids used in the examples were prepared as follows.

[0050] [Comparative Example] Example of designing a random base sequence of approximately 1000 bases (A) We performed approximately 6000 BLAST trials and obtained several artificial sequences with low homology to known sequences of about 30 bases in length. By combining these sequences, we designed a sequence of about 1000 bases. When we performed BLAST on this 1000-base-length sequence, sequences with high homology to known sequences were formed at the junctions, and we were unable to obtain a sequence with an E-value > 30. In conclusion, this procedure could not obtain a sequence of about 1000 bases with sufficiently low homology to known sequences. [ka]

[0051] Design example of a random base sequence of approximately 1000 bases (B) Manufacturing example We ran blastn approximately 6000 times and obtained artificial sequences of about 1000 base pairs in length with low homology to known sequences. Of the 6000 sequences obtained, about 10 had an E-value > 30. However, even the sequences with the highest E-values ​​still contained regions with relatively high homology to known sequences. Therefore, we extracted regions with lower homology to known sequences from the two sequences with the highest E-values ​​and combined them. Furthermore, while checking for uniqueness with blastn, we used text editing software to correct for five or more consecutive base pairs and biases in GC content, adjusting them to 40% to 60%. When we ran the resulting nucleotide sequence (referred to as sequence number 1) through blastn, the number of sequences with the highest E-value among the obtained sequences was 348 (as of November 7, 2024). In conclusion, this procedure allowed us to obtain sequences of about 1000 base pairs with sufficiently low homology to known sequences. [ka]

[0052] The DNA shown in Sequence ID No. 1 was synthesized using an artificial gene synthesis service. The DNA, delivered embedded in a vector, was amplified using PCR to amplify only the region corresponding to sequence 1. Furthermore, the DNA of Sequence ID No. 1 was purified from the amplified reaction mixture to prepare an internal control nucleic acid.

[0053] [Preparation of primers and probes] The forward primer for internal control nucleic acid detection shown in SEQ ID NO: 2, the reverse primer for internal control nucleic acid detection shown in SEQ ID NO: 3, and the probe (Cy5 labeled) for internal control nucleic acid detection shown in SEQ ID NO: 4 were synthesized using an oligoDNA synthesis service.

[0054] [Example 1] Use of internal control nucleic acids in gene detection of microbial specimens 1. Materials and Methods [Preparation of bacterial genomic DNA] Pseudomonas aeruginosa (possessing the blaVIM gene) and Escherichia coli (not possessing the blaVIM gene) strains were cultured on SCD agar. Genomic DNA was purified from the cultured cells using column chromatography and prepared at 1 ng / μL in TE buffer as the sample. TE buffer was used as the negative control.

[0055] The sequence of primers and probes used Forward primers for detecting the blaVIM gene GCTTCGGTCC AGTAGA [SEQ ID NO: 5] Reverse primers for detecting the blaVIM gene GTTGTGTACG TCCCGTCTGC [Sequence ID 6] blaVIM gene detection probe (VIC labeled) VIC-TTCTATCCTG GTGCTGCGCA TTCG-BHQ-1 [Sequence ID 7]

[0056] Reaction solution A 20 μL PCR reaction mixture containing the following reagents was prepared using Roche's AptaTaq DNA Master (5×Conc.). Sample (1 ng / μL) or negative control 5 μL blaVIM gene detection forward primer 400nM blaVIM gene detection reverse primer 400nM Internal control nucleic acid detection forward primer 400nM Reverse primer for internal control nucleic acid detection, 400 nM blaVIM gene detection probe (VIC labeled) 100nM Internal control nucleic acid detection probe (Cy5 labeled) 40nM AptaTaq DNA Master (5×Conc.) 4μL Internal control nucleic acid 2.5 fg

[0057] [Real-time PCR] The prepared reaction solution was subjected to a Thermo Fisher Scientific QuantStudio 5 Dx real-time PCR instrument, and real-time PCR was performed under the following conditions. Real-time PCR was performed according to the instrument's instruction manual.

[0058] Nucleic acid amplification and fluorescence detection conditions using real-time PCR (i) 94℃·15 seconds (ii) 60°C for 60 seconds (fluorescence detection) ((i) and (ii) for 35 cycles)

[0059] Capillary electrophoresis The reaction mixture after real-time PCR was subjected to capillary electrophoresis using a QIAgen capillary electrophoresis system (QIAxcel Advanced System), and capillary electrophoresis was performed using the reagents listed below. Capillary electrophoresis was performed according to the instrument's instruction manual. Gel Cartridge:QIAxcel DNA High Resolution Kit QX Alignment Marker:QX Alignment Marker 15 bp / 3 kb QX DNA Size Marker:QX DNA Size Marker 50 - 800

[0060] 2.Results The results of real-time PCR are shown in Figure 1 as a graph with fluorescence intensity on the vertical axis and cycle number on the horizontal axis, for Pseudomonas aeruginosa (possessing the blaVIM gene), Figure 2 as a graph with Escherichia coli (not possessing the blaVIM gene), and Figure 3 as a graph with negative control results. In Figure 1, an increase in the fluorescence intensity of VIC, indicating amplification of the blaVIM gene, and an increase in the fluorescence intensity of Cy5, indicating amplification of the internal control nucleic acid (IC), were observed. In Figures 2 and 3, only an increase in the fluorescence intensity of Cy5, indicating amplification of the internal control nucleic acid (IC), was observed. These results indicate that there was no reaction inhibition by the sample and that the real-time PCR reaction was performed normally. In other words, the results in Figures 2 and 3, which were judged to be negative for the blaVIM gene, are not false negatives.

[0061] Furthermore, the reaction mixture after real-time PCR was analyzed by capillary electrophoresis, and a graph was created with fluorescence intensity (RFU) on the vertical axis and size (bp) on the horizontal axis. Figure 4 shows the analysis results for Pseudomonas aeruginosa (possessing the blaVIM gene), Figure 5 shows the analysis results for Escherichia coli (not possessing the blaVIM gene), and Figure 6 shows the analysis results for the negative control. The expected sizes of the amplification byproducts are 76 bp for blaVIM and 67 bp for the internal control nucleic acid. Note that the peaks at 15 bp and 3000 bp are alignment markers used in the capillary electrophoresis analysis and do not indicate the presence of amplification products of 15 bp and 3000 bp in the reaction mixture. In Figure 4, clear peaks were detected at 67 bp and 76 bp, which are the amplification sizes for the internal control nucleic acid and blaVIM, and no peaks of obvious nonspecific amplification products were detected. Note that a peak was detected between 15 bp and 67 bp, but this is a primer dimer peak and does not indicate nonspecific amplification products. In Figures 5 and 6, only the peak with an amplification size of approximately 67 bp, which is that of the internal control nucleic acid, was clearly detected, and no peaks of obvious nonspecific amplification products were detected. These results demonstrate that the internal control nucleic acid, the primer for detecting the internal control nucleic acid, and the probe for detecting the internal control nucleic acid can be used to detect the blaVIM gene from microorganisms. In other words, the internal control nucleic acid of the present invention has been shown to be able to distinguish whether the PCR result is false negative.

[0062] [Example 2] Use of internal control nucleic acids in gene detection in animal cell samples 1. Materials and Methods [Preparation of mouse genomic DNA] Genomic DNA was purified from BALB / c mouse hybridoma cells using column chromatography and prepared at 100 ng / μL in TE buffer as the sample. TE buffer was used as the negative control.

[0063] The sequence of primers and probes used Forward primer for β-globin gene detection GTGAGCTCCA CTGTGACAAG [Sequence ID 8] Reverse primer for β-globin gene detection CACACACCAT CATCGAAACT [SEQ ID NO: 9] β-globin gene detection probe (FAM labeled) FAM-CTTCCCCTGG CTATTCTGCT CA-BHQ-1 [Sequence ID 10]

[0064] Reaction solution A 20 μL reaction solution containing the following reagents was prepared using Roche's AptaTaq DNA Master (5×Conc.). Sample (100 ng / μL) or negative control 5 μL β-globin gene detection forward primer 200 nM Reverse primer for β-globin gene detection, 200 nM Internal control nucleic acid detection forward primer 400nM Reverse primer for internal control nucleic acid detection, 400 nM β-globin gene detection probe (FAM labeled) 100nM Internal control nucleic acid detection probe (Cy5 labeled) 40nM AptaTaq DNA Master (5×Conc.) 4μL Internal control nucleic acid 2.5 fg

[0065] [Real-time PCR] The prepared PCR reaction mixture was subjected to a Thermo Fisher Scientific real-time PCR instrument (QuantStudio 5 Dx) and real-time PCR was performed under the following conditions. Real-time PCR was performed according to the instrument's instruction manual.

[0066] Nucleic acid amplification and fluorescence detection conditions using real-time PCR (i) 94℃·15 seconds (ii) 60°C for 60 seconds (fluorescence detection) ((i) and (ii) for 40 cycles)

[0067] Capillary electrophoresis The reaction mixture after real-time PCR was subjected to capillary electrophoresis using a QIAgen capillary electrophoresis system (QIAxcel Advanced System), and capillary electrophoresis was performed using the reagents listed below. Capillary electrophoresis was performed according to the instrument's instruction manual. Gel Cartridge:QIAxcel DNA High Resolution Kit QX Alignment Marker:QX Alignment Marker 15 bp / 3 kb QX DNA Size Marker:QX DNA Size Marker 50 - 800

[0068] 2.Results The results of real-time PCR are plotted with fluorescence intensity on the vertical axis and cycle count on the horizontal axis. Figure 7 shows the results for BALB / c mouse hybridoma cells (containing the β-globin gene), and Figure 8 shows the results for the negative control. In Figure 7, an increase in the fluorescence intensity of FAM, indicating amplification of the β-globin gene, and an increase in the fluorescence intensity of Cy5, indicating amplification of the internal control nucleic acid (IC), were observed. In Figure 8, only an increase in the fluorescence intensity of Cy5, indicating amplification of the internal control nucleic acid (IC), was observed. These results indicate that there was no inhibition of the reaction by the sample, and the real-time PCR reaction was performed normally. In other words, the result in Figure 8, which was judged to be negative for the β-globin gene, is not a false negative.

[0069] Furthermore, the reaction mixture after real-time PCR was analyzed by capillary electrophoresis, and a graph was created with fluorescence intensity (RFU) on the vertical axis and size (bp) on the horizontal axis. Figure 9 shows the analysis results for BALB / c mouse hybridoma cells (possessing the β-globin gene), and Figure 10 shows the analysis results for the negative control. The expected sizes of the amplified byproducts are 159 bp for the β-globin gene and 67 bp for the internal control nucleic acid. Note that the peaks at 15 bp and 3000 bp are alignment markers used in the capillary electrophoresis analysis and do not indicate the presence of 15 bp and 3000 bp amplification products in the reaction mixture. In Figure 9, clear peaks were detected at 67 bp and 159 bp, which are the amplification sizes of the internal control nucleic acid and the β-globin gene, and no peaks of obvious nonspecific amplification products were detected. Note that a peak was detected between 15 bp and 67 bp, but this is a primer dimer peak and does not indicate nonspecific amplification products. In Figure 10, only the peak with an amplification size of approximately 67 bp, which is that of the internal control nucleic acid, was clearly detected, and no peak of obvious nonspecific amplification product was detected. These results demonstrate that the internal control nucleic acid, the primer for detecting the internal control nucleic acid, and the probe for detecting the internal control nucleic acid can be used to detect the β-globin gene from animal cells. In other words, the internal control nucleic acid of the present invention has been shown to be able to distinguish whether the PCR result is false negative.

[0070] [Example 3] Use of internal control nucleic acids in gene detection of viral samples 1. Materials and Methods [Preparation of positive control nucleic acids] A positive RSV control nucleic acid of the RNA shown in SEQ ID NO: 11 was prepared and diluted to 10 fg / μL with TE buffer as the sample. TE buffer was used as the negative control.

[0071] Sequences of the RSV-positive control nucleic acid, primers, and probes used. RS positive RNA TAATACGACT CACTATAGAG GGTCATCGTC TTTTTCTAGG ACATTGTATT GAACAGCAGC 60 TGTGTATGTG GAGCCTTCGT GAAGCTTGTT CACGTATGTT TCCATATTTG CCCCACCCCT 120 CGAG 124 [Sequence ID 11] Forward primer for RSV detection GGCAAATATG GAAACATACG TGAA [Sequence ID 12] Reverse primer for RSV detection TCTTTTTCTA GGACATTGTA YTGAACAG [Sequence ID 13] RSV detection probe FAM-TCTTTTTCTA GGACATTGTA YTGAACAG-BHQ-1 [Sequence ID 14] (In the sequence, Y represents a mixed base of C and T.)

[0072] Reaction solution A 25 μL RT-PCR solution containing the following reagents was prepared using AgPath-ID® One-Step RT-PCR Reagents from Thermo Fisher Scientific. Sample (10 fg / μL) or negative control 5 μL Forward primer for RSV gene detection, 500 nM Reverse primer for RSV gene detection, 300 nM Internal control nucleic acid detection forward primer 400nM Reverse primer for internal control nucleic acid detection, 400 nM RSV gene detection probe (FAM labeled) 150nM Internal control nucleic acid detection probe (Cy5 labeled) 40nM 2X RT-PCR Buffer 12.5 μL 25X RT-PCR Enzyme Mix 1 μL Internal control nucleic acid 2.5 fg

[0073] [Real-time RT-PCR] The prepared RT-PCR reaction mixture was subjected to a Thermo Fisher Scientific QuantStudio 5 Dx real-time PCR instrument, and real-time RT-PCR was performed under the following conditions. Real-time RT-PCR was performed according to the instrument's instruction manual.

[0074] Nucleic acid amplification and fluorescence detection conditions using real-time RT-PCR (i) 48℃ for 10 minutes (ii)95℃・5 minutes (iii)95℃・15 seconds (iii) 55℃・60 seconds (fluorescence detection) ((iii) and (iiii) for 45 cycles)

[0075] Capillary electrophoresis The reaction mixture after real-time RT-PCR was subjected to capillary electrophoresis using a QIAgen capillary electrophoresis system (QIAxcel Advanced System), and capillary electrophoresis was performed using the reagents listed below. Capillary electrophoresis was performed according to the instrument's instruction manual. Gel Cartridge:QIAxcel DNA High Resolution Kit QX Alignment Marker:QX Alignment Marker 15 bp / 3 kb QX DNA Size Marker:QX DNA Size Marker 50 - 800

[0076] 2.Results The results of real-time RT-PCR are shown in Figure 11 (RSV-positive control) and Figure 12 (negative control), with fluorescence intensity on the vertical axis and cycle count on the horizontal axis. In Figure 11, an increase in the fluorescence intensity of FAM, indicating amplification of the RSV-positive control, and an increase in the fluorescence intensity of Cy5, indicating amplification of the internal control nucleic acid (IC), were observed. In Figure 12, only an increase in the fluorescence intensity of Cy5, indicating amplification of the internal control nucleic acid (IC), was observed. These results indicate that there was no reaction inhibition by the sample and that the real-time RT-PCR reaction proceeded normally. In other words, the result in Figure 12, which indicates that the RSV-positive control was judged negative, is not a false negative.

[0077] Furthermore, the reaction mixture after real-time RT-PCR was analyzed by capillary electrophoresis, and a graph was created with fluorescence intensity (RFU) on the vertical axis and size (bp) on the horizontal axis. Figure 13 shows the analysis results for the RSV-positive control, and Figure 14 shows the analysis results for the negative control. The expected sizes of the amplification byproducts are 84 bp for the RSV-positive control and 67 bp for the internal control nucleic acid. The peaks at 15 bp and 3000 bp are alignment markers used in the capillary electrophoresis analysis and do not indicate the presence of 15 bp and 3000 bp amplification products in the reaction mixture. In Figure 13, clear peaks were detected at 67 bp and 84 bp, which are the amplification sizes for the internal control nucleic acid and the RSV-positive control, and no peaks of obvious nonspecific amplification products were detected. A peak was detected between 15 bp and 67 bp, but this is a primer dimer peak and does not indicate nonspecific amplification products. In Figure 14, only the peak with an amplification size of approximately 67 bp, which corresponds to the internal control nucleic acid, was clearly detected, and no peaks of obvious nonspecific amplification products were detected. These results demonstrate that the internal control nucleic acid, the primer for detecting the internal control nucleic acid, and the probe for detecting the internal control nucleic acid can be used to detect genes from viruses. [Industrial applicability]

[0078] Because the internal control nucleic acid of the present invention has low homology to known nucleic acid sequences, it can be used in nucleic acid amplification methods to detect various genes regardless of the sample type (animal, plant, microorganism, virus), and can be used to determine whether or not false negatives occur, thus enabling its use in quality control of genetic testing. Furthermore, while it was previously necessary to design internal control nucleic acids and primers / probes for detecting internal control nucleic acids for each sample type and target gene, the present invention allows for the common use of internal control nucleic acids and primers / probes for detecting internal control nucleic acids, which can contribute to the rapid development of genetic testing kits and cost reduction.

Claims

1. An internal control nucleic acid containing a sequence that is at least 90% identical to a sequence of 50 or more consecutive bases selected from the base sequences shown in Sequence ID No.

1.

2. The nucleic acid according to claim 1, wherein the internal control nucleic acid includes a sequence of 50 or more consecutive bases selected from the base sequences shown in Sequence ID No.

1.

3. The nucleic acid according to claim 1, wherein the internal control nucleic acid consists of a sequence of 50 or more consecutive bases selected from the base sequences shown in Sequence ID No.

1.

4. The nucleic acid according to claim 1, wherein the internal control nucleic acid consists of the base sequence shown in Sequence ID No.

1.

5. The internal control nucleic acid according to any one of claims 1 to 4; Forward primers and reverse primers for amplifying part or all of the nucleic acid; and, A composition containing a sample for the detection and / or quantification of nucleic acids.

6. The composition according to claim 5, further comprising an oligonucleotide probe complementary to the internal control nucleic acid in the region sandwiched between the forward primer and the reverse primer.

7. The composition according to claim 5, wherein the oligonucleotide of the forward primer is identical to the base sequence shown in Sequence ID No. 2 by at least 95%.

8. The composition according to claim 5, wherein the oligonucleotide of the reverse primer is identical to the base sequence shown in Sequence ID No. 3 by at least 95%.

9. The composition according to claim 3, wherein the oligonucleotide probe is identical to the base sequence shown in Sequence ID No. 4 by at least 95%.

10. A method for detecting and / or quantifying nucleic acids in a sample, a) A step of mixing an internal control nucleic acid containing at least 90% of a sequence identical to 50 or more consecutive bases selected from the base sequences shown in Sequence ID No. 1 with a forward primer, a reverse primer, and a sample for amplifying part or all of the nucleic acid. b) extending the forward primer and the reverse primer to generate at least one target amplicon, and c) A step of detecting and / or quantifying the amplification of the target amplicon by electrophoresis. The method, including the method described above.

11. A method for detecting and / or quantifying nucleic acids in a sample, a) A step of mixing an internal control nucleic acid containing at least 90% of a sequence identical to 50 or more consecutive bases selected from the base sequences shown in Sequence ID No. 1 with a forward primer, a reverse primer, an oligonucleotide probe, and a sample for amplifying part or all of the nucleic acid. b) The step of extending the forward primer and the reverse primer to generate at least one target amplicon, c) The step of binding the oligonucleotide probe to the at least one internal control nucleic acid or target amplicon, and d) Step of detecting a signal proportional to the amount of at least one internal control nucleic acid or target amplicon, The method, including the method described above.

12. The method according to claim 11, wherein the internal control consists of a sequence of 50 or more consecutive bases selected from the base sequences shown in Sequence ID No.

1.

13. The method according to claim 12, wherein the internal control includes a sequence consisting of the nucleotide sequence shown in SEQ ID NO:

1.

14. The method according to claim 10 or 11, wherein the oligonucleotide of the forward primer is identical to the base sequence shown in SEQ ID NO: 2 by at least 95%.

15. The method according to claim 10 or 11, wherein the oligonucleotide of the reverse primer is identical to the base sequence shown in SEQ ID NO: 3 by at least 95%.

16. The method according to claim 10 or 11, wherein the oligonucleotide of the forward primer is the nucleotide sequence shown in SEQ ID NO: 2, and the oligonucleotide of the reverse primer is the nucleotide sequence shown in SEQ ID NO:

3.

17. The method according to claim 10 or 11, wherein the internal control nucleic acid comprises a sequence of 50 or more consecutive bases selected from the base sequences shown in Sequence ID No.

1.

18. The method according to claim 10 or 11, wherein the internal control nucleic acid consists of a sequence of 50 or more consecutive bases selected from the base sequences shown in Sequence ID No.

1.

19. The method according to claim 10 or 11, wherein the oligonucleotide probe is the base sequence shown in Sequence ID No.

4.

20. The method according to claim 10 or 11, further comprising the step of extracting nucleic acids from the sample before or after step a).

21. A method for designing an internal control nucleic acid in the detection and / or quantification of nucleic acids, below: a) A step of creating a random base sequence having a length of 500 to 1500 bases, and b) A step of selecting sequences with low homology by comparing the random base sequences with a database, wherein the comparison with the database is performed using BLAST, and the E-value of the BLAST selection criterion is E-value > 30. The method, including the method described above.

22. The process further includes a step of joining together regions with high E-values ​​from one base sequence selected in b), or The method according to claim 21, further comprising the step of joining together regions with high E-values ​​from two or more base sequences selected in (b).

23. The method according to claim 21, further comprising the step of removing from the base sequence selected in (b) a base sequence in which five or more identical bases are consecutive.

24. The method according to claim 21, further comprising the step of adjusting the GC content to 40-60% in the base sequence selected in (b).

25. The method according to any one of claims 21 to 24, wherein the BLAST E-value of the designed inner control nucleic acid is 300 or more, and its length is 500 to 1500 bases.

26. A method for designing an internal control nucleic acid in the detection and / or quantification of nucleic acids, below: a) A step of creating a random base sequence having a length of 500 to 1500 bases, b) A step of selecting a sequence with low homology from the random base sequence by comparing it with a database, wherein the comparison with the database is performed using BLAST, and the E-value of the BLAST selection criterion is E-value > 30. c) Step of removing from the base sequence selected in b) a base sequence in which the same base is repeated for five or more consecutive bases, d) b) A step of adjusting the GC content to 40-60% in the selected base sequence, and / or e) Further comprising the step of selecting a region with a high E-value from one base sequence selected in b) and joining the selected regions, or selecting regions with a high E-value from two or more base sequences selected in b) and joining the selected regions. Includes, The method wherein the BLAST E-value of the designed inner control nucleic acid is 300 or more, and its length is 500 to 1500 bases.

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