Oligonucleotide probes for target nucleic acid detection

Abstract

To provide a probe with a stem-loop structure capable of detecting a target nucleic acid with high sensitivity.SOLUTION: The above problem is solved by setting the base sequence of a stem region to one of (i) CCCTgTg on one end and CACAggg on the other end, (ii) CCCTgTgTg on one end and CACACAggg on the other end, and (iii) CTCggg on one end and CCCgAg on the other end.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to an oligonucleotide probe for detecting a target nucleic acid. In particular, the present invention relates to an oligonucleotide probe having a stem-loop structure and capable of detecting a target nucleic acid with high sensitivity.

Background Art

[0002] In the examination of viral infections, when a method of detecting a viral nucleic acid contained in a sample as a target is adopted, generally, since the viral nucleic acid contained in the sample is extremely微量, a method of amplifying and detecting the viral nucleic acid is used.

[0003] As a method for amplifying a target nucleic acid, the polymerase chain reaction method (PCR method) is widely known. This method is a method of amplifying DNA by repeating a cycle consisting of heat denaturation - primer annealing - extension reaction in the presence of a set of primers having a sequence complementary or homologous to a part of a specific base sequence in the target DNA and a heat-resistant DNA polymerase. Also, when the target nucleic acid is RNA, a so-called RT-PCR method is known in which cDNA is once synthesized by reverse transcriptase and then the PCR method is performed. However, the PCR method and the RT-PCR method require rapid and repeated increases and decreases in the reaction temperature, which has been a barrier to labor saving and cost reduction of the reaction apparatus during automation.

[0004] On the other hand, methods such as NASBA (Nucleic Acid Sequence-Based Amplification), TMA (Transcription-Mediated Amplification), and TRC (Transcription Reverse-transcription Concerted reaction) are known for amplifying target RNA at a constant temperature. These methods amplify nucleic acids at a constant temperature without raising or lowering the reaction temperature, making them convenient for nucleic acid amplification. Therefore, they are preferable methods for automation as they allow for labor savings and cost reduction of the reaction equipment. In recent years, digital nucleic acid amplification methods such as ddPCR (Droplet Digital PCR) have also become known. Digital nucleic acid amplification methods involve forming a reaction solution containing the target nucleic acid into droplets and performing nucleic acid amplification.

[0005] The target nucleic acid amplified by the method described above is detected using an oligonucleotide probe having a sequence complementary or homologous to a specific base sequence of the nucleic acid. Examples of such probes include fluorescently labeled probes utilizing FRET (fluorescence resonance energy transfer), oligonucleotide probes labeled with intercalator fluorescent dyes, TaqMan (trade name) probes, and molecular beacon probes. Among these, when detecting the target nucleic acid using a digital nucleic acid amplification method such as ddPCR, the detection is based on the fluorescence intensity at the endpoint, so molecular beacon probes, which have high fluorescence intensity and a high signal-to-noise ratio, are preferred.

[0006] A Molecular Beacon probe is a probe having a stem-loop structure, consisting of a loop region having a sequence complementary or homologous to a portion of the target nucleic acid (or the amplification product of the target nucleic acid if nucleic acid amplification is performed), and double-stranding regions (stem regions) attached to both ends of the loop region. Furthermore, a fluorescent dye is attached to one end of the stem region, and a quencher is attached to the other end (Figure 1). When the loop region is not hybridized with the target nucleic acid or its amplification product, the fluorescent dye and the quencher are brought close together by the stem region, and therefore no fluorescence is emitted from the fluorescent dye. However, when the loop region hybridizes with the target nucleic acid or its amplification product, the stem region dissociates, and the fluorescent dye moves away from the quencher, causing fluorescence from the fluorescent dye to be emitted. As an example, Patent Document 1 discloses an example in which hepatitis C virus nucleic acid contained in a sample is amplified by a digital nucleic acid amplification method, and the amplified nucleic acid is detected with a Molecular Beacon probe. [Prior art documents] [Patent Documents]

[0007] Japanese Patent Publication No. 2020-162591 [Overview of the Initiative] [Problems that the invention aims to solve]

[0008] The object of the present invention is to provide a probe having a stem-loop structure capable of detecting target nucleic acids with high sensitivity. [Means for solving the problem]

[0009] The inventors of this invention have diligently conducted research to solve the above problems and have completed the present invention by optimizing the base sequence of the double-strand formation region (stem region) that constitutes the molecular beacon probe.

[0010] In other words, the first aspect of the present invention is An oligonucleotide probe for detecting a target nucleic acid, comprising a loop region having a sequence complementary or homologous to a portion of the target nucleic acid, and stem regions attached to both ends of the loop region, The probe is such that the base sequence of the stem region is one of the following (i) to (iii); (i) One end is CCCTgTg and the other end is CACAggg, (ii) One end is CCCTgTgTg and the other end is CACACAggg, (iii) One end is CTCggg and the other end is CCCgAg.

[0011] A second aspect of the present invention is the probe described in the first aspect, wherein a fluorescent dye is further added to one end of the probe and a quencher to the other end.

[0012] A third aspect of the present invention is the probe according to the first or second aspect, wherein the target nucleic acid is adeno-associated virus nucleic acid and the base sequence of the loop region is the sequence described in SEQ ID NO: 27 or its complementary sequence.

[0013] A fourth aspect of the present invention is the probe according to the first or second aspect, wherein the target nucleic acid is hepatitis C virus nucleic acid and the base sequence of the loop region is the sequence described in Sequence ID No. 28 or its complementary sequence.

[0014] Furthermore, a fifth aspect of the present invention is a target nucleic acid detection reagent comprising: a first primer having a sequence complementary to a portion of a specific base sequence of a target nucleic acid; a second primer having a sequence homologous to a portion of the specific base sequence; a nucleic acid amplification reagent; and a probe according to any of the first to fourth aspects, having a loop region having a sequence complementary or homologous to a portion of the specific base sequence and stem regions attached to both ends of the loop region.

[0015] The present invention will be described in detail below.

[0016] In the probe of the present invention, the loop region has a sequence that is complementary or homologous to a part of the target nucleic acid. In the present invention, a complementary sequence is a sequence that can specifically hybridize to the target nucleic acid under stringent conditions, and a homologous sequence is a sequence that can specifically hybridize to the complementary sequence of the target nucleic acid under stringent conditions. An example of "stringent conditions" here is a condition in which a so-called specific hybrid is formed and a nonspecific hybrid is not formed.

[0017] For example, this refers to conditions in which polynucleotides with high homology (e.g., identity or similarity), such as 70% or more, preferably 80% or more, more preferably 90% or more, and even more preferably 95% or more, hybridize, while polynucleotides exhibiting lower homology do not hybridize. While not limited to these conditions, specific examples of hybridization conditions include those at 42°C containing 50% (v / v) formamide, 0.1% (w / v) bovine serum albumin, 0.1% (w / v) Ficol (trade name), 0.1% (w / v) polyvinylpyrrolidone, 50 mM sodium phosphate buffer (pH 6.5), 150 mM sodium chloride, and 75 mM sodium citrate, as well as the nucleic acid amplification and detection conditions described in the examples of this specification. Furthermore, examples of washing conditions include washing once, preferably two to three times, at a salt concentration and temperature equivalent to stringent conditions such as 60°C, 1×SSC (Saline Sodium Citrate Buffer), 0.1% (w / v)SDS (Sodium Dodecyl Sulfate), preferably 0.1×SSC, 0.1% (w / v)SDS, more preferably 65°C, 0.1×SSC, 0.1% (w / v)SDS, and more preferably 68°C, 0.1×SSC, 0.1% (w / v)SDS.

[0018] Preferred examples of the loop region include, when the target nucleic acid is adeno-associated virus nucleic acid, an oligonucleotide consisting of the sequence described in SEQ ID NO: 27 or its complementary sequence, and when the target nucleic acid is hepatitis C virus nucleic acid, an oligonucleotide consisting of the sequence described in SEQ ID NO: 28 or its complementary sequence.

[0019] The probe of the present invention is characterized in that the base sequence of the stem region attached to both ends of the aforementioned loop region is one of the following: (i) one end is CCCTgTg and the other end is CACAggg, (ii) one end is CCCTgTgTg and the other end is CACACAggg, and (iii) one end is CTCggg and the other end is CCCgAg. There are no limitations on the terminal position of the loop region to which these base sequences are attached; for example, in case (i), You may also add CCCTgTg to the 5' end of the loop region and CACAggg to the 3' end of the loop region. CCCTgTg may be added to the 3' end of the loop region, and CACAggg may be added to the 5' end of the loop region.

[0020] Adding a fluorescent dye to one end of the aforementioned stem region and a quencher to the other end is preferable because it allows for simple and highly sensitive detection of the target nucleic acid. There are no limitations on the terminal positions of the stem region to which the fluorescent dye and quencher are added. That is, A fluorescent dye may be added to the 5' end of the stem region, and a quencher may be added to the 3' end of the stem region. A fluorescent dye may be added to the 3' end of the stem region, and a quencher may be added to the 5' end of the stem region.

[0021] The fluorescent dye and quenching agent to be added to the probe of the present invention may be, for example, a combination of fluorescent dye and quenching agent used in molecular beacon probes. Specifically, When using any of 6-FAM (6-Carboxyfluorescein), JOE (6-Carboxy-4’,5’-Dichloro-2’,7’-Dimethoxyfluorescein), TET (5’-Tetrachlorofluorescein), and HEX (5’-Hexachlorofluorescein) as the fluorescent dye, BHQ-1 (manufactured by LGC Biosearch Technologies) or DABCYL (4-((4-(dimethylamino)phenyl)azo)benzoic Acid) can be used as the quencher. When using any of Cy3 (Cyanine 3), ROX (Rhodamine Red X), and Texas Red (Sulforhodamine 101 acid chloride) as the fluorescent dye, BHQ-2 (manufactured by LGC Biosearch Technologies) or DABCYL can be used as the quencher. When using Cy5 (Cyanine 5) or Cy5.5 (Cyanine 5.5) as the fluorescent dye, BHQ-3 (manufactured by LGC Biosearch Technologies) or DABCYL can be used as the quencher.

[0022] When detecting the target nucleic acid contained in the sample using the probe of the present invention, a plurality of probes may be added as long as the base sequences of the loop regions of the probes do not form complementary sequences with each other.

[0023] When detecting a target nucleic acid using the probe of the present invention, it is preferable to amplify the specific base sequence using a first primer having a sequence complementary to a part of the specific base sequence of the target nucleic acid in advance, a second primer having a sequence homologous to a part of the specific base sequence, and a nucleic acid amplification enzyme, and then detect it, because the target nucleic acid can be detected with higher sensitivity. In the present specification, the specific base sequence refers to the base sequence from the 3'-end of the complementary region with the first primer to the 5'-end of the homologous region with the second primer in the target nucleic acid. That is, the nucleic acid containing the specific base sequence or the complementary sequence of the specific base sequence is amplified by the first and second primers and the nucleic acid amplification enzyme.

[0024] In addition, among the target nucleic acids, the sequence (complementary sequence) that can hybridize with the loop region of the probe of the present invention is located within the specific base sequence or the complementary sequence of the specific base sequence, and the amplified nucleic acid can be detected with the probe of the present invention. When the specific base sequence is amplified, the loop region of the probe of the present invention becomes a sequence complementary to a part of the specific base sequence, and when the complementary sequence of the specific base sequence is amplified, the loop region of the probe of the present invention becomes a sequence homologous to a part of the specific base sequence.

[0025] Furthermore, if an RNA polymerase promoter is added to the 5' end of either the first or second primer, nucleic acids containing a specific base sequence with the RNA polymerase promoter added, or a complementary sequence of the specific base sequence, can be synthesized using the RNA polymerase corresponding to the promoter. This is preferable because RNA can be amplified from these nucleic acids using methods that amplify RNA at a constant temperature, such as NASBA (Nucleic Acid Sequence-Based Amplification), TMA (Transcription-Mediated Amplification), and TRC (Transcription Reverse-transcription Concerted reaction). The promoter added to the 5' end of the primer should be one that corresponds to the RNA polymerase used for RNA amplification (for example, T7 RNA polymerase, T3 RNA polymerase, or SP6 RNA polymerase, which are commonly used in the field of molecular biology). In addition, a transcription start region known to affect transcription efficiency may be further added to the promoter. A specific example of a promoter (T7 promoter) to be attached to the 5' end of a primer when using T7 RNA polymerase as the RNA polymerase for RNA amplification is the oligonucleotide consisting of the nucleotide sequence described in Sequence ID No. 4.

[0026] When detecting the target nucleic acid as adeno-associated virus nucleic acid with a probe of the present invention having a loop sequence consisting of the sequence described in SEQ ID NO: 27 or its complementary sequence, it is preferable to use an oligonucleotide consisting of the nucleotide sequence described in SEQ ID NO: 2 as the first primer and an oligonucleotide consisting of the nucleotide sequence described in SEQ ID NO: 3 as the second primer. Furthermore, when detecting the target nucleic acid as hepatitis C virus with a probe of the present invention having a loop sequence consisting of the sequence described in SEQ ID NO: 28 or its complementary sequence, it is preferable to use an oligonucleotide consisting of the nucleotide sequence described in SEQ ID NO: 22 as the first primer and an oligonucleotide consisting of the nucleotide sequence described in SEQ ID NO: 23 as the second primer.

[0027] In this invention, the target nucleic acid is not limited to adeno-associated virus nucleic acid or hepatitis C virus nucleic acid; it may also be viral nucleic acid such as influenza virus or hepatitis B virus, nucleic acid of organisms other than viruses, or artificially produced nucleic acid. The target nucleic acid can be detected with high sensitivity by designing a probe that has a loop region having a sequence complementary or homologous to a part of the target nucleic acid, and double-strand forming regions (stem regions) attached to both ends of the loop region, and the base sequence of the stem region is optimized. The reaction temperature for amplifying a specific base sequence or its complementary sequence depends on the heat resistance and activity of the nucleic acid amplification enzyme used, as well as the Tm (melting temperature) of the primer / probe. However, when using AMV (Avian Myeloblastosis Virus) reverse transcriptase, T7 RNA polymerase, and 96-7 DNA polymerase as nucleic acid amplification enzymes, and the length of the loop regions of the first and second primers and the probe of the present invention is between 10 and 30 bases, the reaction temperature can be set between 35°C and 65°C, and more preferably between 40°C and 50°C. [Effects of the Invention]

[0028] The probe of the present invention has a loop region having a sequence complementary or homologous to a portion of the target nucleic acid, and double-strand forming regions (stem regions) attached to both ends of the loop region, and the base sequence of the stem region is optimized, enabling highly sensitive detection of the target nucleic acid.

[0029] Furthermore, in an embodiment of a target nucleic acid detection reagent that includes a first primer having a sequence complementary to a portion of the specific base sequence of the target nucleic acid, a second primer having a sequence homologous to the portion of the specific base sequence, a nucleic acid amplification reagent, and a probe of the present invention having a loop region having a sequence complementary or homologous to the portion of the specific base sequence, the target nucleic acid can be detected with higher sensitivity because the specific base sequence or its complementary sequence of the target nucleic acid is amplified before detection with the probe of the present invention. [Brief explanation of the drawing]

[0030] [Figure 1] This is a diagram illustrating the molecular beacon probe. [Examples]

[0031] The embodiments of the present invention will be described in detail below using examples, but these examples are for illustrating one form of implementation of the present invention and do not limit the present invention.

[0032] Example 1: Preparation of adeno-associated virus (AAV) single-stranded DNA Using AAV single-stranded DNA as the target nucleic acid, we investigated the effect of differences in the stem region of molecular beacon probes on the detection of the target nucleic acid.

[0033] A single-stranded DNA (SEQ ID NO: 1) solution (hereinafter also referred to as "NS3") prepared by the method described in Japanese Patent Publication No. 2021-170975 was subjected to a 3.0 × 10⁻¹⁶ test using TE (Tris-EDTA) buffer containing 0.01% (w / v) sodium cholate and 0.01% (w / v) sodium azide. 4 The sample was diluted to a copy / 2μL ratio and used as the DNA sample.

[0034] (2) 10 μL of the reaction solution having the following composition was dispensed into a 0.5 mL PCR tube (Individual Dome Cap PCR Tube, manufactured by SSI), and then 2 μL of the DNA sample prepared in (1) above was added. The loop region (sequence number 27) of the Molecular Beacon probe (sequence numbers 5 to 20) is an oligonucleotide consisting of the nucleotide sequence from 1477 to 1492 of NS3 (sequence number 1), with FAM added to the 5' end of the stem region and IBFQ added to the 3' end. The first primer is an oligonucleotide consisting of the complementary sequence of the nucleotide sequence from 1620 to 1637 of NS3 (sequence number 1), with the T7 promoter (sequence number 4) added to its 5' end. The second primer is an oligonucleotide consisting of the nucleotide sequence from 1429 to 1446 of NS3 (sequence number 1).

[0035] Composition of the reaction solution: The concentration is the final concentration after adding the initiator (in 20 μL), as described below. 60mM Tris-HCl buffer (pH8.65) 0.3mM each dATP, dCTP, dGTP, dTTP 3.0mM each ATP, CTP, GTP, UTP 3.4mM ITP 67 mM Trehalose 75nM Molecular Beacon probe (one of SEQ ID NOs. 5 to 20, synthesized by Integrated DNA Technologies) 1.0 μM First primer (SEQ ID NO: 2) 1.0 μM Second primer (SEQ ID NO: 3) 1.33U 96-7 DNA polymerase 1.42U AMV reverse transcriptase 95U T7 RNA polymerase (3) After the above reaction solution was kept warm at 46°C for 3 minutes, 8 μL of an initiator having the following composition was added.

[0036] Initiator composition: Concentration is the final concentration after adding the initiator (in 20 μL). 19.0 mM magnesium chloride 80.0 mM potassium chloride 3.8% (w / v) glycerol 10.5% (v / v) DMSO (4) Subsequently, using a temperature-controlled fluorescence spectrophotometer capable of directly measuring the PCR tubes, the reaction was carried out at 46°C, and the fluorescence intensity of the reaction solution (excitation wavelength 470 nm, fluorescence wavelength 520 nm) was measured over time for 30 minutes.

[0037] (5) The fluorescence intensity ratio was calculated by dividing the fluorescence intensity value of the reaction solution 10 minutes after the addition of the initiator by the initial fluorescence intensity value (the average of the fluorescence intensity values ​​at the time of initiator addition and immediately after addition).

[0038] (6) Except for adding a negative control (0 copies) instead of the DNA sample, the fluorescence intensity ratio was calculated in the same manner as in (2) to (5), and the difference from the fluorescence intensity ratio calculated in (5) was calculated.

[0039] The results are shown in Tables 1 to 3. Comparing the results (difference in fluorescence intensity ratio) with the stem region (CCCggg / CCCggg [meaning (5' end base sequence / 3' end base sequence), same hereinafter], SEQ ID NO: 5) of the Molecular Beacon probe disclosed in Japanese Patent Publication No. 2021-170975, it can be seen that the difference in fluorescence intensity ratio is significantly increased when the stem region is CCCTgTg / CACAggg (SEQ ID NO: 6, Tables 1 to 3), CCCTgTgTg / CACACAggg (SEQ ID NO: 10, Table 1), or CTCggg / CCCgAg (SEQ ID NO: 13, Table 2). Therefore, it is suggested that AAV single-stranded DNA can be detected with higher sensitivity by using these base sequences as the stem region of the Molecular Beacon probe.

[0040] On the other hand, based on the findings in Japanese Patent Publication No. 2021-170975 (stem region: CCCggg / CCCggg), we designed multiple stem regions consisting only of cytosine (C) and guanine (G), but we could not find any stem regions in which the difference in fluorescence intensity ratio was significantly amplified (SEQ ID NOs: 15 to 20, Table 3).

[0041] [Table 1]

[0042] [Table 2]

[0043] [Table 3]

[0044] Example 2: Detection of Hepatitis C Virus RNA We confirmed whether the probe of the present invention is effective even when the target nucleic acid is changed from AAV single-stranded DNA to hepatitis C virus RNA.

[0045] (1) The standard RNA (SEQ ID NO: 21) was prepared by in vitro transcription from a plasmid into which the hepatitis C virus standard RNA (hereinafter also simply referred to as "standard RNA") gene was inserted. The standard RNA was transcribed in 3 × 10⁻⁶ units using sterile water for injection. 4 The RNA sample was diluted to a copy / 2μL ratio and used as the RNA sample.

[0046] (2) 12 μL of the reaction solution having the following composition was dispensed into a 0.5 mL PCR tube (Individual Dome Cap PCR Tube, SSI Corporation), and 2 μL of the RNA sample prepared in (1) above was added. The loop region (sequence number 28) of the Molecular Beacon probes (sequence numbers 24 to 26) is an oligonucleotide consisting of the base sequence from nucleotide positions 108 to 122 of the standard RNA (sequence number 21) (uracil (U) is converted to thymine (T), the same applies hereafter), except that the 7th position of the loop region (13th position in sequence number 24, 14th position in sequence number 25, and 15th position in sequence number 26) is a mixed base (Y) of cytosine and thymine. The first primer (sequence number 22) is an oligonucleotide consisting of the complementary sequence of the base sequence from nucleotide positions 125 to 145 of the standard RNA (sequence number 21), and a T7 promoter (sequence number 4) is attached to its 5' end. The second primer is an oligonucleotide consisting of the nucleotide sequence from the 1st to the 16th base of the standard RNA (SEQ ID NO: 21).

[0047] Composition of the reaction solution: The concentration is the final concentration after adding the initiator (in 20 μL), as described below. 66mM Tris-HCl buffer (pH8.36) 0.33mM each dATP, dCTP, dGTP, dTTP 2.0mM each ATP, CTP, GTP, UTP 3.3mM ITP 150 mM Trehalose 50nM Molecular Beacon probe (one of sequence numbers 24 to 26) 1.0 μM First primer (SEQ ID NO: 22) 1.0 μM Second primer (SEQ ID NO: 23) 12.8U AMV reverse transcriptase 166U T7 RNA polymerase (3) After the above reaction solution was kept warm at 46°C for 3 minutes, 6 μL of an initiator having the following composition was added.

[0048] Initiator composition: Concentration is the final concentration after adding the initiator (in 20 μL). 18.4 mM magnesium chloride 90.0 mM potassium chloride 2.5% (w / v) glycerol 9.0% (v / v) DMSO 0.1% (w / v) Tween 20 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) (4) The fluorescence intensity was measured using the method described in Example 1(4), the fluorescence intensity ratio was calculated using the method described in Example 1(5), and the difference in fluorescence intensity ratio was calculated using the method described in Example 1(6).

[0049] The results are shown in Table 4. Similar to Example 1, changing the stem region of the Molecular Beacon probe from CCCggg / CCCggg (SEQ ID NO: 24) to CCCTgTg / CACAggg (SEQ ID NO: 25), which is one embodiment of the probe of the present invention, significantly increased the difference in fluorescence intensity ratio. On the other hand, using a stem region consisting only of cytosine and guanine other than CCCggg (CCCCgggg / CCCCgggg, SEQ ID NO: 26) significantly reduced the difference in fluorescence intensity ratio.

[0050] [Table 4]

Claims

1. An oligonucleotide probe for detecting a target nucleic acid, comprising a loop region having a sequence complementary or homologous to a portion of the target nucleic acid, and double-strand forming regions (stem regions) attached to both ends of the loop region, The base sequence of the aforementioned stem region is one of the following (i) to (iii): (i) One end is CCCTgTg and the other end is CACAggg, (ii) One end is CCCTgTgTg and the other end is CACACAggg, (iii) One end is CTCggg and the other end is CCCgAg, The probe further has a fluorescent dye attached to one end and a quencher attached to the other end.

2. The probe according to claim 1, wherein the target nucleic acid is adeno-associated virus nucleic acid, and the base sequence of the loop region is the sequence described in SEQ ID NO: 27 or its complementary sequence.

3. The probe according to claim 1, wherein the target nucleic acid is hepatitis C virus nucleic acid, and the base sequence of the loop region is the sequence described in SEQ ID NO: 28 or its complementary sequence.

4. A target nucleic acid detection reagent comprising: a first primer having a sequence complementary to a portion of a specific base sequence of a target nucleic acid; a second primer having a sequence homologous to a portion of the specific base sequence; a nucleic acid amplification reagent; and a probe according to any one of claims 1 to 3, having a loop region having a sequence complementary or homologous to a portion of the specific base sequence and stem regions attached to both ends of the loop region.

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