WO2021230204A1 - Kit et procédé de détection nouveau coronavirus sars-cov-2 - Google Patents

Kit et procédé de détection nouveau coronavirus sars-cov-2 Download PDF

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WO2021230204A1
WO2021230204A1 PCT/JP2021/017709 JP2021017709W WO2021230204A1 WO 2021230204 A1 WO2021230204 A1 WO 2021230204A1 JP 2021017709 W JP2021017709 W JP 2021017709W WO 2021230204 A1 WO2021230204 A1 WO 2021230204A1
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sequence
cov
sars
circular dna
stranded circular
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PCT/JP2021/017709
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Japanese (ja)
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正靖 ▲桑▼原
博仁 藤田
尚志 河島
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学校法人日本大学
学校法人東京医科大学
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Publication of WO2021230204A1 publication Critical patent/WO2021230204A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/08—RNA viruses
    • C07K14/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to a kit for easily and efficiently detecting the new coronavirus SARS-CoV-2 and a detection method using the same.
  • Patent Document 1 a simple method for detecting an RNA sequence. Specifically, the target RNA is hybridized with the single-stranded circular DNA and the primer to form a three-way complex, and the primer is amplified by the rolling circle amplification (RCA) method, and the detection reagent contained in the amplification product is used.
  • RCA rolling circle amplification
  • a binding sequence such as a guanine quadruplex-containing sequence
  • ThT thioflavin T
  • the rolling circle amplification (RCA) method (also called the SATIC method) using single-stranded circular DNA, primers, etc. described in Patent Document 1 has made it possible to efficiently detect the target nucleic acid, but SARS-CoV-2 It was unclear if it could be used to detect.
  • An object of the present invention is to provide a kit and a method for detecting SARS-CoV-2 easily and efficiently.
  • the present inventor has made diligent studies to solve the above problems. As a result, they have found that SARS-CoV-2 can be efficiently detected by improving the SATIC method, using a primer bound to a carrier, and devising a pretreatment method for a biological sample. Completed.
  • the present invention (I) A sequence of 10 to 30 bases complementary to the first site of SARS-CoV-2 derived nucleic acid, The 7-8 base first primer binding sequence adjacent to the 5'side of the sequence and The second single-stranded circular DNA binding sequence and First single-stranded circular DNA containing (Ii) A sequence of 8 to 15 bases of SARS-CoV-2-derived nucleic acid complementary to the second site adjacent to the 3'side of the first site. A sequence of 7 to 8 bases adjacent to the 3'side of the sequence and complementary to the first primer binding site of the first single-stranded circular DNA.
  • a sequence complementary to the second primer binding sequence of the second single-stranded circular DNA adjacent to the 3'side of the sequence With a second oligonucleotide primer, including Including The first oligonucleotide primer is bound to the carrier via its 5'end.
  • the second oligonucleotide primer is bound to the carrier to which the first oligonucleotide primer is bound via its 5'end.
  • a kit for detecting SARS-CoV-2 is provided.
  • the first site of the SARS-CoV-2-derived nucleic acid and the second site adjacent to the 3'side of the first site are within the region of base number 2000 to 3000 or base number 21000 to 22000 of SEQ ID NO: 1. It is preferably set, and more preferably it is set in the region of base numbers 2410 to 2440 or base numbers 21666 to 21696 of SEQ ID NO: 1.
  • the SARS-CoV-2 detection kit is The first oligonucleotide primer has its 5'end modified with biotin and is bound to a carrier on which avidin is immobilized via the biotin.
  • the 5'end of the second oligonucleotide primer is modified with biotin, and the second oligonucleotide primer is bound to the carrier to which the first oligonucleotide primer is bound via the biotin. This is a preferred embodiment.
  • the SARS-CoV-2 detection kit preferably has a molar ratio of 1:10 to 1: 100 between the first oligonucleotide primer and the second oligonucleotide primer bound to the carrier. ..
  • the SARS-CoV-2 detection kit contains (v) a detection reagent, and the preferred embodiment is that the second single-stranded circular DNA contains a sequence complementary to the detection reagent binding sequence.
  • the detection reagent binding sequence is a guanine quadruple chain forming sequence and the detection reagent is a guanine quadruple chain binding reagent.
  • the SARS-CoV-2 detection kit preferably comprises a sequence complementary to the guanine quadruple chain forming sequence containing a C 3 N 1-10 C 3 N 1-10 C 3 N 1-10 C 3 sequence. It is supposed to be.
  • the SARS-CoV-2 detection kit has another preferred embodiment in which the guanine quadruplex binding reagent is the following ThT-PEG-ThT.
  • the PEG chain portion of ThT-PEG-ThT may have a branched structure or may be immobilized on a carrier together with a polyethylene glycol chain. Further, a compound having a spermine linker can be used instead of the PEG linker.
  • ThT-PEG-ThT N is an integer from 4 to 50.
  • the SARS-CoV-2 detection kit preferably contains polyethylene glycol (PEG) together with ThT-PEG-ThT as a detection reagent.
  • PEG polyethylene glycol
  • the SARS-CoV-2 detection kit preferably contains a crown ether and a nonionic surfactant as a pretreatment reagent for a biological sample containing a SARS-CoV-2-derived nucleic acid.
  • the crown ether is 18-crown-6 or 15-crown-5 and the nonionic surfactant is polyoxyethylene sorbitan monolaurate or octylphenol ethoxylate. It is supposed to be.
  • the present invention is a method for detecting SARS-CoV-2-derived nucleic acid using the above kit.
  • Provide methods including.
  • the pretreatment step is a crown on the biological sample. It is more preferable to include an operation of adding an ether and a nonionic surfactant and heating.
  • the detection step includes detection using ThT-PEG-ThT and PEG.
  • RCA in the presence of the sequence of the target nucleic acid SARS-CoV-2, RCA produces a first amplification product, and an oligonucleotide is added to the complex of the first amplification product and the second single-stranded circular DNA.
  • Primers hybridize and RCA produces a second amplification product, eg, a DNA strand in which detection reagent binding sequences such as multiple guanine quadruplex-containing sequences are linked in series, which can be referred to as ThT (derivative).
  • the nucleic acid sequence of SARS-CoV-2 can be specifically detected by staining with a detection reagent.
  • the detection sensitivity can be significantly improved, and by using ThT-PEG-ThT and PEG as detection reagents, the presence or absence of an amplification product is visually confirmed. And the inspection can be performed easily. Furthermore, by using a nonionic surfactant and crown ether for the pretreatment of the biological sample, the detection sensitivity can be remarkably improved.
  • the schematic diagram of the SARS-CoV-2 detection method which concerns on this invention The figure which shows the result of Example 1 (the drawing substitute photograph). The figure which shows the result of Example 2-1 (the drawing substitute photograph). It is a figure (drawing substitute photograph) which shows the result of Example 2-2. It is a figure (drawing substitute photograph) which shows the result of Example 2-3 (when 0.05% Tween20 and 300 mM 18-crown-6ether are used as additives). It is a figure (drawing substitute photograph) which shows the result of Example 2-3 (when 0.1% Tween20 and 300mM18-crown-6ether were used as additives).
  • FIG. corresponds to the sequences of single-stranded circular DNA1, 2 (cT1, cT2) and primers 1, 2 (P1, P2) used when detecting region 1 as a target site, and each site in FIG. 1 in those sequences. It is a figure which showed the arrangement with reference to the reference numeral of FIG.
  • the SARS-CoV-2 detection kit is (I) A sequence of 10 to 30 bases complementary to the first site of SARS-CoV-2 derived nucleic acid, The 7-8 base first primer binding sequence adjacent to the 5'side of the sequence and The second single-stranded circular DNA binding sequence and First single-stranded circular DNA containing (Ii) A sequence of 8 to 15 bases of SARS-CoV-2-derived nucleic acid complementary to the second site adjacent to the 3'side of the first site. A sequence of 7 to 8 bases adjacent to the 3'side of the sequence and complementary to the first primer binding site of the first single-stranded circular DNA.
  • a sequence complementary to the second primer binding sequence of the second single-stranded circular DNA adjacent to the 3'side of the sequence With a second oligonucleotide primer, including Including The first oligonucleotide primer is bound to the carrier via its 5'end.
  • the second oligonucleotide primer is bound to the carrier to which the first oligonucleotide primer is bound via its 5'end.
  • SARS-CoV-2 detection kit SARS-CoV-2 detection kit.
  • the target nucleic acid is a nucleic acid derived from SARS-CoV-2, preferably genomic RNA.
  • examples of the sequence of genomic RNA derived from SARS-CoV-2 include the sequence set forth in SEQ ID NO: 1.
  • SARS-CoV-2 also includes variants and variants. Therefore, the sequence of the genomic RNA derived from SARS-CoV-2 may be a sequence having 95% or more, 98% or more, or 99% or more identity with the base sequence set forth in SEQ ID NO: 1.
  • the first single-stranded circular DNA contains a sequence of 10 to 30 bases complementary to the first site of the SARS-CoV-2-derived nucleic acid.
  • the second single-stranded circular DNA binding sequence and including.
  • the first single-stranded circular DNA 20 has a sequence 201 complementary to the first site 211 of the target nucleic acid 21 (SARS-CoV-2-derived nucleic acid), a primer-binding sequence 202 linked to the 5'side thereof, and a second sequence. Includes double-stranded circular DNA binding sequence 203.
  • the length of sequence 201 is usually 10 to 30 bases, preferably 15 to 25 bases, and the GC content is preferably 30 to 70%.
  • the length of the sequence 202 is 7 or 8 bases, and the sequence is not particularly limited, but the GC content is preferably 30 to 70%.
  • the total length of the first single-stranded circular DNA 20 is preferably 35 to 100 bases.
  • the first single-stranded circular DNA 20 can be obtained by circularizing the single-stranded DNA (ssDNA). Circulation of single-stranded DNA can be performed by any means, for example, CircLigase®, CircLigaseII®, ssDNALigase (Epicentre), ThermoPhageligase® single-stranded. It can be performed using DNA (Prokzyme).
  • the first oligonucleotide primer 22 is a sequence 221 of 8 to 15 bases complementary to the second site 212 adjacent to the 3'side of the first site 211 of the SARS-CoV-2 derived nucleic acid 21 and its 3 Includes a 7-8 base sequence 222 complementary to the primer binding site 202 of the first single-stranded circular DNA 20 linked to the'side.
  • the region containing the first site 211 and the second site 212 adjacent to the 3'side thereof, that is, the first single-stranded circular DNA and the first oligonucleotide primer are hybridized.
  • a region suitable for the SATIC reaction in the SARS-CoV-2 derived nucleic acid sequence can be selected, but preferably the region of base number 2000 to 3000 of SEQ ID NO: 1 or the region of base number 21000 to 22000.
  • the first oligonucleotide primer 22 is attached to the carrier via its 5'end.
  • the first oligonucleotide primer 22 can bind to the carrier via its 5'end, the first oligonucleotide primer 22.
  • the nucleic acid amplification reaction based on the target nucleic acid 21 can be carried out by the rolling circle amplification (RCA) method described later using the above-mentioned, those embodiments are not limited.
  • Examples of the 5'end of the first oligonucleotide primer 22 include those modified with a biotin, an amino group, an aldehyde group, or an SH group.
  • Examples of the carrier include a carrier capable of binding to each of these, and examples thereof include a carrier on which avidin (including a derivative thereof, that is, for example, streptavidin, neutravidin, etc.) is immobilized, and an amino group and an aldehyde group.
  • the carrier preferably has a carrier capable of fixing the first oligonucleotide primer 22 and the second oligonucleotide primer 25, which will be described later, in the vicinity thereof.
  • the presence of both in the vicinity allows the first oligonucleotide primer 22 to amplify the first amplification product 23 and the second oligonucleotide primer 25 to amplify the second amplification product 26 in the solution. This is because it is performed more efficiently than the method described in, for example, Patent Document 1 using two kinds of primers in a free state, and as a result, a significant improvement in detection sensitivity can be achieved.
  • Preferred carriers include, for example, beads and flat carriers such as substrates used in sensors and the like.
  • the beads are particulate insoluble carriers, and the average particle size thereof is, for example, 10 nm to 100 ⁇ m, preferably 30 nm to 10 ⁇ m, more preferably 30 nm to 1 ⁇ m, and further preferably 30 nm to 500 nm.
  • the material of the beads is not particularly limited. Examples thereof include magnetic materials (magnetic materials such as iron oxide such as ferrite and magnetite, chromium oxide and cobalt), silica, agarose and sepharose. Magnetic beads are sometimes referred to as "magnetic beads". Further, metal colloid particles such as gold colloid can also be used.
  • the second single-stranded circular DNA 24 has the same sequence 241 as the second single-stranded circular DNA binding sequence 203 of the first single-stranded circular DNA 20.
  • the second primer binding sequence 242 adjacent to the 5'side of the sequence and including.
  • the length of sequence 203 is usually 10 to 30 bases, preferably 15 to 25 bases, and the GC content is preferably 30 to 70%.
  • the length of the sequence 242 is 7 or 8 bases, and the sequence is not particularly limited, but the GC content is preferably 30 to 70%.
  • the length of the sequence 242 is 7 or 8 bases, and the sequence is not particularly limited, but the GC content is preferably 30 to 70%.
  • the total length of the second single-stranded circular DNA 24 is preferably 35 to 100 bases.
  • the second single-stranded circular DNA 24 can be obtained by circularizing the single-stranded DNA (ssDNA) by the method described above.
  • the second single-stranded circular DNA 24 preferably contains a sequence complementary to the detection reagent binding sequence.
  • the detection reagent binding sequence include a guanine quadruple chain forming sequence.
  • the sequence 243 complementary to the guanine quadruplex forming sequence is any sequence before and after it, that is, between the second single-stranded circular DNA binding sequence 203 and the same sequence 241 and between the second primer binding sequence 242. May include.
  • the second single-stranded circular DNA 24 contains a sequence 243 complementary to the guanine quadruplex forming sequence
  • the second single-stranded circular DNA 24 contains a reagent-binding sequence for detection.
  • the sequence complementary to the above is contained, and the sequence complementary to the detection reagent binding sequence is the sequence 243 complementary to the guanine quadruplex chain forming sequence.
  • the detection reagent binding sequence is an aptamer sequence or a molecular beacon (hairpin-like oligonucleotide having a fluorescent group (donor) and a quenching group (acceptor) that causes FRET), and the detection reagent is an aptamer. It is also possible to detect using a binding color-developing molecule or a molecular beacon (ChemBioChem 2007, 8, 1795-1803; J. Am. Chem. Soc. 2013, 135, 7430-7433).
  • the second single-stranded circular DNA 24 does not contain a sequence complementary to the detection reagent binding sequence, it can be detected by a known detection method capable of detecting the second amplification product 26. That is, it can be detected by a known detection method without using a detection reagent that binds to the detection reagent binding sequence.
  • a method of labeling the second amplification product 26 with a fluorescent reagent that does not bind to the first amplification product 23 but specifically binds to the second amplification product 26 and measuring the fluorescence intensity for detection is mentioned. Be done.
  • the second oligonucleotide primer 25 has the same sequence 251 as the site 204 adjacent to the 5'side of the second single-stranded circular DNA binding sequence 203 of the first single-stranded circular DNA 20 (preferably a sequence of 8 to 15 bases).
  • a sequence 252 (preferably a sequence of 7 to 8 bases) complementary to the second primer binding sequence 242 of the second single-stranded circular DNA 24 adjacent to the 3'side of the sequence. including.
  • the second oligonucleotide primer 25 is bound to the carrier to which the first oligonucleotide primer 22 is bound via its 5'end.
  • the description in the column of the first oligonucleotide primer is incorporated, but preferably the first oligonucleotide primer 22, respectively.
  • the 5'end is the same as that of the carrier. That is, for example, the first oligonucleotide primer 22 has its 5'end modified with biotin and is bound to a carrier on which avidin is immobilized via the biotin, and the second oligonucleotide primer 22 is bound. It is preferable that the 5'end of 25 is modified with biotin and is bound to the carrier to which the first oligonucleotide primer 22 is bound via the biotin.
  • the amount ratio of the first oligonucleotide primer 22 and the second oligonucleotide primer 25 immobilized on the carrier reflects the concentration ratio when each primer is immobilized on the carrier. For example, as in the examples described later, after washing the carrier, the supernatant is removed, and a mixed solution containing the first oligonucleotide primer 22 having a concentration of 1 ⁇ M and the second oligonucleotide primer 25 having a concentration of 20 ⁇ M. Can be treated as 1:20 in the amount ratio (molar ratio) of the first oligonucleotide primer 22 and the second oligonucleotide primer 25 immobilized on the carrier.
  • the quantitative ratio of the first oligonucleotide primer 22 and the second oligonucleotide primer 25 immobilized on the carrier is a molar ratio, preferably 1:10 to 1: 100, and more preferably 1:10 to 1: 1. It is 30, more preferably 1:10 to 1:25.
  • the concentration of the first oligonucleotide primer 22 during the amplification reaction (during use) is preferably 0.0025 pmol / ⁇ L or more, more preferably 0.005 pmol / ⁇ L or more, and preferably 0.04 pmol / ⁇ L or more in terms of molar ratio. Below, it is more preferably 0.02 pmol / ⁇ L or less.
  • the concentration of the second oligonucleotide primer 25 during the amplification reaction (during use) is preferably 0.0125 pmol / ⁇ L or more, more preferably 0.025 pmol / ⁇ L or more in terms of molar ratio, while 0.8 pmol / ⁇ L. Below, it is more preferably 0.4 pmol / L or less.
  • the amount ratio of the first single-stranded circular DNA 20 to the second single-stranded circular DNA 24 during the amplification reaction (during use) is a molar ratio, preferably 1: 2 to 1: 1000, more preferably 1: 3 to 1. : 500, more preferably 1: 4 to 1: 400.
  • the lower limit of the concentration of the first single-stranded circular DNA 20 during the amplification reaction (during use) is, for example, 0.1 nM or more, 1 nM or more, 10 nM or more, 50 nM or more, while the upper limit is, for example, 500 nM or less, 200 nM or less. Is.
  • the lower limit of the concentration of the second single-stranded circular DNA 24 during the amplification reaction (during use) is, for example, 20 nM or more, 40 nM or more, 100 nM or more, 200 nM or more, while the upper limit is, for example, 1000 nM or less, 500 nM or less. be.
  • ⁇ Amplification method> As shown in FIG. 1, first, the target nucleic acid 21 is hybridized with the first single-stranded circular DNA 20 and the primer 22 to form a three-way complex, and then the target nucleic acid is formed by the rolling circle amplification (RCA) method. A nucleic acid amplification reaction based on 21 is performed.
  • RCA rolling circle amplification
  • the RCA method is described in Lizardi et al., Nature Genet. 19: 225-232 (1998); US Pat. Nos. 5,854,033 and 6,143,495; PCT application WO 97/19193 and the like. ..
  • the RCA method is, for example, phi29 polymerase, KlenowDNA Polymerase (5'-3', 3'-5'exominus), Sequence (registered trademark) Version 2.0 T7 DNA Polymerase (USB), Bus DNA Polymerase, Large Fragment ( NEB) and other medium-temperature strand-replacement DNA synthases, Bst DNA Polymerase (Large Fragment), Bsm DNA Polymerase, Large Fragment (Fermentas), BcaBEST DNA polymerase (TakaraBio), Vent DNA polymerase (NEB) , DeepVent DNA polymerase (NEB), DisplaceAce (registered trademark) DNA Polymerase (Epicentre), etc. can be used.
  • the DNA elongation reaction by RCA does not require the use of a thermal cycler and is carried out, for example, at a constant temperature in the range of 25 ° C to 65 ° C.
  • the reaction temperature is appropriately set by a usual procedure based on the optimum temperature of the enzyme and the denaturation temperature based on the primer chain length (the temperature range in which the primer binds (anneals) / dissociates to DNA). Furthermore, it is also carried out at a constant relatively low temperature.
  • the reaction is preferably at 25 ° C to 42 ° C, more preferably at about 30 to 37 ° C.
  • RCA amplifies the first amplification product 23 from primer 22 along the first single-stranded circular DNA 20 in a manner dependent on the target nucleic acid 21.
  • the amplification product 23 contains the sequence 233 complementary to the second single-stranded circular DNA binding sequence 203 of the first single-stranded circular DNA 20, the second single-stranded circular DNA 24 containing the same sequence 241 as this sequence 203. Hybridizes to sequence 233 of the first amplification product 23 via sequence 241.
  • the second oligonucleotide primer 25 hybridizes to the complex of the first amplification product 23 and the second single-stranded circular DNA 24 thus formed to form a tripartite complex.
  • the second oligonucleotide primer 25 has the same sequence 251 as the site 204 adjacent to the 5'side of the second single-stranded circular DNA-binding sequence 203 of the first single-stranded circular DNA 20, the first amplification product. It hybridizes to region 234 complementary to site 204 of the first single-stranded circular DNA 20 of 23 via sequence 251. Further, since the second oligonucleotide primer 25 has a sequence 252 complementary to the second primer binding sequence 242 of the second single-stranded circular DNA 24 on the 3'side of the sequence 251, the second single-stranded circular DNA 24 has a sequence 252. Also hybridizes via sequence 252.
  • the second amplification product 26 is amplified by RCA from the tripartite complex of the first amplification product 23, the second single-stranded circular DNA 24, and the second oligonucleotide primer 25.
  • the second amplification product 26 contains, for example, sequence 261 containing a guanine quadruple chain and is detected by the guanine quadruple chain detection reagent 262.
  • the second single-stranded circular DNA 24 hybridizes to each of the regions 231 contained in the first amplification product 23, and an RCA reaction occurs.
  • the first oligonucleotide primer 22 amplifies the first amplification product 23, and the second oligonucleotide primer 25. Since the step of amplifying the second amplification product 26 from the above is performed in the vicinity, the detection sensitivity is higher than that of the method described in Patent Document 1 using two types of primers that are in a free state in the solution. Significant improvements can be achieved.
  • biological samples include, for example, blood, urine, stool, saliva, sputum, tears, nasal discharge; nose, rectal, pharynx, and urethral swabs, excretions, and secretions, as well as cells, tissues (eg, biopsy). Tissue sample) and the like.
  • ⁇ Pretreatment> When a biological sample is used for detection, it is preferable to pretreat the biological sample before subjecting it to the SATIC reaction.
  • a pretreatment liquid containing crown ether and a nonionic surfactant examples include 18-crown-6 and 15-crown-5.
  • the final concentration of crown ether during the pretreatment is, for example, 100 to 500 mM, preferably 200 to 400 mM.
  • the nonionic surfactant include polyoxyethylene sorbitan monolaurate (Tween 20) and octylphenol ethoxylate (Triton X-100, Nonidet P-40).
  • the final concentration of the nonionic surfactant at the time of pretreatment is preferably 0.01 to 0.5 v / v%, more preferably 0.02 to 0.2 v / v%.
  • the heating time is not particularly limited, but is, for example, 30 to 300 seconds, preferably 60 to 180 seconds.
  • the second amplification product 26 obtained by RCA can be detected by a known detection method as described above, but the second single-stranded circular DNA 24 contains a sequence complementary to the detection reagent binding sequence. It is preferred that the second amplification product 26 obtained by RCA contains the detection reagent binding sequence.
  • the detection reagent binding sequence is a guanine quadruple chain forming sequence or the like
  • the amplification product obtained by RCA can be detected using a guanine quadruple chain binding reagent.
  • the guanine quadruple chain binding reagent include the following reagents.
  • Thioflavin T or its derivative
  • H-aggregate "Yan, JW; Ye, WJ; Chen, SB; Wu, WB; Hou, JQ; Ou, TM; Tan, JH; Li, D .; Gu, LQ; Huang, ZS Anal. Chem . 2012, 84, 6288-6292. ”
  • TMPyP4 "Yaku, H .; Fujimoto, T .; Murashima, T .; Miyoshi, D .; Sugimoto, N. Chem. Commun. 2012, 48, 6203-6216.”
  • PPIX "Li, T .; Wang, E .; Dong, S. Anal. Chem.
  • R 1 represents hydrogen or a hydrocarbon group having 1 to 10 carbon atoms (preferably 1 to 5) which may contain one or more selected from O, S and N.
  • the hydrocarbon group may be a straight chain or a branched chain, may be saturated or unsaturated, may be an aliphatic hydrocarbon group such as an alkyl group, or may be an aromatic hydrocarbon group such as an aryl group or an arylalkyl group.
  • “It may contain one or more selected from O, S and N” means that the hydrocarbon group is an amino group (-NR 2 ) (R is independently hydrogen or an alkyl group having 1 to 5 carbon atoms). ), Nitro group (-NO 2 ), cyano group (-CN), isocyanate group (-NCO), hydroxyl group (-OH), aldehyde group (-CHO), carboxyl group (-COOH), mercapto group (-SH) ), A functional group containing a nitrogen atom such as a sulfonic acid group (-SO 3 H), an oxygen atom, a sulfur atom and the like may be contained, and an ether group (-O-) and an imino group (-).
  • a nitrogen atom such as a sulfonic acid group (-SO 3 H), an oxygen atom, a sulfur atom and the like may be contained, and an ether group (-O-) and an imino group (-).
  • R 2 , R 3 and R 4 each independently represent a (aliphatic) hydrocarbon group having 1 to 5 carbon atoms, more preferably a hydrocarbon group having 1 to 3 carbon atoms, and a methyl group is particularly preferable.
  • the hydrocarbon group having 1 to 5 carbon atoms may be a straight chain or a branched chain, and may be saturated or unsaturated.
  • n indicates an integer of 0 to 5, more preferably an integer of 0 to 3, and particularly preferably 1.
  • X represents O, S or NH, more preferably O.
  • ThT derivatives containing the following PEG chains can also be used.
  • R 5 is an amino group, a hydroxyl group, an alkyl group, or a carboxyl group
  • n is an integer of 4 to 50, preferably an integer of 5 to 20, and more preferably an integer of 8 to 15. , Particularly preferably 11.
  • ThT-PEG a compound having an amino group of R 5 is more preferable.
  • ThT-PEG-ThT A ThT derivative (ThT-PEG-ThT) in which the following ThTs are linked by a PEG chain can also be used.
  • n is an integer of 4 to 50, preferably an integer of 5 to 20, more preferably an integer of 8 to 15, and particularly preferably 11.
  • the PEG chain of ThT-PEG-ThT may be replaced with a spermine linker.
  • Detection is performed, for example, by contacting a sample containing an RCA product with a compound represented by the general formula (I) or a salt thereof, and detecting a compound bound to a guanine quadruplex structure based on the fluorescence emitted by the compound. ,
  • the guanine quadruplex structure in the test DNA can be detected.
  • the detection operation itself is the same as that of a known method except that the compound represented by the general formula (I) or a salt thereof is used, and a solution in which the compound is dissolved in a buffer solution is used as a sample containing the test DNA. It can be carried out by contacting, incubating, washing, and detecting the fluorescence of the fluorescent dye bound to the test DNA after washing.
  • ThT-PEG or ThT-PEG-ThT is used as the guanine quadruplex binding reagent in the method of the present invention
  • specific aggregation occurs when ThT-PEG or ThT-PEG-ThT binds to the RCA product. Therefore, by visually observing the aggregation, the presence or absence of RCA amplification can be easily confirmed without using a fluorescence detection device.
  • ThT-PEG and ThT-PEG-ThT may be used at the same time.
  • the concentration of ThT-PEG or ThT-PEG-ThT is, for example, 5 to 50 ⁇ M, preferably 5 to 20 ⁇ M.
  • ThT-PEG-ThT When ThT-PEG-ThT is used as the guanine quadruplex binding reagent, PEG may coexist.
  • the PEG is, for example, PEG 800 or more, preferably PEG 900 or more, while, for example, PEG 4000 or less, preferably PEG 2000 or less, more preferably PEG 1500 or less, still more preferably PEG 1200 or less.
  • the final concentration of PEG in the reaction system is, for example, 8 w / v% or more, preferably 10 w / v% or more, while, for example, 30 w / v%. Hereinafter, it is preferably 20 w / v% or less.
  • the ratio of ThT-PEG-ThT to PEG is preferably 1: 10000 to 1: 25000.
  • the reaction time is, for example, 15 minutes or more, preferably 20 minutes or more, while, for example, 3 hours or less.
  • ThT-PEG or ThT-PEG-ThT when used as the guanine quadruplex binding reagent, ThT-PEG or ThT-PEG-ThT may be added to the reaction product in a form immobilized on a carrier. Immobilization of ThT-PEG or ThT-PEG-ThT on a carrier is carried out, for example, by adding biotin to ThT-PEG or ThT-PEG-ThT and reacting biotin with streptavidin introduced into the carrier. PEG or ThT-PEG-ThT can be immobilized on the carrier.
  • ThT-PEG-ThT When immobilizing ThT-PEG-ThT on a carrier, it is preferable to provide a branched chain in the PEG chain portion, add biotin to the branched chain, and react with streptavidin introduced into the carrier.
  • ThT-PEG or ThT-PEG-ThT and PEG chain immobilized on a carrier can be used in combination with ThT-PEG and / or ThT-PEG-ThT.
  • An example of synthesis of a ThT derivative and an experimental example of guanine quadruple chain detection using a ThT derivative as an example of a guanine quadruple chain detection reagent that can be used in the method of the present invention are described in Patent Document 1. , Known.
  • ThT-PEG is described as ThT-P42 in the examples of JP-A-2018-154564. The method for synthesizing ThT-PEG-ThT is shown in Examples described later.
  • the amplified product can be confirmed by fluorescence or visual detection, it can be determined to be SARS-CoV-2 positive.
  • Primer was immobilized on nanoparticles and washed.
  • the tube was set in a magnetic rack (tube with magnet), and the magnetic beads and supernatant were separated (5 minutes). The supernatant was removed. 4 ⁇ L of the above P 1 and P 2 mixed solution was added to the washed FG beads. Incubated at 25 ° C for 30 minutes. At that time, vortex was performed every 5 minutes.
  • the tube was placed in a magnetic rack (vertical tube with magnet) and the magnetic beads and supernatant were separated (5 minutes). The supernatant was withdrawn, and 40 ⁇ L of 1 ⁇ ⁇ 29 DNA polymerase reaction buffer was added for pipetting. The above operation was performed twice more.
  • the tube was placed in a magnetic rack (vertical tube with magnet) and the magnetic beads and supernatant were separated (5 minutes). The supernatant was withdrawn, 40 ⁇ L of water was added, and pipetting was performed. The above operation was performed twice more. Refrigerated until used.
  • Result 1 (Fig. 2) In experiments using 40 mer target RNA (about 10 copies / tube), specific detection was confirmed with all detection reagents. In the experiment using the full-length target RNA (about 100 copies / tube), specific detection was confirmed in all the detection reagents. In the experiment using the full-length target RNA (about 10 copies / tube), specific detection was confirmed with the reagents for region 1 and region 2 detection.
  • RNA extraction method by heat treatment of virus 2-1-1) Preparation of primer-immobilized nanoparticles a) biotinylated primers prepared biotinylated primer (P 1 -1 (corresponding to the target region 1), P 1 -2 (corresponding to the target region 2)) and 1 ⁇ biotinylated primer (P 2) ⁇ 29 DNA polymerase reaction It was dissolved in a buffer to prepare a P 1 and P 2 mixed solution of P 1 (1 ⁇ M) and P 2 (20 ⁇ M).
  • Primer was immobilized on nanoparticles and washed.
  • the tube was set in a magnetic rack (tube with magnet), and the magnetic beads and supernatant were separated (5 minutes). The supernatant was removed. 4 ⁇ L of the above P 1 and P 2 mixed solution was added to the washed FG beads. Incubated at 25 ° C for 30 minutes. At that time, vortex was performed every 5 minutes.
  • the tube was placed in a magnetic rack (vertical tube with magnet) and the magnetic beads and supernatant were separated (5 minutes). The supernatant was withdrawn, and 40 ⁇ L of 1 ⁇ ⁇ 29 DNA polymerase reaction buffer was added for pipetting. The above operation was performed twice more.
  • the tube was placed in a magnetic rack (vertical tube with magnet) and the magnetic beads and supernatant were separated (5 minutes). The supernatant was withdrawn, 40 ⁇ L of water was added, and pipetting was performed. The above operation was performed twice more. Refrigerated until used.
  • sample for sample (sputum): 90 ⁇ L of pretreatment solution (0.055% Tween 20, 330 mM 18-crown-6 ether) was mixed with 10 ⁇ L of sample.
  • sample for sample (nasal discharge): 500 ⁇ L of pretreatment solution (0.05% Tween 20, 300 mM 18-crown-6 ether) was mixed. Each was heated at 95 ° C. for 120 seconds.
  • Result 2-1 (Fig. 3) SARS-CoV-2 was added to the sample at a concentration of 0.05% Tween 20, 300 mM 18-crown-6 ether and heated at 95 ° C for 120 seconds. was detected.
  • Primer was immobilized on nanoparticles and washed.
  • the tube was set in a magnetic rack (tube with magnet), and the magnetic beads and supernatant were separated (5 minutes). The supernatant was removed. 4 ⁇ L of the above P 1 and P 2 mixed solution was added to the washed FG beads. Incubated at 25 ° C for 30 minutes. At that time, vortex was performed every 5 minutes.
  • the tube was placed in a magnetic rack (vertical tube with magnet) and the magnetic beads and supernatant were separated (5 minutes). The supernatant was withdrawn, and 40 ⁇ L of 1 ⁇ ⁇ 29 DNA polymerase reaction buffer was added for pipetting. The above operation was performed twice more.
  • the tube was placed in a magnetic rack (vertical tube with magnet) and the magnetic beads and supernatant were separated (5 minutes). The supernatant was withdrawn, 40 ⁇ L of water was added, and pipetting was performed. The above operation was performed twice more. Refrigerated until used.
  • Result 2-2 (Fig. 4) A sample was added to the sample at a concentration of 0.05% Tween 20, 300 mM 18-crown-6 ether and heated at 95 ° C for 60 seconds to 300 seconds. And 180 seconds) or SARS-CoV-2 was detected in sample B2 nasal discharge (heating time 180 seconds).
  • Primer was immobilized on nanoparticles and washed.
  • the tube was set in a magnetic rack (tube with magnet), and the magnetic beads and supernatant were separated (5 minutes). The supernatant was removed. 4 ⁇ L of the above P 1 and P 2 mixed solution was added to the washed FG beads. Incubated at 25 ° C for 30 minutes. At that time, vortex was performed every 5 minutes.
  • the tube was placed in a magnetic rack (vertical tube with magnet) and the magnetic beads and supernatant were separated (5 minutes). The supernatant was withdrawn, and 40 ⁇ L of 1 ⁇ ⁇ 29 DNA polymerase reaction buffer was added for pipetting. The above operation was performed twice more.
  • the tube was placed in a magnetic rack (vertical tube with magnet) and the magnetic beads and supernatant were separated (5 minutes). The supernatant was withdrawn, 40 ⁇ L of water was added, and pipetting was performed. The above operation was performed twice more. Refrigerated until used.
  • Sample pretreatment Sample 10 ⁇ L to 90 ⁇ L pretreatment solution (0.055% Tween 20, 330mM 18-crown-6 ether or 0.11% Tween 20, 330mM 18-crown-6 ether or 0.055% Nonidet P-40, 330mM 18-crown-6 ether or 0.11% Nonidet P-40, 330mM 18-crown-6 ether or 0.055% Tween 20) was mixed. Each was heated at 95 ° C. for 60 to 300 seconds.
  • Target RNA (Region 1: 40base) CUCAAAGGGAUUGUACAGAAAGUGUUAAAUCCAGAGAA (SEQ ID NO: 2)
  • Target RNA (Region 2: 40base) UCACACGUGGUGUUUAUUACCCUGACAAAGUUUUCAGAUC (SEQ ID NO: 3)
  • Target RNA (Region 3: 40base) UCACUUCUAUUCUAAAUGGUAUAUUAGAGUAGGAGCUAGA (SEQ ID NO: 4)
  • cT1 (for area 1: 67base) CCCAAAAAATAACACACTTTCTGTACAATAAAAAGAAGCTGTTGTATTGTTGTCGAAGAAGAAAAGT (SEQ ID NO: 5)
  • cT1 (for area 2: 67base) CCCAAAAAATTTGTCAGGGTAATAAACACAAAAAGAAGCTGTTGTATTGTTGTCGAAGAAGAAAAGT (SEQ ID NO: 6)
  • cT1 (for area 2: 67base
  • FIG. 8 shows the sequences of the single-stranded circular DNAs 1 and 2 (cT1, cT2) and primers 1 and 2 (P1 and P2) used when the region 1 is detected as the target site in each site of FIG.
  • the corresponding sequences are shown with reference to the reference numerals in FIG.
  • Primer was immobilized on nanoparticles and washed.
  • the tube was set in a magnetic rack (tube with magnet), and the magnetic beads and supernatant were separated (5 minutes). The supernatant was removed. 4 ⁇ L of the above P 1 and P 2 mixed solution was added to the washed FG beads. Incubated at 25 ° C for 30 minutes. At that time, vortex was performed every 5 minutes.
  • the tube was placed in a magnetic rack (vertical tube with magnet) and the magnetic beads and supernatant were separated (5 minutes). The supernatant was withdrawn, and 40 ⁇ L of 1 ⁇ ⁇ 29 DNA polymerase reaction buffer was added for pipetting. The above operation was performed twice more.
  • the tube was placed in a magnetic rack (vertical tube with magnet) and the magnetic beads and supernatant were separated (5 minutes). The supernatant was withdrawn, 40 ⁇ L of water was added, and pipetting was performed. The above operation was performed twice more. Refrigerated until used.
  • Result 3 The detection result by the SATIC method is shown in FIG.
  • RT-qPCR a calibration curve was prepared using SARS-CoV-2 with a known number of copies, and the number of copies of SARS-CoV-2 in each sample was quantified. The results are shown in Table 7.
  • NC1 TAGGTGAAACATTTGTCACG (SEQ ID NO: 13)
  • PCR-RP NC1 GGCTTTTAGAGGCATGAGTA (SEQ ID NO: 14)
  • TaqMan Probe NC1 GATTGTACAGAAAGTGTGTTAAATC (SEQ ID NO: 15)
  • viruses other than SARS-CoV-2 (HcoV (human coronavirus) -229E, HcoV-NL63, HcoV-OC43, HcoV-HKU1, HcoV-229E , Human Respirovirus 1, Human Respirovirus 3, Human Orthoneumovirus, Human Metapneumovirus, ADV (Adenovirus) 4, ADV7) were used for the amplification reaction.
  • the reagents used in the reaction are as shown in Table 2, and the procedure is also as described in "2-2. Examination of RNA extraction method by heat treatment of virus (examination of heating time, use of actual sample)". rice field.
  • ThT-PEG-ThT was synthesized according to the following scheme.
  • the method for synthesizing ThT-AE is described in References (Kataoka, Y .; Fujita, H .; Afanaseva, A .; Nagao, C .; Mizuguchi, K .; Kasahara, Y .; Obika, S .; Kuwahara, M. .Biochemistry, 2019, 58, 493.).
  • ThT-PEG-ThT [Synthesis of ThT-PEG-ThT] Add dry DMF (0.3 mL) to ThT-PEG (10 mg, 10 ⁇ mol) and stir, then add HOBt ⁇ H 2 O (4.2 mg, 26 ⁇ mol) and PyBOP (14 mg, 26 ⁇ mol). After that, DIPEA (14 ⁇ L, 80 ⁇ mol) was added. Compound T1 (5.6 mg, 13 ⁇ mol) dissolved in dry DMF (0.2 mL) was added thereto, and the mixture was stirred at room temperature for 5 hours. After distilling off the reaction mixture under reduced pressure, the residue was dissolved in CH 2 Cl 2 and washed with water. The organic layer was distilled off under reduced pressure, and the mixture was subjected to solid-liquid extraction with diethyl ether and then purified by HPLC to obtain ThT-PEG-ThT. Yield: 0.82 mg Yield: 6.1%

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Abstract

La présente invention concerne un kit de détection du SARS-CoV-2 comprenant (i) un premier ADN cyclique simple brin qui comprend une séquence qui est complémentaire d'un premier site dans un acide nucléique dérivé d'un nouveau coronavirus SARS-CoV-2 et qui est composée de 10 à 30 nucléotides, une première séquence de liaison d'amorce qui est contiguë au côté 5' de la séquence et qui est composée de 7 à 8 nucléotides et une seconde séquence de liaison à l'ADN cyclique simple brin ; (ii) une première amorce oligonucléotidique qui comprend une séquence qui est complémentaire d'un second site contigu au côté 3' du premier site dans l'acide nucléique dérivé du SARS-CoV-2 et qui est composée de 8 à 15 nucléotides, ainsi qu'une séquence qui est contiguë au côté 3' de la séquence, est complémentaire du premier site de liaison d'amorce dans le premier ADN cyclique simple brin et est composée de 7 à 8 nucléotides ; (iii) un second ADN cyclique simple brin qui comprend une séquence qui est identique à la seconde séquence de liaison d'ADN cyclique simple brin dans le premier ADN cyclique simple brin et une seconde séquence de liaison d'amorce qui est contiguë au côté 5' de la séquence ; et (iv) une seconde amorce oligonucléotidique qui comprend une séquence qui est identique à un site contigu au côté 5' de la seconde séquence de liaison d'ADN cyclique simple brin dans le premier ADN cyclique simple brin et une séquence qui est contiguë au côté 3' de la séquence et qui est complémentaire de la seconde séquence de liaison d'amorce dans le second ADN cyclique simple brin, dans laquelle la première amorce oligonucléotidique est liée à un support par l'intermédiaire de la terminaison 5' correspondante ; et la seconde amorce oligonucléotidique est liée, par l'intermédiaire de la terminaison 5' correspondante, au support ayant la première amorce oligonucléotidique liée à celle-ci.
PCT/JP2021/017709 2020-05-11 2021-05-10 Kit et procédé de détection nouveau coronavirus sars-cov-2 WO2021230204A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016152936A1 (fr) * 2015-03-24 2016-09-29 国立大学法人 群馬大学 Procédé simple pour la détection de séquences d'arn
WO2018020831A1 (fr) * 2016-07-25 2018-02-01 国立大学法人群馬大学 Méthode simple permettant de détecter une séquence polynucléotidique présentant une mutation génétique
WO2018168895A1 (fr) * 2017-03-15 2018-09-20 国立大学法人 群馬大学 Procédé de détection d'une molécule cible dans lequel une amplification par cercle roulant est utilisée
WO2020213700A1 (fr) * 2019-04-19 2020-10-22 学校法人日本大学 Procédé simple pour la détection d'une séquence d'acide nucléique, etc.

Patent Citations (4)

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Publication number Priority date Publication date Assignee Title
WO2016152936A1 (fr) * 2015-03-24 2016-09-29 国立大学法人 群馬大学 Procédé simple pour la détection de séquences d'arn
WO2018020831A1 (fr) * 2016-07-25 2018-02-01 国立大学法人群馬大学 Méthode simple permettant de détecter une séquence polynucléotidique présentant une mutation génétique
WO2018168895A1 (fr) * 2017-03-15 2018-09-20 国立大学法人 群馬大学 Procédé de détection d'une molécule cible dans lequel une amplification par cercle roulant est utilisée
WO2020213700A1 (fr) * 2019-04-19 2020-10-22 学校法人日本大学 Procédé simple pour la détection d'une séquence d'acide nucléique, etc.

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FUJITA, H., ET AL. ET AL.: "Novel one-tube-one-step real- time methodology for rapid transcriptomic biomarker detection: signal amplification by ternary initiation complexes", ANAL. CHEM., vol. 88, 2016, pages 7137 - 7144, XP055602580, DOI: 10.1021/acs.analchem.6b01192 *

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