Triggered reverse cleavage activity and application of Ago protein and binary complex thereof

Abstract

The invention discloses an Ago protein with triggered reverse cleavage activity and application of the Ago protein. The Ago protein with the triggered reverse cleavage activity is combined with long single-stranded guide nucleic acid (LgDNA, long guide DNA / RNA) to be assembled into a binary complex, and when the binary complex is in contact with target nucleic acid, the activity of reverse cleavage of the LgDNA by the Ago protein can be stimulated. The invention also provides a composition, a cleavage system, a reagent / kit and a nucleic acid detection method using the Ago protein and the long single-stranded guide nucleic acid. The invention further provides a nucleic acid rapid detection method based on the Ago protein triggered reverse cleavage activity, and a wide application prospect is created.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to a triggered reverse cleavage active Ago protein, namely a non-classical nuclease active Ago protein, a binary complex thereof and applications thereof. Background Art

[0002] Nucleic acid testing, as a molecular diagnostic technique, has broad applications in various fields, including molecular medical diagnosis and infectious disease detection. Nucleic acid testing technologies include conventional PCR, real-time fluorescence PCR, isothermal nucleic acid amplification, and nucleic acid hybridization. "Nucleic acid testing" generally refers to real-time fluorescence PCR, the most widely used nucleic acid testing technology for pathogen nucleic acid detection.

[0003] Currently, a nucleic acid detection technology based on CRISPR technology is a nucleic acid detection molecule designed using the collateral cleavage ability of the Cas12 and Cas13 families. The principle is that after the Cas-crRNA complex recognizes and binds to the substrate, it cuts the substrate, uses the trans-cutting ability, and then cuts the reporter molecule, freeing the fluorescent group and detecting it, thereby realizing a method for nucleic acid detection. A nucleic acid detection technology based on the cutting ability of the PfAgo protein uses the PfAgo protein to target and cut the nucleic acid to be tested under the guidance of the guide DNA. The PfAgo protein can use the new guide DNA generated by cutting the nucleic acid to be tested, and then cut the reporter molecule, freeing the fluorescent group and detecting it, thereby realizing a method for nucleic acid detection.

[0004] Nucleic acid detection technology based on CRISPR technology and nucleic acid detection technology based on PfAgo cleavage ability are both secondary cleavage methods. In addition to fluorescent reporter molecules, they also require the addition of guide nucleic acids, which increases reaction time and cost. Summary of the Invention

[0005] In order to address the deficiencies in the prior art, the present invention innovatively proposes for the first time a triggered reverse cleavage activity of an Ago protein, that is, a non-classical nuclease activity of an Ago protein. Specifically, the Ago protein of the present invention has a triggered reverse cleavage activity. The present invention also proposes the application of the Ago protein. The present invention also innovatively proposes a one-step rapid nucleic acid detection method based on the Ago protein, that is, after Ago binds to a long single-stranded guide nucleic acid (LgDNA, longguide DNA / RNA) and targets the target nucleic acid, it activates the non-classical cleavage activity / triggered reverse cleavage activity of the Ago protein to cut the LgDNA, and a one-step rapid nucleic acid detection method based on the non-classical nuclease activity / triggered reverse cleavage activity of the Ago protein, which does not require the addition of an additional guide nucleic acid except for the fluorescent reporter molecule, thereby reducing the reaction time and cost.

[0006] The present invention proposes for the first time that Ago protein and its binary complex have triggered reverse cleavage activity. Specifically, the Ago protein is combined with LgDNA to form a binary complex, and the binary complex targets and binds to the target nucleic acid. Preferably, the target nucleic acid is specifically cut into n sequences, where n≥2; the LgDNA is also specifically cut into m sequences, where m≥2. In a specific embodiment, the Ago protein with triggered reverse cleavage activity is combined with a long single-stranded guide nucleic acid (LgDNA, longguide DNA / RNA) to form a binary complex. When the binary complex contacts the target nucleic acid, it will stimulate the activity of the Ago protein to reversely cut the LgDNA.

[0007] In this invention, "Argonaute" or Ago proteins are a family of nucleic acid-binding proteins commonly found in bacteria, plants, archaea, and animal cells. "PIWI domain-containing proteins" are proteins that have been shown to contain a PIWI-like domain, as determined by protein sequence alignment in major databases (e.g., NCBI); the PIWI-like domain is used to bind nucleic acids. Ago proteins and "PIWI domain-containing proteins" can exist independently or contain overlapping regions.

[0008] In the present invention, the Ago protein includes but is not limited to an Ago protein that can promote or improve the cleavage of LgDNA. In some specific embodiments, it includes but is not limited to a protein that can promote or improve the speed, efficiency, accuracy, sensitivity, etc. of cleavage of LgDNA. In some specific embodiments, it includes but is not limited to any protein that is structurally or functionally identical or similar to an Ago protein. It can also be a protein that includes a nucleic acid sequence or amino acid sequence encoding a PIWI domain.

[0009] In the present invention, the expressions Ago protein, Argonaute protein, and Argonaute nuclease are used interchangeably and have the same meaning, including naturally occurring or engineered proteins.

[0010] In the present invention, the Ago protein may be an Ago protein having protein properties formed by connecting amino acids, or an Ago protein derived from an expression element including its encoding RNA, an expression cassette integrated into a host chromosome, or an expression plasmid.

[0011] Preferably, the Ago protein includes eukaryotic Ago protein and prokaryotic Ago protein.

[0012] Preferably, the Ago protein includes a protein indicated to contain a PIWI-like domain by protein sequence alignment in major databases (such as NCBI); the PIWI-like domain is used to bind to nucleic acid.

[0013] Preferably, the Ago protein can form a binary complex with the LgDNA, the LgDNA is ≥21 nt in length, and its GC content is 10% to 80%; more preferably, the LgDNA is 26 to 60 nt in length, and its GC content is 20% to 60%.

[0014] Preferably, the Ago protein can specifically cut the target nucleic acid into n sequences, wherein n≥2 (for example, n is an integer of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more).

[0015] Preferably, the Ago protein can cut LgDNA into m sequences, wherein m≥2 (for example, m is an integer of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more).

[0016] Specifically, the Ago protein binds to the long single-stranded guide nucleic acid and assembles into a binary complex. When the binary complex recognizes the target nucleic acid, the Ago protein reversely cuts the long single-stranded guide nucleic acid.

[0017] Preferably, the Ago protein includes a protein indicated to contain a PIWI-like domain by comparison with a protein sequence in a database; the PIWI-like domain is used to bind to the target nucleic acid.

[0018] The Ago protein has a triggered reverse cutting activity and can cut two complementary long single-stranded nucleic acids that exist simultaneously. The two complementary long single-stranded nucleic acids serve as guide nucleic acids and target nucleic acids for each other.

[0019] Specifically, the amino acid sequence of the Ago protein includes one or more of the sequences shown in SEQ ID NO.1 to 23; and / or, one or more amino acid sequences having at least 50% sequence homology with the sequence shown in any one of SEQ ID NO.1 to 23; and / or, an amino acid sequence in which one or more amino acid residues are deleted, substituted, added or inserted into the amino acid sequence shown in any one of SEQ ID NO.1 to 23.

[0020] Preferably, the Ago protein includes AaAgo, TheAgo, ThtAgo, TaAgo, and PfAgo.

[0021] Specifically, the long single-stranded guide nucleic acid includes DNA and RNA, and in some specific embodiments, is DNA.

[0022] Specifically, the target nucleic acid includes dsDNA, ssDNA and RNA. The target nucleic acid source includes in vitro amplification products, plants, animals, microorganisms, viruses, or a combination thereof.

[0023] Specifically, the length of the long single-stranded guide nucleic acid is within the range of being able to bind to the Ago protein and be cleaved by the Ago protein.

[0024] The present invention innovatively provides a one-step rapid nucleic acid detection method based on the non-classical nuclease activity / triggered reverse cleavage activity of the Ago protein, wherein the Ago protein is an Ago protein with triggered reverse cleavage activity; and / or, the detection method is a nucleic acid detection method based on a one-step cleavage system established by the triggered reverse cleavage activity of the Ago protein; and / or, the reaction system of the detection method comprises: the Ago protein, a modified long single-stranded nucleic acid probe and a detection reagent; and / or, the steps of the detection method comprise: when the nucleic acid to be detected is present, triggering the triggered reverse cleavage activity of the Ago protein, cleaving the long single-stranded nucleic acid probe, and generating a detectable signal, such as Figure 2 or Figure 4 shown.

[0025] Specifically, the reaction system of the detection method includes: Ago protein, a modified long single-stranded nucleic acid probe and a detection reagent; the reaction process of the detection technology is: when the nucleic acid to be detected is present, the triggered reverse cleavage activity of the Ago protein is triggered, the long single-stranded nucleic acid probe is cut, and a detectable signal is generated.

[0026] In the one-step rapid nucleic acid detection method of the present invention, the steps are further repeated at least once, and the reaction is repeated multiple times, and the Ago protein cleaves the LgDNA fluorescent probe to achieve nucleic acid detection. In a specific embodiment, the steps are repeated once or more until the desired nucleic acid detection level is achieved.

[0027] The one-step rapid nucleic acid detection method of the present invention includes, but is not limited to, detecting in vitro amplification products, plants, animals, microorganisms, viruses, or combinations thereof.

[0028] In the present invention, the Ago protein includes but is not limited to an Ago protein that can promote or improve the cleavage of LgDNA. In some specific embodiments, it includes but is not limited to a protein that can promote or improve the speed, efficiency, accuracy, sensitivity, etc. of cleavage of LgDNA. In some specific embodiments, it includes but is not limited to any protein that is structurally or functionally identical or similar to an Ago protein. It can also be a protein that includes a nucleic acid sequence or amino acid sequence encoding a PIWI domain.

[0029] Preferably, the Ago protein includes eukaryotic Ago protein and prokaryotic Ago protein.

[0030] Preferably, the Ago protein includes a protein indicated to contain a PIWI-like domain by protein sequence alignment in major databases (such as NCBI); the PIWI-like domain is used to bind to nucleic acid.

[0031] In the present invention, the expressions Ago protein, Argonaute protein, and Argonaute nuclease are used interchangeably and have the same meaning, including naturally occurring or engineered proteins.

[0032] Wherein, the Argonaute protein is one or more selected from the sequences shown in SEQ ID NO.1 to 23; and / or, one or more selected from the amino acid sequences having at least 50% sequence homology with the sequences shown in any one of SEQ ID NO.1 to 23; and / or, an amino acid sequence in which one or more amino acid residues are deleted, substituted, added or inserted into the amino acid sequence shown in any one of SEQ ID NO.1 to 23. In a specific embodiment, the Argonaute protein comprises one or more selected from the sequences shown in SEQ ID NO. 1-23 have at least about 50% sequence homology (such as at least about 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, 3%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more). In a specific embodiment, the Argonaute protein comprises an amino acid sequence selected from SEQ ID 1 has at least about 50% sequence identity (such as at least about 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, %, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more of the amino acid sequence.

[0033] Preferably, the Argonaute protein is one or more of AaAgo, NgAgo, TheAgo, ThtAgo, MbAgo, MjAgo, TaAgo, PfAgo, TtAgo, FpAgo, MpAgo, ToAgo, TeAgo, StAgo, CalAgo, KpAgo, PoAgo, TvAgo, BeAgo, ThaAgo, ThbAgo, TfAgo, and AraAgo.

[0034] More preferably, it is one or more of AaAgo, TheAgo, ThtAgo, TaAgo, PfAgo, TtAgo, FpAgo, MpAgo, ToAgo, etc.

[0035] In some specific embodiments, specifically, the Argonaute protein is AaAgo, TheAgo, ThtAgo, TaAgo, or PfAgo.

[0036] The Argonaute protein is derived from, but is not limited to, one or more organisms selected from the group consisting of those listed in Table A below. The Argonaute protein includes, but is not limited to, one or more of the proteins listed in Table A below, and the corresponding protein sequences include, but are not limited to, one or more of SEQ ID NOs. 1-23; preferably, it is derived from a hyperthermophile, Thermococcus, Thermus, or Methanothermococcus.

[0037] Table A

[0038]

[0039]

[0040] SEQ ID NO.1:*

[0041] SEQ ID NO.2. TWKITSFRKDIDVSPNAVEEIILKVEDPSRYNAELKRINEYFITRTSLEPIDDSKYPDKYNMILITKGKRYSYHPSRAMLIRPVEGELQKKIHHSGEFLKALNIAAAEYIKDIIESKRATFEGVQIPSQDIYYKATFLENGRIIRKKIAPYYNIKNTFNWLFKNEPFEKTYSLIVPTKTPKFIKDKKIAI YLLYPKEFSKERTTLVSDVKSALDRVSEYFAYLRTISMTSSSLSLPVEITTPLIAVDYTNLAQRLTKIFEKYENYDYHLAITFVPSMSREQFDRIKKFFFERGIIHKAINIDNLNSKFGKNKQRIIESVILQALYAFGIYFYSLDNLPYDIIIGIDVTREKDKNGKYYGISGAAVVQNKNGEIIKIVPITQPQSSSETVDVEKIFESLQPELDQIVETKSNVDILILRDGRIPSREIEQLKRLSLRRPYTFTLVGIIKRPLVRFKGTDRHLFGPKPNYYFRIGDTYYLTSHFLEHYLKVPLKLERKYVISRGKLATSPLNHEDILAITKLTKLNYSQPKNPDRMKLPAPVHLSHRLINYERRGLRFARPEFLQEGALYFL

[0042] SEQ ID NO.3:MLMKVLTNMVKLNQDIIPNEIYLYKIFNKPEDGMNIYKIAYRNHGIVIDPQNRIIATPSELEYSGKFAIEDEISFNELPENYQNRLVLRILRDNGISDHALSRTLQKYRKPKPFGDFEVIPEIRSSVIKHGGDFYLVLHLSHQIRSKKTLWELVGRNKDALRDFLKEHRGTILLRDIASEHKVV YKPIFKRYNGDPDLIEDNSNDVEHWYDYHLERYWNTPELKKEFYKKFGPVDLNQPIILAKPLRQHNRGDLVHLLPQFVVPYNAEQNLDILASEILEYLKLTSNQRISLLSRLINDIKTNTNIIVSSLTLEANTFDVDLNDMLQVRNADNVKVTLSELEISKTRLFTWMKSRKYPVILPYDIPQKLKKI EKIPVFIIVDSALSRDIQTFAKDEFRYLISSLQKSLSNWVDFPILDIRDKYIFTIDLTSDKDIVNLSIKLVNLMKNAELGLALIATRTKLPNETFDEVKKRLFSVNIISQVVNEATLYKRDCKYNESRLNLYVQHNLLFQILSKLGIKYYVLRHKFSYDYIVGIDVTPMKLSHGYIGGSAVMFDSQGYIRKIIPVEIGEQMGESIDMKEFFKDMVVQFGKFGIDLEGKSILILRDGKITKDEEEGLAYISKVFGIKITTFNIVKRHLLRIFANRKLYRLANSVYLLPHRIKQSVGTPVPLKLSEKRLILDGTITSQEITYNDIFEILLSELNYGSISADMKLPAPVHYAHKFVRALRKGWRIREELLAEGFLYFVSEQ ID NO.4:.

[0043] SEQ ID NO.5:MVLNKVTYKINAYKIKEEFIPKEVHFYRIKSFVNEAFNFYRFVNFYGGMIINKKDKSFVLPYKVDNKVLKYKDGNNEIPIDIEYIKSLKLEYVKPEIAEKLVRGYLKSVHKIEPELSRIIKNIRKHKVVENIKVESYCEYEVKKHDGDYYLILNFRHTASITKHLWDFVNRDKALLEEYVGKKIIFKPNPKVRYTISLVDAPNPQKIEEIMSHIIKYYKWSEDMVKSTFGEIDYNQPIMYCEEILEPFAPQFCNLVFYMDELDSYILKELQSYWRLSNENKGKIINEIAKKLRFIDNTPKELEFMKFNNTPLLVKDVNKNPTKIYSTNTLFTWIYNQNAKIYLPYDVPEIIRNKNLLTYILIDEEIKDELKAIKDKVNKMFRNYNKIANKTELPKFNYANRWKYFSTDDIRGIIKEIKSEFNDEICFALIIGKEKYKDNDYYEILKKQLFDLKIISQNILWENWRKDDKGYMTNNLLIQIMGKLGIKYFILDSKTPYDYIMGLDTGLGIFGNHRVGGCTVVYDSEGKIRRIQPIETPAPGERLHLPYVIEYLENKANIDMENKNILFLRDGFIQNSERNDLKEISKELNSNIEVISIRKNNKYKVFTSDYRIGSVFGNDGIFLPHKTPFGSNPVKLSTWLRFNCGNEEGLKINESIMQLLYDLTKMNYSALYGEGRYLRIPAPIHYADKFVKALGKNWKIDEELLKHGFLYFI

[0044] SEQ ID NO.6: MKMDTNFWFRWDGWNVLELPEDISHEILQLEVYPYSSKKEAYEHLPEELKDELNKKLLIKYSDGEYYYYFFYDKLNFLARIKDFRPMSLLPVKISKYPQKTAISLMSAYVKKILGKKLKAYVDSAKIQGFLENPDIGVRIRRIKTPQIKVVGFNEKSVYVAIKYSSETETKLWELPEKSLDQVMALIEEHVQ NEEEIKSKSPKIKSNSVFSYIVKILKDKEAYDFLLQLKNSTRIKERNAYLRIEESIKSLNPAKSPLDAKYVILAVPKSKYSRSSFSKYQARFSSYLQDLFIFTELNSELIIDPKLESILFNKQEEDVVKNYANAVKKAEFVLKFKSTPYVTRLPEIKNILMAQTEKGPIFGWGVRGLLLTKETFREDLIPFVNHKGSL KILLYYPEHLERKLKRSIFYKRVLSLKEGVYGKAFKNGAEIVDRPLEKGVWKELQTTSALDKYSEKIIEDFESMKKDQSDTFLLLTLLPKDKNIVKFHKMRSYLLKKPNLRVAIQGINESGLDDNYKSYSALLQTAPKLGVYMYSLNPEVISPKYDLILGIDVTRQYEATQRGVAASVVLMTPNGIPKGGFAISQNTNNKETVNLFEVFQELFSAPQVKKELKNRNSEEIGILLARDGFFTISEREDLSTILESGIFEFGNSGVTLNGVEIIKDTGIRIWKHKKFLPSYTSLPSGNLTQYVVLGHKGMNIQKGTSYARPYTVRGYYYSKDGYIRGPETLPRNLLHYLIYLQRLNYTTYLDAVISLPAPVHFAHKCSNFVRKFEISRVAIENALFFVT

[0045] SEQ ID NO.7:MKAKVVINLVKINKKIIPDKIYVYRLFNDPEEELQKEGYSIYRLAYENVGIVIDPENLIIATTKELEYEGEFIPEGEISFSELRNDYQSKLVLRLLKENGYEELSKLLRKFRKPKTFGDYKVIPSVEMSVIKHDEDFYLVIHIIHQIQSMKTLWELVNKDPKELEEFLMTHKENLMLKDIASPLKT VYKPCFEEYTKKPKLDHNQEIVKYWYNHIERYWNTPEAKLEFYRKFGQVDLKQPAILAKFASKIKKNKNYKIYLLPQLVVPTYNAEQLESDVAKEILEYTKLMPEERKELLENILAEVDSDIIDKSLSEIEVEKIAQELENKIRVRDDKGNSVPISQLNVQKSQLLLWTNYSRKYPVILPYEVPEKFRKIREI PMFIILDSGLLADIQNFATNEFRELVKSMYYSLAKKYNSLAKKARSTNEIGLPFLDFRGKEKVITEDLNSDKGIIEVVEQVSSFMKGKELGLAFIAARNLKLSSEKFEEIKRRLFNLNVISQVVNEDTLKNKRDKYDRNRLDLFVRHNLLFQVLSKLGVKYYVLDYRFNYDYIIGIDVAPMKRSEGYIGGSAVMFDSQGYIRKIVPIKIGEQRGESVDMNEFFKEMVDKFKEFNIKLDNKKILLLRDGRITNNEEEGLKYISEMFDIEVVTMDVIKNHPVRAFANMKMYFNLGGAIYLIPHKLKQAKGTPIPIKLAKKRIIKNGKVEKQSITRQDVLDIFILTRLNYGSISADMRLPAPVHYAHKFANAIRNEWKIKEEFLAEGFLYFV

[0046] SEQ ID NO.8:MNHLGKTEVFLNRFALRPLNPEELRPWRLEVVLDPPPGREEVYPLLAQVARRAGGVTVRMGDGLASWSPPEVLVLEGTLARMGQTYAYRLYPKGRRPLDPKDPGERSVLSALARRLLQERLRRLEGVWVEGLAVYRREHARGPGWRVLGGAVLDLWVSDSGAFLLEVDPAYRILCEMSLEAWLAQGHPLPKRVRNAYDRRTWELLRLGEEDPKELPLPGGLSLLDYHASKGRLQGREGGRVAWVADPKDPRKPIPHLTGLLVPVLTLEDLHEEEGSLALSLPWEERRRRTREIASWIGRRLGLGTPEAVRAQAYRLSIPKLMGRRAVSKPADALRVGFYRAQETALALLRLDGAQGWPEFLRRALLRAFGASGASLRLHTLHAHPSQGLAFREALRKAKEEGVQAVLVLTPPMAWEDRNRLKALLLREGLPSQILNVPLREEERHRWENALLGLLAKAGLQVVALSGAYPAELAVGFDAGGRESFRFGGAACAVGGDGGHLLWTLPEAQAGERIPQEVVWDLLEETLWAFRRKAGRLPSRVLLLRDGRVPQDEFALALEALAREGIAYDLVSVRKSGGGRVYPVQGRLADGLYVPLEDKTFLLLTVHRDFRGTPRPLKLVHEAGDTPLEALAHQIFHLTRLYPASGFAFPRLPAPLHLADRLVKEVGRLGIRHLKEVDREKLFFV*

[0047] SEQ ID NO.9:*

[0048] SEQ ID NO.10:YLNLYKIDIPKKIKRLYFYNPDMEPKLFARNLSRVNNFKFQDSNDLVWIEIPDIDFQITPKNVFQYKVEKEEIIKEEEDKKLFVKTLYKYIKKLFLDNDFYFKKGNNFISNSEVFSLDSNENVNAHLTYKIKIHNISNEYYLSILPKFTFLSKEPALESAIKSGYLYNIKSGKSFPYISGLDGILKIDIGNNQIVEVAYPENYLFNFTTRDAEKYGFSKEVHEIYKNKVFEGFKKIPKTLGFLNKITNLENYQLKDGYKIFINVIYKFKNGESRYAKDVFKYSFYKNEQPLKAIFFFSSKKQFFEVQKSLKELFH NKHSVFYRAAAELGFSKVEFLRDSKTKSSAFLYNPEEFTTVKNTEFINQIEDNVMAIVLLDKYIGNIDPLVRNFPDNLILQPILKEKLEDIKPFIIKSYVYKMGNFIPECKPFILKKMEDKEKNLYIGIDLSHDTYARKTNLCIAAVDNTGDILYIGKHKNLELNEKMNLDILEKEYIKAFEKYIEKFNVSPENVFILRDGRFIEDIEIIKNFISYNDTKYTLVEVNKNTNINSYDDLKEWIIKLDENTYIYYPKTFLNQKGVEVKILENNTDYTIEEIIEQIYLLTRVAHSTPYTNYKLPYPLHIANKVALTDYEWKLYIPY

[0049] SEQ ID NO.11:*

[0050] SEQ ID NO.12:PRETCYDKRTTPSQYGWLPIDSLSVMPTQFQEVEVILNRFFVKKLSRPDLTFHEYQCQFTQVPEQGSEQKAISSVCYKLGVTAVRLGSCIITREPIDPERMRTKDWQLQLIGCRELSCQNYRERQALETFERKILEEKLKETFKKTIIEKDYELGLIWWISGEEGLEKTGHGWEVHRGRQIDLKIETDEKLYLEIDIHHRFYTPFKLEWWLSEYPNIQIKYVRNTYKDKKKWILENFADKSPNEIQIEALGISLAEYHRQEGATQQEIDESRVVIVKKISDYKAKPVYHLSQRLSPILTMETLAQIAEQGREKKEIQGVFDYIRKNIGTRLQESQKIAQVIFKNVYNLSSQPEIMKVNGFVMPRAKLLARNNKEVNQTARIKSFGCAKIGETKFGCLNLFDNKPEYPEEVHKCLLAIARSSGVQIKIDSYFTGSDYPKDDLAQQRFWQQWAAQGIKTVLVVMPWSPHEEKTRLRIQALKAGIATQFMIPTPQDNPYKALNVALGLLCKAKWQPVYLKPLDDPQAADLIIGFDTSTNRRLYYGTSAFAILANGQSLGWELPDIQRGETFSGQSIWQVVSKLVLKFQDNYDSYPKKILLMRDGLVQDGEFEQTIRELTHQGIDVDILSVRKSGSGRMGRELTSGNTAITYDDAEVGTVIFYSATDSFILQTTEVIKTKTGPLGSARPLRVVRHYGNTPLELLALQTYHLTQLHPASGFRSCRLPWVLHLADRSSKEFQRIGQISLLQNVDREKLIAV*

[0051] SEQ ID NO.13:MEILYNLYENCKVYYRMGSSGTNLVDSRYYIIRFGPYSKASPTDPNINVDHYKKVLIICNRKDKRLCDNLKNDLEKGLPNNLIFKPFNEIFGTKLDFQVVEYDSDRYDEEKIMKVYEDEYEPRTFPLVITPKLAKSQIDSIYYRVKASFLDSGLEERATPTQIVTVDLLKQRDENKNTNYSWSLLPIAVQMFTKMGGIPYALKQSCINIGSEFNVHFIGLGLTSDPRNKLKRVGFVTIFNDNGSLSYMDSNILEDNKTESYGRIIYNAINRIVNLSNTNKSSNYNILVVHYSGKSLSKDDDQLIRNAIQIALSKKRNIEVYVAKVSKSNIFLIDTDSKTTDKQGSLTYHPRIGIVLKLKDNLYLMNMGGAITDRKINLPTGGFPSTLLISIHKELSSGESLDDDDLVKSVFIMSRVSYSNLSNPVLSEPITIKYSRSIAYLTLRLSYLNEKIELPEHIKKVMWFI

[0052] SEQ ID NO.14:*

[0053] SEQ ID NO.15:LEIFKSNIQLKEQKFHEYTLSGVGSVDNFYEAVGKAQYALSSQNKFEFVSRMGDMYLYSLKKLENMPEIKGVKIEYTGCKSLDIKNHKEVYSNLIVYYINKILKNKKIYDKTEEEKEVEKYKIKFRKSILNPVILQKEENKKIYTSIVNRDGIRVYRVFDIMPFIDDHGYAYLRCDINHIFESDKTIYDMMKENKNVIGLKVDYLWTNNTNSTGIIEGILDKTISDKLSELKGQSLIDYFINNKQGYRVNKFTEEDKKANVIKVKGHKNTFYFIPHSLKQVIDRESLKSIDSKFSSDIEKYIKMDMNYRMRSLSDFLDDIGEIKELENLRFDFNLNSIDKLGFKQGKIDKPCFLVSNGKVIKNKKEVFCKGYYL KPNNKIRVAIMCPEGYVEESKNALSKILSFNKNGYIVSNDKTEQKNFAIKSLLDLEYTGIFCKYKLGDITEYKRIAGNLIKSEKEFDLVLCVIPNKSDESNPYSEFKKIWAKHNIPSQMICIDSLKIINNCKDLNNYNGIYYIYEISLGILAKSGGVPWGLKEINSDVDCFVGLDVGNPYKGIRYPSSSVVFDKNGTLINYFKPEIPQTGEIIVEGILQEIFDEIILSYDRKFKQKPKHIVIHRDGFARENINWYINYFSQKNIKFDIVEVKKQGAVKFAIKDKEIFKNPEAGCYLTNGGKYAYLITNDIKIGSPRPLKIEKVYGDTSLDDIVKQIFYLSELHVGSFKKTRLPITTEYADRISKNIMYIPSGCLSEKLFFL

[0054] SEQ ID NO.16:MEQFFLSELFLDTRSNQIPLYFYHVPVLDLEKIDQYHYKTLRLLEKQNPDQTIHFYRHLIGSFHPIKYWGEMGDQTLVHRPIKTNIIEERKLLERLLIKNIEQAQNRDHFYTYRGRITKKKPETSSTILSFRYLTLHTTIDHTSRISIGFNMGHTLLHKMNLYQLLQEKTNILHEGMEVFDPYNRSTYKFIGQSQATVSDPILPGNQSILEYLRQQGRTNLKIPQNTPAVTVESKGRSKKQFHFAPQLLKLICSFDQVSIPQQIQQQIKLNAHKRTEMMRKFVEHVLSNWQNKAPFPVSFSMNSTQPKQHGYHIKQFKDPFLLFGRGFTTQDQKNGLKQGGSYTRPAQKVKYQYLIQPEIIDRLVNQKNHFQFAKSLEQQSSKWGVELDIHKTGRKELDFSNSTKLRDSLKEIAPTLQYPTVVIIDEQYRTNKAIYRIIKQECGREKNIPTQVVYLDTTHNKYAYANILLGLYAKAGIQPWILKKPLHSPCFIGLDVSRDDGKHSTGVVQAVGQDGRILFAQSLTSTERGEIISEDSLQQIISTIRYHYNKEFGHNPPHITFHRDGRGYEGELRAITRVMNEMGIPFDYISVVKNCQRRMALYNEKQGKYRNALGYTYIKDNFAYLCTTNPRDALGMARPIKIEQQYGHLTMDQVLEDIYHLTFMNTHSTLRPRLPVTVNYADKSSTYYNRGLLPLGESNGLPFV

[0055] SEQ ID NO.17:MNTPLTHYVLTEWESDTNTNVLNIHLYTLPVRNVFEQHTENGNASFDLRKLNRSLVIDFFNHYIASWQPIENWGEYTFTQYEIRSINPTILAERTILERLLLRTIESVQPKKEIAAGSRKFTWLKAEKVVENISIHRVIQCDITVDYTGKITIGFDLNHSYRTNASVYDLIKSNAIFKGDRVIDIYNNLHYEFVEISNSTINDAIPELNQSVVSYFTKERKQAWKVDKLEQSMPVVYLKAFNGTRIAYAPAMLQKELTFESLPSSVVRQTSEIYKQNANQKIKTLLDEIQKILARTDKIKFNRQKLLVQQAGYEILELSNPTLQFGKNVTQTQLKYGLDKGGVVASKPLSVNLLVYPELIDTKLDVITDFNDKLNTLSHKWGVPLSILKKSGAYRNKSIDFTNPHQLAILLKELTKNSFQELTLVIIPEKISGMWYDTVKKEFGGNSSVPTQFITMETLQKANDYILGNILLGLYSKSGIQPWILNSFLSSDCFIGLDVSHEAGRHSTGIVQIVGKDGRVLSSRANTSNEAGEKIRHETMCQIVYSAIDQYHQHYNERPKHITFHRDGFCREDLDSLDEVMNSLGVEYDMVEIIKKTNRRMALNVDKQGWETKPGLCYVRDDLAYLIATNPHPRVGTAQPIKIIKKKGSLPIETIIQDIYHLSFMHIGSLLKCRLPVTTYYADLSSTFFNRQWLPLDSGKSLHFV

[0056] SEQ ID NO.18:MDNYILTEYKAGIHASEIKIHIYRMPVKDLEKIDYEYGKYTRDLRQKNRKTISFYRSLIGSFEKLTIVPKGYEKYERSIKLDQSEESLQERKLLERLIFDGLRDSNNHFMSTEQSIIEKEPIKSLSKCKIHRGIYIDITVKEKGDIFIGFELKHSIQSTHTIIKALKEKKLNKGDKVFDFLNSAHYEFEGISDKTISDPLPELGNKSIIQHYKTKPSIYCHLVKKPNMPAILVRSKSGKVYPYPPQLLKKECLMKDVPAKEHSSIKLNPNDKINYSIEIMKRIIDAFENRYFPIGFEKNNLNIAKLGYRRRLVPDPLLRIGNGATCNHRDLKAGAFLRHKIYDSVSSPIY YQLLLDQPFEREWQKKMSEAFITKMENRSRQWGIKLQCTGNQILPTSNPYALRLHLKDINLDTDIISVVLLDETKQEGEEVYSTIKKELGGTRGAHTQVILIDSLKNEYTIPQILLGIYTKAGLQPWVLHQPLHADCYVGYDVSHENGRHTTGIVQVFGKDGSQIFSQPISSAEAGEKV SKETIQTMVIHVLYYYQKKVGKMPQHIVFHRDGRGYVEEIDWIKDILSNRDLTNGQSIAFDYISVIKECGRRMAYFDDIKKKYVNVPGIAYLDDNAQKAYLCSTNPYEKVGMSKPIKIVKKIGEMTLEQIVEDIYHLSFMNIDTDRKVRLPVTTNYADKSSTFFSRGYLSSQKKGIGFV

[0057] SEQ ID NO.19:MNQYYLSEWLSGTNAAELPVHIYTIPLSMEENRHSMGGKVCYGLSKQNQFQTMVFFDQYIASFKAITNWQGYHCLHYENRPIKLAASYERTLLERLIKKELEQRSRLTHVIDRGAFRLKQSKPMNAAELLIYPAIQMNIEVQVNGDIIAGFDYVHRLEYKENVQAYMTDQKINMTGRSVVDTTHARTYEYEFVEVAPYKAGEVSPYLKESVIDYYIRKKEERKLNGVSNQSPVIHVKNREGAIFPYLPHLLKLSCSFDSLPSHLSKAASKTIKMKPHEKMTELYKEAYRLLRKLPMITFPQDNIRAVKLGYEVGYASPPLLQFGNKVTNKKVTEGLSKGGVYKGGNVHVSFFVDPLLQQNPTARKNIGQFIQLLIQQSAKLGVNLIVSDKPRALRGQLSPQLLTSEEFPYQLKSISRYFDGTVIVIASQQTIDQVYRLIKKEFGGKQDMITQFVTYTEQLIDITKSFYLIQNLLLGIFVKSGKQPWVLGDSLHSDCFIGLDVSHESGNHACGMIQTISRDGTLIQQKSLSMPETGEKIHSATIEELLYDTIHFYQEHYKEPPRHITFHRDGFCREDLAFIERQLAKANIAFDYVEILKNVNRRMAVYHQERWTTEQSLYYRKNNVAYLCSTSPKEYVGMAKVMKIVQKTNHLPFDHIISDVYKLSFMHIHSMLKTRLPITIHYADLSSTFHNRDMLHPATQHTKSLPFV

[0058] SEQ ID NO.20:MEAKWVYLNRFVLRPLEERERKPLLLGVSWEPTPSLEENPHRLLARVANRVGGLAVPWKGGLLAWSPPHLREGEVGGLEGPYRFALRLEGEALLDPSRPEEREALSRLAQRRLEVGLAAAYGRGGSHEVEGSLLLGKEVRAGEGWRVRKGAYLRALVDGGGRMLLEVDIVHRILPTLTLEAWLDRYPAPRRVRSTYPAPSGRRQTWTLVAVEPGLSPEGVDLGGVSLLDYHVGRGRLREKGQAGRVVRVRDERGKEVYHLSGLLQPVLTLEDLAELGLEGALALQIPPSERFSLARKVAGHVASRVYGLRDPRPVEARGHLLSPPQLKALRGQRIAKPAEALRRGVLQGRPAKVGLLVVEGEPAWPAPLRKALLEVARASEVPLALAEPVAVARRDLLGLDLAAILERLSREAGVDALLVQTPPLSPEERNRLKRACLQRGLPSQFFNPPLEGHKLDNVLLGLVSKLGWRVMALDGAYPAELAVGFDAGADGSRSLRYGGAACAVTADGGLLSWLLPEAQRGERIHGEVVWGMLQEALVAFQRGAGRWPRHVLLLRDGKTQREEFTLALRELAKAGIGYDLVSVRKSGGGRIYPKEGERLRDGLYVPLPEAEGKTAFLLLTAYPGEGRMRATPRPLKVVHEEGSTPVRELARQLYHLSRLYPPSGYRFPSLPAPLHLADRLVREVGRVGLSSLQGLGREKLFFV

[0059] SEQ ID NO.21:MMGDSRSLEVRLNRFLLRPLRPEEREPWLLVSELNPPPSREDVHALLALLANRAGGRTARMGDSLLTWSPPESLLLEGTLSWRGNTYTYRLRPLARRVLNPRNPSERDALSALARRLLREVLEQFRREGFWVEGWAFYRKEHARGPGWRVLKGAALDLWVSAEGAMVLEVDPTYRILCDMTLEAWLAQGHPPPKRVKNAYNDRTWELLGLGEEDPQGILLPGGLNLVEYHASKGRIRDGGWGRVAWVANPKDAKEKIPHLTSLLIPVLTLEDLHEEGGSNLALSIPWNQRQEETLKVALSVARRLGVEHPKPVEAKAWRMRMPELRARRRVGKPADALRVGLYRAQETTLALLRLDGGRGWPDFLLKALENAFRASQARLHVREIHADPSQPLAFREALEEAKEAGVQAVLVLTPPLSWEERHRLKALFLKEGLPSQLLNVPIQREERHRLENALLGLLAKAGLQVVALEGAYPADLTVGFDAGGRKSFRFGGAACAVGSDGGHLLWSLPEAQAGERIPGEVVWDLLEEALLVFKRKRGRLPSRVLLLRDGRLPKDEFTLALAKLRQLGIGFDLVSVRKSGGGRIYPTRGRLLDGLLVPVEERTFLLLTVHREFRGTPRPLKLVHEEGETPLEALAEQIYHLTRLYPASGFAFPRLPAPLHLADRLVKEVGRLGVRHLKEVDREKLFFV

[0060] SEQ ID NO.22:MNRSKQIGSKVPVYLNRFPLRPLSPDELRPRLFRPELSPPPDREEAHPLLAQVARQLGVPAIPWQGAFLLTWEEPKRMQGEVRRGERVYAFSLEAQGRAELDPARPQDREALSRLASRRLEERLARLRLRRDMQDMNVEERRIYRKEVASGPGWRFLQGAELDLLVDQKGRLVLEVDLFHRILPSTLEAWLREGYPLPRRARNAYGGPRRVWDVVRLGEEAEPAAVLLPGGKNLLDYHRERGRLEGEAGQVVWVQDPRRPREEVPHLTGLLLPVLSLEEVHDLGEVPRLQIPPGERLEGARRRTARWVAKHLNLGAPPSLEPLMDQALRLPLPSLVAAGGKRRVKK PADALEVGPFRSRPAKVALLRLDGGRGWPPFLERLERLGLETGTLEADLQDELALRRKLSALREAGFSALLVLTPPLSWEVRNRLKALCLKEGLPTQLLNTPLKEAKEEEHRLANALLGLLVKAGFQLLALEGEYPAELVVGFDAGGQNSFRFGGAACAVGQDGSLLTWALPEA QPGERIPEEVVWDLLQEALLAFSPEGRLSRKVLLLRDGKVPPGEFARALEELRRRGVAYDLLSVRKSGGGRLYPVRGRLADGLFLPLEEETFLLLTVHREGRGTPRPLKLVREEGNTPLVDLALQIYHLTRLYPASGFLFPRLPAPLHLADRLVREVGRLGVRHLKEVPRDRLFFV

[0061] SEQ ID NO.23:*

[0062] Specifically, the reaction system of the detection method includes: the Ago protein with triggered reverse cleavage activity; a long single-stranded guide nucleic acid that introduces a small molecule marker, called a long single-stranded nucleic acid probe, which serves as a guide nucleic acid and / or a reporter nucleic acid; and the detection reagent, which includes a buffer solution, salt ions, divalent metal ions, and a detection enhancer; wherein the long single-stranded guide nucleic acid serves as a guide nucleic acid and / or a reporter nucleic acid.

[0063] Specifically, the target nucleic acid (nucleic acid to be detected) includes dsDNA, ssDNA and RNA; the source of the target nucleic acid includes in vitro amplification products, plants, animals, microorganisms, viruses, or a combination thereof.

[0064] Specifically, the long single-stranded guide nucleic acid can also be cleaved by Ago protein when it is modified with 5'-end phosphorylation, hydroxylation, fluorescent group, quenching group, biotin, etc.

[0065] The length of the long single-stranded guide nucleic acid fluorescent probe is ≥21nt; the GC content of the long single-stranded guide nucleic acid fluorescent probe is 10% to 80%; preferably, the length of the long single-stranded guide nucleic acid fluorescent probe is 26 to 60nt, and the GC content of the long single-stranded guide nucleic acid fluorescent probe is 20% to 60%.

[0066] Specifically, the detection reagent includes: a buffer solution, salt ions, metal ions, and a detection enhancer.

[0067] Specifically, the method should be carried out under the condition of a reagent that maintains the targeting and cleavage activity of the Ago protein; the main components of the reagent are a buffer solution to provide a pH environment, preferably pH 6-9; salt ions, preferably K+, Na+, preferably at a concentration of 10mM-200mM; divalent metal ions, preferably Mn2+, Mg2+, Co2+, Ni2+, preferably at a concentration of 0.1mM-10mM; detection enhancers, preferably betaine, glycerol, DTT, ammonium sulfate, polysucrose, tween-20, BSA, mannitol, PEG, sorbitol.

[0068] Specifically, the method is carried out at a temperature that maintains the targeting and cleavage activity of the Ago protein, which is divided into (1) a high temperature condition that can open the DNA double helix; and (2) a non-high temperature condition that requires a helicase to open the DNA double helix.

[0069] Specifically, the helicase is a helicase that can open double-stranded DNA while maintaining the targeting and cutting activity of the Ago protein.

[0070] The present invention provides a rapid nucleic acid detection system and detection product based on the triggered reverse cleavage activity of Ago protein. In the detection system and detection product, the Ago protein is an Ago protein with triggered reverse cleavage activity, and the Ago protein has the activity of specifically targeting the nucleic acid to be detected and cleaving long single-stranded nucleic acid probes.

[0071] The present invention provides a "quenching-fluorescence" nucleic acid detection system / detection product based on the Ago protein-triggered reverse cleavage activity, which comprises:

[0072] (1) Ago protein; (2) LgDNA fluorescent probe; (3) Detection reagent.

[0073] The detection method implemented based on the detection system / detection product includes the following steps: ① labeling a set of "quenching-fluorescence" groups on the long single-stranded guide nucleic acid, so that the long single-stranded guide nucleic acid becomes a fluorescent probe, called a long single-stranded nucleic acid fluorescent probe. The intact long single-stranded nucleic acid fluorescent probe has no fluorescence, but can emit fluorescence after being broken; ② Ago protein binds to the long single-stranded nucleic acid fluorescent probe, and when the nucleic acid to be detected is present, the triggered reverse cleavage activity of the Ago protein is triggered, and the long single-stranded nucleic acid fluorescent probe is cut to generate a detectable fluorescent signal, such as Figure 8 shown.

[0074] The long single-stranded nucleic acid fluorescent probe is a long single-stranded guide nucleic acid with a fluorescent group and a quencher group, and the long single-stranded nucleic acid fluorescent probe serves as a guide nucleic acid and / or a reporter nucleic acid; and / or,

[0075] In the detection method, the long single-stranded nucleic acid fluorescent probe serves as a guide nucleic acid to guide the Ago protein to target the nucleic acid to be detected, and is cut by the Ago protein to generate fluorescence, thereby realizing nucleic acid detection.

[0076] in,

[0077] Preferably, the Ago protein includes eukaryotic Ago protein and prokaryotic Ago protein,

[0078] Preferably, it is an Ago protein with triggered reverse cleavage activity,

[0079] More preferably, it is the gene editing enzyme AaAgo.

[0080] The length of the LgDNA fluorescent probe is ≥21 nt, and the GC content is 10% to 80%.

[0081] Preferably, the length of the LgDNA fluorescent probe is 26 nt to 60 nt.

[0082] Preferably, the GC content of the LgDNA fluorescent probe is 20% to 60%.

[0083] Wherein, the LgDNA fluorescent probe is labeled with a fluorescent group and a quenching group.

[0084] The intact LgDNA fluorescent probe does not emit light, but can emit light after being broken.

[0085] Wherein, the detection system also contains a buffer solution containing 1mM-12mM MnCl2.

[0086] The samples to be tested include non-amplified samples, amplified (or nucleic acid amplified) samples, and cell samples.

[0087] The present invention further provides a nucleic acid detection system / detection product based on flow chromatography test paper based on the Ago protein-triggered reverse cleavage activity, which comprises:

[0088] (1) Ago protein; (2) long single-stranded nucleic acid probe; (3) detection reagent; (4) flow chromatographic test paper.

[0089] The detection method implemented based on the detection system / detection product includes the following steps: ① introducing multiple small molecule markers on the long single-stranded nucleic acid, called long single-stranded nucleic acid probes, and the long single-stranded nucleic acid probes serve as guide nucleic acids and / or reporter nucleic acids; ② Ago protein binds to the long single-stranded nucleic acid probe, and when the nucleic acid to be detected is present, the triggered reverse cleavage activity of the Ago protein is triggered, cutting the long single-stranded nucleic acid probe. The intact long single-stranded probe will be captured on the control line of the flow chromatography test paper. After the breakage, one small molecule marker is captured on the control line, and another small molecule marker is captured on the detection line, thereby achieving the purpose of quickly and conveniently detecting nucleic acids.

[0090] Preferably, the Ago protein includes eukaryotic Ago protein and prokaryotic Ago protein,

[0091] Preferably, it is an Ago protein with triggered reverse cleavage activity,

[0092] More preferably, it is the gene editing enzyme AaAgo.

[0093] The length of the long single-stranded nucleic acid probe is ≥21 nt, and the GC content is 10% to 80%.

[0094] Preferably, the length of the long single-stranded nucleic acid probe is 26 nt to 60 nt.

[0095] Preferably, the GC content of the long single-stranded nucleic acid probe is 20% to 60%.

[0096] The long single-stranded nucleic acid probe is a long single-stranded nucleic acid into which two small molecule markers are introduced.

[0097] Preferably, the two introduced small molecule markers are FAM and biotin, respectively.

[0098] Wherein, the detection system also contains a buffer solution containing 1mM-12mM MnCl2.

[0099] The samples to be tested include non-amplified samples, amplified (or nucleic acid amplified) samples, and cell samples.

[0100] The present invention also provides an Ago protein-long single-stranded nucleic acid probe binary complex, which includes an Ago protein with triggered reverse cleavage activity or a nucleic acid encoding an Ago protein, and a long single-stranded nucleic acid probe with multiple different small molecule markers.

[0101] The present invention also provides a composition / reagent / kit, which includes Ago protein, and / or a cleavage system based on Ago protein, and / or an Ago protein-long single-stranded nucleic acid probe binary complex, etc.

[0102] The present invention also provides an application, which is: the Ago protein, the Ago protein-long single-stranded guide nucleic acid binary complex, the rapid nucleic acid detection method based on the Ago protein-triggered reverse cleavage activity, the nucleic acid detection reaction system based on the Ago protein, the Ago protein-long single-stranded nucleic acid probe binary complex, and the application of the composition / reagent / kit containing the Ago protein in nucleic acid detection, preoperative detection, rapid quarantine, and disease monitoring.

[0103] The present invention also provides a nucleic acid cleavage system, which includes the above-mentioned Ago protein with triggered reverse cleavage activity, a long single-stranded guide nucleic acid, and a target nucleic acid.

[0104] The beneficial effects of the present invention include but are not limited to: the present invention innovatively proposes for the first time a non-classical nuclease activity Ago protein, preferably an Ago protein with triggered reverse cleavage activity. And the present invention innovatively proposes for the first time a one-step rapid nucleic acid detection method based on the non-classical nuclease activity triggered reverse cleavage activity of the Ago protein, that is, a rapid nucleic acid detection method established based on the non-classical cleavage of the Ago protein triggered reverse cleavage activity. Compared with the current nucleic acid detection technology based on CRISPR technology and the nucleic acid detection technology based on PfAgo cleavage ability, the present invention also proposes the application of the Ago protein, the nucleic acid detection reaction system based on the Ago protein, the Ago protein-long single-stranded nucleic acid probe binary complex, and the composition / reagent / kit containing the Ago protein in nucleic acid detection, preoperative detection, rapid quarantine, and disease monitoring, including but not limited to applications such as rapid access to community nucleic acid testing during sudden outbreaks; rapid and highly sensitive detection before emergency surgery to prevent occupational exposure of medical staff to hospital rashes or emergency operations; rapid quarantine in fields, farms, and customs sites; early home monitoring of genetic diseases, cancers, and other diseases.

[0105] The present invention has high sensitivity and can detect trace amounts of target nucleic acid amplification products. It has a short detection time of 3-10 minutes and low cost. It only requires fluorescent reporter molecules and primary cutting, and does not require other guiding nucleic acids.

[0106] In combination with the above beneficial effects, the one-step rapid nucleic acid detection method provided by the present invention can be applied to many application scenarios. BRIEF DESCRIPTION OF THE DRAWINGS

[0107] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without paying any creative work.

[0108] Figure 1 The non-classical nuclease activity / triggered reverse cleavage activity of the Ago protein of the present invention: Ago protein binds to a long single-stranded guide nucleic acid (LgDNA) to assemble into a binary complex. The binary complex specifically targets and cleaves the target nucleic acid, and the LgDNA is also specifically cleaved. The LgDNA is modified with a quencher group at the 5' end and a fluorescent group at the 3' end. Then, the LgDNA serves as a guide nucleic acid and / or a reporter nucleic acid. The Ago protein binds to the LgDNA and specifically targets the nucleic acid to be detected, triggering the breakage of the LgDNA, thereby generating a detectable fluorescent signal.

[0109] Figure 2 Schematic diagram of the binding and cleavage of the Ago protein of the present invention to the target DNA.

[0110] Figure 3 This is a diagram showing the experimental results of the Ago protein of the present invention binding to single-stranded guide nucleic acids of different lengths to target and cut the target DNA.

[0111] Figure 4 This is a schematic diagram of the Ago protein of the present invention binding to the long single-stranded guide nucleic acid and targeting the target nucleic acid, and the long single-stranded guide nucleic acid being cut.

[0112] Figure 5 This is a diagram showing the results of the Ago protein of the present invention cutting single-stranded guide nucleic acids of different lengths.

[0113] Figure 6 This is a diagram showing the experimental results of the Ago protein of the present invention binding to a long single-stranded guide nucleic acid labeled with different groups at the 5' end to target and cleave the target nucleic acid.

[0114] Figure 7 This is a diagram showing the results of multiple Ago proteins binding to long single-stranded guide nucleic acids of the present invention and targeting target nucleic acids, and the long single-stranded guide nucleic acids being cut.

[0115] Figure 8 Schematic diagram of the theoretical model of the "quenching-fluorescence" Ago nucleic acid detection system / detection product of the present invention.

[0116] FIG9 is a diagram showing the experimental results of the theoretical model of the "quenching-fluorescence" Ago nucleic acid detection system / detection product of the Ago protein of the present invention.

[0117] FIG10 is a graph showing the sensitivity results of the “quenching-fluorescence” Ago nucleic acid detection system / detection product of the Ago protein of the present invention.

[0118] Figure 11 The various Ago proteins of the present invention can be used for rapid nucleic acid detection.

[0119] Figure 12 The Ago protein of the present invention cuts long single-stranded guide nucleic acids at different temperatures.

[0120] Figure 13 This is a schematic diagram of the Ago nucleic acid detection system / detection product based on flow chromatographic test paper of the present invention: Ago protein is combined with a long single-stranded guide nucleic acid (LgDNA) to assemble into a binary complex. The binary complex specifically targets and shears the target nucleic acid, and the LgDNA will also be specifically cut. A long single-stranded nucleic acid that introduces two small molecule (FAM, biotin) labels to LgDNA is a long single-stranded probe. At this time, LgDNA serves as both a guide nucleic acid and a reporter nucleic acid. When the nucleic acid to be detected is present in the detection system, the triggered reverse cleavage activity of the Ago protein is triggered, cutting the long single-stranded nucleic acid probe. The intact long single-stranded probe will be captured on the control line of the flow chromatographic test paper. After breaking, the end labeled with biotin is captured on the control line, and the end labeled with FAM is captured on the detection line, achieving the purpose of rapid and convenient nucleic acid detection.

[0121] Figure 14 This is a result diagram of the Ago nucleic acid detection system / detection product based on flow chromatography test paper of the present invention. DETAILED DESCRIPTION

[0122] The invention is further described in detail with reference to the following specific examples and accompanying drawings. The processes, conditions, experimental methods, etc. for implementing the present invention, except for those specifically mentioned below, are common knowledge and common common sense in the art and are not particularly limited by the present invention.

[0123] The non-classical nuclease activity of the Ago protein of the present invention: Ago protein binds to a long guide nucleic acid (LgDNA) and assembles into a binary complex. The binary complex specifically targets and cleaves the target nucleic acid, and the LgDNA will also be specifically cleaved. The LgDNA is modified with a quenching group at the 5' end and a fluorescent group at the 3' end. Then, the LgDNA serves as a guide nucleic acid and / or a reporter nucleic acid. The Ago protein binds to the LgDNA and specifically targets the nucleic acid to be detected, triggering the breakage of the LgDNA, thereby generating a detectable fluorescent signal, such as Figure 1 shown.

[0124] Example 1: Ago protein binds to single-stranded guide nucleic acids of different lengths and targets and cleaves target DNA

[0125] The 5' fluorescently modified 91nt Target ssDNA was used as the target DNA. Ago protein was added to 2x quickbuffer, and 5' phosphorylated single-stranded guide nucleic acids of different lengths were added respectively. After mixing, the mixture was reacted at 95℃ for 20-30min, and the sample was subjected to denaturing PAGE gel electrophoresis and imaged with a Bio-Rad imager.

[0126] The Ago protein used in this example is AaAgo protein, and the lengths of the single-stranded guide nucleic acids are 10 nt, 11 nt, 14 nt, 16 nt, 26 nt, 36 nt, and 46 nt, respectively.

[0127] The nucleic acid sequences used are as follows:

[0128] 5' fluorescently modified 91nt Target ssDNA:

[0129] Target ssDNA (SEQ ID NO. 24):

[0130] 5'Alexa Fluor 488-TTTTTCCTACCAGAATCGCCCAGTGGCTGCTAATACCTTGGACTTTG GACAGAATGATACTGAATTTGTATGTTTTGATAATTTTTTTTTT-3'

[0131] 5' phosphorylated gDNA of different lengths:

[0132] pLGC-10(SEQ ID NO.25):5'P-TTATCAAAAC-3'

[0133] pLGC-11(SEQ ID NO.26):5'P-TTATCAAAACA-3'

[0134] pLGC-14(SEQ ID NO.27):5'P-TTATCAAAACATAC-3'

[0135] pLGC-16(SEQ ID NO.28):5'P-TTATCAAACATACAA-3'

[0136] pLGC-26(SEQ ID NO.29):5'P-TTATCAAAACATACAAATTCAGTATC-3'

[0137] pLGC-36 (SEQ ID NO.30):5'P-TTATCAAAACATACAAATTCAGTATCATTCTGTCCA-3'

[0138] pLGC-46 (SEQ ID NO. 31):

[0139] 5'P-TTATCAAAACATACAAATTCAGTATCATTCTGTCCAAAGTCCAAGG-3'

[0140] The specific experimental steps are as follows:

[0141] (1) 300nM AaAgo protein and 200nM gDNA of different lengths (pLGC-10, pLGC-11, pLGC-14, pLGC-16, pLGC-26, pLGC-36, pLGC-46) and 100nM Target ssDNA were added to 2x quick buffer (1x buffer components: 20mM Tris-HCl pH 8.8, 10mM (NH4)2SO4, 150mM KCl, 2mM MgCl2, 1.25mM betaine, 0.1% Tween 20, 1mM DTT, 0.2% Ficoll-400) with a final concentration of 1mM MnCl2, supplemented with ddH2O to a final volume of 20μl, and reacted at 95℃ for 20-30min.

[0142] (2) Add 2x RNA loading buffer (1x TBE, 6%) to the reaction product The reaction was terminated with 5 mM EDTA, 0.05% SDS, 85% Formamide) at 95°C for 5 min. The reaction products were detected by 12% denaturing PAGE gel. Figure 3 As shown, Ago protein binds to gDNA of different lengths and targets ssDNA for cleavage. The longer the gDNA, the shorter the target ssDNA product, which can be cleaved multiple times.

[0143] Example 2 Ago protein cleavage of single-stranded guide nucleic acids of different lengths

[0144] The 91nt target-DNA without fluorescent modification was used as the target DNA. Ago protein was added to 2x quick buffer, and single-stranded guide nucleic acids with different lengths of 5' phosphorylated modification and 3' fluorescent modification were added respectively. After mixing, the mixture was reacted at 95℃ for 20-30min, and the sample was subjected to denaturing PAGE gel electrophoresis and imaged with a Bio-Rad imager.

[0145] The Ago protein used in this example is AaAgo protein, and the lengths of the single-stranded guide nucleic acids are 16 nt, 26 nt, 36 nt, and 46 nt, respectively. The nucleic acid sequences used are as follows:

[0146] 91nt Target-DNA without fluorescent modification:

[0147] target-DNA (SEQ ID NO.32):

[0148] 5'-TTTTTCCTACCAGAATCGCCCAGTGGCTGCTAATACCTTGGACTTTGGACAGAATGATA CTGAATTTGTATGTTTTGATAATTTTTTTT-3'

[0149] Single-stranded guide nucleic acids with different lengths of 5' phosphorylation modification and 3' fluorescent modification:

[0150] pLGC-16-3'488(SEQ ID NO.33):5'P-TTATCAAAACATACAA-3'AlexaFluor 488

[0151] pLGC-26-3'488(SEQ ID NO.34):5'P-TTATCAAAACATACAAATTCAGTATC-3'AlexaFluor 488 pLGC-36-3'488(SEQ ID NO.35):5'P-TTATCAAAACATACAAATTCAGTATCATTCTGTCCA-3'Alexa Fluor 488

[0152] pLGC-46-3'488 (SEQ ID NO. 36):

[0153] The specific experimental steps for 5'P-TTATCAAAACATACAAATTCAGTATCATTCTGTCCAAAGTCCAAGG-3'AlexaFluor 488 are as follows:

[0154] (1) 300 nM AaAgo protein and 200 nM gDNA of different lengths (pLGC-16-3'488, pLGC-26-3'488, pLGC-36-3'488, pLGC-46-3'488) and 100 nM target-DNA were added to 2x quickbuffer (1xbuffer components: 20 mM Tris-HCl pH 8.8, 10 mM (NH4)2SO4, 150 mM KCl, 2 mM MgCl2, 1.25 mM betaine, 0.1% Tween 20, 1 mM DTT, 0.2% Ficoll-400) with a final concentration of 1 mM MnCl2, supplemented with ddH2O to a final volume of 20 μl, and reacted at 95°C for 20 to 30 min.

[0155] (2) Add 2x RNA loading buffer (1x TBE, 6%) to the reaction product The reaction was terminated with 5 mM EDTA, 0.05% SDS, 85% Formamide) at 95°C for 5 min. The reaction products were detected by 12% denaturing PAGE gel. Figure 5 As shown, Ago protein binds to gDNA of different lengths and targets cleaves ssDNA. The longer the gDNA, the more likely it is to be cleaved. Example 3: Ago protein binds to long single-stranded guide nucleic acids labeled with different groups at the 5' end and targets the target nucleic acid. Target ssDNA with 91 nt of 5' fluorescently modified target DNA was used as the target DNA. 2x quickbuffer was added with Ago protein, and long single-stranded guide nucleic acids labeled with different groups at the 5' end were added separately. After mixing, the mixture was reacted at 95°C for 20-30 minutes, and the sample was subjected to denaturing PAGE gel electrophoresis and imaged with a Bio-Rad imager.

[0156] The Ago protein used in this example is AaAgo protein, and the 5' end of 36nt gDNA is modified with phosphorylation, hydroxylation, fluorescent group, and quenching group.

[0157] The nucleic acid sequences used are as follows:

[0158] 5' fluorescently modified 91nt Target ssDNA:

[0159] Target ssDNA (SEQ ID NO.37):

[0160] 5'Alexa Fluor 488-TTTTTCCTACCAGAATCGCCCAGTGGCTGCTAATACCTTGGACTTTG GACAGAATGATACTGAATTTGTATGTTTTGATAATTTTTTTTTT-3'

[0161] 36nt gDNA 5' phosphorylation modification:

[0162] pLGC-36 (SEQ ID NO.38):5'P-TTATCAAAACATACAAATTCAGTATCATTCTGTCCA-3'

[0163] 36nt gDNA 5' hydroxylation modification:

[0164] LGC-36 (SEQ ID NO.39):5'OH-TTATCAAAACATACAAATTCAGTATCATTCTGTCCA-3'

[0165] 36nt gDNA 5' fluorescent group modification:

[0166] F-LGC-36-B (SEQ ID NO.40):

[0167] 5'6-FAM-TTATCAAAACATACAAATTCAGTATCATTCTGTCCA-3'BHQ1

[0168] 36nt gDNA 5' quencher modification:

[0169] B-LGC-36(SEQ ID NO.41):5'BHQ1-TTATCAAAACATACAAATTCAGTATCATTCTGTCCA-3'

[0170] The specific experimental steps are as follows:

[0171] (1) 300 nM Aa Ago protein and 200 nM 36 nt gDNA (pLGC-36, LGC-36, F-LGC-36-B, B-LGC-36) labeled with different 5' groups and 100 nM target ssDNA were added to 2x quickbuffer (1xbuffer components 20 mM Tris-HCl pH 8.8, 10 mM (NH4)2SO4, 150 mM KCl, 2 mM MgCl2, 1.25 mM betaine, 0.1% Tween20, 1 mM DTT, 0.2% Ficoll-400) with a final concentration of 1 mM MnCl2, supplemented with ddH2O to a final volume of 20 μl, and reacted at 95°C for 20 to 30 min.

[0172] (2) Add 2x RNA loading buffer (1x TBE, 6% The reaction was terminated with 5 mM EDTA, 0.05% SDS, 85% Formamide) at 95°C for 5 min. The reaction products were detected by 12% denaturing PAGE gel. Figure 6 As shown, Ago protein binds to 36nt long gDNA with different modifications at the 5' end to target and cut ssDNA. The target ssDNA is cut and the gDNA is also cut.

[0173] Example 4: Multiple Ago proteins can bind to long single-stranded guide nucleic acids and target nucleic acids, and the long single-stranded guide nucleic acids are cleaved.

[0174] A 91nt target-DNA without fluorescent modification was used as the target DNA. Long single-stranded guide nucleic acids modified with different types of Ago protein 5' fluorescent groups were added to quick buffer. After mixing, the mixture was reacted at 95℃ for 30min, and then subjected to denaturing PAGE gel electrophoresis and imaged with a Bio-Rad imager.

[0175] The Ago proteins used in this example are TheAgo, TthAgo, AaAgo, TaAgo, PfAgo, TtAgo, FpAgo, MpAgo, ToAgo, TeAgo, StAgo, MbAgo, and MjAgo.

[0176] The nucleic acid sequences used are as follows:

[0177] 91nt Target-DNA without fluorescent modification:

[0178] target-DNA (SEQ ID NO.42):

[0179] 5'-TTTTTCCTACCAGAATCGCCCAGTGGCTGCTAATACCTTGGACTTTGGACAGAATGA TACTGAATTTGTATGTTTTGATAATTTTTTTTTT-3'

[0180] 5' fluorescent group modified long single-stranded guide nucleic acid:

[0181] F-LGC-36-B (SEQ ID NO.43):

[0182] 5'6-FAM-TTATCAAAACATACAAATTCAGTATCATTCTGTCCA-3'BHQ1

[0183] The specific experimental steps are as follows:

[0184] (1) 300 nM of different types of Ago proteins (TheAgo, TthAgo, AaAgo, TaAgo, PfAgo, TtAgo, FpAgo, MpAgo, ToAgo, TeAgo, StAgo, MbAgo, MjAgo) and 200 nM F-LGC-36-B, as well as 100 nM target-DNA, were added to 2x quickbuffer (1xbuffer components: 20 mM Tris-HCl pH 8.8, 10 mM (NH4)2SO4, 150 mM KCl, 2 mM MgCl2, 1.25 mM betaine, 0.1% Tween 20, 1 mM DTT, 0.2% Ficoll-400) with a final concentration of 1 mM MnCl2, supplemented with ddH2O to a final volume of 20 μl, and reacted at 95°C for 30 min.

[0185] (2) Add 2x RNA loading buffer (1x TBE, 6%) to the reaction product The reaction was terminated with 5 mM EDTA, 0.05% SDS, 85% Formamide) at 95°C for 5 min. The reaction products were detected by 12% denaturing PAGE gel. Figure 7 As shown, various Ago proteins can bind to 36nt 5'-end FAM-modified long gDNA to target and cleave ssDNA, and the gDNA is cleaved.

[0186] Example 5 Establishment of a Theoretical Model for Rapid Nucleic Acid Detection of Ago Protein

[0187] A 91nt target DNA without fluorescent modification was used as the DNA to be detected. A set of "fluorescence-quenching" LgDNA fluorescent probes labeled with Ago protein was added to 2x quick buffer. After mixing, the mixture was reacted at 95°C for 3-10 minutes. Fluorescence detection was performed on a Bio-Rad real-time fluorescence quantitative PCR instrument. After the reaction was completed, the detection was performed under a blue light instrument and on a denaturing PAGE gel, and the photo was taken with a Bio-Rad gel scanner.

[0188] The Ago proteins used in this example are AaAgo.

[0189] The nucleic acid sequences used are as follows:

[0190] 91nt Target-DNA without fluorescent modification:

[0191] target-DNA (SEQ ID NO.44):

[0192] 5'-TTTTTCCTACCAGAATCGCCCAGTGGCTGCTAATACCTTGGACTTTGGACAGAATGAT ACTGAATTTGTATGTTTTGATAATTTTTTTTTT-3'

[0193] LgDNA fluorescent probes labeled with a set of "fluorescence-quenching"

[0194] F-LGC-36-B (SEQ ID NO.45):

[0195] 5'6-FAM-TTATCAAAACATACAAATTCAGTATCATTCTGTCCA-3'BHQ1

[0196] The specific experimental steps are as follows:

[0197] (1) 300 nM AaAgo protein, 50 nM F-LGC-36-B, and 50 nM target-DNA were added to 2xquickbuffer (1xbuffer components: 20 mM Tris-HCl pH 8.8, 10 mM (NH4)2SO4, 150 mM KCl, 2 mM MgCl2, 1.25 mM betaine, 0.1% Tween 20, 1 mM DTT, 0.2% Ficoll-400) with a final concentration of 1 mM MnCl2, and ddH2O was added to the final volume of 20 μl. The reaction was carried out at 95℃ for 3-10 min and the reaction was carried out at 10 s / cycle in a Bio-Rad real-time fluorescence quantitative PCR instrument. Figure 9A .

[0198] (2) After the reaction is completed, the test is performed under a blue light instrument. Figure 9C , the reaction product was added with 2x RNA loading buffer (1xTBE, 6% The reaction was terminated with 5 mM EDTA, 0.05% SDS, 85% Formamide) at 95°C for 5 min. The reaction products were detected by 12% denaturing PAGE gel. Figure 9B As shown in Figure 9, Ago protein binds to 36nt long gDNA and targets ssDNA for cleavage. The 36nt LgDNA is modified with 5'-6-FAM and 3'-BHQ 1. Ago protein binds to LgDNA and targets cleavage of ssDNA. LgDNA is also cleaved to generate a detectable fluorescent signal.

[0199] Example 6: One-step rapid nucleic acid detection method based on the non-classical nuclease activity / triggered reverse cleavage activity of Ago protein. After Ago-PCR amplification with different template amounts of DNA, 10 μl of the amplification system was added to Ago protein, 2xquickbuffer, and LgDNA fluorescent probe. After mixing, the mixture was reacted at 95°C for 3-10 minutes. After cooling to room temperature, the sample was observed on a blue light analyzer with the naked eye or in professional mode with a mobile phone.

[0200] In this example, the Ago protein used for Ago-PCR amplification was CalAgo protein, the Ago protein used for amplification product detection was AaAgo protein, and the amplification template was ssM13mp18. The reaction system concentrations were 3000, 300, 30, 3, and 0 copies / 30 μl, respectively. The amplification primers used were as follows:

[0201] pM13-3-30 (SEQ ID NO. 46):

[0202] 5'P-TTAAATATGCAACTAAAGTACGTGTCTGG-3'

[0203] pM13-7-40 (SEQ ID NO. 47):

[0204] 5'P-TTATTGACCATTTGCGAAATGTATCTAATGGTCAAACTAA-3'

[0205] LgDNA fluorescent probe:

[0206] M13-3-7 (SEQ ID NO.48): 5'6-FAM-GGGAATCAACTGTTATATGGAATGAAACTTCCC-3'BHQ1

[0207] The specific experimental steps are as follows:

[0208] (1) Specific system and reaction procedure of Ago-PCR isothermal amplification:

[0209]

[0210]

[0211] 1x Buffer components: 1x buffer components 20mM Tris-HCl pH 8.8, 10mM (NH4)2SO4, 150mM KCl, 2mM MgCl2, 1.25mM betaine, 0.1% Tween 20, 1mM DTT, 0.2% Ficoll-400.

[0212] Take 10 μl of Ago-PCR product and perform electrophoresis on 2.5% agarose gel containing EB. Figure 10A shown.

[0213] (2) One-step rapid nucleic acid detection method based on AaAgo protein

[0214] In the experimental group PCR tube, add 1.5 μl of 1 μM LgDNA fluorescent probe (M13-3-7), 5 μl 2xquickbuffer (1xbuffer components 20 mM Tris-HCl pH 8.8, 10 mM (NH4)2SO4, 150 mM KCl, 2 mM MgCl2, 1.25 mM betaine, 0.1% Tween 20, 1 mM DTT, 0.2% Ficoll-400), add 0.8 μl 25 mM nCl2, add 10 pmol AaAgo protein, add ddH2O to the total system of 10 μl, add 10 μl Ago-PCR product, and react at 95°C for 3-10 minutes.

[0215] (3) Observe and take photos in the professional mode of mobile phone on the blue light meter. Figure 10B As shown, Ago-PCR products were directly detected, 36nt LgDNA was modified with 5'-6-FAM and 3'-BHQ 1, Ago protein bound to LgDNA and targeted cleavage of Ago-PCR products, and LgDNA was also cleaved to generate detectable fluorescent signals.

[0216] The advantages of the present invention include but are not limited to high sensitivity, ability to detect trace amounts of target nucleic acid amplification products, short detection time (reaction time is 3-10 minutes), low cost, and only requires LgDNA fluorescent probes without the need for additional guide nucleic acids.

[0217] Example 7: Multiple Ago proteins can be used for rapid nucleic acid detection

[0218] According to the Ago-PCR amplification system in Example 6, the amplified products were detected using different types of Ago according to the nucleic acid detection system in Example 6. The results are as follows: Figure 11 TheAgo, ThtAgo, TaAgo, PfAgo, TtAgo, FpAgo, MpAgo, and ToAgo shown can be used for rapid nucleic acid detection.

[0219] Example 8: Ago protein cleaves long single-stranded guide nucleic acids at different temperatures

[0220] A 91nt target-DNA without fluorescent modification was used as the target DNA. A long single-stranded guide nucleic acid modified with the 5' fluorescent group of Ago protein was added to 2x quick buffer. After mixing, the mixture was reacted at different temperatures for 30 minutes. The samples were subjected to denaturing PAGE gel electrophoresis and imaged with a Bio-Rad imager.

[0221] The nucleic acid sequences used are as follows:

[0222] 91nt Target-DNA without fluorescent modification:

[0223] Target-DNA (SEQ ID NO.49):

[0224] 5'-TTTTTCCTACCAGAATCGCCCAGTGGCTGCTAATACCTTGGACTTTGGACAGAATGAT ACTGAATTTGTATGTTTTGATAATTTTTTTTTT-3'

[0225] 5' fluorescent group modified long single-stranded guide nucleic acid:

[0226] F-LGC-36-B (SEQ ID NO.50):

[0227] 5'6-FAM-TTATCAAAACATACAAATTCAGTATCATTCTGTCCA-3'BHQ1

[0228] The specific experimental steps are as follows:

[0229] (1) 300nM AaAgo protein and 200nM F-LGC-36-B, as well as 100nM target-DNA, were added to 2xquickbuffer (1xbuffer components: 20mM Tris-HCl pH 8.8, 10mM (NH4)2SO4, 150mM KCl, 2mM MgCl2, 1.25mM betaine, 0.1% Tween 20, 1mM DTT, 0.2% Ficoll-400) with a final concentration of 1mM MnCl2, supplemented with ddH2O to a final volume of 20μl, and reacted at different temperatures (37℃, 45℃, 50℃, 55℃, 60℃, 65℃, 70℃, 75℃, 80℃, 85℃, 90℃, 95℃) for 30min.

[0230] (2) Add 2x RNA loading buffer (1x TBE, 6%) to the reaction product The reaction was terminated with 5 mM EDTA, 0.05% SDS, 85% Formamide) at 95°C for 5 min. The reaction products were detected by 12% denaturing PAGE gel. Figure 12 The cleavage of LgDNA at different temperatures is shown.

[0231] Example 9 Ago nucleic acid detection system / detection product result diagram of flow chromatography test paper

[0232] After Ago-PCR amplification with different template amounts of DNA, the amplified product was added to Ago protein, 2xquickbuffer, and long single-linked nucleic acid probe, mixed, reacted at 95℃ for 3-5 minutes, and the final product was added to the sample well of the test card and left at room temperature for 10-15 minutes to observe the results. The principle is as follows Figure 13 shown.

[0233] In this example, Ago-PCR amplification was performed according to the Ago-PCR amplification system in Example 6. The Ago protein used for amplification product detection was AaAgo protein. The final reaction product was added to the sample well of the detection card and placed at room temperature for 10 to 15 minutes to observe the results.

[0234] The long single-stranded nucleic acid probe used in this example is:

[0235] FAM-LgDNA-Biotin (SEQ ID NO.51):

[0236] 5'6-FAM-GGGAATCAACTGTTATATGGAATGAAACTTCCC-3'Biotin

[0237] The specific experimental steps are as follows:

[0238] (1) The nucleic acid to be tested was amplified using the Ago-PCR system described in Example 6.

[0239] (2) Add 4.5 μl of 1 μM long single-stranded nucleic acid probe (FAM-LgDNA-Biotin) and 15 μl 2x quickbuffer (1xbuffer components 20 mM Tris-HCl pH 8.8, 10 mM (NH4)2SO4, 150 mM KCl, 2 mM MgCl2, 1.25 mM betaine, 0.1% Tween 20, 1 mM DTT, 0.2% Ficoll-400) to the experimental group PCR tube, add 2.4 μl 25 mM MnCl2, add 5 pmol AaAgo protein, add ddH2O to the total system of 30 μl, add Ago-PCR product, and react at 95°C for 3-5 minutes. (3) Add the final reaction product to the sample well of the test card and let it stand at room temperature for 10-15 minutes. Observe the results, such as Figure 14 As shown in ① positive: that is, when the nucleic acid to be tested is present in the detection system, the triggered reverse cleavage activity of the Ago protein is triggered, and the long single-stranded nucleic acid probe is cut. The complete long single-stranded probe will be captured on the control line (C line) of the flow chromatography test paper. After the break, the end labeled with biotin is captured on the control line (C line), and the end labeled with FAM is captured on the detection line (T line). At this time, both the control line (C line) and the detection line (T line) show color bands, and the color can be deep or light, which is positive. ② negative: that is, when there is no nucleic acid to be tested in the detection system, the triggered reverse cleavage activity of the Ago protein cannot be triggered, and the long single-stranded nucleic acid probe cannot be cut. The complete long single-stranded nucleic acid probe will be captured on the control line (C line) of the flow chromatography test paper. At this time, only the control line (C line) shows a color band, and no band appears on the detection line (T line).

[0240] As used in the present invention, the term "comprising" is an open expression, that is, including the contents specified in the present invention, but not excluding other aspects.

[0241] As used herein, the term "about" when applied to a value means that some slight imprecision in the value is allowed in the calculation or measurement (approximately the accuracy of the value by some method; approximately or reasonably close to the value; nearly). If for some reason the imprecision specified by "about" is not understood in this conventional sense in the art, then "about" as used herein at least indicates the variation that may result from conventional methods of measuring or using such parameters.

[0242] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items and is not intended to limit the present invention.

[0243] The protection content of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be thought of by those skilled in the art are included in the present invention and are protected by the appended claims.

Claims

1. An Ago protein having triggered reverse cleavage activity, characterized in that: The Ago protein combines with the long single-stranded guide nucleic acid to assemble into a binary complex. When the binary complex contacts the target nucleic acid, it can stimulate the activity of the Ago protein to reversely cut the long single-stranded guide nucleic acid.

2. The Ago protein having triggered reverse cleavage activity according to claim 1, wherein The Ago protein includes a protein indicated to contain a PIWI-like domain by comparison with a database protein sequence; the PIWI-like domain is used to bind to the target nucleic acid; and / or, The Ago protein has a triggered reverse cleavage activity, and can cleave two complementary long single-stranded nucleic acids that exist simultaneously, wherein the two complementary long single-stranded nucleic acids serve as guide nucleic acids and target nucleic acids for each other; and / or, The amino acid sequence of the Ago protein includes one or more of the sequences shown in SEQ ID NOs. 1 to 23; and / or one or more amino acid sequences having at least 50% sequence homology with the sequence shown in any one of SEQ ID NOs. 1 to 23; and / or an amino acid sequence in which one or more amino acid residues are deleted, substituted, added or inserted into the amino acid sequence shown in any one of SEQ ID NOs. 1 to 23; Preferably, the amino acid sequence of the Ago protein has at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity; Preferably, the Ago protein and the protein containing the PIWI-like domain exist independently and / or contain overlapping portions; preferably, the Ago protein includes an Ago protein that can promote or improve the cleavage of a long single-stranded guide nucleic acid, a protein that can promote or improve the speed, efficiency, accuracy, and sensitivity of cleaving a long single-stranded guide nucleic acid, any protein that is structurally or functionally identical or similar to an Ago protein, and a protein that contains a nucleic acid sequence or amino acid sequence encoding a PIWI domain; Preferably, the Ago protein includes an Ago protein having protein properties formed by connecting amino acids, an Ago protein encoding RNA, or an expression cassette in which the encoding RNA is integrated into a host chromosome, or an Ago protein including an expression plasmid encoding the RNA.

3. A reaction method for an Ago protein having triggered reverse cleavage activity, characterized in that: The Ago protein is the Ago protein with triggered reverse cleavage activity according to claim 1; The Ago protein and the long single-stranded guide nucleic acid are incubated in advance and then targeted to the target nucleic acid; or, Without prior incubation, the Ago protein, the long single-stranded guide nucleic acid and the target nucleic acid react together, and the Ago protein binds to the long single-stranded guide nucleic acid and targets the target nucleic acid simultaneously; and / or, The Ago protein binds to the long single-stranded guide nucleic acid and assembles into a binary complex. When the binary complex recognizes the target nucleic acid, the Ago protein reversely cuts the long single-stranded guide nucleic acid.

4. The reaction method according to claim 3, wherein The length of the long single-stranded guide nucleic acid is ≥21 nt, and the GC content is 10% to 80%; and / or, The long single-stranded guide nucleic acid includes DNA and RNA; the length of the long single-stranded guide nucleic acid is such that it can bind to the Ago protein and be cleaved by the Ago protein; and / or, The target nucleic acid includes dsDNA, ssDNA and RNA; the target nucleic acid source includes in vitro amplification products, plants, animals, microorganisms, viruses, or a combination thereof.

5. The reaction method according to claim 3, wherein The Ago protein is capable of specifically cutting the target nucleic acid into n sequences, wherein n≥2; and / or, The Ago protein is capable of cutting the long single-stranded guide nucleic acid into m sequences, where m≥2.

6. A binary complex formed by an Ago protein and a long single-stranded guide nucleic acid, characterized in that: The Ago protein is the Ago protein with triggered reverse cleavage activity as described in claim 1; the Ago protein is combined with the long single-stranded guide nucleic acid to assemble into a binary complex, and the binary complex targets and binds to the target nucleic acid, thereby triggering the triggered reverse cleavage activity of the Ago protein, cutting the target nucleic acid and / or the long single-stranded guide nucleic acid.

7. A rapid nucleic acid detection method based on Ago protein-triggered reverse cleavage activity, characterized in that: The Ago protein is the Ago protein with triggered reverse cleavage activity according to claim 1; and / or, The detection method is a nucleic acid detection method based on a one-step cleavage system established by the Ago protein-triggered reverse cleavage activity; and / or, The reaction system of the detection method comprises: the Ago protein, a modified long single-stranded nucleic acid probe and a detection reagent; and / or, The detection method comprises the following steps: when the nucleic acid to be detected is present, triggering the triggered reverse cleavage activity of the Ago protein to cleave the long single-stranded nucleic acid probe to generate a detectable signal.

8. The detection method according to claim 7, wherein The reaction system of the detection method includes: the Ago protein with triggered reverse cleavage activity; a long single-stranded guide nucleic acid introduced with a small molecule marker, called a long single-stranded nucleic acid probe, which serves as a guide nucleic acid and / or a reporter nucleic acid; and the detection reagent, which includes a buffer solution, salt ions, divalent metal ions, and a detection enhancer; and / or, The long single-stranded nucleic acid probe can be bound and cleaved by the Ago protein when it is modified with 5'-end phosphorylation, hydroxylation, fluorescent group, quenching group, or biotin; and / or, The length of the long single-stranded nucleic acid probe is ≥21 nt; the GC content of the long single-stranded nucleic acid probe is 10% to 80%; and / or, The nucleic acid to be detected includes dsDNA, ssDNA and RNA; the source of the nucleic acid to be detected includes in vitro amplification products, plants, animals, microorganisms, viruses, or a combination thereof; and / or, The detection method is carried out in the presence of a reagent that maintains the targeting and cleavage activity of the Ago protein; and / or, The detection method is carried out at a temperature that maintains the targeting and cleavage activity of the Ago protein; wherein, The temperature is divided into (1) high temperature conditions that can open the DNA double strands; (2) non-high temperature conditions that require helicase to open the DNA double strands; wherein, The helicase is a helicase that can open double-stranded DNA without affecting the targeting and cutting activities of the Ago protein.

9. A nucleic acid detection method based on "quenching-fluorescence" and relying on Ago protein, characterized in that: The Ago protein is the Ago protein with triggered reverse cleavage activity as described in claim 1; the detection method comprises the following steps: ① labeling a set of "quenching-fluorescence" groups on a long single-stranded guide nucleic acid, so that the long single-stranded guide nucleic acid becomes a fluorescent probe, called a long single-stranded nucleic acid fluorescent probe. The intact long single-stranded nucleic acid fluorescent probe has no fluorescence, but can emit fluorescence after being broken; ② The Ago protein binds to the long single-stranded nucleic acid fluorescent probe, and when the nucleic acid to be detected is present, the triggered reverse cleavage activity of the Ago protein is triggered, the long single-stranded nucleic acid fluorescent probe is cut, and a detectable fluorescent signal is generated.

10. The nucleic acid detection method according to claim 9, wherein The long single-stranded nucleic acid fluorescent probe is a long single-stranded guide nucleic acid with a fluorescent group and a quencher group, and the long single-stranded nucleic acid fluorescent probe serves as a guide nucleic acid and / or a reporter nucleic acid; and / or, In the detection method, the long single-stranded nucleic acid fluorescent probe serves as a guide nucleic acid to guide the Ago protein to target the nucleic acid to be detected, and is cut by the Ago protein to generate fluorescence, thereby realizing nucleic acid detection.

11. A nucleic acid detection method based on flow chromatographic test paper and relying on Ago protein, characterized in that: The Ago protein is the Ago protein with triggered reverse cleavage activity as described in claim 1; the detection method comprises the following steps: ① introducing two small molecule markers on the long single-stranded guide nucleic acid, called long single-stranded nucleic acid probes; ② the Ago protein binds to the long single-stranded nucleic acid probe, and when the nucleic acid to be detected is present, the triggered reverse cleavage activity of the Ago protein is triggered, cutting the long single-stranded nucleic acid probe, and the intact long single-stranded probe will be captured on the control line of the flow chromatography test paper. After the breakage, one small molecule marker is captured on the control line, and the other small molecule marker is captured on the detection line, thereby achieving the purpose of quickly and conveniently detecting nucleic acids.

12. The nucleic acid detection method according to claim 11, wherein The long single-stranded nucleic acid probe is a long single-stranded nucleic acid with multiple different small molecule markers, and the long single-stranded nucleic acid probe serves as a guide nucleic acid and / or a reporter nucleic acid.

13. A rapid nucleic acid detection system and detection product based on Ago protein-triggered reverse cleavage activity, characterized in that: In the detection system and detection product, the Ago protein is the Ago protein with triggered reverse cleavage activity as claimed in claim 1, and the Ago protein has the activity of specifically targeting the nucleic acid to be detected and cleaving the long single-stranded nucleic acid probe.

14. A binary complex formed by an Ago protein and a long single-stranded nucleic acid probe, characterized in that: The method comprises the Ago protein with triggered reverse cleavage activity or the nucleic acid encoding the Ago protein as claimed in claim 1, and a long single-stranded nucleic acid probe with multiple different small molecule markers.

15. A nucleic acid cleavage system, characterized in that: The nucleic acid cleavage system comprises the Ago protein with triggered reverse cleavage activity as claimed in claim 1, a long single-stranded guide nucleic acid, and a target nucleic acid.

16. A composition / reagent / kit, characterized in that: It includes the Ago protein as described in claim 1, the Ago protein-long single-stranded guide nucleic acid binary complex as described in claim 6, the Ago protein-long single-stranded nucleic acid probe binary complex as described in claim 14, and / or the nucleic acid cleavage system as described in claim 15.

17. An application, characterized in that: The applications include the Ago protein according to claim 1, the Ago protein-long single-stranded guide nucleic acid binary complex according to claim 6, the rapid nucleic acid detection method based on Ago protein-triggered reverse cleavage activity according to claims 7 and 8, the rapid nucleic acid detection system according to claim 13, the Ago protein-long single-stranded nucleic acid probe binary complex according to claim 14, the nucleic acid cleavage system according to claim 15, or the composition / reagent / kit according to claim 16 in nucleic acid detection, preoperative detection, rapid quarantine, and disease monitoring.

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