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WO2023246032A1 - 一锅法单链DNA环化扩增和CRISPR/Cas介导的核酸分子检测方法 - Google Patents

一锅法单链DNA环化扩增和CRISPR/Cas介导的核酸分子检测方法 Download PDF

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WO2023246032A1
WO2023246032A1 PCT/CN2022/140596 CN2022140596W WO2023246032A1 WO 2023246032 A1 WO2023246032 A1 WO 2023246032A1 CN 2022140596 W CN2022140596 W CN 2022140596W WO 2023246032 A1 WO2023246032 A1 WO 2023246032A1
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nucleic acid
stranded dna
dna
acid molecule
sequence
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杨立桃
朱早兵
郭永坤
张大兵
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Shanghai Jiao Tong University
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Definitions

  • the invention belongs to the field of biological nucleic acid molecule detection, and specifically relates to a one-pot single-stranded DNA circular amplification and CRISPR/Cas-mediated nucleic acid molecule detection method.
  • the invention is a new nucleic acid isothermal amplification and target signal.
  • Readout integrated detection technology uses DNA ligase, strand displacement DNA polymerase and CRISPR/Cas protein to quickly, one-step and single-tube amplify and detect specific DNA or RNA under normal temperature conditions.
  • RNA sample needs to be reverse transcribed into cDNA, and then the cDNA sample needs to be subjected to conventional qPCR amplification detection and analysis.
  • RT-qPCR reverse transcription fluorescent quantitative polymerase chain reaction
  • RT-qPCR testing requires expensive thermal cyclers, experienced operators, and has high requirements on the quality of extracted RNA.
  • POCT point of care testing
  • nucleic acid isothermal amplification technology mainly takes advantage of the displacement of amplification enzymes at constant temperature and the characteristics of polymerases, which can be used in primers. Achieve efficient amplification of specific targets under the spontaneous action of
  • nucleic acid isothermal amplification methods such as loop-mediated isothermal amplification technology (LAMP), which uses 4-6 pairs of primers to identify target-specific sites at 60-65°C. Utilizes Bst DNA polymerase with displacement enzyme activity to achieve efficient (within 1 hour) amplification detection of nucleic acids.
  • LAMP loop-mediated isothermal amplification technology
  • Recombinase Polymerase Amplification (RPA) technology simulates the in vivo nucleic acid replication mechanism at a constant temperature of 37-42°C and consists of three key enzymes or proteins: recombinase, single-stranded binding protein and DNA polymerase Participate and assist in DNA polymerase amplification technology. The entire reaction generally obtains a detectable level product within 20-30 minutes.
  • Nucleic acid sequence-based amplification (NASBA) technology consists of reverse transcriptase (RT), T7 RNA polymerase and RNase H, and two oligonucleotide primers. It can complete rapid amplification of RNA in about 60 minutes.
  • LAMP amplification requires a large number of primers (4-6), and aerosol contamination in on-site testing can easily lead to false positive results. Amplification of mutated sites is almost inevitable.
  • the enzyme components of the RPA method are relatively complex and cannot detect mutation sites.
  • a reverse transcription step is still required for RNA samples.
  • Rolling circle amplification is an isothermal amplification reaction catalyzed by DNA polymerase (Phi29) with strand displacement activity. This method only requires a padlock probe to hybridize with the target sequence and then connect to a circular template.
  • the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) system is a natural "immune system” that is widely found in bacteria or archaeal microorganisms. As an adaptive immune mechanism, the CRISPR system can recognize foreign The genetic material is integrated into the CRISPR sequence of its own genome. When foreign genetic material invades again, the foreign nucleic acid is precisely cut by Cas nuclease. Cas nuclease is an important related protein in CRISPR. At present, multiple CRISPR related proteins have been discovered, such as Cas9, Cas12, Cas13, Cas14, etc. Among them, the latter three (Cas12, Cas13, Cas14) have cis and trans functions.
  • the purpose of the present invention is to overcome the above-mentioned defects of the prior art.
  • the present invention provides a one-pot single-stranded DNA circular amplification and CRISPR/Cas-mediated nucleic acid molecule detection method, which is a single-tube, one-pot method.
  • Ultra-sensitive nucleic acid molecule detection technology called: OPERATOR.
  • OPERATOR Ultra-sensitive nucleic acid molecule detection technology
  • the present invention enables connection, amplification, and detection reactions to be carried out in the same reaction tube, and a single tube realizes the detection of RNA, ssDNA, and dsDNA samples.
  • This method has the advantages of ultra-sensitivity, specificity, and fast detection speed.
  • OPERATOR technology directly amplifies and detects RNA molecules without the need for reverse transcription steps.
  • Embodiments of the present invention provide a one-pot single-stranded DNA circular amplification and CRISPR/Cas-mediated nucleic acid molecule detection method.
  • the nucleic acid molecule detection method includes the following steps:
  • reaction system mixture which includes: single-stranded DNA probe, dual fluorescently labeled single-stranded DNA probe (cis probe CP, trans probe TP), oligonucleotide primer, DNA Ligase or a variant thereof, strand displacement DNA polymerase or a variant thereof, guide RNA (crRNA) or a derivative thereof, CRISPR-related Cas protein or a variant thereof, OPERATOR reaction buffer; wherein, the guide RNA or a derivative thereof
  • the object contains the same sequence as the target sequence of the nucleic acid molecule to be detected, the single-stranded DNA probe is specifically complementary to one strand of the nucleic acid molecule to be detected; and the backbone sequence of the single-stranded DNA refers to the sequence except the complementary target part or its derivatives In addition, it also includes PAM site sequences and random connection sequences;
  • the single-stranded DNA probe forms a single-stranded circular DNA probe under the action of DNA ligase; the single-stranded circular DNA probe hybridizes with the nucleic acid molecule to be tested and is converted into circular DNA under the action of DNA ligase; oligonucleotide
  • the primer uses circular DNA as a template, can randomly bind to the circular DNA template, and continuously extends under the action of strand-displacement DNA polymerase to form a long long DNA containing a repeated single-stranded DNA probe sequence.
  • the nucleic acid molecules to be detected in the nucleic acid sample include one or more of single-stranded DNA, double-stranded DNA, and single-stranded RNA. If the nucleic acid to be detected is dsDNA, pre-denature the dsDNA before the reaction.
  • the single-stranded DNA probe includes a 5' end, a 3' end, and a backbone sequence.
  • the 5' end and 3' end are respectively complementary to the sequence of the nucleic acid molecule to be detected;
  • the backbone sequence refers to the sequence except the complementary target part or its derivatives, including PAM site sequences and random connection sequences.
  • the random connection sequence is generally 40-80bp in length and has a GC content of 30%-70%.
  • the single-stranded DNA probe introduces a “TTT” PAM site at the 3’ end of the sequence. Templates used to identify target sequences, circularize, and amplify so that the amplified products are not bound by PAM.
  • the oligonucleotide primer is a base-modified random primer (6-10nt) or a primer (10-20nt) consistent with the sequence of the nucleic acid molecule to be detected.
  • the number of modified bases is 1-10.
  • Random primers are random hexamer primers. Random hexamer primers are random sequence primers containing 6 bases.
  • the sequence of the double fluorescently labeled single-stranded DNA probe is complementary to the sequence of the nucleic acid molecule to be detected, the 5' end of the probe is labeled with a fluorescent group, and the 3' end is labeled with a quenching group;
  • the fluorescent group at the 5' end of the probe includes one of FAM, HEX, VIC, Cy5, Cy3, ROX, FITC, and Joe, and the fluorescent quenching group labeled at the 3' end includes one of TAMRA, BHQ1, MGB, and BHQ2. A sort of.
  • the DNA ligase is a ligase that connects single-stranded DNA gaps in double-stranded DNA molecules or RNA/DNA hybrid double-strands.
  • DNA ligase includes one of T4 DNA ligase, E.coli DNA ligase, SplintR ligase, and HiFi Taq DNA ligase.
  • the enzyme may include wild-type, engineered, codon-optimized, evolved, thermophilic, chimeric, engineered, and/or a mixture of more than one DNA ligase.
  • the DNA ligase is preferably T4 DNA ligase. DNA ligase can specifically ligate the phosphodiester bonds of ssDNA that are complementary to the target.
  • the strand-displacement DNA polymerase includes one of Phi29, Klenow, and Vent.
  • the DNA polymerase is preferably Phi29 DNA polymerase.
  • the enzyme may include wild-type, engineered, codon-optimized, evolved, thermophilic, chimeric, engineered, and/or a mixture of more than one reverse transcriptase enzyme.
  • DNA polymerase is able to recognize and generate ssDNA in random primers that trigger a strand displacement amplification reaction.
  • the CRISPR-related Cas protein is a CRISPR-Cas nuclease with double-stranded DNA or single-stranded DNA recognition and cutting function and trans-DNA single-stranded cutting function.
  • the CRISPR-Cas nuclease includes one of SpyCas9, FnCas9, FnCas12a, LbCas12, BhCas12b, Bs3Cas12b, LsCas12b, SbCas12b, AaCas12b, AkCas12, AmCas12b, BsCas12b, DiCas12b, TcCas12b, AacCas12b, LwCas13, Cas14 or a variant thereof in body kind of.
  • the enzyme may include wild-type, engineered, codon-optimized, evolved, thermophilic, chimeric, engineered, and/or a mixture of more than one Cas protein.
  • the CRISPR-Cas nuclease is preferably Cas12a.
  • CRISPR-Cas nuclease combined with guide RNA can be specifically activated by the target nucleic acid sequence and has non-specific DNA nuclease activity to achieve the shearing of DNA fluorescent probes.
  • the spacer sequence of the guide RNA or derivative thereof in step (2) is complementary to the sequence of the target nucleic acid molecule.
  • the OPERATOR reaction buffer includes 1-5mM dNTP, 10-100mM Tris-HCl, 5-25mM MgCl 2 , 0.01-20mM ATP, 0.5-10mM DTT and 0.5-1.5mg/ml.
  • Bovine serum albumin the pH value of the buffer is between 6.5-8.0.
  • the circularized key probe triggers an efficient strand displacement amplification reaction under the triggering of random primers.
  • the length of the random primers is 6nt DNA random primers, and the final concentration used is not less than 10 ⁇ M. Random primers can trigger efficient rolling circle amplification, which is much more efficient than traditional single primer-triggered amplification.
  • the temperature of the isothermal reaction is 37°C, and the reaction time is 1 hour.
  • the single-stranded DNA probe forms a single-stranded circular DNA probe under the action of DNA ligase; during a constant temperature reaction, the single-stranded circular DNA probe hybridizes with the nucleic acid molecule to be measured and is replaced by circular DNA under the action of DNA ligase;
  • Oligonucleotide primers use circular DNA as a template, can randomly bind to the circular DNA template, and continuously extend under the action of strand displacement DNA polymerase to form long long DNA containing repeated single-stranded DNA probe sequences; long
  • the stranded DNA combines with the dual fluorescently labeled single-stranded DNA probe to form complementary double-stranded DNA; the formed double-stranded DNA is recognized by the crRNA and Cas protein complex, and the dual fluorescently labeled single-stranded DNA probe is cut to produce detectable fluorescence. Signal.
  • the second object of the present invention is to provide an isothermal nucleic acid detection kit based on the nucleic acid molecule detection method. It can achieve accurate, rapid and highly sensitive detection of specific RNA or DNA molecules under normal temperature and isothermal conditions.
  • the kit includes an enzyme mixture, a single-stranded DNA probe, a guide RNA, a dual fluorescently labeled single-stranded DNA probe, an oligonucleotide primer and an OPERATOR reaction buffer; the enzyme mixture includes CRISPR-Cas Nuclease, DNA ligase, strand-displacement DNA polymerase.
  • the CRISPR-Cas nuclease is FnCas12a.
  • the key probe (single-stranded DNA probe) consists of a sequence complementary to the target sequence and a loop backbone sequence. A "TTT" PAM site is introduced at the 3' end of the key probe sequence; the oligonucleotide primer is a random hexamer Primer; the DNA ligase is T4 DNA ligase; the DNA polymerase is Phi29 DNA polymerase; the DNA fluorescent probe is a single fluorescent probe labeled with a fluorescent group at the 5' end and a fluorescent quenching group at the 3' end. strand DNA.
  • the buffer includes 1-5mM dNTP, 10-100mM Tris-HCl, 5-25mM MgCl2, 0.01-20mM ATP and 0.5-10mM DTT, 0.1-1.5mg/ml bovine serum albumin, the buffer
  • the liquid pH is between 6.5-8.0.
  • Random primer 6Ns (10 ⁇ M-100 ⁇ M); FAM-labeled fluorescent probe 1-4nM; enzyme mixture (T4 DNA ligase, 5U-200U; Phi29 DNA polymerase, 5U-20U; Cas12a protein, 0.1ug-5ug).
  • target DNA, guide RNA and Cas12a protein form a complex, which will cleave other single-stranded DNA molecules in the system.
  • the detection method and kit of the present invention can detect nucleic acid molecules of bacteria, mycotoxins, human or other animal and plant tissues.
  • the invention also provides a reaction system, which has: a single-stranded DNA probe, a dual fluorescently labeled single-stranded DNA probe, an oligonucleotide primer, a DNA ligase and its variants, a strand-displacement DNA polymerase and its variants. body, clustered regularly interspaced short palindromic repeats (CRISPR) RNA (crRNA) or its derivatives, CRISPR-associated (Cas) protein or its variants, OPERATOR reaction buffer. Wherein the crRNA or its derivative contains the same target sequence as the nucleic acid molecule to be detected.
  • CRISPR clustered regularly interspaced short palindromic repeats
  • Cas CRISPR-associated protein or its variants
  • OPERATOR reaction buffer wherein the crRNA or its derivative contains the same target sequence as the nucleic acid molecule to be detected.
  • the invention can quickly complete the detection of DNA or RNA molecules under normal temperature and isothermal conditions.
  • the RNA, single-stranded DNA or double-stranded DNA of the sample to be detected is obtained through nucleic acid extraction; and then the ligase, amplification enzyme and CRISPR-related protein are used.
  • the combined enzyme, single-stranded DNA probe and nucleic acid fluorescent probe react isothermally with the nucleic acid to be detected, and finally the fluorescence signal is detected to determine whether the target nucleic acid is present in the sample to be detected.
  • Figure 1 is a schematic flow chart of sample detection according to the present invention.
  • Figure 2 is a schematic flow chart of detecting ssDNA or RNA samples according to the present invention.
  • Figure 3 is a schematic flowchart of the present invention for detecting DNA samples.
  • Figure 4 shows the detection of RNA, dsDNA, and ssDNA molecules according to the present invention.
  • Figure 5 shows the detection sensitivity of the present invention for single-stranded RNA samples.
  • Figure 6 shows the detection of the N gene of the new coronavirus according to the present invention.
  • Figure 7 is a comparison diagram of the step-by-step and one-pot detection methods of the present invention.
  • the invention is a one-pot single-stranded DNA circular amplification and CRISPR/Cas-mediated nucleic acid molecule detection method. The process is shown in Figure 1.
  • sequence list of primers, probes, etc. used in the examples is as follows:
  • Target 1 As the target sequence.
  • the Target 1 sequence is shown in SEQ ID NO.1, which is:
  • Preparation of guide RNA Synthesize the reverse complementary long primer crRNA-target-R containing the T7 sequence, as shown in SEQ ID NO.2, which is: TGTAAAACCTTTCTTTTTACGTTATCTACAACAGTAGAAATTACCCTATAGTGAGTCGTATTAATTTC, and the forward primer crRNA-F of T7, as shown in SEQ ID NO.3, is: GAAATTAATACGACTCACTATAGGG, DNA is made into incomplete double-stranded DNA by annealing of double primers. After preparation, store at -20 degrees or -80 degrees.
  • the single-stranded DNA probe sequence of Target 1 is PL target 1, 2, and 3, such as SEQ ID NO.4, which is:
  • amplification and detection reaction first anneal 100nM single-stranded DNA probe and double-stranded DNA to be detected at high temperature (85-95°C) for 5 minutes, then naturally cool and add to the reaction system.
  • the reaction system includes buffer
  • the solution (1 ⁇ ) includes 4mM dNTP, 40mM Tris-HCl, 10mM MgCl2, 0.5mM ATP and 10mM DTT, 0.5mg/ml bovine serum albumin.
  • the pH value of the buffer is 7.5.
  • Random primer 6Ns (NpNpNpNpNpNpsNs) (10 ⁇ M); guide RNA 100nM; FAM dual fluorescent labeled probe 200nM; enzyme mixture (T4 DNA ligase, 5U; Phi29 DNA polymerase, 10U; Cas12a protein, 250nM).
  • Fluorescence detection After the reaction is mixed, set the temperature to 37 degrees in the 7900 HT Fast Real-Time RCR system, the fluorescence detection probe is FAM, the TP sequence is as shown in SEQ ID NO.9: TTATTATT, and the CP sequence is as SEQ ID NO. .10 shows: TTTAACGTAAAAAGAAGGTTTTACACTT.
  • the fluorescence signal collection time interval is 1 min, and the detection time is 1 hour.
  • the specific reaction process is as follows: anneal the above-mentioned single-stranded DNA probe and the double-stranded DNA to be detected at high temperature (85-95°C) for 5 minutes, then naturally cool and add to the integrated amplification and reaction system, and react at a constant temperature of 37°C for 1 hour.
  • the 7900 HT Fast Real-Time RCR system is used simultaneously for fluorescence signal detection.
  • the fluorescence signal collection time interval is 1 minute, and the detection time is 1 hour.
  • Target 2 As the target sequence.
  • the Target 2 sequence is SEQ ID NO.5, which is:
  • the preparation method of the target single-stranded DNA is to synthesize a primer (Target 2) such as SEQ ID NO. 5, which is: TATTGTTAACGTGAGTCTTGTAAAACCTTCTTTTTACGTTTACTCTCGGTTAAAAAT, dissolve it in water and dilute it to 10uM.
  • a primer such as SEQ ID NO. 5, which is: TATTGTTAACGTGAGTCTTGTAAAACCTTCTTTTTACGTTTACTCTCGGTTAAAAAT, dissolve it in water and dilute it to 10uM.
  • Preparation of guide RNA Synthesize the reverse complementary long primer crRNA-target-R containing the T7 sequence as shown in SEQ ID NO.2, which is: TGTAAAACCTTCTTTTTACGTTATCTACAACAGTAGAAATTAC CCTATAGTGAGTC GTATTA ATTTC, and the forward primer crRNA-F of T7 as shown in SEQ ID NO.3 As shown, it is: GAAATTAATACGACTCACTATAGGG, DNA is made into incomplete double strands by melting double primers. After preparation, store at -20 degrees or -80 degrees.
  • the single-stranded DNA probe sequence of Target 2 is PL target 1, 2, 3.
  • SEQ ID NO.4 is: AAGGTTTTACActttccgtctttatagtctgtcgtattaatttctctttAACGTAAAAAG
  • amplification and detection reaction Add the ssDNA to be tested into the reaction system.
  • the reaction system includes buffer (1 ⁇ ) including 4mM dNTP, 40mM Tris-HCl, 10mM MgCl2, 0.5mM ATP, 10mM DTT, 0.5 mg/ml bovine serum albumin
  • the buffer pH value is 7.5. 100nM single-stranded DNA probe; random primer 6Ns (NpNpNpNpNpsNs) (10 ⁇ M); guide RNA 100nM; FAM dual fluorescent label probe 200nM; enzyme mixture (T4 DNA ligation Enzyme, 5U; Phi29 DNA polymerase, 10U; Cas12a protein, 250nM).
  • Fluorescence detection After the reaction is mixed, set the temperature to 37 degrees in the 7900 HT Fast Real-Time RCR system, and the fluorescence detection probe is FAM. As shown in the table, the fluorescence signal collection time interval is 1 min, and the detection time is 1 hour.
  • Target 3 As the target sequence.
  • the Target 3 sequence such as SEQ ID NO.6, is:
  • Preparation of guide RNA Synthesize the reverse complementary long primer crRNA-target-R containing the T7 sequence as shown in SEQ ID NO.2, which is: TGTAAAACCTTTCTTTTTACGTTATCTACAACAGTAGAAATTACCCTATAGTGAGTCGTATTAATTTC, and the forward primer crRNA-F of T7 as shown in SEQ ID NO.3: GAAATTAATACGACTCACTATAGGG, DNA is made into incomplete double-stranded DNA by annealing of double primers. After preparation, store at -20 degrees or -80 degrees.
  • SEQ ID NO.4 The single-stranded DNA probe sequence of Target 3 is shown in SEQ ID NO.4, which is:
  • Amplification and detection reaction Add the RNA to be tested into the reaction system.
  • the reaction system includes buffer (1 ⁇ ) including 4mM dNTP, 40mM Tris-HCl, 10mM MgCl2, 0.5mM ATP, 10mM DTT, and 0.5mg/ml bovine serum albumin.
  • the pH value of the buffer is 7.5. 100nM single-stranded DNA probe; random primer 6Ns (NpNpNpNpNpsNs) (10 ⁇ M); guide RNA 100nM; FAM dual fluorescent labeled probe 200nM; enzyme mixture (T4 DNA ligase, 5U; Phi29 DNA Polymerase, 10U; Cas12a protein, 250nM).
  • Fluorescence detection After the reaction is mixed, set the temperature to 37 degrees in the 7900 HT Fast Real-Time RCR system, and the fluorescence detection probe is FAM. As shown in the table, the fluorescence signal collection time interval is 1 min, and the detection time is 1 hour.
  • the specific reaction process is as follows: Add the above-mentioned single-stranded DNA probe and RNA to be detected into the integrated amplification and reaction system, react at a constant temperature of 37°C for 1 hour, and simultaneously use the 7900 HT Fast Real-Time RCR system for fluorescence signal detection.
  • the signal collection time interval is 1 minute, and the detection time is 1 hour.
  • Example 4 Using the present invention to detect new coronavirus
  • the new coronavirus is an RNA virus.
  • Total RNA is extracted from the nasopharyngeal samples to be tested, and the extracted total RNA is used as the RNA to be tested;
  • the SARS-CoV-2 gene sequence was selected as the target sequence.
  • the conserved region sequence of COVID-19 N is shown in SEQ ID NO.7, which is: AAUGGCUGGCAAUGGCGGUGAU.
  • the key probe sequence of the new coronavirus N gene sequence is selected as shown in SEQ ID NO.8, PL-N: TGCCAGCCATTctttccgtctttatagtctgtcgtattaatttctctttATCACCGCCAT.
  • Preparation of guide RNA Synthesize the reverse complementary long primer crRNA-N-R containing the T7 sequence as shown in SEQ ID NO.9: ATCACCGCCATTGCCAGCCATTATCTACAACAGTAGAAATTACCCTATAGTGAGTCGTATTAATTTC, and the forward primer crRNA-F of T7 as shown in SEQ ID NO.3: GAAATTAATACGACTCACTATAGGG, by double Primer quenching results in incomplete double-stranded DNA. After preparation, store at -20 degrees or -80 degrees.
  • Amplification and detection reaction Add the RNA to be tested into the reaction system.
  • the reaction system includes buffer including 1-5mM dNTP, 10-100mM Tris-HCl, 5-25mM MgCl2, 0.01-20mM ATP, 0.5-10mM DTT, 0.1- 1.5mg/ml bovine serum albumin, the pH value of the buffer is between 6.5-8.0.
  • Random primer 6N s (10 ⁇ M-100 ⁇ M); single-stranded DNA probe (100nM-400nM); guide RNA (100nM-400nM); FAM dual fluorescently labeled probe 1-4nM; enzyme mixture (T4 DNA ligase, 5U-200U ; Phi29 DNA polymerase, 5U-20U; Cas12a protein, 0.1ug-5ug).
  • Fluorescence detection After the reaction is mixed, set the temperature to 37 degrees in the 7900 HT Fast Real-Time RCR system, and the fluorescence detection probe is FAM. As shown in the table, the fluorescence signal collection time interval is 1 min, and the detection time is 1 hour.
  • the specific reaction process is as follows: Add the above-mentioned single-stranded DNA probe and RNA to be detected into the integrated amplification and reaction system, react at a constant temperature of 37°C for 1 hour, and simultaneously use the 7900 HT Fast Real-Time RCR system for fluorescence signal detection.
  • the signal collection time interval is 1 minute, and the detection time is 1 hour.
  • results As shown in Figure 6, the present invention can be used to detect the new coronavirus.
  • the steps of the one-pot single-stranded DNA circular amplification and CRISPR/Cas-mediated nucleic acid molecule detection method in Comparative Example 1 are basically the same as those in the Examples. The only difference is that the single-stranded DNA is circularized in separate steps.
  • Buffer 1 (B1) used: 40mM Tris-HCl, 10mM MgCl 2, 10mM DTT, 0.5mM ATP, pH 7.8 at 25°C; Phi29 amplification Buffer 2 (B2): 50mM Tris-HCl, 10mM MgCl2, 10mM (NH4) 2SO4, 4mM DTT, pH 7.5@25°C and CRISPR/Cas-mediated nucleic acid detection Buffer 3 (B3): 50mM NaCl, 10mM Tris-HCl, 10mM MgCl2, 100 ⁇ g/ml bovine serum albumin, pH 7.9 @25°C, optimized One-pot reaction Buffer (B): 4mM dNTP, 40mM Tris-HCl, 10mM MgCl2, 0.5mM ATP and 10mM DTT, 0.5mg/ml bovine serum albumin, pH 7.5@7.5). In order to verify the optimized effect, the above RNA was selected (Target 3) As the
  • Preparation of guide RNA Synthesize the reverse complementary long primer crRNA-target-R containing the T7 sequence as shown in SEQ ID NO.2: TGTAAAACCTTCTTTTTACGTTATCTACAACAGTAGAAATTACCCTATAGTGAGTCGTATTAATTTC, and the forward primer crRNA-F of T7 as shown in SEQ ID NO.3: GAAATTAATACGACTCACTATAGGG, Incomplete double-stranded DNA is produced by double-primer quenching. After preparation, store at -20 degrees or -80 degrees.
  • SEQ ID NO.4 The single-stranded DNA probe sequence of Target 3 is shown in SEQ ID NO.4, which is:
  • the one-pot B1 reaction system includes the buffer (1 ⁇ ) including 4mM dNTP, 40mM Tris-HCl, 10mM MgCl2, 10mM DTT, and 0.5mM ATP.
  • the buffer The liquid pH is 7.8.
  • the one-pot B2 reaction system includes, the buffer (1 ⁇ ) includes 4mM dNTP, 50mM Tris-HCl, 10mM MgCl2, 10mM (NH4)2SO4, 4mM DTT, 0.5mM ATP, and the pH value of the buffer is 7.5.
  • the one-pot B3 reaction system includes, the buffer (1 ⁇ ) includes 4mM dNTP, 50mM NaCl, 10mM Tris-HCl, 10mM MgCl2, 0.5mM ATP, and the pH value of the buffer is 7.9.
  • Buffer (B) includes, buffer (1 ⁇ ) includes 4mM dNTP, 40mM Tris-HCl, 10mM MgCl2, 0.5mM ATP and 10mM DTT, 0.5mg/ml bovine serum albumin buffer pH value is 7.5.
  • each system also includes: random primer 6Ns (NpNpNpNpNpsNs) (10 ⁇ M); FAM-labeled fluorescent probe 200nM, as shown in the table; enzyme mixture (T4 DNA ligase, 5U; Phi29 DNA polymerase, 10U; Cas12a protein , 250nM).
  • enzyme mixture T4 DNA ligase, 5U; Phi29 DNA polymerase, 10U; Cas12a protein , 250nM.
  • the specific reaction process is as follows: Add the above-mentioned single-stranded DNA probe and RNA to be detected into an integrated amplification and reaction system, react at a constant temperature of 37°C for 1.5 hours, and simultaneously use a 7900 HT Fast Real-Time RCR system for fluorescence signal detection.
  • the signal collection time interval is 1 minute, and the detection time is 1.5 hours.
  • Comparative analysis results As shown in Figure 7, this comparative analysis method can be used to detect single-stranded RNA in one pot.
  • the optimized one-pot detection buffer is better than B1, B2, and B3.
  • One-pot testing time can be shortened to 30 minutes.
  • the present invention has the following beneficial effects:
  • the present invention can be used to detect DNA or RNA
  • Multi-channel detection can be achieved and multiple samples can be detected at one time;
  • the present invention can complete the detection in as little as 30 minutes;
  • the present invention realizes the isothermal reaction of a single buffer in a single tube, with convenient operation and simple steps;
  • the reaction system of the present invention includes an amplification step, the detection and amplification products are RNA and DNA respectively. Only when the RNA target is present, the single-stranded DNA probe can be circularized to trigger amplification. reaction, overcoming the easy contamination characteristics of LAMP and fluorescence quantitative PCR. At the same time, this method is a closed-tube reaction and is physically isolated, which minimizes the possibility of contamination;
  • Normal temperature isothermal detection 3 types of engineering enzymes and chemical components work together to create an environment that simulates nucleic acid amplification in organisms to the greatest extent, and each engineering enzyme performs its own duties at its optimal reaction temperature. work, and therefore work most efficiently;

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Abstract

本发明公开了一锅法单链DNA环化扩增和CRISPR/Cas介导的核酸分子检测方法，所述方法包括:单链DNA探针环化、滚环扩增、单链DNA的双探针CRISPR/Cas剪切识别、以及单管一锅法检测反应。本发明主要针对环化、扩增、剪切识别不能同步进行的难题，采用一种同时衔接环化、扩增和剪切识别三步反应的OPERATOR技术，在体外单一反应管体系中对核酸分子精确、灵敏、快速检测。本发明相比传统的核酸分子检测方法，具有如下优点:适用于单链DNA、双链DNA和RNA分子的灵敏快速检测。特别是，检测RNA时不需要逆转录，直接进行一锅法检测，而不需要分步反应。

Description

一锅法单链DNA环化扩增和CRISPR/Cas介导的核酸分子检测方法 技术领域
本发明属于生物学核酸分子检测领域，具体涉及一种一锅法单链DNA环化扩增和CRISPR/Cas介导的核酸分子检测方法，本发明是一种新的核酸等温扩增与靶标信号读出一体化检测技术，利用DNA连接酶、链置换DNA聚合酶和CRISPR/Cas蛋白在常温条件下，可快速、一步法、单管完成特定DNA或者RNA的扩增和检测反应。
背景技术
脱氧核糖核酸(DNA)用荧光定量聚合酶链式反应(qPCR)进行扩增核检测反应是目前分子诊断的金标准。对于核糖核酸(RNA)的体外检测，往往需要分为两个分步骤，首先，需要将RNA样本逆转录成cDNA，然后，对cDNA样本进行常规的qPCR扩增检测分析。虽然，已经有商业化的逆转录荧光定量聚合酶链式反应(RT-qPCR)试剂盒，然而，目前RT-qPCR试剂盒参差不齐，单次测试灵敏度在45％～60％之间(Al-Tawfiq&Memish,2020)。此外，RT-qPCR检测需要昂贵的热循环仪、经验丰富的操作员、且对提取的RNA质量要求较高，同时，由于RT-qPCR的测试时间较长(从样本处理到结果读出，至少2-4小时)，种种因素限制了其在即时检测(Point of Care Test，POCT)中的应用。
近年来，随着核酸等温扩增技术的发展，其在核酸分子诊断中的作用越来越凸显，等温扩增技术主要利用了扩增酶在恒温中的置换性以及聚合酶特性，能够在引物的自发作用下实现对特定靶标的高效扩增。目前，已经衍生出多种核酸等温扩增方法，例如：环介导等温扩增技术(LAMP),它是一种利用4-6对引物识别靶标特异的位点，在60-65℃下，利用置换酶活性的Bst DNA聚合酶实现核酸的高效(1小时以内)扩增检测。重组酶聚合酶扩增(Recombinase Polymerase Amplification，RPA)技术，是在恒温37-42℃条件下，模拟体内核酸复制机制，由3种关键酶或蛋白：重组酶、单链结合蛋白和DNA聚合酶参与，协助DNA聚合酶扩增技术，整个反应一般在20-30min内获得可以检测水平的产品。基于核酸序列的扩增(NASBA)技术，由逆转录酶(RT)，T7RNA聚合酶和RNase H以及两个寡核苷酸引物组成。能够在60分钟左右，完成对RNA的快速扩增。
然而，以上几种等温扩增技术都存在各自的不足，例如：LAMP法扩增，需要的引物较多(4-6条)，现场检测中容易产生气溶胶污染导致假阳性的结果。对突变位点的扩增几乎无法避免。RPA法的酶组分较为复杂，无法对突变位点进行检测，同时，对于RNA样本仍需要逆转录步骤。滚环扩增方法(RCA)是一种具有链置换活性DNA聚合酶(Phi29)催化的等温扩增反应，该方法仅需要一条锁式探针与靶序列杂交后连接成环状模板，采用通用型引物与环状模板匹配后在DNA聚合酶作用下沿环延伸并不断置换先前产生的延伸链，最终产生重复的长单链DNA产物，具有在30-37℃直接高效扩增的优点。但是对于扩增的单链DNA的产物的检测缺乏有效和特异性的方法。
CRISPR(成簇的规律间隔的短回文重复序列)系统是一类广泛存在细菌或者古生菌微生物体内的一种天然“免疫系统”，CRISPR系统作为一种适应性免疫机制，其能够识别外来的遗传物质并且将其整合到自身基因组的CRISPR序列中，当外来遗传物质再次侵入时，通过Cas核酸酶精准剪切外源核酸。Cas核酸酶是CRISPR中一种重要的相关蛋白，目前，已发现多个CRISPR相关蛋白，如Cas9、Cas12、Cas13、Cas14等，其中后三者(Cas12、Cas13、Cas14)因其具有顺、反式切割活性，已被广泛用于核酸诊断领域。2019年，王金课题组利用Cas12结合LAMP等温开发出了HOLMES(S.Y.Li et al.,2018)，HOLMESv2(L.Li et al.,2019)检测系统，该系统灵敏度可达到10-8nM左右。但HOLMES方法对于无法对RNA样本直接检测，同时，由于扩增与检测的温度差异，无法实现一体化、单管检测。
发明内容
本发明的目的是为了克服现有技术上述缺陷，本发明了提供了一种一锅法单链DNA环化扩增和CRISPR/Cas介导的核酸分子检测方法，是一种单管一锅法超灵敏的核酸分子检测技术，名为：OPERATOR。涉及单链DNA探针环化、滚环复制和CRISPR/Cas12a剪切信号放大检测，具体涉及DNA连接酶和置换性扩增酶滚环扩增与CRISPR-Cas核酸酶剪切相结合的检测新方法，在完成扩增的同时进行检测反应。本发明通过反应缓冲液体系优化，使得连接、扩增、检测反应可以在同一个反应管中进行，单管实现了对RNA、ssDNA、dsDNA样本的检测。该方法具有超灵敏、特异性、检测速度快等优点。针对RNA样本的检测，OPERATOR技术无需逆转录步骤，直接对RNA分子进行扩增检测。
本发明采用以下技术方案实现上述目的：
本发明的实施例提供了一种一锅法单链DNA环化扩增和CRISPR/Cas介导的核酸 分子检测方法，所述核酸分子检测方法包括以下步骤：
(1)从检测样本中提取核酸样本；
(2)配制反应系统混合液，所述混合液包括：单链DNA探针、双荧光标记单链DNA探针(顺式探针CP，反式探针TP)、寡核苷酸引物、DNA连接酶或其变体、链置换DNA聚合酶或其变体、向导RNA(crRNA)或其衍生物、CRISPR相关Cas蛋白或其变体、OPERATOR反应缓冲液；其中，所述向导RNA或其衍生物包含与待检测核酸分子的靶序列相同的序列，单链DNA探针与待检测核酸分子的一条链特异性的互补；且单链DNA的骨架序列指除互补靶标部分的序列或其衍生物以外，还包括PAM位点序列和随机连接序列；
(3)将核酸样本加入反应体系混合液中，进行恒温反应；
(4)荧光探针被剪切后产生可以检测的荧光信号，读取和记录产生的荧光信号，得核酸检测结果。
单链DNA探针在DNA连接酶作用下形成单链环状DNA探针；单链环状DNA探针与待测核酸分子杂交，在DNA连接酶作用下换成为环状DNA；寡核苷酸引物以环状DNA为模板，能够随机结合在环状DNA模板上，并在链置换DNA聚合酶作用下连续延伸形成长的含有重复单链DNA探针序列的长链DNA。
进一步地，步骤(1)中，所述核酸样本中待检测核酸分子包括单链DNA、双链DNA、单链RNA中的一种或多种。若待检测的核酸为dsDNA，在反应之前对dsDNA预变性处理。
进一步地，步骤(2)中，所述单链DNA探针包括5’端、3’端、骨架序列。所述5’端和3’端分别和待检核酸分子序列互补；所述骨架序列指除互补靶标部分的序列或其衍生物，包括PAM位点序列和随机连接序列。随机连接序列一般长度为40-80bp，GC含量在30％-70％。单链DNA探针在序列3’末端引入“TTT”PAM位点。用于识别靶标序列、环化以及扩增的模板，使得扩增后的产物不受PAM约束。
进一步地，步骤(2)中，所述寡核苷酸引物为碱基修饰后的随机引物(6-10nt)或与待检测核酸分子序列一致的引物(10-20nt)。修饰碱基个数为1-10。随机引物为随机六聚体引物。随机六聚体引物是含有6个碱基的随机序列引物。
进一步地，步骤(2)中，所双荧光标记单链DNA探针的序列和待检核酸分子序列互补，探针的5’端标记有荧光基团，3’端标记有淬灭基团；探针的5’端的荧光基团包括FAM、HEX、VIC、Cy5、Cy3、ROX、FITC、Joe中的一种，3’端标记的荧光淬灭基团 包括TAMRA、BHQ1、MGB、BHQ2中的一种。
进一步地，步骤(2)中，所述DNA连接酶为连接双链DNA分子或者RNA/DNA杂交双链的单链DNA缺口的连接酶。DNA连接酶包括T4 DNA连接酶、E.coli DNA连接酶、SplintR连接酶、HiFi Taq DNA ligase中的一种。该酶可以包括野生型、改造过的、密码子优化过的、进化过的、嗜热的、嵌合的、工程化的和/或一种以上DNA连接酶的混合物。所述的DNA连接酶优选为T4 DNA连接酶。DNA连接酶能够特异连接与靶标互补的ssDNA的磷酸二酯键。
进一步地，步骤(2)中，所述链置换DNA聚合酶包括Phi29、Klenow、Vent中的一种。所述DNA聚合酶优选为Phi29 DNA聚合酶。该酶可以包括野生型、改造过的、密码子优化过的、进化过的、嗜热的、嵌合的、工程化的和/或一种以上逆转录酶的混合物。DNA聚合酶能够识别并在随机引物的触发链置换扩增反应产生ssDNA。
进一步地，步骤(2)中，所述CRISPR相关Cas蛋白为具有双链DNA或单链DNA识别剪切功能和反式DNA单链剪切功能的CRISPR-Cas核酸酶。所述CRISPR-Cas核酸酶包括SpyCas9、FnCas9、FnCas12a，LbCas12、BhCas12b、Bs3Cas12b、LsCas12b、SbCas12b、AaCas12b、AkCas12、AmCas12b、BsCas12b、DiCas12b、TcCas12b、AacCas12b、LwCas13、Cas14中的一种或其变体中的一种。该酶可以包括野生型、改造过的、密码子优化过的、进化过的、嗜热的、嵌合的、工程化的和/或一种以上Cas蛋白的混合物。CRISPR-Cas核酸酶优选为Cas12a。CRISPR-Cas核酸酶结合向导RNA可以被目标核酸序列特异性激活从而具有非特异性的DNA核酸酶活性，实现对DNA荧光探针的剪切。
进一步地，步骤(2)中所述向导RNA或其衍生物的间隔序列与目标核酸分子序列互补。
进一步地，步骤(2)中，所述OPERATOR反应缓冲液包括1-5mM dNTP、10-100mM Tris-HCl、5-25mM MgCl 2、0.01-20mM ATP、0.5-10mM DTT和0.5-1.5mg/ml牛血清蛋白，所述缓冲液pH值在6.5-8.0之间。
所述环化后的锁匙探针在随机引物的触发下引发高效的链置换扩增反应，所述随机引物长度为6nt DNA随机引物，使用的终浓度不低于10μM。随机引物能够触发高效的滚环扩增，效率远高于传统的单引物触发扩增。
进一步地，步骤(3)中，所述恒温反应的温度为37℃，反应时间为1h。单链DNA探针在DNA连接酶作用下形成单链环状DNA探针；恒温反应时，单链环状DNA探针与待测核酸分子杂交，在DNA连接酶作用下换成为环状DNA；寡核苷酸引物以环状 DNA为模板，能够随机结合在环状DNA模板上，并在链置换DNA聚合酶作用下连续延伸形成长的含有重复单链DNA探针序列的长链DNA；长链DNA与双荧光标记单链DNA探针结合形成互补的双链DNA；形成的双链DNA被crRNA和Cas蛋白复合物识别，并剪切双荧光标记单链DNA探针，产生可以检测的荧光信号。
本发明的第二个目的是提供一种基于所述核酸分子检测方法的等温核酸检测试剂盒。可以实现常温等温条件下对特定RNA或DNA分子的精准、快速和高灵敏度检测。
优选地，所述试剂盒包括酶混合液、单链DNA探针、向导RNA、双荧光标记单链DNA探针、寡核苷酸引物和OPERATOR反应缓冲液；所述酶混合液包括CRISPR-Cas核酸酶、DNA连接酶、链置换DNA聚合酶。
进一步地，所述CRISPR-Cas核酸酶为FnCas12a。锁匙探针(单链DNA探针)由与靶序列互补的序列与环骨架序列组成，锁匙探针序列3’末端引入“TTT”PAM位点；所述寡核苷酸引物为随机六聚体引物；所述DNA连接酶为T4 DNA连接酶；所述DNA聚合酶为Phi29 DNA聚合酶；所述DNA荧光探针为5‘端标记荧光基团、3’端标记荧光淬灭基团的单链DNA。
在一个具体实施例中，缓冲液包括1-5mM dNTP、10-100mM Tris-HCl、5-25mM MgCl2、0.01-20mM ATP和0.5-10mM DTT、0.1-1.5mg/ml牛血清蛋白，所述缓冲液pH值在6.5-8.0之间。随机引物6Ns(10μM-100μM)；FAM标记的荧光探针1-4nM；酶混合物(T4 DNA连接酶，5U-200U；Phi29 DNA聚合酶，5U-20U；Cas12a蛋白，0.1ug-5ug)。
在本发明试剂盒中，靶标DNA、向导RNA和Cas12a蛋白形成复合物，该复合物会切割体系中其他的单链DNA分子。
本发明的检测方法和试剂盒可以检测细菌、真菌毒、人或其他动植物组织的核酸分子。
本发明还提供了反应系统，所述系统具有：单链DNA探针、双荧光标记单链DNA探针、寡核苷酸引物、DNA连接酶及其变体、链置换DNA聚合酶及其变体、成簇的规律间隔的短回文重复(CRISPR)RNA(crRNA)或其衍生物、CRISPR相关(Cas)蛋白或其变体、OPERATOR反应缓冲液。其中所述crRNA或其衍生物包含与待检测核酸分子的靶序列相同。
本发明能在常温等温条件下，快速完成DNA或RNA分子的检测，首先通过核酸提取得到待检测样本的RNA、单链DNA或双链DNA；再利用连接酶、扩增酶、CRISPR 相关蛋白的组合酶、单链DNA探针以及核酸荧光探针与待检核酸在等温进行反应，最后通过检测荧光信号，判断待检测样本中是否存在目标核酸。
附图说明
通过阅读参照以下附图对非限制性实施例所作的详细描述，本发明的其它特征、目的和优点将会变得更明显：
图1为本发明检测样本的流程示意图。
图2为本发明检测ssDNA或RNA样本的流程示意图。
图3为本发明检测DNA样本的流程示意图。
图4为本发明对RNA、dsDNA、ssDNA分子的检测。
图5为本发明对单链RNA样本的检测灵敏度。
图6为本发明检测新冠病毒N基因。
图7为本发明分步骤与一锅法检测的对比图。
具体实施方式
下面结合附图和具体实施例对本发明进行详细说明。以下实例在本发明技术方案的前提下进行实施，提供了详细的实施方式和具体的操作过程，将有助于本领域的技术人员进一步理解本发明。需要指出的是，本发明的保护范围不限于下述实施例，在本发明的构思前提下做出的若干调整和改进，都属于本发明的保护范围。
本发明是一种一锅法单链DNA环化扩增和CRISPR/Cas介导的核酸分子检测方法，流程如图1所示。
实施例所用到的引物、探针等序列列表如下:
Figure PCTCN2022140596-appb-000001
实施例1：使用本发明检测dsDNA靶标
选用dsDNA(Target 1)作为靶标序列，Target 1序列如SEQ ID NO.1所示，为：
Figure PCTCN2022140596-appb-000002
向导RNA的制备：合成包含T7序列的反向互补长引物crRNA-target-R如SEQ ID NO.2所示，为:TGTAAAACCTTCTTTTTACGTTATCTACAACAGTAGAAATTACCCTATAGTGAGTCGTATTAATTTC，T7的正向引物crRNA-F如SEQ ID NO.3，为：GAAATTAATACGACTCACTATAGGG，通过双引物褪火制成DNA不完全双链。制备完成后保存于-20度或者-80度。
Target 1的单链DNA探针序列为PL target 1，2，3如SEQ ID NO.4，为：
Figure PCTCN2022140596-appb-000003
如图3所示，扩增和检测反应：首先将100nM单链DNA探针与待检双链DNA高温(85-95℃)退火5分钟，然后自然冷却后加入反应体系，反应体系包括，缓冲液(1×)包括4mM dNTP、40mM Tris-HCl、10mM MgCl2、0.5mM ATP和10mM DTT、0.5mg/ml牛血清蛋白所述缓冲液pH值为7.5。随机引物6Ns(NpNpNpNpNpsNs)(10μM)；向导RNA 100nM；FAM双荧光标记探针200nM；酶混合物(T4 DNA连接酶，5U；Phi29 DNA聚合酶，10U；Cas12a蛋白，250nM)。
荧光检测：反应混合后，在7900 HT Fast Real-Time RCR系统中设置温度为37度，荧光检测探针为FAM，TP序列如SEQ ID NO.9所示：TTATTATT，CP的序列如SEQ ID NO.10所示：TTTAACGTAAAAAGAAGGTTTTACACTT。
荧光信号采集时间间隔为1min，检测时间为1小时。
具体反应过程如下：将上述单链DNA探针与待检双链DNA高温(85-95℃)退火5分钟，然后自然冷却后加入扩增和反应的一体化体系中，37℃恒温反应1小时，同步采用7900 HT Fast Real-Time RCR系统进行荧光信号检测，荧光信号采集时间间隔为1min，检测时间为1小时。
结果：如图4所示，应用此方法可以检测双链DNA。
实施例2：使用本发明检测ssDNA靶标
选用ssDNA(Target 2)作为靶标序列，Target 2序列为如SEQ ID NO.5，为：
Figure PCTCN2022140596-appb-000004
靶标单链DNA的制备方法为，合成引物(Target 2)如SEQ ID NO.5，为：TATTGTTAACGTGAGTCTTGTAAAACCTTCTTTTTACGTTTACTCTCGTGTTAAAAAT，溶解于水中稀释至10uM。
向导RNA的制备：合成包含T7序列的反向互补长引物crRNA-target-R如SEQ ID NO.2所示，为:TGTAAAACCTTCTTTTTACGTTATCTACAACAGTAGAAATTAC CCTATAGTGAGTC GTATTAATTTC，T7的正向引物crRNA-F如SEQ ID NO.3所示，为：GAAATTAATACGACTCACTATAGGG，通过双引物褪火制成DNA不完全双链。制备完成后保存于-20度或者-80度。
Target 2的单链DNA探针序列为PL target 1，2，3，如SEQ ID NO.4为：AAGGTTTTACActttccgtctttatagtctgtcgtattaatttctctttAACGTAAAAAG
如图2所示，扩增和检测反应：将待检ssDNA加入反应体系，反应体系包括，缓冲液(1×)包括4mM dNTP、40mM Tris-HCl、10mM MgCl2、0.5mM ATP和10mM DTT、0.5mg/ml牛血清蛋白所述缓冲液pH值为7.5。100nM单链DNA探针；随机引物6Ns(NpNpNpNpNpsNs)(10μM)；向导RNA 100nM；FAM双荧光标记探针200nM；酶混合物(T4 DNA连接酶，5U；Phi29 DNA聚合酶，10U；Cas12a蛋白，250nM)。
荧光检测：反应混合后，在7900 HT Fast Real-Time RCR系统中设置温度为37度，荧光检测探针为FAM，如表所示，荧光信号采集时间间隔为1min，检测时间为1小时。
结果：如图4所示，应用此方法可以检测单链DNA。
实施例3：使用本发明检测RNA靶标
选用RNA(Target 3)作为靶标序列，Target 3序列如SEQ ID NO.6，为：
Figure PCTCN2022140596-appb-000005
Figure PCTCN2022140596-appb-000006
向导RNA的制备：合成包含T7序列的反向互补长引物crRNA-target-R如SEQ ID NO.2所示，为:TGTAAAACCTTCTTTTTACGTTATCTACAACAGTAGAAATTACCCTATAGTGAGTCGTATTAATTTC，T7的正向引物crRNA-F如SEQ ID NO.3所示：GAAATTAATACGACTCACTATAGGG，通过双引物褪火制成DNA不完全双链。制备完成后保存于-20度或者-80度。
Target 3的单链DNA探针序列如SEQ ID NO.4所示，为：
Figure PCTCN2022140596-appb-000007
扩增和检测反应：将待检RNA加入反应体系，反应体系包括，缓冲液(1×)包括4mM dNTP、40mM Tris-HCl、10mM MgCl2、0.5mM ATP和10mM DTT、0.5mg/ml牛血清蛋白所述缓冲液pH值为7.5。100nM单链DNA探针；随机引物6Ns(NpNpNpNpNpsNs)(10μM)；向导RNA 100nM；FAM双荧光标记探针200nM；酶混合物(T4 DNA连接酶，5U；Phi29 DNA聚合酶，10U；Cas12a蛋白，250nM)。
荧光检测：反应混合后，在7900 HT Fast Real-Time RCR系统中设置温度为37度，荧光检测探针为FAM，如表所示，荧光信号采集时间间隔为1min，检测时间为1小时。
具体反应过程如下：将上述单链DNA探针与待检RNA加入扩增和反应的一体化体系中，37℃恒温反应1小时，同步采用7900 HT Fast Real-Time RCR系统进行荧光信号检测，荧光信号采集时间间隔为1min，检测时间为1小时。
结果：如图4所示，应用此方法可以检测单链RNA；如图5所示，应用此方法可以检测低至1.625拷贝/微升的单链RNA分子。
实施例4：使用本发明检测新冠病毒
新冠病毒为RNA病毒，从待检鼻咽样本中提取总RNA，提取的总RNA作为待检RNA；
选用新冠N基因序列作为靶标序列，新冠N的保守区序列如SEQ ID NO.7所示，为：AAUGGCUGGCAAUGGCGGUGAU。
选用新冠N基因序列锁匙探针序列如SEQ ID NO.8所示，PL-N:TGCCAGCCATTctttccgtctttatagtctgtcgtattaatttctctttATCACCGCCAT。
向导RNA的制备：合成包含T7序列的反向互补长引物crRNA-N-R如SEQ ID NO.9所示：ATCACCGCCATTGCCAGCCATTATCTACAACAGTAGAAATTACCCTATAGTGAGTCGTATTAATTTC，T7的正向引物crRNA-F如SEQ ID NO.3所示：GAAATTAATACGACTCACTATAGGG，通过双引物褪火制成DNA不完全双链。制备完成后保存于-20度或者-80度。
扩增和检测反应：将待检RNA加入反应体系，反应体系包括，缓冲液包括1-5mM dNTP、10-100mM Tris-HCl、5-25mM MgCl2、0.01-20mM ATP和0.5-10mM DTT、0.1-1.5mg/ml牛血清蛋白，所述缓冲液pH值在6.5-8.0之间。随机引物6N s(10μM-100μM)；单链DNA探针(100nM-400nM)；向导RNA(100nM-400nM)；FAM双荧光标记探针1-4nM；酶混合物(T4 DNA连接酶，5U-200U；Phi29DNA聚合酶，5U-20U；Cas12a蛋白，0.1ug-5ug)。
荧光检测：反应混合后，在7900 HT Fast Real-Time RCR系统中设置温度为37度，荧光检测探针为FAM，如表所示，荧光信号采集时间间隔为1min，检测时间为1小时。
具体反应过程如下：将上述单链DNA探针与待检RNA加入扩增和反应的一体化体系中，37℃恒温反应1小时，同步采用7900 HT Fast Real-Time RCR系统进行荧光信号检测，荧光信号采集时间间隔为1min，检测时间为1小时。
结果：如图6所示，本发明可用于新冠病毒进行检测。
对比例1
本对比例1中的一锅法单链DNA环化扩增和CRISPR/Cas介导的核酸分子检测方法，步骤与实施例基本相同，不同之处仅在于：将分步骤中单链DNA环化所用Buffer 1(B1)：40mM Tris-HCl,10mM MgCl 2,10mM DTT,0.5mM ATP,pH 7.8 at 25℃；Phi29扩增Buffer 2(B2)：50mM Tris-HCl,10mM MgCl2,10mM(NH4)2SO4,4mM DTT,pH 7.5@25℃和CRISPR/Cas介导的核酸检测Buffer 3(B3)：50mM NaCl,10mM Tris-HCl,10mM MgCl2,100μg/ml牛血清蛋白,pH  7.9 @25℃，优化为一锅反应Buffer(B)：4mM dNTP、40mM Tris-HCl、10mM MgCl2、0.5mM ATP和10mM DTT、0.5mg/ml牛血清蛋白,pH 7.5@7.5),为了验证优化后效果，选用上述RNA(Target 3)作为靶标序列分别对B1，B2，B3以及一锅法反应B对比，Target 3序列如SEQ ID NO.7所示，为：
Figure PCTCN2022140596-appb-000008
向导RNA的制备：合成包含T7序列的反向互补长引物crRNA-target-R如SEQ ID NO.2所示:TGTAAAACCTTCTTTTTACGTTATCTACAACAGTAGAAATTACCCTATAGTGAGTCGTATTAATTTC，T7的正向引物crRNA-F如SEQ ID NO.3所示：GAAATTAATACGACTCACTATAGGG，通过双引物褪火制成DNA不完全双链。制备完成后保存于-20度或者-80度。
Target 3的单链DNA探针序列如SEQ ID NO.4所示，为：
Figure PCTCN2022140596-appb-000009
一锅法检测反应：将待检RNA加入反应体系，一锅法B1反应体系包括，缓冲液(1×)包括4mM dNTP、40mM Tris-HCl、10mM MgCl2、10mM DTT、0.5mM ATP，所述缓冲液pH值为7.8。一锅法B2反应体系包括，缓冲液(1×)包括4mM dNTP、50mM Tris-HCl、10mM MgCl2、10mM(NH4)2SO4、4mM DTT、0.5mM ATP，所述缓冲液pH值为7.5。一锅法B3反应体系包括，缓冲液(1×)包括4mM dNTP、50mM NaCl、10mM Tris-HCl、10mM MgCl2、0.5mM ATP，所述缓冲液pH值为7.9。一锅法优化反应体系Buffer(B)包括，缓冲液(1×)包括4mM dNTP、40mM Tris-HCl、10mM MgCl2、0.5mM ATP和10mM DTT、0.5mg/ml牛血清蛋白所述缓冲液pH值为7.5。此外，各体系中还包括：随机引物6Ns(NpNpNpNpNpsNs)(10μM)；FAM标记的荧光探针200nM，如表所示；酶混合物(T4 DNA连接酶，5U；Phi29 DNA聚合酶，10U；Cas12a蛋白，250nM)。各反应体系混合后，在7900 HT Fast Real-Time RCR系统中设置温度为37度，荧光检测探针为FAM，荧光信号采集时间间隔为1min，检测时间为1.5小时。
具体反应过程如下：将上述单链DNA探针与待检RNA加入扩增和反应的一体化体系中，37℃恒温反应1.5小时，同步采用7900 HT Fast Real-Time RCR系统进行荧光信号检测，荧光信号采集时间间隔为1min，检测时间为1.5小时。
对比分析结果：如图7所示，应用此对比分析方法可以一锅法检测单链RNA，优化后的一锅法检测buffer比B1，B2，B3更佳。一锅法检测时间可缩短至30分 钟。
与现有技术相比，本发明具有如下有益效果：
(1)高灵敏度：应用本发明检测核酸分子(ssDNA、dsDNA和RNA)，可以实现对单拷贝级别的核酸分子检测；
(2)通用性：应用本发明可以实现对DNA或RNA的检测；
(3)多通道：可以实现多通道的检测，一次性检测多个样本；
(4)快速：本发明可以短至30分钟内完成检测；
(5)便捷：本发明实现了单管单个缓冲液的等温反应，操作方便，步骤简便；
(6)假阳性低：本发明的反应体系虽然包含扩增步骤，但检测和扩增产物分别为RNA和DNA，只有当RNA靶标存在时，单链DNA探针才能被环化从而引发扩增反应，克服了LAMP、荧光定量PCR等易污染的特点。同时，本方法是闭管反应，在物理上进行隔绝，最大可能降低了污染的可能性；
(7)常温等温检测：由3种工程酶和化学组分一起创造出一个最大程度模拟生物体内核酸扩增的环境，而且每种工程酶都各司其职，在其最适的反应温度上工作，因此工作效率最高；
(8)一步法：构建了三种反应兼容的缓冲液，将单链DNA探针环化、扩增、Cas12a剪切检测反应放置再同一反应管中进行，通过一次性加样完成样本的检测，因此操作更简单。
以上对本发明的具体实施例进行了描述。需要理解的是，本发明并不局限于上述特定实施方式，本领域技术人员可以在权利要求的范围内做出各种变形或修改，这并不影响本发明的实质内容。

Claims (10)

  1. 一锅法单链DNA环化扩增和CRISPR/Cas介导的核酸分子检测方法，其特征在于，所述核酸分子检测方法包括如下步骤：
    (1)从检测样本中提取核酸样本；
    (2)配制反应系统混合液，所述混合液包括：单链DNA探针、双荧光标记单链DNA探针、寡核苷酸引物、DNA连接酶或其变体、链置换DNA聚合酶或其变体、向导RNA或其衍生物、CRISPR相关Cas蛋白或其变体、OPERATOR反应缓冲液；其中，所述向导RNA或其衍生物包含与待检测核酸分子的靶序列相同的序列，单链DNA探针与待检测核酸分子的一条链特异性的互补，且单链DNA的骨架序列指除互补靶标部分的序列或其衍生物以外，还包括PAM位点序列和随机连接序列；
    (3)将核酸样本加入反应体系混合液中，进行恒温反应；
    (4)荧光探针被剪切后产生可以检测的荧光信号，读取和记录产生的荧光信号，得核酸检测结果。
  2. 根据权利要求1所述的核酸分子检测方法，其特征在于，步骤(1)中所述核酸样本中待检测核酸分子包括单链DNA、双链DNA、单链RNA中的一种或多种。
  3. 根据权利要求1所述的核酸分子检测方法，其特征在于，步骤(2)中所述单链DNA探针的5’端和3’端分别和待检核酸分子序列互补，其中所述单链DNA的骨架序列指除互补靶标部分的序列或其衍生物以外，还包括PAM位点序列和随机连接序列。
  4. 根据权利要求1所述的核酸分子检测方法，其特征在于，步骤(2)中所述寡核苷酸引物为碱基修饰后的随机引物或与待检测核酸分子序列一致的引物。
  5. 根据权利要求1所述的核酸分子检测方法，其特征在于，所双荧光标记单链DNA探针的序列和待检核酸分子序列互补，探针的5’端、3’端标记有荧光基团；探针的5’端的荧光基团包括FAM、HEX、VIC、Cy5、Cy3、ROX、FITC、Joe中的一种，3’端标记的荧光淬灭基团包括TAMRA、BHQ1、MGB、BHQ2中的一种。
  6. 根据权利要求1所述的核酸分子检测方法，其特征在于，步骤(2)中所述DNA连接酶为连接双链DNA分子或者RNA/DNA杂交双链的单链DNA缺口的连接酶；DNA连接酶包括T4DNA连接酶、E.coli DNA连接酶、SplintR连接酶、HiFi Taq DNA ligase中的一种；所述链置换DNA聚合酶包括Phi29、Klenow、Vent中的一种。
  7. 根据权利要求1所述的OPERATOR等温检测方法，其特征在于，步骤(2)中 所述CRISPR相关Cas蛋白为具有双链DNA或单链DNA识别剪切功能和反式DNA单链剪切功能的CRISPR-Cas核酸酶；所述CRISPR-Cas核酸酶包括SpyCas9、FnCas9、FnCas12a，LbCas12、BhCas12b、Bs3Cas12b、LsCas12b、SbCas12b、AaCas12b、AkCas12、AmCas12b、BsCas12b、DiCas12b、TcCas12b、AacCas12b、LwCas13、Cas14中的一种或其变体中的一种。
  8. 根据权利要求1所述的核酸分子检测方法，其特征在于，步骤(2)中所述向导RNA或其衍生物的间隔序列与目标核酸分子序列互补。
  9. 根据权利要求1所述的核酸分子检测方法，其特征在于，步骤(2)中所述OPERATOR反应缓冲液包括1-5mM dNTP、10-100mM Tris-HCl、5-25mM MgCl 2、0.01-20mM ATP、0.5-10mM DTT和0.5-1.5mg/ml牛血清蛋白，所述缓冲液pH值在6.5-8.0之间。
  10. 一种基于权利要求1所述的核酸分子检测方法的等温核酸检测试剂盒，其特征在于，所述试剂盒包括酶混合液、单链DNA探针、向导RNA、双荧光标记单链DNA探针、寡核苷酸引物和OPERATOR反应缓冲液；所述酶混合液包括CRISPR-Cas核酸酶、DNA连接酶、链置换DNA聚合酶。
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