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WO2023246033A1 - 一锅法滚环转录和CRISPR/Cas介导的核酸检测方法及试剂盒 - Google Patents

一锅法滚环转录和CRISPR/Cas介导的核酸检测方法及试剂盒 Download PDF

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WO2023246033A1
WO2023246033A1 PCT/CN2022/140597 CN2022140597W WO2023246033A1 WO 2023246033 A1 WO2023246033 A1 WO 2023246033A1 CN 2022140597 W CN2022140597 W CN 2022140597W WO 2023246033 A1 WO2023246033 A1 WO 2023246033A1
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nucleic acid
rna
stranded
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dna
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杨立桃
郭永坤
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Shanghai Jiao Tong University
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Definitions

  • the invention belongs to the field of rapid nucleic acid detection in molecular biology, and specifically relates to a one-pot rolling circle transcription and CRISPR/Cas-mediated nucleic acid detection method and kit.
  • it involves a new integrated detection technology of nucleic acid isothermal amplification and signal output, which uses DNA ligase, RNA polymerase and CRISPR/Cas protein under normal temperature and isothermal conditions to quickly, one-step and single-tube complete the synthesis of specific DNA or RNA. Amplification and detection.
  • qPCR Fluorescent quantitative polymerase chain reaction
  • Nucleic acid isothermal amplification technology has emerged as a promising alternative method that can achieve rapid and efficient amplification of target nucleic acid molecules under isothermal conditions without the need for a thermal cycler required for qPCR.
  • isothermal amplification can be performed under simple conditions, such as room temperature, water bath, etc., especially for rapid detection of nucleic acid molecules in areas with limited resources, which has unique advantages that qPCR cannot match.
  • LAMP loop-mediated isothermal amplification
  • RPA Recombinase polymerase amplification
  • a variety of key enzymes or proteins assist DNA polymerase amplification to achieve exponential amplification of nucleic acids. The entire reaction It usually takes 20-30 minutes.
  • Nucleic acid sequence-based amplification (NASBA) technology uses reverse transcriptase, T7 RNA polymerase and RNase H and two oligonucleotide primers to achieve rapid and continuous amplification of RNA at around 41°C (about 60 minutes) .
  • Rolling circle amplification (RCA) technology uses DNA ligase and DNA polymerase with strand displacement activity to perform strand displacement of a circular template under the guidance of one or more primers at 30°C to generate multiple repeats of the target sequence. For long single chains, the whole process usually takes about 2 hours.
  • LAMP amplification requires a lot of primers (4-6) and has relatively high requirements for primer design.
  • the detection of point mutations or modification sites is often Cannot meet the requirement.
  • LAMP-amplified products can easily form aerosol contamination when the lid is opened, leading to false-positive results.
  • the enzyme components of the RPA method are relatively complex and require high buffer solutions.
  • the stability of the reaction system is not strong, which can easily lead to poor reproducibility. In addition, it cannot detect mutation sites.
  • LAMP and RPA still require reverse transcription for RNA sample detection.
  • the NASBA method is not suitable for the detection of DNA.
  • the reaction components are complex and have poor stability, and are easily affected by the matrix.
  • Rolling circle transcription is an isothermal amplification reaction catalyzed by RNA polymerase with in vitro transcription activity. After one strand of the target nucleic acid molecule hybridizes with a single-stranded DNA probe, it is ligated into a circular template under the action of DNA ligase. Under the action of RNA polymerase, it is continuously transcribed along the circular template into a long, repeating single-stranded sequence containing the target sequence. stranded RNA products to achieve efficient amplification at room temperature.
  • RCT Rolling circle transcription
  • the CRISPR (clustered regularly interspaced short palindromic repeats) system is an immune system evolved by bacteria or archaea to resist viral infections. It can recognize foreign genetic material and integrate it 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.
  • Cas13a is a newly identified CRISPR nuclease in recent years. This nuclease has the activity of being activated by specific RNA to obtain non-specific RNA nuclease, thereby cutting other single-molecule RNA nuclease. Cas13a can detect specific RNA products when combined with an RNA fluorescence reporter system.
  • Cas13a has low sensitivity when acting alone, and can only achieve fM to pM level nucleic acid detection.
  • the sensitivity for molecular diagnosis is not good, such as without nucleic acid amplification.
  • Potential tumor markers miR-19b and miR-20a were directly detected under increased conditions, with a detection limit of 10 pM.
  • Zhang Feng and others used Cas13a protein combined with isothermal amplification technology RPA to develop SHERLOCK, a new method that can detect nucleic acids. Its sensitivity can achieve the detection of aM-level samples.
  • the SHERLOCK method cannot achieve one-step single-tube detection for RNA detection, and the RPA method has complex components and poor stability, which increases the difficulty of operation.
  • Embodiments of the present invention provide one-pot rolling circle transcription and CRISPR/Cas-mediated nucleic acid detection methods and kits, and specifically provide a nucleic acid based on single-stranded DNA circularization, transcription and CRISPR-related Cas proteins or variants thereof Detect new methods (PROTRACTOR).
  • DNA ligase is used to circularize single-stranded DNA probes and convert target molecule signals into circular DNA signals;
  • RNA polymerase uses circular DNA as a template to complete efficient transcription; CRISPR-related Cas proteins or their variants are guided by crRNA The transcript product is recognized and cut, and the detection reaction is performed while completing the amplification.
  • RNA, ssDNA, or dsDNA target nucleic acid molecules
  • One embodiment of the present invention provides a PROTRACTOR isothermal nucleic acid detection method, which includes the following steps:
  • a reaction system which includes a single-stranded DNA probe, an RNA fluorescent probe, a DNA ligase or a variant thereof, an RNA polymerase or a variant thereof, a guide RNA or its derivatives, a CRISPR-related Cas protein, or Its variant, PROTRACTOR reaction buffer; wherein the single-stranded DNA probe is specifically complementary to one strand of the target nucleic acid molecule;
  • step S3 Add the total nucleic acid extracted in step S2 to the reaction system in step S1 to perform a constant temperature reaction and generate a fluorescent signal; during the constant temperature reaction, the single-stranded DNA probe forms a single-stranded loop under the action of DNA ligase or its variant. shaped DNA probe;
  • the single-stranded DNA probe is composed of a DNA sequence complementary to one strand of the target nucleic acid molecule sequence and a connecting sequence containing T7p.
  • the single-stranded DNA probe sequence is specifically complementary to one strand of the target nucleic acid molecule, and the single-stranded DNA probe forms a single-stranded circular DNA probe under the action of DNA ligase or a variant thereof;
  • the single-stranded DNA probe is used to specifically identify and bind the target sequence on one strand of the target nucleic acid molecule, and is used as a template for amplification after circularization.
  • the single-stranded DNA probe is composed of one strand of the target nucleic acid molecule sequence. It consists of a complementary DNA sequence and a connecting sequence.
  • the connecting sequence contains a T7 promoter complementary sequence (T7p), which can be recognized and combined by RNA polymerase to start transcription;
  • the single-stranded circular DNA probe is continuously transcribed into long single-stranded RNA under the action of RNA polymerase or its variants, which contains multiple repeats of the target sequence of the target nucleic acid molecule;
  • RNA amplicons are formed through transcription and are recognized by the binary complex formed by guide RNA or its derivatives and CRISPR-related Cas protein or its variants, and cut the RNA fluorescent probe to generate a detectable fluorescent signal. ;
  • a fluorescence detector is used to read and record the fluorescence signal generated by PROTRACTOR, and the fluorescence signal is used to determine the presence or absence of the target nucleic acid molecule in the sample to be detected.
  • the target nucleic acid molecule is dsDNA, pre-denature the dsDNA before the reaction.
  • the PROTRACTOR is a universal nucleic acid detection platform that can detect different types of nucleic acid molecules, including one or more of single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), and single-stranded RNA (ssRNA).
  • ssDNA single-stranded DNA
  • dsDNA double-stranded DNA
  • ssRNA single-stranded RNA
  • the CRISPR-related Cas protein or its variant is a nuclease with single-stranded RNA recognition and cutting function and trans-RNA single-strand cutting function; the CRISPR-related Cas protein or its variant includes LbaCas13, LbuC13a, LwaCas13a, AspCas13b , any one or a variant of any one of BzoCas13b, CcaCas13b, PsmCas13b, PinCas13b, Pin2Cas13b, Pin3Cas13b, PbuCas13b, PguCas13b, PigCas13b, PsaCas13b, RanCas13b, PspCas13b, EsCas13d, RspCas13d.
  • DNA ligase refers to an ATP-dependent DNA ligase that uses the energy of ATP to catalyze the formation of phosphodiester bonds between two nucleotide chains. It is used to connect double-stranded DNA molecules or single-stranded DNA gaps in RNA/DNA hybrid double strands. ;
  • the DNA ligase or its variant includes any one or a variant of any 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.
  • the DNA ligase can specifically connect the phosphodiester bond of a single-stranded DNA probe hybridized to one strand of a target nucleic acid molecule sequence to form a circular DNA template.
  • the single-stranded DNA probe is composed of three parts: a 5' end arm, a connecting sequence and a 3' end arm connected in series; the sequences of the 5' end and 3' end arm are complementary to one strand of the target nucleic acid molecule sequence; the connecting sequence is A DNA sequence containing the complementary sequence of the T7 promoter (T7p).
  • RNA fluorescent probe is labeled with any one of FAM, HEX, VIC, Cy5, Cy3, TET, ROX, FITC, and Joe at the 5' end, and labeled with TAMRA, BHQ1, MGB, or BHQ2 at the 3' end.
  • FAM FAM
  • HEX HEX
  • VIC Cy5
  • Cy3 Cy3
  • ROX ROX
  • FITC Fluorescence-Activated RNA
  • Joe labeled with TAMRA, BHQ1, MGB, or BHQ2 at the 3' end.
  • the guide RNA or its derivative is complementary to the sequence of the target nucleic acid molecule.
  • the main components of the PROTRACTOR reaction buffer include 0.1mM-5mM NTPs, 10mM-100mM Tris-HCl, 0.5mM-10mM MgCl2, 0.01mM-10mM ATP and 0.5mM-10mM DTT, with a pH value between 6.5-8.0.
  • the RNA polymerase is selected from one of T7 RNA polymerase, E.coli RNA polymerase, T3 RNA polymerase, and SP6 RNA polymerase.
  • the RNA polymerase is preferably T7 RNA 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.
  • the RNA polymerase can recognize and bind to the T7p region of the circular DNA template, initiate efficient transcription, and produce a large amount of repetitive long single-stranded RNA products containing target sequences.
  • one embodiment of the present invention also provides a reaction system for nucleic acid detection, including a single-stranded DNA probe, an RNA fluorescent probe, a DNA ligase or a variant thereof, an RNA polymerase or a variant thereof, and a guide RNA. Or its derivatives, CRISPR-related Cas protein or its variants, PROTRACTOR reaction buffer.
  • the kit including the reaction system also belongs to the protection scope of the present invention. It can achieve accurate, rapid and highly sensitive detection of specific target nucleic acid molecules (RNA, ssDNA or dsDNA) under normal temperature and isothermal conditions.
  • the invention can quickly complete the detection of DNA or RNA molecules under normal temperature and isothermal conditions.
  • the nucleic acid of the sample to be detected is obtained through rapid nucleic acid extraction; and then the combined enzyme of ligase, transcriptase, CRISPR-related protein and single-stranded DNA probe are used.
  • the nucleic acid fluorescent probe reacts with the nucleic acid to be detected in an isothermal manner, and finally by detecting the fluorescence signal, it is judged whether there is a target nucleic acid molecule in the sample to be detected.
  • the CRISPR-related Cas protein or its variant forms a binary complex with the guide RNA, and specifically combines with the target RNA sequence on the amplicon to form a ternary complex, thereby activating non-specific RNA nuclease activity and ultra-sensitive cutting.
  • Single-stranded RNA molecules in the reaction system; the CRISPR-related Cas protein or its variants may include wild-type, modified, codon-optimized, evolved, thermophilic, chimeric, engineered, and/or Or a mixture of more than one Cas protein.
  • the CRISPR-related Cas protein or variant thereof is preferably Cas13a.
  • the PROTRACTOR reaction buffer is optimized to be 0.1mM-5mM NTPs, 10mM-50mM Tris-HCl, 1mM-10mM MgCl2, 0.01mM-10mM ATP and 0.5mM-5mM DTT, with a pH value between 7.0-8.0.
  • the PROTRACTOR reaction buffer includes 0.1mM-5mM NTPs, 40mM Tris-HCl, 10mM MgCl2, 0.01mM-10mM ATP and 1mM DTT, with a pH value between 7.0-8.0.
  • FAM and BHQ1 double-labeled RNA fluorescent probe 0.1pM-4pM, guide RNA, 0.1pM-5pM, enzyme mixture (T4 DNA ligase, 1U-20U; T7 RNA polymerase, 10U-100U; LwaCas13a protein, 0.01pM -5pM).
  • the detection method and kit of the present invention are a universal and universal rapid detection platform that can detect nucleic acid molecules of viruses, bacteria, fungi, animals, plants and other organisms.
  • Figure 1 is a schematic flow chart for detecting ssDNA or RNA samples according to the present invention
  • Figure 2 is a schematic flow chart for detecting dsDNA samples according to the present invention
  • Figure 3 shows the detection of RNA, dsDNA, and ssDNA molecules by this method
  • Figure 4 shows the detection sensitivity of single-stranded RNA samples in this method
  • Figure 5 shows the use of the present invention to detect the N gene of the new coronavirus
  • Figure 6 shows the use of the present invention to distinguish subtypes of the new coronavirus, W strain and delta strain
  • Figure 7 shows a comparison of the one-pot method with different buffer solutions.
  • Example 1 A method for detecting dsDNA targets
  • Target 1 Select dsDNA (Target 1) as the target sequence.
  • the Target 1 sequence is:
  • T7 promoter complementary sequence T7p-F
  • crRNA-F CCTATAGTGAGTCGTATTAAGAA
  • SEQ ID NO.3 the forward primer of the T7 promoter complementary sequence
  • the incomplete double-stranded crDNA is used as a template for in vitro transcription of guide RNA.
  • T7 transcriptase is used to perform an overnight reaction at 37 degrees, and then the RNA Clean&Concentrator 100 kit is used to purify the crRNA and stored at -20 degrees or -80 degrees.
  • the single-stranded DNA probe sequence (padlock1) of Target 1 is: TACCACCAACCTCCAACCTA AACCCTATAGTGAGTCGTATTAATCCCGCCTACAGGTAATTATAAT, such as (SEQ ID NO.4);
  • the buffer includes 0.5mM NTPs, 40mM Tris-HCl, 10mM MgCl2, 0.01mM-10mM ATP and 1mM DTT, pH value between 7.0-8.0.
  • RNA fluorescent probe 0.8pM, guide RNA, 0.5pM, enzyme mixture (T4 DNA ligase, 10U; T7 RNA polymerase, 10U; LwaCas13a protein, 0.5pM); among them, RNA fluorescent probe is 6-FAM-mArArUrGrGrCmAmArUrGrGrCmA-BHQ1;
  • Fluorescence detection After the reaction is mixed, set the temperature to 37 degrees in the fluorescence real-time quantitative PCR instrument (7900 HT Fast Real-Time PCR).
  • the fluorescent group of the fluorescence detection probe of the double-labeled RNA is FAM, and the fluorescence signal is collected.
  • the time interval is 1 minute, and the detection time is 30 minutes.
  • Target 2 Select ssDNA (Target 2) as the target sequence.
  • the Target 2 sequence is:
  • Target 2 The target single-stranded DNA (Target 2) was synthesized by Shanghai Sangon with the primer GATTCTAAGGTTGGTGGTAATTATAATTACCTGTATAGATTGTTTAGGAAGTCTAATCTCA, as shown in (SEQ ID NO.5), and then dissolved in enzyme-free sterile water and diluted to 10uM;
  • Preparation of guide RNA Synthesize the primer crRNA-target-R including the T7 promoter sequence: TTCTTAATACG ACTCACTATAGGGATTTAGACTACCCCAAAAACGAAGGGGACTAAAACGGTAATTATAATTACCACCAACCT, as shown in (SEQ ID NO.2); the forward primer of the T7 promoter complementary sequence (T7p-F): crRNA- F:CCTATAGTGAGTCGTATTAAGAA, as shown in (SEQ ID NO.3), incomplete double-stranded crDNA is made by double primer annealing, used as a template for in vitro transcription of guide RNA, and T7 transcriptase is used to perform an overnight reaction at 37 degrees, and then used Purify crRNA using the RNA Clean&Concentrator 100 kit and store it at -20 degrees or -80 degrees.
  • the single-stranded DNA probe sequence (padlock2) of Target 2 is:
  • amplification and detection reaction First, add the single-stranded DNA probe and ssDNA to be detected into the reaction system.
  • the buffer includes 0.5mM NTPs, 40mM Tris-HCl, 10mM MgCl2, 0.01mM-10mM ATP and 1mM DTT, pH value between 7.0-8.0.
  • RNA fluorescent probe 0.8pM, guide RNA, 0.5pM, enzyme mixture (T4 DNA ligase, 10U; T7 RNA polymerase, 10U; LwaCas13a protein, 0.5pM); among them, RNA fluorescent probe is 6-FAM-mArArUrGrGrCmAmArUrGrGrCmA-BHQ1;
  • Fluorescence detection After the reaction is mixed, set the temperature in the fluorescence real-time quantitative PCR instrument (7900 HT Fast Real-Time PCR) to 37 degrees.
  • the fluorescent group of the fluorescence detection probe of the double-labeled RNA is FAM, and the fluorescence signal collection time interval is 1min, the detection time is 30 minutes;
  • Target3 Select RNA (Target3) as the target sequence.
  • the Target 3 sequence is:
  • T7 promoter complementary sequence T7p-F
  • crRNA-F CCTATAGTGAGTCGTATTAAGAA
  • SEQ ID NO.3 the forward primer of the T7 promoter complementary sequence
  • the incomplete double-stranded crDNA is used as a template for in vitro transcription of guide RNA.
  • T7 transcriptase is used to perform an overnight reaction at 37 degrees, and then the RNA Clean&Concentrator 100 kit is used to purify the crRNA and stored at -20 degrees or -80 degrees.
  • the single-stranded DNA probe sequence (padlock3) of Target 3 is:
  • Amplification and detection reaction First, add the single-stranded DNA probe and RNA to be detected into the reaction system.
  • the buffer includes 0.5mM NTPs, 40mM Tris-HCl, 10mM MgCl2, 0.01mM-10mM ATP and 1mM DTT, pH The value is between 7.0-8.0.
  • FAM and BHQ1 double-labeled RNA fluorescent probe 0.8pM, guide RNA, 0.5pM, enzyme mixture (T4 DNA ligase, 10U; T7 RNA polymerase, 10U; LwaCas13a protein, 0.5pM).
  • the RNA fluorescent probe is 6-FAM-mArArUrGrGrCmAmArArUrGrGrCmA-BHQ1.
  • Fluorescence detection After the reaction is mixed, set the temperature in the fluorescence real-time quantitative PCR instrument (7900 HT Fast Real-Time PCR) to 37 degrees.
  • the fluorescent group of the fluorescence detection probe of the double-labeled RNA is FAM, and the fluorescence signal collection time interval is 1min, the detection time is 30 minutes.
  • the new coronavirus is an RNA virus.
  • Nasopharyngeal swab samples from healthy people and patients are collected, and total RNA is extracted from them as the RNA to be tested;
  • the conserved region of the N gene of the new coronavirus was selected as the target binding region, and the sequence is: ACCGAAGAGCUACCAGACGAAUUC, as shown in (SEQ ID NO.7).
  • Preparation of guide RNA Synthesize the primer crRNA-N-R including the T7 promoter sequence: TTCTTAATACGACTCACTATAGGGATTTAGACTACCCCAAAAACGAAGGGGACTAAAACGAATTCGTCTGGTAGCTCTTCGGT, as shown in (SEQ ID NO.2); the forward primer (T7p-F) of the T7 promoter complementary sequence: crRNA-F: CCTATAGTGAGTCGTATTAAGAA , as shown in (SEQ ID NO.3); incomplete double-stranded crDNA is made by annealing double primers and used as a template for in vitro transcription of guide RNA. T7 transcriptase is used to perform an overnight reaction at 37 degrees, and then RNA Clean&Concentrator 100 is used The crRNA is purified with the kit and stored at -20 degrees or -80 degrees.
  • the single-stranded DNA probe sequence of the N gene is: TAGCTCTTCGGTCCCACCTAAACCCTATAGTGAGTCGTATTAATCCCGCCTACAGAATTCGTCTGG, as shown in (SEQ ID NO.8);
  • Amplification and detection reaction First, add the single-stranded DNA probe (padlock-N) and the RNA to be detected into the reaction system.
  • the buffer includes 0.5mM NTPs, 40mM Tris-HCl, 10mM MgCl2, 0.01mM-10mM ATP. and 1mM DTT, pH value between 7.0-8.0.
  • FAM and BHQ1 double-labeled RNA fluorescent probe 0.8pM, guide RNA, 0.5pM, enzyme mixture (T4 DNA ligase, 10U; T7 RNA polymerase, 10U; LwaCas13a protein, 0.5pM).
  • the RNA fluorescent probe is 6-FAM-mArArUrGrGrCmAmArArUrGrGrCmA-BHQ1.
  • Fluorescence detection After the reaction is mixed, set the temperature in the fluorescence real-time quantitative PCR instrument (7900 HT Fast Real-Time PCR) to 37 degrees.
  • the fluorescent group of the fluorescence detection probe of the double-labeled RNA is FAM, and the fluorescence signal collection time interval is 1min, the detection time is 30 minutes.
  • results As shown in Figure 5, the present invention can be used for the detection of new coronavirus clinical samples.
  • This example detects the L452R site mutation, T478K site and P681R site mutation of the new coronavirus S gene, and uses SNP typing to distinguish the two subtypes of the new coronavirus, W strain and delta strain.
  • L452R-W The original sequence of the W strain of L452R (L452R-W) is AUUAUAAUUACCUGUAUAGAUUGU, as shown in (SEQ ID NO.9);
  • T478K-W The original sequence of the W strain of T478K (T478K-W) is AGGCCGGUAGCACACCUUGUAAUG, as shown in (SEQ ID NO.11);
  • the original sequence of the W strain of P681R is AGACUAAUUCUCCUCGGCGGGCAC, as shown in (SEQ ID NO.13);
  • the mutation sequence of the delta strain of P681R is AGACUAAUUCUCGUCGGCGGGCAC, as shown in (SEQ ID NO.14);
  • T7p-F Forward primer of T7 promoter complementary sequence
  • Incomplete double-stranded crDNA is made by annealing double primers and used as a template for in vitro transcription of guide RNA.
  • T7 transcriptase is used to perform an overnight reaction at 37 degrees, and then the RNA Clean&Concentrator 100 kit is used to purify the crRNA and store it at -20 degrees. Or -80 degrees.
  • the L452R typing single-stranded DNA probe sequence (padlock-L452R) of the new coronavirus delta strain is GGTAATTATAATCCCAAATCCTCCCTATAGTGAGTCGTATTAATCCCAAACAAAACAATCTATAAC, such as (SEQ ID NO. 18);
  • the T478K typing single-stranded DNA probe sequence (padlock-T478K) of the new coronavirus delta strain is: TGCTACCGGCCTCCCAAACCCACCCTATAGTGAGTCGTATTAATCCCAAACAAACATTACAAGGAT, such as (SEQ ID NO. 19);
  • the P681R typing single-stranded DNA probe sequence of the new coronavirus delta strain is: GAGAATTAGTCTAACAAACAAACCCTATAGTGAGTCGTATTAATCCCGCCTACAGTGCCCGCCGCC, such as (SEQ ID NO. 20).
  • Amplification and detection reaction First, add the single-stranded DNA probe and RNA to be detected into the reaction system.
  • the buffer includes 0.5mM NTPs, 40mM Tris-HCl, 10mM MgCl2, 0.01mM-10mM ATP and 1mM DTT, with a pH value of 7.0 -8.0.
  • FAM and BHQ1 double-labeled RNA fluorescent probe 0.8pM, guide RNA, 0.5pM, enzyme mixture (T4 DNA ligase, 10U; T7 RNA polymerase, 10U; LwaCas13a protein, 0.5pM).
  • the RNA fluorescent probe is 6-FAM-mArArUrGrGrCmAmArArUrGrGrCmA-BHQ1.
  • Fluorescence detection After the reaction is mixed, set the temperature in the fluorescence real-time quantitative PCR instrument (7900 HT Fast Real-Time PCR) to 37 degrees.
  • the fluorescent group of the fluorescence detection probe of the double-labeled RNA is FAM, and the fluorescence signal collection time interval is 1min, the detection time is 30 minutes.
  • Figure 6 shows the subtype detection of the new coronavirus. Using this method, two mutation types of the new coronavirus, W strain and delta strain, can be distinguished based on the single base mutation site.
  • Example 3 The difference between this comparative example and Example 3 is that the one-pot method PROTRACTOR buffer (B): 0.5mM NTP mixture, 40mM Tris-HCl, 10mM MgCl2, 10mM DTT, 0.5mM ATP, pH 7.5@25°C is replaced by Buffer 1 (B1) used for step-by-step single-stranded DNA circularization: 50mM Tris-HCl, 10mM MgCl2, 10mM DTT, 0.5mM ATP, pH 7.5@25°C, rolling circle transcription Buffer 2 (B2): 40mM Tris-HCl, 6mM MgCl2, 10mM (NH4)2SO4, 1mM DTT, 2mM Spermidine, pH 7.9@25°C and CRISPR/Cas-mediated nucleic acid detection Buffer 3 (B3): 50mM NaCl, 10mM Tris-HCl, 10mM MgCl2, 100 ⁇ g/ml bovine serum Protein, pH 7.9@25
  • RNA fluorescent probe After the reaction is mixed, set the temperature to 37 degrees in the fluorescence real-time quantitative PCR instrument (7900 HT Fast Real-Time PCR).
  • the fluorescent group of the double-labeled RNA fluorescence detection probe is FAM, and the fluorescence signal collection time interval is 1min, the detection time is 30 minutes.
  • the RNA fluorescent probe is 6-FAM-mArArUrGrGrCmAmArArUrGrGrCmA-BHQ1.
  • this comparative analysis method can be used to detect single-stranded RNA in one pot.
  • the optimized one-pot detection buffer B is better than B1, B2, and B3.
  • One-pot detection time can be shortened to 10 minutes.
  • Example 3 The difference between this comparative example and Example 3 is that no buffer (NC) is added and distilled water is used instead of buffer.
  • RNA fluorescent probe After the reaction is mixed, set the temperature to 37 degrees in the fluorescence real-time quantitative PCR instrument (7900 HT Fast Real-Time PCR).
  • the fluorescent group of the double-labeled RNA fluorescence detection probe is FAM, and the fluorescence signal collection time interval is 1min, the detection time is 30 minutes.
  • the RNA fluorescent probe is 6-FAM-mArArUrGrGrCmAmArArUrGrGrCmA-BHQ1.
  • nucleic acid molecules are enriched, amplified and identified at room temperature to achieve accurate, ultra-sensitive and rapid detection of nucleic acid molecules.
  • the present invention Compared with traditional nucleic acid molecule detection methods, the present invention has the following advantages: no need for DNA amplification, no PAM sites, no target sequence-specific primers, and is suitable for different types of nucleic acid molecular targets such as single-stranded DNA, double-stranded DNA, and RNA. In particular, for RNA target detection, no additional reverse transcription step is required.
  • the invention has broad application prospects in the field of rapid detection of nucleic acid molecules.
  • the present invention can be used to detect DNA or RNA, and can distinguish point mutations
  • the present invention does not require DNA amplification or primers, and does not require reverse transcription for the detection of RNA samples;
  • the present invention realizes the isothermal reaction of a single buffer in a single tube, is easy to operate, has simple steps, and is suitable for rapid detection of nucleic acid molecules in areas with limited resources;
  • the reaction of the present invention will be circularized to trigger an amplification reaction after the single-stranded DNA probe specifically binds to one strand of the target nucleic acid molecule.
  • the amplicon will only be associated with the specific guide RNA and CRISPR. Only after recognition by the binary complex formed by the Cas protein or its variants will the cleavage reaction be activated to generate a fluorescent signal, overcoming the problem of false positives from the reaction mechanism.
  • the amplification product of the present invention is RNA instead of DNA.
  • the characteristics of RNA that are easy to degrade and not easy to generate aerosols enable the detection method itself to overcome the easy contamination characteristics of LAMP and qPCR. At the same time, this method is a closed-tube reaction, which is physically isolated and reduces the possibility of contamination to the greatest extent;
  • Isothermal detection Three engineering enzymes, including DNA ligase or its variants, RNA polymerase or its variants, Cas protein, CRISPR-related Cas protein or its variants, and multiple chemical components are used to create a biological simulation to the greatest extent.
  • the environment for nucleic acid amplification in the body, and each engineering enzyme performs its own duties and works at its optimal reaction temperature, so the work efficiency is the highest;
  • One-step method In order to make the operation easier, reduce the pollution caused by opening the cap and adding samples, improve the reaction efficiency and shorten the reaction time, it is more suitable for on-site rapid detection, especially in areas with limited resources; we use single-stranded DNA detection.
  • the three reactions of needle circularization, transcription, and CRISPR-related Cas protein or its variant shearing are innovatively integrated into the same reaction tube for simultaneous reactions, achieving a one-step approach.

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Abstract

本发明公开了一锅法滚环转录和CRISPR/Cas介导的核酸检测方法及试剂盒；所述核酸检测方法，包括S1、从待检测样本中提取总核酸；S2、配置反应体系，反应体系包括单链DNA探针、RNA荧光探针、DNA连接酶或其变体、RNA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、PROTRACTOR反应缓冲液；S3、将提取的总核酸加入反应体系中，进行恒温反应，并产生荧光信号；其中单链DNA探针与目标核酸分子的一条链特异性的互补，并在DNA连接酶或其变体作用下形成单链环状DNA探针；S4、读取和记录PROTRACTOR产生荧光信号，通过荧光信号来判断待检测样本中目标核酸分子的有无。

Description

一锅法滚环转录和CRISPR/Cas介导的核酸检测方法及试剂盒 技术领域
本发明属于分子生物学核酸快速检测领域，具体涉及一种一锅法滚环转录和CRISPR/Cas介导的核酸检测方法及试剂盒。尤其涉及一种新的核酸等温扩增与信号输出一体化检测技术，利用DNA连接酶，RNA聚合酶和CRISPR/Cas蛋白在常温等温条件下，可快速、一步、单管完成特定DNA或者RNA的扩增和检测。
背景技术
荧光定量聚合酶链反应(qPCR)是目前核酸分子诊断的金标准。与脱氧核糖核酸(DNA)检测不同的是，核糖核酸(RNA)的体外诊断往往需要分为两个分步骤：(1)在逆转录酶的作用下将RNA逆转录成cDNA；(2)对cDNA样本进行qPCR扩增和检测分析。虽然，qPCR是非常成熟的一门技术并已广泛应用于医学诊断，但它仍需要大型且昂贵的热循环仪、样本的运输与保存、专业的操作人员和检测时间过长(从样本到结果，至少需要2-4小时)，这在很大程度上限制了qPCR在资源有限环境和即时监测(POC)分析中的应用。
核酸等温扩增技术已经成为一种有前途的替代方法，它可以在恒温条件下实现目标核酸分子的快速、高效扩增，而无需qPCR所需的热循环仪。此外，等温扩增可以在简单的条件下进行，如室温，水浴锅等，尤其是用于资源有限的地区核酸分子的快速检测具有得天独厚的优势，这是qPCR无法比拟的。自20世纪90年代初以来，数十种等温核酸扩增技术被开发出来，其中有些已经商业化，例如环介导等温扩增(LAMP)技术,它是一种利用4-6对引物识别靶标特异的位点，在60-65℃下，利用链置换酶活性的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(RPA)技术，是在恒温37-42℃条件下，模拟体内核酸复制机制，由多种关键酶或蛋白协助DNA聚合酶扩增实现核酸的指数扩增技术，整个反应一般需要20-30分钟。基于核酸序列的扩增(NASBA)技术，利用逆转录酶，T7 RNA聚合酶和RNase H以及两个寡核苷酸引物，在41℃左右实现对RNA的快速和持续扩增(60分钟左右)。滚环扩增(RCA)技术，利用DNA连接酶和链置换活性的DNA聚合酶，在30℃下，一条或多条引物的引导下 进行环形模板的链置换，产生多条具有靶标序列的重复长单链，整个过程一般在2小时左右。
然而，以上几种等温扩增技术都存在各自的不足，如LAMP法扩增，需要的引物较多(4-6条)，对引物设计要求比较高，对于点突变或修饰位点的检测常常不能满足要求。经LAMP扩增后的产物在开盖的情况下极易形成气溶胶污染，从而导致产生假阳性结果。RPA法的酶组分较为复杂，对缓冲液的要求较高，反应体系的稳定性不强，易造成重复性不好，此外，它也无法对突变位点进行检测。同时，LAMP和RPA对于RNA样本检测仍需要逆转录。NASBA法对DNA的检测不太适合，反应成分比较复杂且稳定性较差，易受基质的影响。RCA法的反应时间偏长，在现场检测中的优势不太明显。滚环转录(RCT)是一种具有体外转录活性的RNA聚合酶催化的等温扩增反应。在目标核酸分子的一条链与单链DNA探针杂交后在DNA连接酶的作用下连接成环状模板，在RNA聚合酶的作用下沿着环状模板持续转录为含靶标序列的重复长单链RNA产物，实现常温下的高效扩增。但对于扩增单链RNA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)系统是细菌或者古生菌进化出来用以抵御病毒感染的免疫系统，它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，Cas13a是近年来新鉴定到一种CRISPR核酸酶，这个核酸酶具有能被特异性RNA激活获得非特异性的RNA核酸酶的活性，从而切割其它单链RNA，配合RNA荧光报告系统能够检测特异性的RNA产物，但Cas13a单独作用时灵敏度较低，只能实现fM到pM级别的核酸检测，对于分子诊断的灵敏度欠佳，如在不进行核酸扩增的情况下直接检测潜在肿瘤标志物miR-19b和miR-20a，检出限为10pM。2017年和2019年，张锋等利用Cas13a蛋白结合等温扩增技术RPA，开发了可以检测核酸的新方法SHERLOCK，其灵敏度可以实现对aM级样品的检测。但SHERLOCK方法对于RNA检测无法实现一步单管的检测，且RPA方法成分复杂，稳定性欠佳，增加了操作难度。
发明内容
为了克服现有技术上述缺陷。本发明的实施例提供了一锅法滚环转录和CRISPR/Cas介导的核酸检测方法及试剂盒，具体提供一种基于单链DNA环化、转录和CRISPR相关Cas蛋白或其变体的核酸检测新方法(PROTRACTOR)。利用DNA连接酶实现单链DNA探针的环化，将靶标分子信号转换为环状DNA信号；RNA聚合酶以 环状DNA为模板，完成高效转录；CRISPR相关Cas蛋白或其变体在crRNA引导下对转录产物进行识别剪切，在完成扩增的同时进行检测反应。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，实现对目标核酸分子(RNA、ssDNA或dsDNA)的超灵敏、高特异性和快速检测。该方法摆脱了常规核酸检测中DNA扩增和引物的限制。在进行RNA样本检测时，无需逆转录，能直接对RNA分子进行扩增检测。
<第一方面>
本发明其中一个实施例提供一种PROTRACTOR等温核酸检测方法，包括如下步骤：
S1、从待检测样本中提取总核酸；
S2、配置反应体系，所述反应体系包括单链DNA探针、RNA荧光探针、DNA连接酶或其变体、RNA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、PROTRACTOR反应缓冲液；其中单链DNA探针与目标核酸分子的一条链特异性的互补；
S3、将步骤S2提取的总核酸加入步骤S1的反应体系中，进行恒温反应，并产生荧光信号；恒温反应过程中，单链DNA探针在DNA连接酶或其变体作用下形成单链环状DNA探针；
S4、利用荧光检测仪读取和记录PROTRACTOR产生荧光信号，通过荧光信号来判断待检测样本中目标核酸分子的有无。
进一步，所述单链DNA探针由与目标核酸分子序列的一条链互补的DNA序列和含T7p的连接序列组成。
本发明的技术原理如下：
单链DNA探针序列与目标核酸分子的一条链特异性的互补，所述单链DNA探针在DNA连接酶或其变体作用下形成单链环状DNA探针；
所述单链DNA探针是用于特异性识别并结合目标核酸分子一条链上的靶标序列，在环化后用作扩增的模板，单链DNA探针由与目标核酸分子序列的一条链互补的DNA序列与连接序列组成，其中，连接序列包含一个T7启动子互补序列(T7p)，能被RNA聚合酶识别结合，开启转录；
进行样品检测时，单链环状DNA探针在RNA聚合酶或其变体的作用下持续转录为长的单链RNA，其含有目标核酸分子的靶标序列的多个重复；
通过转录形成大量单链RNA扩增子，并被向导RNA或其衍生物和CRISPR相关Cas蛋白或其变体形成的二元复合物识别，并剪切RNA荧光探针，产生可以检测的荧光信号；
然后利用荧光检测仪读取和记录PROTRACTOR产生荧光信号，通过荧光信号来判断待检测样本中目标核酸分子的有无。
若目标核酸分子为dsDNA，在反应之前对dsDNA进行预变性处理。
所述PROTRACTOR为普适性核酸检测平台，能检测不同类型的核酸分子，包括单链DNA(ssDNA)、双链DNA(dsDNA)、单链RNA(ssRNA)中的一种或几种。
所述CRISPR相关Cas蛋白或其变体为具有单链RNA识别剪切功能和反式RNA单链剪切功能的核酸酶；所述CRISPR相关Cas蛋白或其变体包括LbaCas13、LbuC13a、LwaCas13a、AspCas13b、BzoCas13b、CcaCas13b、PsmCas13b、PinCas13b、Pin2Cas13b、Pin3Cas13b、PbuCas13b、PguCas13b、PigCas13b、PsaCas13b、RanCas13b、PspCas13b、EsCas13d、RspCas13d中的任意一种或任意一种的变体。
DNA连接酶是指利用ATP的能量催化两个核苷酸链之间形成磷酸二酯键的依赖ATP的DNA连接酶，用来连接双链DNA分子或者RNA/DNA杂交双链的单链DNA缺口；所述DNA连接酶或其变体，包括T4 DNA连接酶、E.coli DNA连接酶、SplintR连接酶和HiFi Taq DNA连接酶中的任意一种或任意一种的变体。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。
所述DNA连接酶能够特异性连接与目标核酸分子序列的一条链杂交的单链DNA探针的磷酸二酯键，形成环状DNA模板。
所述单链DNA探针由5'端臂，连接序列和3'端臂三部分串联而成；5'端和3'端臂的序列均与目标核酸分子序列的一条链互补；连接序列是一段包含T7启动子互补序列(T7p)的DNA序列。
所述RNA荧光探针为5'端标记FAM、HEX、VIC、Cy5、Cy3、TET、ROX、FITC、Joe中的任意一种荧光基团，3'端标记TAMRA、BHQ1、MGB、BHQ2中的任意一种荧光淬灭基团的一条单链RNA。
所述向导RNA或其衍生物的与目标核酸分子序列互补。
所述PROTRACTOR反应缓冲液主要成分包括0.1mM-5mM NTPs、10mM-100mM Tris-HCl、0.5mM-10mM MgCl2、0.01mM-10mM ATP和0.5mM-10mM DTT，pH值 在6.5-8.0之间。
所述RNA聚合酶选自T7 RNA聚合酶、E.coli RNA聚合酶、T3 RNA聚合酶、SP6 RNA聚合酶中的一种。所述RNA聚合酶优选为T7 RNA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。
所述RNA聚合酶能够识别并结合环状DNA模板的T7p区域，开启高效转录，产生大量的含靶标序列的重复长单链RNA产物。
<第二方面>
进一步，本发明其中一个实施例还提供一种用于核酸检测的反应体系，包括单链DNA探针、RNA荧光探针、DNA连接酶或其变体、RNA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、PROTRACTOR反应缓冲液。
包括所述的反应体系的试剂盒也属于本发明的保护范围。可以实现常温等温条件下对特定目标核酸分子(RNA、ssDNA或dsDNA)的精准、快速和高灵敏度检测。
本发明能在常温等温条件下，快速完成DNA或RNA分子的检测，首先通过核酸快速提取得到待检测样本的核酸；再利用连接酶、转录酶、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分子。
所述CRISPR相关Cas蛋白或其变体和向导RNA形成二元复合物，与扩增子上的靶标RNA序列特异性结合成三元复合物，从而激活非特异性的RNA核酸酶活性，超灵敏切割反应体系中单链RNA分子；所述CRISPR相关Cas蛋白或其变体可以包括野生型、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相关Cas蛋白或其变体优选为Cas13a。
进一步地，为了缩短PROTRACTOR的反应时间，提高检测效率，更适用于现场快速检测，尤其是在资源有限的地区使用。我们对不同缓冲液一锅法对比，优化了PROTRACTOR反应缓冲液具体成分和浓度。所述PROTRACTOR反应缓冲液优化为0.1mM-5mM NTPs、10mM-50mM Tris-HCl、1mM-10mM MgCl2、0.01mM-10mM ATP和0.5mM-5mM DTT，pH值在7.0-8.0之间。
在一个具体实施案例中，PROTRACTOR反应缓冲液包括0.1mM-5mM NTPs、40mM Tris-HCl、10mM MgCl2、0.01mM-10mM ATP和1mM DTT，pH值在7.0-8.0之间。FAM和BHQ1双标记的RNA荧光探针0.1pM-4pM，向导RNA，0.1pM-5pM，酶混 合液(T4 DNA连接酶，1U-20U；T7 RNA聚合酶，10U-100U；LwaCas13a蛋白，0.01pM-5pM)。
本发明的检测方法和试剂盒是一个普适性和通用型快检平台，可以检测病毒，细菌，真菌、动植物等生物体的核酸分子。
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ssDNA或RNA样本的流程示意图；
图2为本发明检测dsDNA样本的流程示意图；
图3为该方法对RNA、dsDNA、ssDNA分子的检测；
图4为在该方法中对单链RNA样本的检测灵敏度；
图5为利用本发明检测新冠病毒N基因；
图6为利用本发明区分新冠病病毒的亚型，W株和delta株；
图7为不同缓冲液一锅法的对比。
具体实施方式
下面结合实施例对本发明进行详细说明。以下实施例将有助于本领域的技术人员进一步理解本发明，但不以任何形式限制本发明。应当指出的是，对本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干调整和改进。这些都属于本发明的保护范围。
实施例1：一种检测dsDNA靶标的方法
选用dsDNA(Target 1)作为靶标序列，Target 1序列为：
GTGATGAAGTCAGACAAATCGCTCCAGGGCAAACTGGAAAGATTGCTGATTATAATTATAAATTACCAGATGATTTTACAGGCTGCGTTATAGCTTGGAATTCTAACAATCTTGATTCTAAGGTTGGTGGTAATTATAATTACCTGTATAGATTGTTTAGGAAGTCTAATCTCAAACCTTTTGAGAGAGATATTTCAACTGAAATCTATCAGGCCGGTAGCACACCTTGTAATGGTGTTGAAGGT，如(SEQ ID NO.1)所示；
向导RNA的制备：合成包括T7启动子序列的引物crRNA-target-R：
TTCTTAATACGACTCACTATAGGGATTTAGACTACCCCAAAAACGAAGGGGACTAAAACGGTAATTATAATTACCACCAACCT，如(SEQ ID NO.2)所示，T7启动子互补序列的正向引物(T7p-F)：crRNA-F:CCTATAGTGAGTCGTATTAAGAA，如(SEQ ID NO.3) 所示，通过双引物退火制成不完全双链的crDNA，用作向导RNA体外转录的模板，利用T7转录酶在37度进行过夜反应，再利用RNA Clean&Concentrator 100试剂盒进行纯化得到crRNA，保存于-20度或者-80度。
Target 1的单链DNA探针序列(padlock1)为：TACCACCAACCTCCAACCTA AACCCTATAGTGAGTCGTATTAATCCCGCCTACAGGTAATTATAAT，如(SEQ ID NO.4)；
如图2所示，扩增和检测反应：
(1)从待检测样本中提取双链DNA；
(2)将单链DNA探针与待检双链DNA，高温(80-95℃)退火5分钟，然后自然冷却至室温后加入反应体系中，缓冲液包括0.5mM NTPs、40mM Tris-HCl、10mM MgCl2、0.01mM-10mM ATP和1mM DTT，pH值在7.0-8.0之间。FAM和BHQ1双标记的RNA荧光探针0.8pM，向导RNA，0.5pM，酶混合液(T4 DNA连接酶，10U；T7 RNA聚合酶，10U；LwaCas13a蛋白，0.5pM)；其中，RNA荧光探针为6-FAM-mArArUrGrGrCmAmArArUrGrGrCmA-BHQ1；
(3)荧光检测：反应混合后，在荧光实时定量PCR仪(7900 HT Fast Real-Time PCR)中设置温度为37度，双标记RNA的荧光检测探针的荧光基团为FAM，荧光信号采集时间间隔为1min，检测时间为30分钟。
结果：如图3所示，应用此方法可以检测双链DNA。
实施案例2：一种联测检测ssDNA靶标
选用ssDNA(Target 2)作为靶标序列，Target 2序列为：
GATTCTAAGGTTGGTGGTAATTATAATTACCTGTATAGATTGTTTAGGAAGTCTAATCTCA；如(SEQ ID NO.5)所示；
靶标单链DNA(Target 2)由上海生工合成引物GATTCTAAGGTTGGTGGTAATTATAATTACCTGTATAGATTGTTTAGGAAGTCTAATCTCA，如(SEQ ID NO.5)所示，然后溶解于无酶无菌水中稀释至10uM；
向导RNA的制备：合成包括T7启动子序列的引物crRNA-target-R：TTCTTAATACG ACTCACTATAGGGATTTAGACTACCCCAAAAACGAAGGGGACTAAAACGGTAATTATAATTACCACCAACCT，如(SEQ ID NO.2)所示；T7启动子互补序列的正向引物(T7p-F)：crRNA-F:CCTATAGTGAGTCGTATTAAGAA，如(SEQ ID NO.3)所示，通过双引物退火制成不完全双链的crDNA，用作向导RNA体外转录的模板，利用T7 转录酶在37度进行过夜反应，再利用RNA Clean&Concentrator 100试剂盒进行纯化得到crRNA，保存于-20度或者-80度。
Target 2的单链DNA探针序列(padlock2)为：
TACCACCAACCTCCAACCTAAACCCTATAGTGAGTCGTATTAATCCCGCCTACAGGTAATTATAAT，如(SEQ ID NO.4)所示；
如图1所示，扩增和检测反应：首先将单链DNA探针与待检ssDNA加入反应体系中，缓冲液包括缓冲液包括0.5mM NTPs、40mM Tris-HCl、10mM MgCl2、0.01mM-10mM ATP和1mM DTT，pH值在7.0-8.0之间。FAM和BHQ1双标记的RNA荧光探针0.8pM，向导RNA，0.5pM，酶混合液(T4 DNA连接酶，10U；T7 RNA聚合酶，10U；LwaCas13a蛋白，0.5pM)；其中，RNA荧光探针为6-FAM-mArArUrGrGrCmAmArArUrGrGrCmA-BHQ1；
荧光检测：反应混合后，在荧光实时定量PCR仪(7900 HT Fast Real-Time PCR)中设置温度为37度，双标记RNA的荧光检测探针的荧光基团为FAM，荧光信号采集时间间隔为1min，检测时间为30分钟；
结果：如图3所示，应用此方法可以检测单链DNA。
实施案例3一种检测RNA靶标的方法
选取RNA(Target3)作为靶标序列，Target 3序列为：
GUGAUGAAGUCAGACAAAUCGCUCCAGGGCAAACUGGAAAGAUUGCUGAUUAUAAUUAUAAAUUACCAGAUGAUUUUACAGGCUGCGUUAUAGCUUGGAAUUCUAACAAUCUUGAUUCUAAGGUUGGUGGUAAUUAUAAUUACCUGUAUAGAUUGUUUAGGAAGUCUAAUCUCAAACCUUUUGAGAGAGAUAUUUCAACUGAAAUCUAUCAGGCCGGUAGCACACCUUGUAAUGGUGUUGAAGGU，如(SEQ ID NO.6)所示
向导RNA的制备：合成包括T7启动子序列的引物crRNA-target-R：
TTCTTAATACGACTCACTATAGGGATTTAGACTACCCCAAAAACGAAGGGGACTAAAACGGTAATTATAATTACCACCAACCT，如(SEQ ID NO.2)所示，T7启动子互补序列的正向引物(T7p-F)：crRNA-F:CCTATAGTGAGTCGTATTAAGAA，如(SEQ ID NO.3)，所示通过双引物退火制成不完全双链的crDNA，用作向导RNA体外转录的模板，利用T7转录酶在37度进行过夜反应，再利用RNA Clean&Concentrator 100试剂盒进行纯化得到crRNA，保存于-20度或者-80度。
Target 3的单链DNA探针序列(padlock3)为：
TACCACCAACCTCCAACCTAAACCCTATAGTGAGTCGTATTAATCCCGCCTACAGGTAATTATAAT，如(SEQ ID NO.4)所示；
扩增和检测反应：首先将单链DNA探针与待检RNA加入反应体系中，缓冲液包括缓冲液包括0.5mM NTPs、40mM Tris-HCl、10mM MgCl2、0.01mM-10mM ATP和1mM DTT，pH值在7.0-8.0之间。FAM和BHQ1双标记的RNA荧光探针0.8pM，向导RNA，0.5pM，酶混合液(T4 DNA连接酶，10U；T7 RNA聚合酶，10U；LwaCas13a蛋白，0.5pM)。其中，RNA荧光探针为6-FAM-mArArUrGrGrCmAmArArUrGrGrCmA-BHQ1。
荧光检测：反应混合后，在荧光实时定量PCR仪(7900 HT Fast Real-Time PCR)中设置温度为37度，双标记RNA的荧光检测探针的荧光基团为FAM，荧光信号采集时间间隔为1min，检测时间为30分钟。
结果：如图3所示，应用此方法可以检测单链RNA；如图4所示，应用此方法可以检测低至1拷贝每反应的单链RNA分子。
实施案例4：一种检测新冠病毒的方法
新冠病毒为RNA病毒，采集健康人群和患者的鼻咽拭子样品，从中分别提取总RNA作为待检RNA；
选取新冠病毒N基因的保守区域作为靶标结合区域，序列为：ACCGAAGAGCUACCAGACGAAUUC，如(SEQ ID NO.7)所示。
向导RNA的制备：合成包括T7启动子序列的引物crRNA-N-R：TTCTTAATACGACTCACTATAGGGATTTAGACTACCCCAAAAACGAAGGGGACTAAAACGAATTCGTCTGGTAGCTCTTCGGT，如(SEQ ID NO.2)所示；T7启动子互补序列的正向引物(T7p-F)：crRNA-F:CCTATAGTGAGTCGTATTAAGAA，如(SEQ ID NO.3)所示；通过双引物退火制成不完全双链的crDNA，用作向导RNA体外转录的模板，利用T7转录酶在37度进行过夜反应，再利用RNA Clean&Concentrator 100试剂盒进行纯化得到crRNA，保存于-20度或者-80度。
N基因的单链DNA探针序列(padlock-N)为：TAGCTCTTCGGTCCCACCTAAACCCTATAGTGAGTCGTATTAATCCCGCCTACAGAATTCGTCTGG，如(SEQ ID NO.8)所示；
扩增和检测反应：首先将单链DNA探针(padlock-N)与待检RNA加入反应体系中，缓冲液包括缓冲液包括0.5mM NTPs、40mM Tris-HCl、10mM MgCl2、0.01mM-10mM ATP和1mM DTT，pH值在7.0-8.0之间。FAM和BHQ1双标记的RNA荧光探针 0.8pM，向导RNA，0.5pM，酶混合液(T4 DNA连接酶，10U；T7 RNA聚合酶，10U；LwaCas13a蛋白，0.5pM)。其中，RNA荧光探针为6-FAM-mArArUrGrGrCmAmArArUrGrGrCmA-BHQ1。
荧光检测：反应混合后，在荧光实时定量PCR仪(7900 HT Fast Real-Time PCR)中设置温度为37度，双标记RNA的荧光检测探针的荧光基团为FAM，荧光信号采集时间间隔为1min，检测时间为30分钟。
结果：如图5所示，本发明可用于新冠病毒临床样本的检测。
实施案例5：一种检测新冠病毒基因突变的方法
本实施例检测新冠病毒S基因的L452R位点突变，T478K位点和P681R位点三个单碱基突变位点，利用SNP分型来区分新冠病毒的两个亚型，W株和delta株。
L452R的W株(L452R-W)原有序列AUUAUAAUUACCUGUAUAGAUUGU，如(SEQ ID NO.9)所示；
L452R的delta株(L452R-Delta)突变序列AUUAUAAUUACCGGUAUAGAUUGU，如(SEQ ID NO.10)所示；
T478K的W株(T478K-W)原有序列AGGCCGGUAGCACACCUUGUAAUG，如(SEQ ID NO.11)所示；
T478K的delta株(T478K-Delta)突变序列AGGCCGGUAGCAAACCUUGUAAUG，如(SEQ ID NO.12)所示；
P681R的W株(P681R-W)原有序列为AGACUAAUUCUCCUCGGCGGGCAC，如(SEQ ID NO.13)所示；
P681R的delta株(P681R-Delta)突变序列AGACUAAUUCUCGUCGGCGGGCAC，如(SEQ ID NO.14)所示；
向导RNA的制备：合成包括T7启动子序列的引物
crRNA-L452R-R：
TTCTTAATACGACTCACTATAGGGATTTAGACTACCCCAAAAACGAAGGGGACTAAAACACAATCTATACCGGTAATTATAAT，如(SEQ ID NO.15)所示；
crRNA-T478K-R：
TTCTTAATACGACTCACTATAGGGATTTAGACTACCCCAAAAACGAAGGGGACTAAAACCATTACAAGGTTTGCTACCGGCCT，如(SEQ ID NO.16)所示；
crRNA-P681R-R：
TTCTTAATACGACTCACTATAGGGATTTAGACTACCCCAAAAACGAAGGGGACTAAAACGTGCCCGCCGACGAGAATTAGTCT，如(SEQ ID NO.17)所示；
T7启动子互补序列的正向引物(T7p-F)：
crRNA-F:CCTATAGTGAGTCGTATTAAGAA，如(SEQ ID NO.3)所示
通过双引物退火制成不完全双链的crDNA，用作向导RNA体外转录的模板，利用T7转录酶在37度进行过夜反应，再利用RNA Clean&Concentrator 100试剂盒进行纯化得到crRNA，保存于-20度或者-80度。
新冠病毒delta株的L452R分型单链DNA探针序列(padlock-L452R)为GGTAATTATAATCCCAAATCCTCCCTATAGTGAGTCGTATTAATCCCAAACAAAACAATCTATAAC，如(SEQ ID NO.18)；
新冠病毒delta株的T478K分型单链DNA探针序(padlock-T478K)为：TGCTACCGGCCTCCCAAACCCACCCTATAGTGAGTCGTATTAATCCCAAACAAACATTACAAGGAT，如(SEQ ID NO.19)；
新冠病毒delta株的P681R分型单链DNA探针序列为：GAGAATTAGTCTAACAAACAAACCCTATAGTGAGTCGTATTAATCCCGCCTACAGTGCCCGCCGCC，如(SEQ ID NO.20)。
扩增和检测反应：首先将单链DNA探针与待检RNA加入反应体系中，缓冲液包括0.5mM NTPs、40mM Tris-HCl、10mM MgCl2、0.01mM-10mM ATP和1mM DTT，pH值在7.0-8.0之间。FAM和BHQ1双标记的RNA荧光探针0.8pM，向导RNA，0.5pM，酶混合液(T4 DNA连接酶，10U；T7 RNA聚合酶，10U；LwaCas13a蛋白，0.5pM)。其中，RNA荧光探针为6-FAM-mArArUrGrGrCmAmArArUrGrGrCmA-BHQ1。
荧光检测：反应混合后，在荧光实时定量PCR仪(7900 HT Fast Real-Time PCR)中设置温度为37度，双标记RNA的荧光检测探针的荧光基团为FAM，荧光信号采集时间间隔为1min，检测时间为30分钟。
结果：如图6所示为新冠病毒的亚型检测，应用此方法可以根据单碱基突变位点区分出新冠病毒的两种突变类型，W株和delta株。
对比例1
本对比例与实施例3的区别在于，将一锅法中PROTRACTOR缓冲液(B)：0.5mM NTP混合物，40mM Tris-HCl,10mM MgCl2,10mM DTT,0.5mM ATP,pH 7.5@25℃替换为分步骤单链DNA环化所用Buffer 1(B1)：50mM Tris-HCl,10mM MgCl2,10mM DTT, 0.5mM ATP,pH 7.5@25℃，滚环转录Buffer 2(B2)：40mM Tris-HCl,6mM MgCl2,10mM(NH4)2SO4,1mM DTT,2mM Spermidine，pH 7.9@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℃，来验证一锅法PROTRACTOR缓冲液的优势。
荧光检测：反应混合后，在荧光实时定量PCR仪(7900 HT Fast Real-Time PCR)中设置温度为37度，双标记RNA的荧光检测探针的荧光基团为FAM，荧光信号采集时间间隔为1min，检测时间为30分钟。其中，RNA荧光探针为6-FAM-mArArUrGrGrCmAmArArUrGrGrCmA-BHQ1。
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缓冲液B比B1，B2，B3更佳。一锅法检测时间可缩短至10分钟。
对比例2
本对比例与实施例3的区别在于，不添加任何缓冲液(NC)，用蒸馏水代替缓冲液。
荧光检测：反应混合后，在荧光实时定量PCR仪(7900 HT Fast Real-Time PCR)中设置温度为37度，双标记RNA的荧光检测探针的荧光基团为FAM，荧光信号采集时间间隔为1min，检测时间为30分钟。其中，RNA荧光探针为6-FAM-mArArUrGrGrCmAmArArUrGrGrCmA-BHQ1。
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，不添加任何缓冲液，反应基本不进行。
其包括了单链DNA探针环化，转录、CRISPR相关Cas蛋白或其变体识别剪切及单管一锅法检测反应。本发明针对单链DNA探针环化，转录、产物识别剪切及其很难在单一反应体系中同步进行的难题，建立了同时整合这三步反应的PROTRACTOR技术。在体外单一反应管体系中常温下对核酸分子进行富集、扩增和识别，实现核酸分子精确、超灵敏、快速检测。本发明相比传统的核酸分子检测方法，具有以下优点：无需DNA扩增，无需PAM位点，无需靶序列特异性引物，适应于单链DNA、双链DNA和RNA等不同类型核酸分子靶标。特别是，针对RNA靶标检测，不需要额外的逆转录步骤。本发明在核酸分子快速检测领域具有广泛的应用前景。
本发明的主要优点在于：
1、高灵敏度：应用本发明检测目标核酸分子(ssDNA、dsDNA和RNA)，可以实现单分子(单拷贝)检测；
2、通用性：应用本发明可以实现对DNA或RNA的检测，并且能够区分点突变；
3、成分稳定：本发明无需DNA扩增，无需引物，对于RNA样本的检测，无需逆转录；
4、快速：本发明可以短至10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，适合于资源有限地区核酸分子的快速检测；
6、假阳性低：本发明的反应在单链DNA探针与目标核酸分子的一条链特异性结合后才会被环化从而引发扩增反应，扩增子只有被特异的向导RNA和CRISPR相关Cas蛋白或其变体形成的二元复合物识别后才会激活剪切反应产生荧光信号，从反应机制上克服了假阳性的问题。此外本发明的扩增产物为RNA而非DNA，RNA易降解和不易产生气溶胶的特性使得检测方法本身克服了LAMP和qPCR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，最大程度上降低了污染的可能性；
7、等温检测：由DNA连接酶或其变体，RNA聚合酶或其变体与Cas蛋白CRISPR相关Cas蛋白或其变体3种工程酶和多种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，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：为了操作更简便，减少开盖加样带来的污染，提升反应效率和缩短反应时间，更适用于现场快速检测，尤其是在资源有限的地区使用；我们将单链DNA探针环化、转录和CRISPR相关Cas蛋白或其变体剪切三种反应创新性的整合到同一反应管中同时进行反应，实现了一步法的跨越。
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. 一种PROTRACTOR等温核酸检测方法，其特征在于，包括如下步骤：
    S1、从待检测样本中提取总核酸；
    S2、配置反应体系，所述反应体系包括单链DNA探针、RNA荧光探针、DNA连接酶或其变体、RNA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、PROTRACTOR反应缓冲液；其中单链DNA探针与目标核酸分子的一条链特异性的互补；
    S3、将步骤S1提取的总核酸加入步骤S2的反应体系中，进行恒温反应，并产生荧光信号；恒温反应过程中，单链DNA探针在DNA连接酶或其变体作用下形成单链环状DNA探针；
    S4、读取和记录PROTRACTOR产生荧光信号，通过荧光信号来判断待检测样本中目标核酸分子的有无。
  2. 如权利要求1所述的核酸检测方法，其特征在于，所述PROTRACTOR为普适性核酸检测平台，能检测不同类型的核酸分子，包括单链DNA、双链DNA、单链RNA中的一种或几种。
  3. 如权利要求1所述的核酸检测方法，其特征在于，所述CRISPR相关Cas蛋白或其变体为具有单链RNA识别剪切功能和反式RNA单链剪切功能的核酸酶；所述CRISPR相关Cas蛋白或其变体包括LbaCas13、LbuC13a、LwaCas13a、AspCas13b、BzoCas13b、CcaCas13b、PsmCas13b、PinCas13b、Pin2Cas13b、Pin3Cas13b、PbuCas13b、PguCas13b、PigCas13b、PsaCas13b、RanCas13b、PspCas13b、EsCas13d、RspCas13d中的任意一种或任意一种的变体。
  4. 如权利要求1所述的核酸检测方法，其特征在于，所述单链DNA探针由5'端臂，连接序列和3'端臂三部分串联而成；5'端和3'端臂的序列均与目标核酸分子序列的一条链互补；连接序列是一段包含T7启动子互补序列(T7p)的DNA序列。
  5. 如权利要求1所述的核酸检测方法，其特征在于，所述RNA荧光探针为5'端标记FAM、HEX、VIC、Cy5、Cy3、TET、ROX、FITC、Joe中的任意一种荧光基团，3'端标记TAMRA、BHQ1、MGB、BHQ2中的任意一种荧光淬灭基团的一条单链RNA。
  6. 如权利要求1所述的核酸检测方法，其特征在于，DNA连接酶是指利用ATP的 能量催化两个核苷酸链之间形成磷酸二酯键的依赖ATP的DNA连接酶，用来连接双链DNA分子或者RNA/DNA杂交双链的单链DNA缺口；所述DNA连接酶或其变体包括T4 DNA连接酶、E.coli DNA连接酶、SplintR连接酶和HiFi Taq DNA连接酶中的任意一种或任意一种的变体。
  7. 如权利要求1所述的核酸检测方法，其特征在于，所述向导RNA或其衍生物与目标核酸分子序列互补。
  8. 如权利要求1所述的核酸检测方法，其特征在于，所述PROTRACTOR反应缓冲液主要成分包括0.1mM-5mM NTPs、10mM-100mM Tris-HCl、0.5mM-10mM MgCl2、0.01mM-10mM ATP和0.5mM-10mM DTT，pH值在6.5-8.0之间。
  9. 如权利要求1所述的核酸检测方法，其特征在于，所述RNA聚合酶选自T7 RNA聚合酶、E.coli RNA聚合酶、T3 RNA聚合酶、SP6 RNA聚合酶中的一种。
  10. 一种用于核酸检测试剂盒，其特征在于，所述试剂盒包括单链DNA探针、RNA荧光探针、DNA连接酶或其变体、RNA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、PROTRACTOR反应缓冲液。
PCT/CN2022/140597 2022-06-21 2022-12-21 一锅法滚环转录和CRISPR/Cas介导的核酸检测方法及试剂盒 Ceased WO2023246033A1 (zh)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109055499A (zh) * 2018-08-30 2018-12-21 杭州杰毅麦特医疗器械有限公司 基于CRISPR-Cas的等温核酸检测方法及试剂盒
CN111363860A (zh) * 2020-05-27 2020-07-03 吴江近岸蛋白质科技有限公司 一种检测新型冠状病毒covid-19的核酸组合物及应用

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109055499A (zh) * 2018-08-30 2018-12-21 杭州杰毅麦特医疗器械有限公司 基于CRISPR-Cas的等温核酸检测方法及试剂盒
CN111363860A (zh) * 2020-05-27 2020-07-03 吴江近岸蛋白质科技有限公司 一种检测新型冠状病毒covid-19的核酸组合物及应用

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
FENG WEI, NEWBIGGING ASHLEY M., TAO JEFFREY, CAO YIREN, PENG HANYONG, LE CONNIE, WU JINJUN, PANG BO, LI JUAN, TYRRELL D. LORNE, ZH: "CRISPR technology incorporating amplification strategies: molecular assays for nucleic acids, proteins, and small molecules", CHEMICAL SCIENCE, ROYAL SOCIETY OF CHEMISTRY, UNITED KINGDOM, vol. 12, no. 13, 8 April 2021 (2021-04-08), United Kingdom , pages 4683 - 4698, XP093102174, ISSN: 2041-6520, DOI: 10.1039/D0SC06973F *
WANG GAOTING, TIAN WEIMIN, LIU XIAOLING, REN WEI, LIU CHENGHUI: "New CRISPR-Derived microRNA Sensing Mechanism Based on Cas12a Self-Powered and Rolling Circle Transcription-Unleashed Real-Time crRNA Recruiting", ANALYTICAL CHEMISTRY, AMERICAN CHEMICAL SOCIETY, US, vol. 92, no. 9, 5 May 2020 (2020-05-05), US , pages 6702 - 6708, XP093120001, ISSN: 0003-2700, DOI: 10.1021/acs.analchem.0c00680 *
WANG YUXI, ZHANG YONG, CHEN JUNBO, WANG MINJIN, ZHANG TING, LUO WENXIN, LI YALUN, WU YANGPING, ZENG BO, ZHANG KAIXIANG, DENG RUIJI: "Detection of SARS-CoV-2 and Its Mutated Variants via CRISPR-Cas13-Based Transcription Amplification", ANALYTICAL CHEMISTRY, AMERICAN CHEMICAL SOCIETY, US, vol. 93, no. 7, 23 February 2021 (2021-02-23), US , pages 3393 - 3402, XP093119999, ISSN: 0003-2700, DOI: 10.1021/acs.analchem.0c04303 *
YAN HE, WEN YUNJIE, HAN SONG, HUGHES STEVEN J., ZENG YONG: "One-Pot Endonucleolytically Exponentiated Rolling Circle Amplification by CRISPR-Cas12a Affords Sensitive, Expedited Isothermal Detection of MicroRNAs", BIORXIV, 1 May 2022 (2022-05-01), XP093120004, [retrieved on 20240116], DOI: 10.1101/2022.05.01.490215 *

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