WO2022100270A1 - 检测样品中靶核酸的方法 - Google Patents

检测样品中靶核酸的方法 Download PDF

Info

Publication number
WO2022100270A1
WO2022100270A1 PCT/CN2021/118280 CN2021118280W WO2022100270A1 WO 2022100270 A1 WO2022100270 A1 WO 2022100270A1 CN 2021118280 W CN2021118280 W CN 2021118280W WO 2022100270 A1 WO2022100270 A1 WO 2022100270A1
Authority
WO
WIPO (PCT)
Prior art keywords
reaction
sgrna
sample
nucleic acid
concentration
Prior art date
Application number
PCT/CN2021/118280
Other languages
English (en)
French (fr)
Inventor
孙阳
王亚蕾
张茁
程雪佳
Original Assignee
北京迅识科技有限公司
浙江迅识生物科技有限公司
北京迅识生物科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 北京迅识科技有限公司, 浙江迅识生物科技有限公司, 北京迅识生物科技有限公司 filed Critical 北京迅识科技有限公司
Priority to EP21890798.8A priority Critical patent/EP4234715A1/en
Priority to US18/037,173 priority patent/US20240002915A1/en
Publication of WO2022100270A1 publication Critical patent/WO2022100270A1/zh
Priority to ZA2023/05265A priority patent/ZA202305265B/en

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6848—Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction

Definitions

  • the invention belongs to the technical field of genetic engineering, and in particular, relates to a method for detecting target nucleic acid in a sample.
  • RPA Recombinase Polymerase Amplification
  • RAA Recombinase Aid Amplification
  • RPA RAA
  • RAA RAA
  • it has the ability to rapidly complete the amplification of nearly 10 orders of magnitude dynamic range at a single temperature step close to normal temperature (37-42 °C), and has the best reaction speed among various amplification technologies. In 10 minutes, it can complete the amount of amplification that ordinary PCR can complete in nearly an hour. Under ideal conditions, the sensitivity close to QPCR can be achieved, reaching the limit of 1-20 copies/reaction.
  • display technologies such as the combination of exonuclease and reporter probes, on a fluorescent platform or a lateral flow test strip platform, it can replace traditional PCR to achieve real-time rapid detection of various microorganisms.
  • RPA/RAA technology is currently the fastest known polymerase amplification system, but its disadvantage is that the primer probe is long and the fault tolerance is strong, resulting in its low specificity of amplification and reporting. Mismatched amplification products are easily generated.
  • RPA/RAA technology itself recombinase relies on ATP fuel characteristics, so that this non-specific product will compete with a large amount of fuel resources for specific products, resulting in specific amplification until the end of amplification can not produce enough products to present to the reporter system, thus affect the detection sensitivity.
  • the microenvironment of the sample is usually very complex due to the interference background of proteins and nucleic acids.
  • the RPA/RAA reaction has a certain tolerance to interference factors such as proteins, the sensitivity and specificity caused by this nucleic acid background interference. The characteristics of sexual instability limit the application and promotion of RPA/RAA alone for high-sensitivity and rapid clinical diagnosis.
  • the trans-cleavage activity of Cas protein in CRISPR-Cas is applied to the downstream of RPA/RAA reaction, which can make up for the above-mentioned problem of insufficient signal caused by insufficient specific amplification products, and can make high-sensitivity detection requirements in one. It is carried out in a more extensive environment and does not require too sophisticated optical equipment.
  • the CDetection system based on the Cas12b system discovered and reported by Li Wei et al. has good buffer system tolerance and temperature tolerance, and it has been confirmed that it can have a certain detection sensitivity in a single-tube step-by-step reaction.
  • composition of enzymes in RPA/RAA is relatively complex and cannot be compatible with large changes in ion concentration, so the concentration of the main salts and additives in the buffer system depends on the design of the amplification system itself, and these factors lead to the conformation of sgRNA
  • the change of sgRNA-Cas complex further affects the effectiveness of sgRNA-Cas complex;
  • the dosage of the Cas12-sgRNA system is simply increased in order to fill the above defects, the template will be cut off by the Cas protein during the RPA/RAA amplification process, resulting in the inability to enter the subsequent exponential amplification, resulting in the amplification linearity and sensitivity of the reaction. affected.
  • the trans-cutting activity of the Cas12-sgRNA system will continuously cut and consume the primers required for the amplification reaction, which directly affects the upper limit of the amplification of RPA and RAA;
  • RPA/RAA contains a large number of single-chain and double-chain binding proteins, which will compete with Cas proteins for substrates. In the system dominated by the former, Cas cis/trans cleavage cannot proceed smoothly; after diluting the RPA/RAA reaction, the As a result, the ratio of single- and double-stranded binding proteins to Cas proteins is reduced, the dominance of the Cas-sgRNA system reaction is restored, and the cleavage signal can be generated quickly.
  • the present invention provides a method for detecting target nucleic acid in a sample.
  • the present invention relates to the following aspects:
  • a method for detecting target nucleic acid in a sample comprising the following steps:
  • the sgRNA-Cas system includes Cas12b protein and sgRNA directed against the target nucleic acid;
  • the recombinase-mediated isothermal amplification system includes primers and single-stranded DNA reporter molecules that generate detectable signals after being cut.
  • divalent cations are selected from one or more of magnesium ions, calcium ions, manganese ions, cesium ions, nickel ions, iron ions, and cobalt ions.
  • the mixed reaction system further comprises suramin and/or tRNA.
  • sample is selected from the group consisting of whole blood, plasma, serum, cerebrospinal fluid, urine, feces, buccal swab, nasopharyngeal swab, saliva, cells or tissue extracts.
  • the present invention can realize fast and effective signal reporting with only one sample addition and mixing and simple temperature change for the recombinant polymerase amplification and CDetection reaction, reducing intermediate interruptions.
  • the second step of the reaction is the addition and mixing operation of the CDetection reaction components, which greatly improves the convenience of the operation and reduces the aerosol pollution of the recombinant polymerase amplification product generated by opening the reaction tube in the middle. Specifically, there are the following advantages:
  • RNA template detection is increased from less than 1E5 to 1E1 per reaction, and the increase ratio is more than 10,000 times.
  • Figure 1 shows the effect of the amount of sgRNA in the mixed reaction system on the intensity of positive signals.
  • Figure 2 shows the effect of primer dosage on the positive signal intensity in the mixed reaction system.
  • Figure 3 shows the effects of magnesium ion concentration and reaction temperature on the intensity of positive signals in the mixed reaction system.
  • Figure 4 shows the effect of reaction time on the intensity of positive signals in a mixed reaction system.
  • Figure 5 shows the effect of additives on the separation of negative and positive in the mixed reaction system: a is the separation of negative and positive with 5E4 copies of RNA per reaction, 5E4 copies of double-stranded DNA per reaction, and the same volume of water as the sample without additives; b is the positive and negative separation of 5E2 copies of RNA per reaction, 5E2 copies of double-stranded DNA per reaction, and the same volume of water as the sample without additives; c is the addition of 200ng of suramin.
  • 5E4 copies of double-stranded DNA per reaction and the positive and negative separation degree of the sample equal volume of water; e is the case of adding 500ngtRNA, respectively, put 5E4 copies of RNA per reaction, 5E4 copies of double-stranded DNA per reaction, and the same volume of water as the sample.
  • f is the separation degree of negative and positive when adding 500ngtRNA, 5E2 copies of RNA per reaction, 5E2 copies of double-stranded DNA per reaction, and the same volume of water as the sample;
  • g is the case of adding 200ng of suramin and 500ng of tRNA, respectively The positive and negative separation of 5E4 copies of RNA per reaction, 5E4 copies of double-stranded DNA per reaction, and the same volume of water as the sample;
  • h is the case of adding 200ng suramin and 500ngtRNA, respectively, adding 5E2 copies RNA per reaction, 5E2 copies of double-stranded DNA Positive and negative resolution per reaction, equal volume of water to sample.
  • FIG. 6 shows the results of virus infection detection using the detection method of the present invention in Test Example 1.
  • Figure 7 shows the effects of different probe concentrations in the mixed reaction system on the intensity of negative and positive signals.
  • FIG. 8 shows the results of virus infection detection using the detection method of the present invention in Test Example 2.
  • the present invention provides a method for detecting target nucleic acid in a sample.
  • the method includes the following steps:
  • the sgRNA-Cas system includes Cas12b protein and sgRNA directed against the target nucleic acid;
  • the recombinase-mediated isothermal amplification system includes primers and single-stranded DNA reporter molecules that generate detectable signals after being cut.
  • sgRNA small guide RNA for short
  • guide RNA is a single-molecule guide RNA, which is an important part of the CRISPR gene knockout and knock-in system. during post-transcriptional modification of RNA editing. It is also a small non-coding RNA. It can be paired with pre-mRNA and some uracil (U) inserted into it, resulting in a functional mRNA.
  • Guide RNA-edited RNA molecules about 60-80 nucleotides in length, are transcribed from a separate gene, with a 3' oligo U tail, a sequence in the middle that is exactly complementary to the edited mRNA, and the 5' end is an anchor sequence that is complementary to the unedited mRNA sequence.
  • the Cas12b protein refers to an RNA-guided sequence-specific nuclease derived from the microbial CRISPR system.
  • Cas12b can target and cleave DNA target sequences under the guidance of guide RNA to form DNA double-strand breaks, also known as canonical dsDNA cleavage activity.
  • canonical dsDNA cleavage activity More importantly, after the complex of Cas12b and gRNA recognizes and binds to the corresponding target DNA sequence, it can activate its non-specific single-stranded DNA cleavage activity, also known as non-canonical bypass ssDNA cleavage activity.
  • non-canonical bypass ssDNA cleavage activity cleavage of a single-stranded DNA reporter molecule that produces a detectable signal upon cleavage, ie, can reflect the presence and/or amount of target DNA.
  • the Cas12b protein may be AaCas12b protein from Alicyclobacillus acidiphilus, AkCas12b protein from Alicyclobacillus kakegawensis, AmCas12b protein from Alicyclobacillus macrosporangiidus, BhCas12b protein from Bacillus hisashii, BsCas12b protein from Bacillus genus, Bs3Cas12b protein from Bacillus genus, Desulfovibrinopinatus protein DiCas12b protein from , LsCas12b protein from Laceyellasediminis, SbCas12b protein from Spirochaetes bacterium, TcCas12b protein from Tubeibacillus calidus.
  • the "single-stranded DNA reporter molecule that generates a detectable signal after being cleaved” is used to indirectly reflect the content of the target nucleic acid contained in the sample.
  • the single-stranded DNA reporter molecule may contain a fluorophore and its quenching group at both ends of the single-stranded DNA, respectively. When the single-stranded DNA is not cleaved, the fluorophore does not fluoresce due to the presence of the quenching group.
  • Suitable fluorophores and their corresponding quenching groups are known in the art. Suitable fluorophores include, but are not limited to, FAM, TEX, HEX, Cy3 or Cy5. Suitable quenching groups include, but are not limited to, BHQ1, BHQ2, BHQ3 or TAMRA.
  • Suitable fluorophore-quencher pairs include, but are not limited to, FAM-BHQ1, TEX-BHQ2, Cy5-BHQ3, Cy3-BHQ1 or FAM-TAMRA.
  • the detectable signal is a fluorescent signal.
  • the fluorophore is FAM and the quenching group is BHQ1.
  • the length of the single-stranded DNA in the single-stranded DNA reporter molecule may be about 2-100 nucleotides, such as 2-5, 2-10, 2-15, 2-20, 2-25, 2-30, 2-40 or 2 to more nucleotides.
  • the single-stranded DNA in the single-stranded DNA reporter molecule may comprise any sequence.
  • the single-stranded DNA in the single-stranded DNA reporter molecule may be selected from polyA (oligoadenylic acid), polyC (polycytidylic acid), or polyT (polythymidylic acid).
  • RPA Recombinase Polymerase Amplification
  • the protein-DNA complex formed by the combination of the recombinase and the primer can search for homologous sequences in double-stranded DNA. Once the primers locate the homologous sequence, a strand exchange reaction takes place and initiates DNA synthesis to amplify the target region on the template by strand displacement. The displaced DNA strand binds to SSB to form a stable single-stranded structure, waiting for the new primer synthesis reaction to proceed.
  • primers anchored on complementary strands with opposite 3' tails are co-initialized to form a double-stranded amplicon and circulate continuously. The whole process is very fast, and generally detectable levels of amplification products can be obtained within ten minutes.
  • RPA detection The sensitivity of RPA detection is very high, and it can amplify a trace amount of nucleic acid (especially DNA) template to a detectable level, and obtain an amplification product with a length of about 1012 from a single template molecule. Moreover, RPA does not require complex sample processing, and is suitable for field detection where nucleic acid cannot be extracted.
  • RPA can amplify both DNA and RNA, and it also eliminates the need for additional cDNA synthesis steps. Not only can you perform endpoint detection of amplification products, but you can also monitor the amplification process in real time, and even read the results with test strips (lateral flow chromatography test strips LFD).
  • RAA Recombinase Aid Amplification
  • the nucleic acid in the sample is amplified by the recombinase-mediated isothermal amplification system, and then the sgRNA-Cas system is used to detect the amount of nucleic acid.
  • the present invention forms a mixed reaction system with the sgRNA-Cas system and the recombinase-mediated isothermal amplification system, and realizes one-step detection of nucleic acids in samples by optimizing the reaction conditions of the mixed reaction system .
  • the contents of sgRNA, primer and single-stranded DNA reporter molecule can be adjusted according to actual needs.
  • the concentration of the sgRNA in the mixed reaction system is 2-80ng/ ⁇ l, such as 2-80ng/ ⁇ l, 10-70ng/ ⁇ l, 20-60ng/ ⁇ l, 30-50ng / ⁇ l, 2ng/ ⁇ l, 4ng/ ⁇ l, 10ng/ ⁇ l, 20ng/ ⁇ l, 30ng/ ⁇ l, 40ng/ ⁇ l, 50ng/ ⁇ l, 60ng/ ⁇ l, 70ng/ ⁇ l, 80ng/ ⁇ l.
  • the concentration of the primers in the mixed system is 300-1200nM, for example, 300-1200nM, 400-1100nM, 500-1000nM, 600-900nM, 700-800nM, 300nM, 400nM, 500nM, 600nM, 700nM, 800nM, 900nM, 1000nM, 1100nM, 1200nM.
  • the concentration of the single-stranded DNA reporter molecule is 200-2000 nM, preferably 700-1500 nM.
  • a one-step reaction is realized by precisely controlling the concentration of the single-stranded DNA reporter molecule in the reaction system.
  • the reaction time is subject to the intensity of signal generation.
  • the fluorescence intensity generated can be reduced in a shorter time. Achieving the detection sensitivity of the instrument can also generate sufficient signal when the number of target DNA fragments in the system is low.
  • the reaction is reacted at a first temperature, and inactivated at a second temperature, wherein the second temperature is higher than the first temperature, that is, the temperature used during inactivation is higher than the temperature during reaction.
  • the second temperature is greater than 47°C, such as 48°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C.
  • the mixed reaction system further comprises divalent cations, and the concentration of the divalent cations is 8mM ⁇ 25mM, such as 8mM ⁇ 25mM, 10mM ⁇ 20mM, 12mM ⁇ 18mM, 8mM, 9mM, 10mM, 11mM, 12mM , 13mM, 14mM, 15mM, 16mM, 17mM, 18mM, 19mM, 20mM, 21mM, 22mM, 23mM, 24mM, 25mM.
  • the concentration of the divalent cations is 8mM ⁇ 25mM, such as 8mM ⁇ 25mM, 10mM ⁇ 20mM, 12mM ⁇ 18mM, 8mM, 9mM, 10mM, 11mM, 12mM , 13mM, 14mM, 15mM, 16mM, 17mM, 18mM, 19mM, 20mM, 21mM, 22mM, 23mM, 24mM, 25mM.
  • the divalent cation is selected from one or more of magnesium ions, calcium ions, manganese ions, cesium ions, nickel ions, iron ions, and cobalt ions.
  • the target nucleic acid of the present invention may include DNA or RNA, wherein when the target nucleic acid is RNA, the mixed reaction system further includes suramin and/or tRNA, that is, it may include suramin, tRNA or both suramin Ming and tRNA.
  • the concentration of the suramin in the mixed reaction system is 3-30 ng/ ⁇ l, preferably 3-12 ng/ ⁇ l.
  • the one-step reaction is realized by precisely controlling the concentration of suramin in the reaction system.
  • the recombinase-mediated isothermal amplification system includes polymerase, RT enzyme , recombinase, etc. all rely on the hydrolysis of triphosphate bonds to generate activity.
  • concentration of suramin By adjusting the concentration of suramin, the sequence of each enzyme reaction in the reaction system can be coordinated.
  • 3-30 ng/ ⁇ l suramin is used to inhibit the DNA polymerase and recombinase that are too active, so that the RT enzyme can combine with RNA template-primer complex earlier to generate cDNA template, so that the whole tandem reaction system can proceed smoothly.
  • the concentration of the tRNA in the mixed reaction system is 10-20 ng/ ⁇ l.
  • it can be 10-20ng/ ⁇ l, 12-18ng/ ⁇ l, 10ng/ ⁇ l, 11ng/ ⁇ l, 12ng/ ⁇ l, 13ng/ ⁇ l, 14ng/ ⁇ l, 15ng/ ⁇ l, 16ng/ ⁇ l, 17ng/ ⁇ l, 18ng/ ⁇ l , 19ng/ ⁇ l, 20ng/ ⁇ l.
  • a one-step reaction is realized by precisely controlling the concentration of tRNA in the reaction system.
  • adding a certain amount of background RNA can improve the sensitivity of RT-PCR, which is mainly to improve the structural diversity of template RNA, to unravel part of the higher-order structure, and to allow trace amounts of template to reduce its interaction with The non-specific adsorption of other substances is more conducive to its amplification reaction.
  • RT-RAA the inventors of the present application found that in the case of insufficient RT enzyme activity, the input of this additive can also have a relatively significant effect.
  • the present invention can improve the amplification of recombinant polymerase by regulating the conditions such as the concentration of sgRNA in the reaction system to be 2-80 ng/ ⁇ l, the concentration of primers to be 300 to 1200 nM, and the concentration of single-stranded DNA reporter molecule to be 200 to 2000 nM.
  • the mutual inhibition of the sgRNA-Cas reaction can be increased, and the recombinant polymerase amplification and CDetection reaction can be realized quickly and effectively with only one sample addition and mixing and a simple temperature change, which greatly improves the convenience of operation and reduces the pollution.
  • the sample described in the present invention can be selected from whole blood, plasma, serum, cerebrospinal fluid, urine, feces, buccal swabs, nasopharyngeal swabs, saliva, cell or tissue extracts.
  • the sequence of the reporter probe is: 5'-FAM-TTTTTTTT-BHQ1-3', wherein the reporter probe is the single-stranded DNA reporter molecule described in the present invention
  • RNA nuclease inhibitor was purchased from promega, catalog number N2515
  • AaCas12b protease was purchased from Nanjing Novizan Biotechnology Co., Ltd., the product number is EN311-PE
  • concentrations of single molecule guide RNA in this example were 20ng/ ⁇ l, 80ng/ ⁇ l, 240ng/ ⁇ l, respectively, wherein 1 ⁇ l of nuclease-free water was used instead of single molecule guide RNA.
  • the recombinase-mediated isothermal amplification system was prepared according to the dosage of each component in Table 2, and after the preparation was completed, it was placed at room temperature for use.
  • Amplification buffer A is 20% PEG35K, purchased from sigma-aldrich, product number 818892 diluted to 20% for use.
  • Magnesium acetate solution was purchased from sigma-aldrich, Cat. No. 63052, diluted to 280mM for use
  • the forward primer sequence is 5'-GTTGTAGCTTGTCACACCGTTTCTATAGATTAGC-3' (SEQ ID NO:2)
  • the reverse primer sequence is 5'-CCTGGTTTAACATATAGTGAACCGCCACACAT-3' (SEQ ID NO:3)
  • 2019-nCoV RdRP pseudovirus extracted using Meckey nucleic acid extraction kit, IVD5412, and diluted to 500 copies per microliter after extraction
  • the 2019-nCoV RdRP gene pseudovirus was purchased from Xiamen Zhishan Biotechnology Co., Ltd., the product number is JBD249.
  • the above mixed solution was added to the enzyme dry powder (RT-RAA dry powder, purchased from Hangzhou Zhongce Biotechnology Co., Ltd., item number S0040ZC). Incubate at 42°C for 1min ⁇ 60cycles, and light once per minute.
  • enzyme dry powder purchased from Hangzhou Zhongce Biotechnology Co., Ltd., item number S0040ZC. Incubate at 42°C for 1min ⁇ 60cycles, and light once per minute.
  • the FAM group is used as the reporter group, and the presence or absence of the target nucleic acid is judged according to the relative fluorescence signal intensity of the FAM group at 30 min.
  • the amount of sgRNA added respectively was 0ng, 20ng sgRNA, 80ng sgRNA, 240ng sgRNA, and the corresponding final molar concentrations in the mixed reaction system were 0, 5.84nM, 23.36nM, and 70.08nM, respectively.
  • the sgRNA-Cas pre-dispensing system was prepared according to the dosage of each component in Table 3, and after the preparation was completed, it was placed at room temperature for use.
  • the recombinase-mediated isothermal amplification system was prepared according to the dosage of each component in Table 4, and was placed at room temperature for use after the preparation was completed.
  • the above system was placed in a real-time fluorescence quantitative PCR instrument, and the incubation program was 45 °C for 1 min ⁇ 60 cycles, and light was collected once per minute.
  • the FAM group is used as the reporter group, and the presence or absence of the target nucleic acid is determined according to its relative fluorescence signal intensity at 60 min.
  • Embodiment 3 Influence of reaction temperature on reaction result
  • the concentration X of single molecule guide RNA was 10ng/ ⁇ l, 50ng/ ⁇ l, 100ng/ ⁇ l and 200ng/ ⁇ l, respectively.
  • the recombinase-mediated isothermal amplification system system was prepared according to the dosage of each component in Table 6, and after the preparation was completed, it was placed at room temperature for use.
  • the amount of magnesium acetate solution added in the above system was 1.429 ⁇ l and 2.5 ⁇ l, respectively.
  • the sequence of the single-molecule guide RNA, the sequence of the reporter probe, and the sequence of the forward and reverse primers are the same as those in Example 1.
  • the sources of components such as amplification buffer A, magnesium acetate solution, RNA nuclease inhibitor, AaCas12b protease, etc., were the same as those in Example 1, and the transfer RNA was purchased from Invitrogen with the product number AM7119.
  • the above system was placed in a real-time fluorescence quantitative PCR instrument, and the incubation program was 45°C or 42°C, 1 min ⁇ 60 cycles, and light was collected once per minute.
  • the FAM group is used as the reporter group, and the presence or absence of the target nucleic acid is judged according to the relative fluorescence signal intensity at 60 min.
  • the height of positive signal is the relative fluorescence signal intensity of FAM group when 100 copies of viral genomic RNA is input and 45A reacts for 60 minutes, that is, the result of subtracting the baseline fluorescence intensity from the fluorescence intensity of FAM group at 60 minutes.
  • **The negative signal height is the relative fluorescence signal intensity of the FAM group when an equal volume of sample elution buffer is used instead of viral genomic RNA and added to the reaction system, and the reaction is performed at 45°C for 60 minutes.
  • the positive-negative separation degree is the ratio of the relative fluorescence signal intensity of the FAM group when the above-mentioned positive reaction and negative reaction are reacted at 45°C for 60 min.
  • the height of positive signal is the relative fluorescence signal intensity of FAM group when 100 copies of viral genomic RNA is input and 42A reacts for 60min, that is, the result of subtracting the baseline fluorescence intensity from the fluorescence intensity of FAM group at 60min.
  • **The negative signal height is the relative fluorescence signal intensity of the FAM group when an equal volume of sample elution buffer is used instead of viral genomic RNA and added to the reaction system, and the reaction is performed at 42°C for 60 minutes.
  • the positive-negative resolution is the ratio of the relative fluorescence signal intensity of the FAM group when the above-mentioned positive reaction and negative reaction react at 42°C for 60 min.
  • 2 50 ⁇ TET buffer is a self-prepared buffer (100mM Tris pH8.0, 5mM EDTA, 0.5% Tween 20)
  • the sequences of the forward and reverse primers were the same as those of Example 1.
  • the sources of components such as amplification buffer A, magnesium acetate solution, and RNA nuclease inhibitors are the same as those in Example 1.
  • the above mixture was placed in a real-time fluorescence quantitative PCR instrument at 45°C and incubated for 1 min ⁇ 60 cycles to observe the shape of the amplification curve.
  • the FAM reporter group was replaced with Evagreen dye to characterize the amplification behavior of the target gene in the system, and the results are shown in Figure 4.
  • the Cas enzyme AaCas12b used in this method can maintain the optimal nuclease activity in a very wide temperature range (31-59 °C); therefore, without affecting the accumulation of RT-RAA reaction products, we will use the first 7 minutes of The reaction conditions were kept at 45 °C, and the reaction temperature for 8-60 min was adjusted to 52 °C.
  • the key enzymes of RT-RAA were inactivated to exclude the influence of RT-RAA reaction components on the cleavage process. The kinetics of the cleavage reaction accelerates the accumulation of the reporter group signal.
  • the recombinase-mediated isothermal amplification system was prepared according to the dosage of each component in Table 11, and after the preparation was completed, it was placed at room temperature for use.
  • the sequence of the single-molecule guide RNA and the sequence of the forward and reverse primers are the same as those in Example 1.
  • the sources of components such as amplification buffer A, magnesium acetate solution, RNA nuclease inhibitor, and AaCas12b protease are the same as those in Example 1.
  • Transfer RNA was purchased from Invitrogen, Cat. No. AM7119.
  • RNA sample is 2019-nCoV RdRP pseudovirus, use Mekey nucleic acid extraction kit, IVD5412 for extraction, and dilute to 50,000 copies per microliter and 500 Copies are used per microliter;
  • the DNA sample is a plasmid containing the 2019-nCovRdRP gene), 5 ⁇ l of C mix.
  • the 2019-nCoV RdRP gene pseudovirus was purchased from Xiamen Zhishan Biotechnology Co., Ltd. with the product number JBD249; the 2019-nCovRdRP gene plasmid was purchased from GenScript (a chemically synthesized product without a product number).
  • the above system was placed in a real-time fluorescence quantitative PCR instrument, and the incubation program was 45 °C for 1 min ⁇ 60 cycles, and light was collected once per minute.
  • the FAM group is used as the reporter group, and the presence or absence of the target nucleic acid is judged according to the relative fluorescence signal intensity at 60 min.
  • the degree of separation between negative and positive refers to the relative fluorescence intensity of the positive reaction reporter group/the relative fluorescence intensity of the negative reaction reporter group.
  • the recombinase-mediated isothermal amplification system was prepared according to the dosage of each component in Table 14, and after the preparation was completed, it was placed at room temperature for use.
  • the sequence of the single-molecule guide RNA, the sequence of the forward and reverse primers, and the sequence of the reporter probe are the same as those in Example 1.
  • the sources of components such as amplification buffer A, magnesium acetate solution, RNA nuclease inhibitor, and AaCas12b protease are the same as those in Example 1.
  • the final concentration experimental gradient of the probe is set to 200nM, 400nM, 700nM, 1000nM, 1500nM, 2000nM, and the corresponding volume (X) added in the system is 0.1 ⁇ l, 0.4 ⁇ l, 0.7 ⁇ l, 1 ⁇ l, 1.5 ⁇ l, respectively. ⁇ l, 2 ⁇ l.
  • the 2019-nCoV RdRP gene pseudovirus was purchased from Xiamen Zhishan Biotechnology Co., Ltd. with the product number JBD249; the 2019-nCovRdRP gene plasmid was purchased from GenScript (a chemically synthesized product without a product number).
  • the above system was placed in a real-time quantitative PCR instrument, the incubation program was 45°C for 7 min, and the fluorescence was collected once, (52°C for 1 min) ⁇ 22 cycles, and the fluorescence was collected every minute, and at 52°C for 1 min, the fluorescence was collected once.
  • the FAM group is used as the reporter group, and the presence or absence of the target nucleic acid is judged according to the relative fluorescence signal intensity at 30 min.
  • the reaction results are shown in Figure 7, and the relative fluorescence signal separation between negative and positive is shown in Table 15.
  • the following conditions were used to detect the target nucleic acid of the sample.
  • the recombinase-mediated isothermal amplification system was prepared according to the dosage of each component in Table 17, and after the preparation was completed, it was placed at room temperature for use.
  • the sequence of the single-molecule guide RNA, the sequence of the reporter probe, and the sequence of the forward and reverse primers are the same as those in Example 1.
  • the sources of components such as amplification buffer A, magnesium acetate solution, RNA nuclease inhibitor, and AaCas12b protease are the same as those in Example 1.
  • Transfer RNA was purchased from Invitrogen, Cat. No. AM7119.
  • Figure 6 shows the CDetection reaction results of four 100-copy viral genomic RNAs and four negative CDetection reaction results (using an equal volume of sample elution buffer instead of viral nucleic acid samples).
  • the gene target POP7 is often used as an internal reference for CRISPR-Cas-related detection methods (James P. Broughton, 2020).
  • the test results of the same method for the second target POP7 gene (NM_005837.3) are provided in this test example. , to verify the versatility of the method described in this patent in the detection of different species and different targets.
  • the sgRNA-Cas pre-formulation system was prepared according to the dosage of each component in Table 18, and after the preparation was completed, it was placed on an ice box or in a 4°C refrigerator for use.
  • AaCas12b protease was purchased from Nanjing Novizan Biotechnology Co., Ltd., the product number is EN311-PE
  • RNA nuclease inhibitor 2 RNA nuclease inhibitor was purchased from promega, catalog number N2515
  • the sequence of the single guide RNA is:
  • the recombinase-mediated isothermal amplification system was prepared according to the dosage of each component in Table 19, and after the preparation was completed, it was placed at room temperature for use.
  • Amplification buffer A is 4x RT-RAA buffer A, purchased from sigma-aldrich, Cat. No. 818892, diluted to 20% for use.
  • Magnesium acetate solution was purchased from sigma-aldrich, Cat. No. 63052, diluted to 280mM for use
  • the forward primer sequence is 5'-TGAGTACTGGACCTCGGACCAGAGCCATGTAAGAA-3' (SEQ ID NO:5)
  • the reverse primer sequence is 5'-GCCCACCAAGAGACAATTACCCCCACCCTCAATGC-3' (SEQ ID NO:6)
  • the reporter probe sequence is: 5'-FAM-TTTTTTTTTT-BHQ1-3'
  • POP7 nucleic acid sample human 293T cell transcriptome, after extraction, diluted to 10ng/ ⁇ l for use, 10 ⁇ l per reaction
  • the above mixed solution was added to the enzyme dry powder (RT-RAA dry powder, purchased from Hangzhou Zhongce Biotechnology Co., Ltd., item number S0040ZC). Incubate at 45°C for 7 minutes, then incubate at 52°C for 1 minute ⁇ 23 cycles, with lighting once per minute.
  • RT-RAA dry powder purchased from Hangzhou Zhongce Biotechnology Co., Ltd., item number S0040ZC.
  • the FAM group is used as the reporter group, and the presence or absence of the target nucleic acid is judged according to the relative fluorescence signal intensity of the FAM group at 30 minutes.
  • the results are shown in Figure 8 and Table 20.
  • the test results show that when 100ng of human transcriptome nucleic acid is put in, the separation degree of fluorescence signal of positive samples and negative samples can reach a level of about 2 after 30 minutes of reaction.
  • Relative fluorescence intensity fluorescence intensity at 32 minutes - initial fluorescence intensity.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biophysics (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

一种检测样品中靶核酸的方法，包括以下步骤：使样品与由sgRNA-Cas体系和重组酶介导等温扩增体系组成的混合反应体系进行反应；反应结束后，检测反应产生的可检测信号；其中，sgRNA-Cas体系包含Cas12b蛋白和针对靶核酸的sgRNA；重组介导等温扩增体系包含引物、被切割后产生可检测信号的单链DNA报告分子。使试剂使用者操作时减少了一个中间停止反应加入第二步反应组分的加样混合操作，操作均一性和连贯性更好，有利于提高反应精密度；省略了在重组聚合酶扩增反应后打开反应管的操作，混合这两个反应时Cas蛋白酶的顺式切割活性会消化扩增产物，使之不会释放到环境中成为污染源，这对高通量高灵敏度检测尤其重要；提升了反应的灵敏度。

Description

检测样品中靶核酸的方法 技术领域
本发明属于基因工程技术领域，具体地，涉及一种检测样品中靶核酸的方法。
背景技术
Piepenburg等于2006年创建了一种新型的全程等温扩增技术，使用酶来打开双链DNA，该技术称为重组酶聚合酶扩增(Recombinase Polymerase Amplification，以下称RPA)。RPA参考了T4噬菌体DNA复制系统，该反应中除了需要一种常温下能工作的DNA聚合酶之外，还包含噬菌体重组酶、辅助蛋白和DNA结合蛋白。RPA以TwistDx公司产品为代表。重组酶介导等温扩增技术(Recombinase Aid Amplification，以下称RAA)则使用从细菌或真菌中获得的重组酶，使整个扩增体系的稳定性得到进一步提升。
无论是RPA或RAA，都具备在一个接近常温的(37～42℃)单一温阶下快速完成接近10个数量级动态范围的扩增能力，在各种扩增技术中具备数一数二的反应速度。在10分钟内就能完成普通PCR接近一个小时才能完成的扩增量。在理想条件下，也能做到接近QPCR的灵敏度，达到1～20拷贝/反应的极限程度。结合各种展现技术，例如内外切酶和报告探针的组合，在荧光平台上或侧向流试纸条平台上，可替代传统PCR实现各种微生物的实时快速检测。
在2018年，多个研发团队陆续发表SHERLOCK、DETECTR、HOLMES等技术，都是基于等温扩增结合CRISPR-Cas系统实现对病原体的微生物的实时快速检测，进一步提高了等温在现场扩增应用的范围，稳定的提高了等温扩增的反应速度、灵敏度、特异性，以及对荧光读取仪器的依赖等。
RPA/RAA技术是目前已知扩增速度最快的聚合酶扩增体系，但其缺点是引物探针较长，容错性较强的特征，导致其扩增和报告的特异性不是很强，容易产生错配扩增产物。RPA/RAA技术本身重组酶依赖ATP燃料的特点，使这种非特异产物会大量的竞争特异产物的燃料资源，导致特异性扩增直至扩增末期都不能产生足够的产物呈递给报告系统，从而影响检测灵敏度。尤其在临床诊断中，样 本的微环境通常由于蛋白和核酸等的干扰背景非常复杂，虽然RPA/RAA反应对蛋白等干扰因子具备一定的耐受能力，但这种核酸背景干扰导致的灵敏度和特异性不稳定的特性，限制了RPA/RAA单独进行临床高灵敏度快速诊断的应用推广。
与RPA/RAA技术联合，CRISPR-Cas中Cas蛋白反式切割活性应用于RPA/RAA反应下游，能补足上述特异性扩增产物不足导致的信号不足的问题，能使高灵敏度的检测需求在一个更粗放的环境下进行，不需要太精密的光学仪器设备。李伟等发现和报道的基于Cas12b系统的CDetection体系，具备较好的缓冲液体系耐受性和温度耐受性，已证实在单管分步反应中能具备一定的检测灵敏度。但CDetection与RPA/RAA的相互干扰问题，没有得到解决，反应还是需要依次等温扩增和CDetection两步反应进行而不能做到同步反应。导致其困难的机制虽没有详细的外部报道，但根据其反应机理上的可能性如下：
a.RPA/RAA中酶的成分比较复杂，不能兼容太大的离子浓度变化，所以缓冲液体系中主要的盐和添加剂的浓度依赖于扩增体系本身的设计，而这些因素导致了sgRNA的构象的改变从而进一步影响sgRNA-Cas复合体的有效性；
b.如果为了填补上述的缺陷简单的加大Cas12-sgRNA体系的用量，RPA/RAA扩增过程中模板又会被Cas蛋白切断导致不能进入后续的指数扩增，导致反应的扩增线性和灵敏度受到影响。此外Cas12-sgRNA体系的反式切割活性，还会对扩增反应所需要的引物等进行不断的切割和消耗，直接影响到RPA和RAA的扩增上限；
c.RPA/RAA中含有大量的单链、双链结合蛋白，都会与Cas蛋白竞争底物，在前者主导的体系里，Cas顺式/反式切割不能顺利进行；稀释RPA/RAA反应后的产物，使单链、双链结合蛋白与Cas蛋白的比例下降，Cas-sgRNA体系反应的主导性才会恢复，切割信号才能较快的产生。
发明内容
针对现有技术存在的不足，本发明提供一种检测样品中靶核酸的方法。
具体来说，本发明涉及如下方面：
1、一种检测样品中靶核酸的方法，其特征在于，所述方法包括以下步骤：
使所述样品与由sgRNA-Cas体系和重组酶介导等温扩增体系组成的混合反应体系进行反应；
反应结束后，检测反应产生的可检测信号；
其中，所述sgRNA-Cas体系包含Cas12b蛋白和针对所述靶核酸的sgRNA；所述重组酶介导等温扩增体系包含引物、被切割后产生可检测信号的单链DNA报告分子。
2、根据项1所述的方法，其特征在于，所述混合反应体系中所述sgRNA的浓度为2～80ng/μl。
3、根据项1或2所述的方法，其特征在于，所述混合体系中所述引物的浓度为300～1200nM。
4、根据项1-3任一项所述的方法，其特征在于，所述单链DNA报告分子的浓度为200～2000nM，优选为700～1500nM。
5、根据项1-4任一项所述的方法，其特征在于，所述反应在第一温度反应，并在第二温度灭活，所述第二温度大于所述第一温度。
6、根据项5所述的方法，其特征在于，所述第二温度大于47℃。
7、根据项1-6任一项所述的方法，其特征在于，所述混合反应体系还包含二价阳离子，所述二价阳离子的浓度为8mM～25mM。
8、根据项7所述的方法，其特征在于，所述二价阳离子选自镁离子、钙离子、锰离子、铯离子、镍离子、铁离子、钴离子中的一种或两种以上。
9、根据项1-8任一项所述的方法，其特征在于，当所述靶核酸为RNA时，所述混合反应体系还包含苏拉明和/或tRNA。
10、根据项9所述的方法，其特征在于，所述混合反应体系中所述苏拉明的浓度为3～30ng/μl，优选为3～12ng/μl。。
11、根据项9所述的方法，其特征在于，所述混合反应体系中所述tRNA的浓度为10～20ng/μl。
12、根据项1-11任一项所述的方法，其特征在于，所述样品选自全血、血浆、血清、脑脊液、尿液、粪便、口腔拭子、鼻咽拭子、唾液、细胞或组织提取物。
本发明过改善重组聚合酶扩增和sgRNA-Cas反应的相互抑制，可以实现重组聚合酶扩增和CDetection反应只需要一次加样混合和简单改变温度就能进行快捷有效的信号报告，减少中间中断反应进行第二步CDetection反应组分的加样混合操作，极大的提高了操作的便捷性和降低了中途打开反应管产生的重组聚合酶扩增产物气溶胶污染。具体来说，有以下优势：
1、使试剂使用者操作时减少操作步骤，操作均一性和连贯性更好，有利于提高反应精密度；
2、省略了在重组聚合酶扩增反应后打开反应管的操作，另外混合这两个反应时Cas蛋白酶的顺式切割活性会消化扩增产物，使之不会释放到环境中成为污染源，这对高通量高灵敏度检测尤其重要。反应产物的气溶胶污染通常是核酸检测中非常令人头疼的一个问题，也是PCR实验室需要提高对环境分区要求的根本原因。污染源不存在了，检测实验室的建设环境要求大大降低了，不再需要分区进行加样和扩增检测反应；
3、通过加入两个添加剂，使RNA模板检验的灵敏度从原有的不足1E5提升至1E1每反应，提升比例超过10000倍。
附图说明
图1为混合反应体系中sgRNA用量对阳性信号强度的影响。
图2为混合反应体系中引物用量对阳性信号强度的影响。
图3为混合反应体系中镁离子浓度和反应温度对阳性信号强度的影响。
图4为混合反应体系中反应时间对阳性信号强度的影响。
图5为混合反应体系中添加剂对阴阳性分离度的影响：a为无添加剂情况下分别投入5E4拷贝RNA每反应、5E4拷贝双链DNA每反应、与样本等体积的水的阴阳性分离度；b为无添加剂情况下分别投入5E2拷贝RNA每反应、5E2拷贝双链DNA每反应、与样本等体积的水的阴阳性分离度；c为添加200ng苏拉明情况下分别投入5E4拷贝RNA每反应、5E4拷贝双链DNA每反应、与样本等体积的水的阴阳性分离度；e为添加500ngtRNA情况下分别投入5E4拷贝RNA每反应、5E4拷贝双链DNA每反应、与样本等体积的水的阴阳性分离度；f为添加500ngtRNA情况下分别投入5E2拷贝RNA每反应、5E2拷贝双链DNA每反应、与样本等体积的水的阴阳性分离度；g为添加200ng苏拉明和500ngtRNA情况下分别投入5E4拷贝RNA每反应、5E4拷贝双链DNA每反应、与样本等体积的水的阴阳性分离度；h为添加200ng苏拉明和500ngtRNA情况下分别投入5E2拷贝RNA每反应、5E2拷贝双链DNA每反应、与样本等体积的水的阴阳性分离度。
图6为试验例1中使用本发明的检测方法检测病毒感染的结果。
图7为混合反应体系中不同探针浓度对阴阳性信号强度的影响。
图8为试验例2中使用本发明的检测方法检测病毒感染的结果。
具体实施方式
下面结合实施例进一步说明本发明，应当理解，实施例仅用于进一步说明和阐释本发明，并非用于限制本发明。
除非另外定义，本说明书中有关技术的和科学的术语与本领域内的技术人员所通常理解的意思相同。虽然在实验或实际应用中可以应用与此间所述相似或相同的方法和材料，本文还是在下文中对材料和方法做了描述。在相冲突的情况下，以本说明书包括其中定义为准，另外，材料、方法和例子仅供说明，而不具限制性。以下结合具体实施例对本发明作进一步的说明，但不用来限制本发明的范围。
本发明提供一种检测样品中靶核酸的方法。
所述方法包括以下步骤：
使所述样品与由sgRNA-Cas体系和重组酶介导等温扩增体系组成的混合反应体系进行反应；
反应结束后，检测反应产生的可检测信号；
其中，所述sgRNA-Cas体系包含Cas12b蛋白和针对所述靶核酸的sgRNA；所述重组酶介导等温扩增体系包含引物、被切割后产生可检测信号的单链DNA报告分子。
其中，sgRNA(small guide RNA简称)即单分子向导RNA，是CRISPR基因敲除敲入系统中重要的组成部分，单分子向导RNA是作用于动质体(kinetoplastid)体内一种称为RNA编辑(RNA editing)的后转录修饰过程中。也是一种小型非编码RNA。可与pre-mRNA配对，并在其中插入一些尿嘧啶(U)，产生具有作用的mRNA。向导RNA编辑的RNA分子，长度大约是60～80个核苷酸，是由单独的基因转录的，具有3'寡聚U的尾巴，中间有一段与被编辑mRNA精确互补的序列，5'端是一个锚定序列，它同非编辑的mRNA序列互补。
Cas12b蛋白指的是一种来自微生物CRISPR系统的RNA指导的序列特异性核酸酶。Cas12b能在向导RNA的指导下靶向并切割DNA靶序列形成DNA双链断裂，也称作经典dsDNA切割活性。更重要的是，Cas12b与gRNA的复 合物在识别并结合相应靶DNA序列后，能够激活其非特异性单链DNA切割活性，也称作非经典旁路ssDNA切割活性。利用非经典旁路ssDNA切割活性，切割在被切割后产生可检测信号的单链DNA报告分子，即可以反映靶DNA的存在和/或量。
所述Cas12b蛋白可以是来自Alicyclobacillus acidiphilus的AaCas12b蛋白、来自Alicyclobacillus kakegawensis的AkCas12b蛋白、来自Alicyclobacillus macrosporangiidus的AmCas12b蛋白、来自Bacillus hisashii的BhCas12b蛋白、来自Bacillus属的BsCas12b蛋白、来自Bacillus属的Bs3Cas12b蛋白、来自Desulfovibrioinopinatus的DiCas12b蛋白、来自Laceyellasediminis的LsCas12b蛋白、来自Spirochaetes bacterium的SbCas12b蛋白、来自Tuberibacilluscalidus的TcCas12b蛋白。
“被切割后产生可检测信号的单链DNA报告分子”用于间接的反映样品中含有的靶核酸的含量。该单链DNA报告分子可以在单链DNA的两端分别包含荧光团和其猝灭基团。当单链DNA未被切割时，由于猝灭基团存在，荧光团不发出荧光。而当Cas12b-gRNA复合物被靶核酸分子激活，通过其非经典旁路ssDNA切割活性切割所述单链DNA报告分子的DNA单链时，荧光团被释放，从而发出荧光。合适的荧光团及其相应猝灭基团，以及其标记核酸分子的方法在本领域是已知的。合适的荧光团包括但不限于FAM、TEX、HEX、Cy3或Cy5。合适的猝灭基团包括但不限于BHQ1、BHQ2、BHQ3或TAMRA。合适的荧光团-猝灭基团对包括但不限于FAM-BHQ1、TEX-BHQ2、Cy5-BHQ3、Cy3-BHQ1或FAM-TAMRA。因此，在一些实施方案中，所述可检测信号是荧光信号。在一些实施方式中，所述荧光团是FAM，所述猝灭基团是BHQ1。
所述单链DNA报告分子中单链DNA的长度可以是大约2-100个核苷酸，例如2-5个、2-10个、2-15个、2-20个、2-25个、2-30个、2-40个或2至更多个核苷酸。所述单链DNA报告分子中单链DNA可以包含任意序列。在一些实施方案中，所述单链DNA报告分子中的单链DNA可以选自polyA(寡聚腺苷酸)、polyC(聚胞苷酸)或polyT(聚胸苷酸)。
重组酶聚合酶扩增(Recombinase Polymerase Amplification，RPA)，被称为是可以替代PCR的核酸检测技术。RPA技术主要依赖于三种酶：能结合单链核酸(寡核苷酸引物)的重组酶、单链DNA结合蛋白(SSB)和链置换DNA 聚合酶。
重组酶与引物结合形成的蛋白-DNA复合物，能在双链DNA中寻找同源序列。一旦引物定位了同源序列，就会发生链交换反应形成并启动DNA合成，对模板上的目标区域进行链置换扩增。被置换的DNA链与SSB结合，形成稳定的单链结构，等待新的引物合成反应的进行。在这个体系中，锚定在互补链上的3’尾尾相对的引物，共同起始后构成双链的扩增子并不断地循环下去。整个过程进行得非常快，一般可在十分钟之内获得可检出水平的扩增产物。
常规PCR必须经过变性、退火、延伸三个步骤，而PCR仪本质上就是一个控制温度升降的设备。RPA反应的最适温度在37℃-42℃之间，无需变性，在常温下即可进行。这无疑能大大加快PCR的速度。此外，由于不需要温控设备，RPA可以真正实现便携式的快速核酸检测。
RPA检测的灵敏度很高，能够将痕量的核酸(尤其是DNA)模板扩增到可以检出的水平，从单个模板分子得到长度大约为1012的扩增产物。而且RPA还用不着复杂的样品处理，适用于无法提取核酸的实地检测。
RPA既可以扩增DNA也可以扩增RNA，还省去了额外的cDNA合成步骤。你不仅可以对扩增产物进行终点检测，还可以对扩增过程进行实时监控，甚至可以通过试纸条(侧流层析试纸条LFD)读取结果。
与RPA类似，重组酶介导等温扩增技术(Recombinase Aid Amplification，以下称RAA)则使用从细菌或真菌中获得的重组酶，使整个扩增体系的稳定性得到进一步提升。如背景技术所介绍，虽然现有技术中已有人尝试将重组酶介导等温扩增体系和sgRNA-Cas体系结合进行核酸的检测。但是其本质上是两步两锅的反应，首先通过重组酶介导等温扩增体系将样品中的核酸进行扩增，然后利用sgRNA-Cas体系检测核酸的量。鉴于现有技术中存在的各种问题，本发明将sgRNA-Cas体系和重组酶介导等温扩增体系组成混合反应体系，并通过优化混合反应体系的反应条件，实现一步法检测样品中的核酸。
上述混合反应体系中，sgRNA、引物、单链DNA报告分子的含量可以根据实际需要进行调整。
在一个具体的实施方式中，所述混合反应体系中所述sgRNA的浓度为2～80ng/μl，例如可以为2～80ng/μl、10～70ng/μl、20～60ng/μl、30～50ng/μl、2ng/μl、4ng/μl、10ng/μl、20ng/μl、30ng/μl、40ng/μl、50ng/μl、60ng/μl、70ng/μl、80ng/μl。
在一个具体的实施方式中，所述混合体系中所述引物的浓度为300～1200nM，例如可以为300～1200nM、400～1100nM、500～1000nM、600～900nM、700～800nM、300nM、400nM、500nM、600nM、700nM、800nM、900nM、1000nM、1100nM、1200nM。
在一个具体的实施方式中，所述单链DNA报告分子的浓度为200～2000nM，优选为700～1500nM。例如可以为200～2000nM、300～1900nM、400～1800nM、500～1700nM、600～1600n、700～1500nM、800～1400nM、900～1300nM、1000～1200nM、200～700nM、1500～2000nM、200nM、300nM、400nM、500nM、600nM、700nM、800nM、900nM、1000nM、1100nM、1200nM、1300nM、1400nM、1500nM、1600nM、1700nM、1800nM、1900nM、2000nM。
在本发明中，通过精确地控制单链DNA报告分子在反应体系中的浓度，从而实现了一步反应。通过深入地研究，本申请的发明人发现反应时间受制于信号产生的强度，通过加大反式切割反应中底物单链DNA报告分子的浓度，可以在更短的时间内使产生的荧光强度达到仪器的检测灵敏度，也可以在体系中靶点DNA片段数量较低的情况下产生足够的信号。
进一步地，所述反应第一温度反应，并在第二温度灭活，其中所述第二温度大于所述第一温度，即灭活时使用的温度大于反应时的温度。在一个具体的实施方式中，所述第二温度大于47℃，例如可以为48℃、50℃、55℃、60℃、65℃、70℃、75℃、80℃、85℃。
进一步地，所述混合反应体系还包含二价阳离子，所述二价阳离子的浓度为8mM～25mM，例如可以为8mM～25mM、10mM～20mM、12mM～18mM、8mM、9mM、10mM、11mM、12mM、13mM、14mM、15mM、16mM、17mM、18mM、19mM、20mM、21mM、22mM、23mM、24mM、25mM。
其中，所述二价阳离子选自镁离子、钙离子、锰离子、铯离子、镍离子、铁离子、钴离子中的一种或两种以上。
本发明所述的靶核酸可以包括DNA或RNA，其中当所述靶核酸为RNA时，所述混合反应体系还包含苏拉明和/或tRNA，即可以包含苏拉明、tRNA或者同时包含苏拉明和tRNA。
当所述混合反应体系包含苏拉明时，所述混合反应体系中所述苏拉明的浓度为3～30ng/μl，优选为3～12ng/μl。例如可以为3～30ng/μl、3～12ng/μl、12～30ng/μl、 5～25ng/μl、10～20ng/μl、3ng/μl、5ng/μl、8ng/μl、10ng/μl、12ng/μl、15ng/μl、18ng/μl、20ng/μl、22ng/μl、25ng/μl、28ng/μl、30ng/μl。
在本发明中，通过精确地控制苏拉明在反应体系中的浓度，从而实现了一步反应。其中，通过深入地研究，本申请的发明人发现由于苏拉明是某些酶三磷酸键水解中心的阻遏剂，重组酶介导等温扩增体系(RT-RAA)中包括聚合酶、RT酶、重组酶等都依赖三磷酸键水解产生活性。通过调整苏拉明的浓度，可以协调反应体系中各酶反应的顺序，在本发明中，就使用了3～30ng/μl苏拉明来抑制活力过强的DNA聚合酶和重组酶，从而让RT酶能更早的与RNA模板-引物复合物结合产生cDNA模板，从而让整个串联反应的体系能顺利进行下去。
当所述混合反应体系包含tRNA时，所述混合反应体系中所述tRNA的浓度为10～20ng/μl。例如可以为10～20ng/μl、12～18ng/μl、10ng/μl、11ng/μl、12ng/μl、13ng/μl、14ng/μl、15ng/μl、16ng/μl、17ng/μl、18ng/μl、19ng/μl、20ng/μl。
在本发明中，通过精确地控制tRNA在反应体系中的浓度，从而实现了一步反应。在多个报道中，加入一定量的背景RNA能提高RT-PCR的灵敏度，这主要是提高了模板RNA的结构多样性，能解开部分的高级结构，也能让痕量的模板降低其与其它物质的非特异吸附从而更利于其扩增反应。同样在RT-RAA中，本申请的发明人发现，在RT酶活性不足的情况下，这种添加剂的投入也能起到比较显著的效果。
综上所述，本发明通过调控反应体系中sgRNA的浓度为2～80ng/μl，引物的浓度为300～1200nM，单链DNA报告分子的浓度为200～2000nM等条件，能够改善重组聚合酶扩增和sgRNA-Cas反应的相互抑制，可以实现重组聚合酶扩增和CDetection反应只需要一次加样混合和简单改变温度就能进行快捷有效的信号报告，极大的提高了操作的便捷性并减少了污染。
本发明所述的样品可以选自全血、血浆、血清、脑脊液、尿液、粪便、口腔拭子、鼻咽拭子、唾液、细胞或组织提取物。
实施例
实施例1 sgRNA用量对反应的影响
1、sgRNA-Cas预配体系预配制(C混液)
按照表1中的各组分用量配制sgRNA-Cas预配体系，配制完成后于常温放 置待用。
表1.sgRNA-Cas预配制体系组分含量及加样顺序
Figure PCTCN2021118280-appb-000001
1单分子向导RNA的序列为：
Figure PCTCN2021118280-appb-000002
2报告探针序列为：5’-FAM-TTTTTTT-BHQ1-3’，其中报告探针即为本发明所述的单链DNA报告分子
3 RNA核酸酶抑制剂购自promega，货号为N2515
4 AaCas12b蛋白酶购自南京诺维赞生物科技有限公司，货号为EN311-PE
其中表1中各组分相关信息，同样适用于后面的表格，除非另有描述。
在本实例中单分子向导RNA的浓度分别为20ng/μl、80ng/μl、240ng/μl，其中使用1μl无核酸酶水代替单分子向导RNA。
2、重组酶介导等温扩增体系体系配制(A混液)
按照表2中的各组分用量配制重组酶介导等温扩增体系体系，配制完成后于室温放置待用。
表2.重组酶介导等温扩增体系组分含量及加样顺序
Figure PCTCN2021118280-appb-000003
1扩增缓冲液A为20％PEG35K，购自sigma-aldrich，货号为818892稀释至20％使用。
2醋酸镁溶液购自sigma-aldrich，货号为63052，稀释至280mM使用
3正向引物序列为5‘-GTTGTAGCTTGTCACACCGTTTCTATAGATTAGC-3’(SEQ ID NO:2)
反向引物序列为5‘-CCTGGTTTAACATATAGTGAACCGCCACACAT-3’(SEQ ID NO:3)
3、体系混合
将1μl病毒核酸样本(2019-nCoV RdRP假病毒，使用美基核酸提取试剂盒，IVD5412进行提取，提取后稀释至500拷贝每微升使用)加入45μl A混液中，其次加入9.8μl C混液。其中，2019-nCoV RdRP基因假病毒,购自厦门致善生物科技股份有限公司，货号为JBD249。
将上述混液加入酶干粉中(RT-RAA干粉，购自杭州众测生物科技有限公司，货号为S0040ZC)。于42℃下孵育1min×60cycles，每分钟采光一次。
本方法采用FAM基团作为报告基团，依据其30min处的FAM基团的相对荧光信号强度判断是否存在靶标核酸。
其中分别加入sgRNA的加入量分别为0ng、20ng sgRNA、80ng sgRNA、240ng sgRNA，其在混合反应体系中对应的终摩尔浓度分别为0、5.84nM、23.36nM、70.08nM。
得到的检测结果如图1所示。
由图1可以看出，提升sgRNA用量以提高有效sgRNA-Cas复合体数量，增加被切割探针数量，使得更多的FAM荧光基团被释放出来远离猝灭基团，从而增加了阳性反应荧光信号强度，拉大了与阴性信号强度的距离，使得整个体系的灵敏度得以提升，在有限反应时间内，能够检测出更低的病毒数量，使得体内病毒含量低的感染者免于假阴性的检测结果。
由于sgRNA内部结构复杂，在进行折叠时可能会发生一些错误结构，使得投入的部分sgRNA成为不能与AaCas12b蛋白形成复合体的无效sgRNA，本专利通过提升sgRNA的投入量以弥补体系中存在的无效sgRNA，从而提高sgRNA-AaCas12b复合体数量，提升了特定反应时间内的信号强度。
实施例2 引物用量对反应的影响(C混液)
1、sgRNA-Cas预配体系预配制
按照表3中的各组分用量配制sgRNA-Cas预配体系，配制完成后于常温放置待用。
表3.sgRNA-Cas预配制体系组分含量及加样顺序
Figure PCTCN2021118280-appb-000004
2、重组酶介导等温扩增体系体系配制(A混液)
按照表4中的各组分用量配制重组酶介导等温扩增体系体系，配制完成后于室温放置待用。
表4.重组酶介导等温扩增体系组分含量及加样顺序
Figure PCTCN2021118280-appb-000005
3、体系混合
向空管子中分别加入45μl A混液，1μl病毒核酸样本(2019-nCoV RdRP假病毒，使用美基核酸提取试剂盒，IVD5412进行提取，提取后稀释至50000拷贝每微升及5000拷贝每微升使用)，5μl C混液、其中，2019-nCoV RdRP基因假病毒,购自厦门致善生物科技股份有限公司，货号为JBD249。
将上述51μl体系加入酶干粉中(RT-RAA干粉，购买自杭州众测生物科技有限公司，货号为S0040ZC)，并震荡混匀8-10秒。
将上述体系放置实时荧光定量PCR仪中，孵育程序为45℃1min×60cycles，每分钟采光1次。本方法采用FAM基团作为报告基团，依据其60min处的相对荧光信号强度进行是否存在靶标核酸。
得到的检测结果如图2所示。
如图2所示当引物在体系中的终浓度由400nM提升至800nM时，50000拷贝和5000拷贝样本量下反应30分钟时的阴阳分离度分别提升了37％(分离度由3.43提升至4.70)和32％(分离度由5.42提升至7.17)。这从侧面反应提高引物浓度能够提升恒温扩增反应的扩增效率，增加体系内特异性扩增产物含量，在阴性稳定的前提下提升阳性样本的阳性信号，有利于区分阴性和阳性样本，提升实际应用过程中的阴阳性检出率。
实施例3 反应温度对反应结果的影响
1、sgRNA-Cas预配体系预配制(C混液)
按照表5中的各组分用量配制sgRNA-Cas预配体系，配制完成后于冰上放置待用。
表5.sgRNA-Cas预配制体系组分含量及加样顺序
Figure PCTCN2021118280-appb-000006
单分子向导RNA的浓度X分别为10ng/μl、50ng/μl、100ng/μl、200ng/μl。
2、重组酶介导等温扩增体系体系配制(A混液)
按照表6中的各组分用量配制重组酶介导等温扩增体系体系，配制完成后于室温放置待用。
表6.重组酶介导等温扩增体系组分含量及加样顺序
Figure PCTCN2021118280-appb-000007
Figure PCTCN2021118280-appb-000008
上述体系中醋酸镁溶液加入量分别为1.429μl和2.5μl。
其中，单分子向导RNA的序列、报告探针序列、正反向引物序列与实施例1相同。扩增缓冲液A、醋酸镁溶液、RNA核酸酶抑制剂、AaCas12b蛋白酶等组分来源均与实施例1相同，转运RNA购自invitrogen，货号为AM7119。
3、体系混合
向空管子中分别加入44μl A混液，1μl病毒核酸样本(2019-nCoV RdRP假病毒，使用美基核酸提取试剂盒，IVD5412进行提取,提取后稀释至100拷贝每微升使用)，5μl C混液。其中，2019-nCoV RdRP基因假病毒,购自厦门致善生物科技股份有限公司，货号为JBD249。
将上述50μl体系加入酶干粉中(RT-RAA干粉，购买自杭州众测生物科技有限公司，货号为S0040ZC)，并震荡混匀8-10秒。
将上述体系放置实时荧光定量PCR仪中，孵育程序为45℃或42℃，1min×60cycles，每分钟采光1次。本方法采用FAM基团作为报告基团，依据其60min处的相对荧光信号强度判断是否存在靶标核酸。
得到的检测结果如图3、表7和表8所示。
表7.高温反应条件下的一步法CDetection反应结果
Figure PCTCN2021118280-appb-000009
*阳性信号高度为投入100拷贝病毒基因组RNA，45A反应60min时FAM基团的相对荧光信号强度，即60min时FAM基团的荧光强度减去基线荧光强度的结果。**阴性信号高度为使用等体积的样本洗脱buffer代替病毒基因组RNA加入反应体系，45℃反应60min时FAM基团的相对荧光信号强度。***阳性阴性分离度为上述阳性反应与阴性反应45℃反应60min时FAM基团的相对荧光信号强度的比值。
表8.低温反应条件下的一步法CDetection反应结果
Figure PCTCN2021118280-appb-000010
*阳性信号高度为投入100拷贝病毒基因组RNA，42A反应60min时FAM基团的相对荧光信号强度，即60min时FAM基团的荧光强度减去基线荧光强度的结果。**阴性信号高度为使用等体积的样本洗脱buffer代替病毒基因组RNA加入反应体系，42℃反应60min时FAM基团的相对荧光信号强度。***阳性阴性分离度为上述阳性反应与阴性反应42℃反应60min时FAM基团的相对荧光信号强度的比值。
由上述数据可知，当使用较高CDetection反应温度时，体系的特异性有提升的趋势，体现在阴性反应信号值持平或降低；与此同时sgRNA在体系正常用量的情况下(200ng)，阳性信号较低温反应的过程有了约3.4倍的提升。
实施例4 反应时间对反应的影响
1、按照下表配制反应体系
表9.重组酶介导等温扩增体系组分含量及加样顺序
Figure PCTCN2021118280-appb-000011
其中，1转运RNA购自invitrogen，货号为AM7119
2 50×TET缓冲液为自配缓冲液(100mM Tris pH8.0，5mM EDTA,0.5％Tween 20)
正反向引物序列与实施例1相同。扩增缓冲液A、醋酸镁溶液、RNA核酸酶抑制剂等组分来源均与实施例1相同。
3 Evagreen染料(20x)购自Biotium
2、上机
在上述混合液中加入1μl 100拷贝每微升的假病毒核酸样本后，将上述混合液置于实时荧光定量PCR仪45℃下孵育1min×60cycles观察扩增曲线形状。
使用Evagreen染料替换了FAM报告基团以表征体系中靶标基因的扩增行为，结果如图4所示。
如上图所示，在一步法反应体系中45℃反应条件下，5-7个扩增循环即会进入线性扩增阶段。
根据上图结果，在一步法反应体系中45℃反应条件下，5-10个扩增循环即会进入线性扩增阶段。同时本方法中采用的Cas酶AaCas12b可以在非常宽泛的温度范围内(31-59℃)维持最优核酸酶活性；因此在不影响RT-RAA反应产物积累的考虑下，我们将前7分钟的反应条件保持在45℃下进行，而8-60min的反应温度调整为52℃，灭活RT-RAA关键酶以排除RT-RAA反应组分对切割过程的影响，同时通过提高切割反应温度以提高切割反应动力学加快报告基团信号积累速度。
实施例5 添加剂对反应的影响
通过添加剂的使用解除反转录抑制。
1、sgRNA-Cas预配体系预配制(C混液)
按照表10中的各组分用量配制sgRNA-Cas预配体系，配制完成后于冰上放置待用。
表10.sgRNA-Cas预配制体系组分含量及加样顺序
Figure PCTCN2021118280-appb-000012
2、重组酶介导等温扩增体系体系配制(A混液)
按照表11中的各组分用量配制重组酶介导等温扩增体系体系，配制完成后于室温放置待用。
表11.重组酶介导等温扩增体系组分含量及加样顺序
Figure PCTCN2021118280-appb-000013
其中，单分子向导RNA的序列、正反向引物序列与实施例1相同。扩增缓冲液A、醋酸镁溶液、RNA核酸酶抑制剂、AaCas12b蛋白酶等组分来源均与实施例1相同。转运RNA购自invitrogen，货号为AM7119。
3、体系混合
向空管子中分别加入45μl A混液，1μl病毒核酸样本(RNA样本为2019-nCoV RdRP假病毒，使用美基核酸提取试剂盒，IVD5412进行提取，提取后分别稀释至50000拷贝每微升及500拷贝每微升使用；DNA样本为含有2019-nCovRdRP基因的质粒)，5μl C混液。其中，2019-nCoV RdRP基因假病毒,购自厦门致善生物科技股份有限公司，货号为JBD249；2019-nCovRdRP基因质粒购自金斯瑞(属化学合成产品无货号)。
将上述51μl体系加入酶干粉中(RT-RAA干粉，购买自众测，货号为S0040ZC)，并震荡混匀8-10秒。
将上述体系放置实时荧光定量PCR仪中，孵育程序为45℃1min×60cycles，每分钟采光1次。本方法采用FAM基团作为报告基团，依据其60min处的相对荧光信号强度进行是否存在靶标核酸的判读。
结果如图5所示。
同时添加200ng苏拉明和500ngtRNA时，使用本发明的检测方法得到的反应30min的阴阳性分离度数据如表12所示。
表12 阴阳分离度结果
Figure PCTCN2021118280-appb-000014
Figure PCTCN2021118280-appb-000015
*阴阳性分离度是指阳性反应报告基团相对荧光强度/阴性反应报告集团相对荧光强度。
如上述数据所示，当在混合反应体系中使用苏拉明和tRNA作为添加剂，以RNA作为底物投入时与阴性反应有了明显的分离度，在一定程度上解除了本发明的一步法检测体系对反转录反应的抑制效应。
实施例7 报告探针用量对检测结果的影响
1、sgRNA-Cas预配体系预配制(C混液)
按照表13中的各组分用量配制sgRNA-Cas预配体系，配制完成后于冰上放置待用。
表13.sgRNA-Cas预配制体系组分含量及加样顺序
Figure PCTCN2021118280-appb-000016
2、重组酶介导等温扩增体系体系配制(A混液)
按照表14中的各组分用量配制重组酶介导等温扩增体系体系，配制完成后于室温放置待用。
表14.重组酶介导等温扩增体系组分含量及加样顺序
Figure PCTCN2021118280-appb-000017
Figure PCTCN2021118280-appb-000018
其中，单分子向导RNA的序列、正反向引物序列、报告探针序列与实施例1相同。扩增缓冲液A、醋酸镁溶液、RNA核酸酶抑制剂、AaCas12b蛋白酶等组分来源均与实施例1相同。本实施例中探针的终浓度实验梯度设置为200nM、400nM、700nM、1000nM、1500nM、2000nM，对应的在体系中的加入体积(X)分别为0.1μl、0.4μl、0.7μl、1μl、1.5μl、2μl。
3、体系混合
向含扩增反应酶干粉的管子中分别加入10μl样本(本次实验中共用到2个样本，一个为阴性对照水，一个为8拷贝/微升的阳性样本，该阳性样本为2019-nCoV RdRP假病毒，使用美基核酸提取试剂盒，IVD5412进行提取，提取后分别稀释至8拷贝每微升)、33μl A混液，并将7μl C混液加入相应管盖中后扣上管盖，对上述体系进行充分震混后离心，离心后再次震混离心，离心后尽快上机进行荧光信号检测。其中，2019-nCoV RdRP基因假病毒,购自厦门致善生物科技股份有限公司，货号为JBD249；2019-nCovRdRP基因质粒购自金斯瑞(属化学合成产品无货号)。
将上述体系放置实时荧光定量PCR仪中，孵育程序为45℃7min，收集一次荧光，(52℃1min)×22cycles，每分钟收集一次荧光，52℃1min，收集一次荧光。本方法采用FAM基团作为报告基团，依据其30min处的相对荧光信号强度进行是否存在靶标核酸的判读。反应结果如图7所示，阴阳性相对荧光信号分离度如表15所示。
表15 不同探针浓度的阴阳性相对荧光信号分离度
Figure PCTCN2021118280-appb-000019
Figure PCTCN2021118280-appb-000020
本实施例中每个探针浓度以及每个样本做了2次生物学重复，图7所示结果为两次反应的均值，如图7所示提升所用探针浓度能够提升阳性样本的信号强度。如表16所示，本实施例结果所示200nM-2000nM的报告探针浓度的阴阳性信号分离度均约等于2或者大于2，符合使用需求，因此推荐使用200nM-2000nM的报告探针浓度。
试验例1 2019-nCoV RdRP基因检测
根据上述各因素的影响分析，采用如下条件检测样品靶核酸。
1、sgRNA-Cas预配体系预配制(C混液)
按照表16中的各组分用量配制sgRNA-Cas预配体系，配制完成后于冰上放置待用。
表16.sgRNA-Cas预配制体系组分含量及加样顺序
Figure PCTCN2021118280-appb-000021
2、重组酶介导等温扩增体系体系配制(A混液)
按照表17中的各组分用量配制重组酶介导等温扩增体系体系，配制完成后于室温放置待用。
表17.重组酶介导等温扩增体系组分含量及加样顺序
Figure PCTCN2021118280-appb-000022
Figure PCTCN2021118280-appb-000023
其中，单分子向导RNA的序列、报告探针序列、正反向引物序列与实施例1相同。扩增缓冲液A、醋酸镁溶液、RNA核酸酶抑制剂、AaCas12b蛋白酶等组分来源均与实施例1相同。转运RNA购自invitrogen，货号为AM7119。
3、体系混合
取10μl提取后的假病毒核酸(2019-nCoV RdRP基因假病毒,购自厦门致善生物科技股份有限公司，货号为JBD249。使用美基核酸提取试剂盒，IVD5412进行提取。稀释至大于等于10拷贝每微升使用)，加入待用反应孔中，在该孔中继续分别加入重组酶介导等温扩增体系A混液35μl、C混液5μl；将上述50μl混合液加入酶干粉(RT-RAA干粉，S0040ZC)中，于45℃下孵育7min，52℃下孵育1min×23cycles。本方法采用FAM基团作为报告基团，依据其30min处的相对荧光信号强度进行是否存在靶标核酸的判读。
得到的检测结果如图6所示。
图6为4个100拷贝的病毒基因组RNA的CDetection反应结果及4个阴性(使用等体积样本洗脱buffer代替病毒核酸样本)CDetection反应结果。
在ABI 7500实时荧光定量PCR仪上，根据以往的测试数据经验及CDetection反应特征，将30min处报告基团相对荧光信号强度的样本判定是否存在靶标核酸。
试验例2 人POP7基因靶点的测试
基因靶点POP7通常被用作CRISPR-Cas相关检测方法的内参检测(James  P.Broughton，2020)，本试验例中提供了第二个靶点POP7基因(NM_005837.3)的同一方法的检测结果，用以验证本专利所述方法在不同物种不同靶点检测应用中的通用性。
1、sgRNA-Cas预配体系预配制(C混液)
按照表18中的各组分用量配制sgRNA-Cas预配体系，配制完成后置于冰盒上或4℃冰箱中待用。
表18.sgRNA-Cas预配制体系组分含量及加样顺序
Figure PCTCN2021118280-appb-000024
1 AaCas12b蛋白酶购自南京诺维赞生物科技有限公司，货号为EN311-PE
2 RNA核酸酶抑制剂购自promega，货号为N2515
3单向导RNA的序列为：
Figure PCTCN2021118280-appb-000025
2、重组酶介导等温扩增体系预配制(A混液)
按照表19中的各组分用量配制重组酶介导等温扩增体系体系，配制完成后于室温放置待用。
表19.重组酶介导等温扩增体系组分含量及加样顺序
Figure PCTCN2021118280-appb-000026
Figure PCTCN2021118280-appb-000027
1扩增缓冲液A为4x RT-RAA buffer A，购自sigma-aldrich，货号为818892，稀释至20％使用。
2醋酸镁溶液购自sigma-aldrich，货号为63052，稀释至280mM使用
3正向引物序列为5‘-TGAGTACTGGACCTCGGACCAGAGCCATGTAAGAA-3’(SEQ ID NO:5)
反向引物序列为5‘-GCCCACCAAGAGACAATTACCCCCACCCTCAATGC-3’(SEQ ID NO:6)
4报告探针序列为：5’-FAM-TTTTTTT-BHQ1-3’
3、体系混合
将10μl POP7核酸样本(人类293T细胞转录组，提取后稀释至10ng/μl使用，每反应使用10μl)，加入35μl A混液中，其次加入5μl C混液。
将上述混液加入酶干粉中(RT-RAA干粉，购自杭州众测生物科技有限公司，货号为S0040ZC)。于45℃下孵育7分钟后于52℃下孵育1分钟×23个循环，每分钟采光一次。
本方法采用FAM基团作为报告基团，依据其30分钟处的FAM基团的相对荧光信号强度判断是否存在靶标核酸。结果如图8和表20所示。检测结果表明当投入100ng人转录组核酸时，反应30分钟时阳性样本与阴性样本的荧光信号信号分离度能达到2左右的水平。
表20.人转录组和吐温稀释组的检测结果
Figure PCTCN2021118280-appb-000028
1相对荧光强度＝第32分钟荧光强度-初始荧光强度。
2阴阳性分离度＝样本相对荧光强度平均值/0.01％吐温20相对荧光强度平均值。

Claims (10)

  1. 一种检测样品中靶核酸的方法，其特征在于，所述方法包括以下步骤：
    使所述样品与由sgRNA-Cas体系和重组酶介导等温扩增体系组成的混合反应体系进行反应；
    反应结束后，检测反应产生的可检测信号；
    其中，所述sgRNA-Cas体系包含Cas12b蛋白和针对所述靶核酸的sgRNA；所述重组酶介导等温扩增体系包含引物、被切割后产生可检测信号的单链DNA报告分子。
  2. 根据权利要求1所述的方法，其特征在于，所述混合反应体系中所述sgRNA的浓度为2～80ng/μl。
  3. 根据权利要求1或2所述的方法，其特征在于，所述混合体系中所述引物的浓度为300～1200nM。
  4. 根据权利要求1-3任一项所述的方法，其特征在于，所述单链DNA报告分子的浓度为200～2000nM，优选为700～1500nM。
  5. 根据权利要求1-4任一项所述的方法，其特征在于，所述反应在第一温度反应，并在第二温度灭活，所述第二温度大于所述第一温度，优选地所述第二温度大于47℃。
  6. 根据权利要求1-5任一项所述的方法，其特征在于，所述混合反应体系还包含二价阳离子，所述二价阳离子的浓度为8mM～25mM，优选地所述二价阳离子选自镁离子、钙离子、锰离子、铯离子、镍离子、铁离子、钴离子中的一种或两种以上。
  7. 根据权利要求1-6任一项所述的方法，其特征在于，当所述靶核酸为RNA时，所述混合反应体系还包含苏拉明和/或tRNA。
  8. 根据权利要求7所述的方法，其特征在于，所述混合反应体系中所述苏拉明的浓度为3～30ng/μl，优选为3～12ng/μl。
  9. 根据权利要求8所述的方法，其特征在于，所述混合反应体系中所述tRNA的浓度为10～20ng/μl。
  10. 根据权利要求1-9任一项所述的方法，其特征在于，所述样品选自全血、 血浆、血清、脑脊液、尿液、粪便、口腔拭子、鼻咽拭子、唾液、细胞或组织提取物。
PCT/CN2021/118280 2019-01-23 2021-09-14 检测样品中靶核酸的方法 WO2022100270A1 (zh)

Priority Applications (3)

Application Number Priority Date Filing Date Title
EP21890798.8A EP4234715A1 (en) 2020-11-16 2021-09-14 Method for testing target nucleic acid in sample
US18/037,173 US20240002915A1 (en) 2020-11-16 2021-09-14 Method for testing target nucleic acid in sample
ZA2023/05265A ZA202305265B (en) 2019-01-23 2023-05-12 Method for testing target nucleic acid in sample

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202011278709 2020-11-16
CN202011278709.X 2020-11-16

Publications (1)

Publication Number Publication Date
WO2022100270A1 true WO2022100270A1 (zh) 2022-05-19

Family

ID=81547953

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/118280 WO2022100270A1 (zh) 2019-01-23 2021-09-14 检测样品中靶核酸的方法

Country Status (4)

Country Link
US (1) US20240002915A1 (zh)
EP (1) EP4234715A1 (zh)
CN (1) CN114507716A (zh)
WO (1) WO2022100270A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116139954A (zh) * 2023-02-20 2023-05-23 首都医科大学 一种用于多通道等温扩增crispr检测的微流控芯片

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117467644A (zh) * 2022-07-22 2024-01-30 上海吐露港生物科技有限公司 一种通过改变离子来降低CRISPR-Cas12a特异切割靶标核酸脱靶率的方法
CN115820818B (zh) * 2022-12-13 2024-02-23 博迪泰（厦门）生物科技有限公司 一种一步法核酸检测方法及其应用
CN116287144B (zh) * 2023-05-22 2024-04-12 江苏为真生物医药技术股份有限公司 核酸检测系统、装置和方法

Citations (7)

* 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
CN109837328A (zh) * 2018-09-20 2019-06-04 中国科学院动物研究所 核酸检测方法
CN110184329A (zh) * 2019-05-31 2019-08-30 华南理工大学 一种基于CRISPR/Cas和恒温扩增的一步法核酸检测方法和试剂盒
CN110551800A (zh) * 2018-06-03 2019-12-10 上海吐露港生物科技有限公司 一种耐高温Cas蛋白的用途及靶标核酸分子的检测方法和试剂盒
WO2020006036A1 (en) * 2018-06-26 2020-01-02 Massachusetts Institute Of Technology Crispr effector system based amplification methods, systems, and diagnostics
CN111593145A (zh) * 2020-06-11 2020-08-28 亚能生物技术（深圳）有限公司 一种CRISPR/Cas12一步核酸检测方法及新型冠状病毒检测试剂盒
CN111630163A (zh) * 2017-11-22 2020-09-04 加利福尼亚大学董事会 用于切割ssdna以及检测靶dna的v型crispr/cas效应蛋白

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111630163A (zh) * 2017-11-22 2020-09-04 加利福尼亚大学董事会 用于切割ssdna以及检测靶dna的v型crispr/cas效应蛋白
CN110551800A (zh) * 2018-06-03 2019-12-10 上海吐露港生物科技有限公司 一种耐高温Cas蛋白的用途及靶标核酸分子的检测方法和试剂盒
WO2020006036A1 (en) * 2018-06-26 2020-01-02 Massachusetts Institute Of Technology Crispr effector system based amplification methods, systems, and diagnostics
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
CN109837328A (zh) * 2018-09-20 2019-06-04 中国科学院动物研究所 核酸检测方法
CN110184329A (zh) * 2019-05-31 2019-08-30 华南理工大学 一种基于CRISPR/Cas和恒温扩增的一步法核酸检测方法和试剂盒
CN111593145A (zh) * 2020-06-11 2020-08-28 亚能生物技术（深圳）有限公司 一种CRISPR/Cas12一步核酸检测方法及新型冠状病毒检测试剂盒

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
TENG, FEI ET AL.: "CDetection: CRISPR-Cas12b-based DNA detection with sub-attomolar sensitivity and single-base specificity", GENOME BIOLOGY, vol. 20, 1 July 2019 (2019-07-01), XP055817045, ISSN: 1474-760X, DOI: 10.1186/s13059-019-1742-z *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116139954A (zh) * 2023-02-20 2023-05-23 首都医科大学 一种用于多通道等温扩增crispr检测的微流控芯片
CN116139954B (zh) * 2023-02-20 2023-08-22 首都医科大学 一种用于多通道等温扩增crispr检测的微流控芯片

Also Published As

Publication number Publication date
EP4234715A1 (en) 2023-08-30
CN114507716A (zh) 2022-05-17
US20240002915A1 (en) 2024-01-04

Similar Documents

Publication Publication Date Title
WO2022100270A1 (zh) 检测样品中靶核酸的方法
US11447821B2 (en) Nucleic acid amplification and testing
EP4202064A1 (en) Kit and method for isothermal rapid detection of sars-cov-2 virus nucleic acid
JP6480511B2 (ja) 試料中の核酸配列を定量するための組成物および方法
Xie et al. Advancing sensing technology with CRISPR: From the detection of nucleic acids to a broad range of analytes–a review
US9617586B2 (en) Nicking and extension amplification reaction for the exponential amplification of nucleic acids
US20240141412A1 (en) Compositions and methods of a nuclease chain reaction for nucleic acid detection
WO2020168710A1 (zh) 一种基于Cas9切口酶偶联DNA聚合酶的恒温核酸检测分析方法及试剂盒
EP2179052B1 (en) Detection of micro-organisms based on their nad-dependent dna ligase activity
JP2013505723A (ja) 粗製のマトリックスにおける核酸の検出
KR20170022854A (ko) Lamp를 이용한 인플루엔자 검출용 프라이머 및 그 용도
JP2009545317A (ja) 分析物および核酸の検出
WO2022077687A1 (zh) 一种新型crispr核酸检测方法及应用
WO2023056451A1 (en) Compositions and methods for assaying for and genotyping genetic variations
CN113308519B (zh) 用于检测单碱基突变位点的引物和探针及检测方法
Chen et al. Research progress of CRISPR-based biosensors and bioassays for molecular diagnosis
CN115011713A (zh) 基于DNAzyme双循环体系的牛结核分枝杆菌检测探针组及其检测方法
WO2023207909A1 (zh) 基于crispr的核酸检测试剂盒及其应用
US8309303B2 (en) Reverse transcription and amplification of RNA with simultaneous degradation of DNA
WO2023203206A1 (en) Multiplexable crispr-cas9-based virus detection method
Arshad et al. CeO2 nanozyme mediated RPA/CRISPR-Cas12a dual-mode biosensor for detection of invA gene in Salmonella
US20240124917A1 (en) Compositions and methods for nucleic acid detection by lateral flow assays
CN115838834A (zh) 一种基于RT-RAA和CRISPR/Cas12a的人鼻病毒A型和C型检测体系
US20230313282A1 (en) Compositions and methods of isothermal nucleic acid amplification and detection
Sakhabutdinova et al. Reverse transcriptase-free detection of viral RNA using Hemo KlenTaq DNA polymerase

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21890798

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 18037173

Country of ref document: US

ENP Entry into the national phase

Ref document number: 2021890798

Country of ref document: EP

Effective date: 20230523

NENP Non-entry into the national phase

Ref country code: DE