CN115029345A - Nucleic acid detection kit based on CRISPR and application thereof - Google Patents

Nucleic acid detection kit based on CRISPR and application thereof Download PDF

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CN115029345A
CN115029345A CN202210435996.3A CN202210435996A CN115029345A CN 115029345 A CN115029345 A CN 115029345A CN 202210435996 A CN202210435996 A CN 202210435996A CN 115029345 A CN115029345 A CN 115029345A
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刘佳
瞿介明
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Ruinjin Hospital Affiliated to Shanghai Jiaotong University School of Medicine Co Ltd
ShanghaiTech University
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Abstract

The invention discloses a detection kit based on CRISPR and application thereof. The detection kit comprises a gene editing system, wherein the gene editing system comprises nuclease and guide RNA; the sequence of the guide RNA is selected from one or more of nucleotide sequences shown as SEQ ID NO 1-4. The kit can improve the detection efficiency in the shortened reaction time and the reduced reaction temperature, simultaneously reduce the detection limit, has no nucleic acid extraction and no uncapping in the operation, and is simple, convenient and quick.

Description

Nucleic acid detection kit based on CRISPR (clustered regularly interspaced short palindromic repeats) and application thereof
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a nucleic acid detection kit based on CRISPR (clustered regularly interspaced short palindromic repeats) and free of extraction and uncovering and application thereof.
Background
The pathogen responsible for COVID-19 has been identified as a β coronavirus, designated SARS-CoV-2. Like other coronaviruses, SARS-CoV-2 is a single-stranded, positive-sense RNA virus. SARS-CoV-2 and SARS-CoV as well as the middle east respiratory syndrome coronavirus (MERS-CoV) have nucleic acid similarities of 79% and 50%, respectively. Existing molecular diagnostic methods for COVID-19 are mainly based on reverse transcription PCR (RT-PCR). E, N and ORF1ab genes of SARS-CoV-2 are target genes of commonly used RT-PCR. High throughput RT-PCR platforms have also been developed for large scale diagnostics. However, RT-PCR is usually dependent on special instruments, so large scale RT-PCR detection is often limited to patients in hospitals. Meanwhile, the current epidemic prevention and control requirements are nucleic acid detection results within 48 hours, and detection means for high-risk environments (such as airports, high-speed rails and other public places) are lacked, so that great uncertainty is brought to epidemic prevention and control.
Recent studies have shown that the Clustered Regulated Interleaved Short Palindromic Repeats (CRISPR) technique can be used for low-cost, portable molecular detection of pathogens. CRISPR and CRISPR-associated genes (Cas) genes are the immune system of bacteria against foreign viral infection. The modular nature of CRISPR makes this technology widely used in genome engineering. The molecular diagnosis of the pathogenic nucleic acid based on the CRISPR depends on the target activity of RNA or DNA of Cas nuclease, and the rapid and accurate detection of the new coronavirus can be realized within 30 minutes. However, CRISPRs are not yet widely used, and one of the main limitations is that the operation of CRISPRs is too complicated.
Disclosure of Invention
The invention aims to solve the technical problem that the prior art is short of a CRISPR reaction system which is simple and convenient to operate, accurate and rapid, and provides a nucleic acid detection kit based on CRISPR and application thereof. The nucleic acid detection kit can quickly and accurately reflect the detection result of CRISPR, for example, when SARS-CoV-2 is detected, the specific crRNA is utilized, and the virus gene is identified with high sensitivity and high specificity; and the detection can be carried out without nucleic acid extraction and cover opening, so that the pollution possibility and the false positive rate are reduced, and the application value is high.
The invention solves the technical problems through the following technical scheme.
A first aspect of the present invention provides a gene editing system comprising a nuclease and a guide RNA; wherein the sequence of the guide RNA is selected from one or more of nucleotide sequences shown as SEQ ID NO 1-4.
In some embodiments of the invention, the guide RNA has a sequence as shown in any one of SEQ ID NOS 1-4.
In the present invention, the nuclease may be an enzyme conventionally used in the art for disrupting phosphodiester bonds in nucleic acids to generate breaks in nucleic acid chains, preferably a Cas protein.
In some embodiments of the invention, the Cas protein is Cas12a or Cas13 a;
in the present invention, when the Cas protein is Cas12a, the Cas12a is preferably AsCas12a (from Acidaminococcus sp. bv3l6), BbCas12a (from Beauveria basisana KA00251), BoCas12a (from bacteroides oral), FnCas12a (from Francisella novicida U112), HkCas12a (from Helcococcus kunzii), Lb4Cas12a (from Lachnospiraceae MC2017), Lb5Cas12a (from Lachnospiraceae bacterial NC2008), LbCas12a (from Lachnospiraceae ND2006), OsCas12a (from Oribacterium sp.) or Cas12a (from thiospira. xssp. 5).
A second aspect of the invention provides an isolated nucleic acid encoding a gene editing system as described in the first aspect.
The third aspect of the present invention provides a detection system for nucleic acid detection, the detection system comprising a gene editing system and a reaction buffer; wherein the gene editing system comprises nuclease and guide RNA, and the reaction buffer solution comprises 10-50 mM NaCl and 5-50 mM MgCl 2 5-50 mM Tris-HCl, 0.1-1.0 mM dithiothreitol and 50-200 mug/mL bovine serum albumin, pH 7-8.
In the present invention, the nuclease is as described in the first aspect, and the guide RNA is an RNA that is conventional in the art and is capable of binding to the nuclease and guiding the nuclease to a gene of interest, for example, sgRNA or crRNA; the gene of interest may be a viral gene, such as a gene of an RNA virus.
In the present invention, the working concentration of the guide RNA and the nuclease can be conventional in the art, for example, the working concentration of the guide RNA is 50-200 nM, such as 100 nM; the working concentration of the nuclease is 25-100 nM, such as 50 nM.
In some embodiments of the invention, the reaction buffer comprises 20-30 mM NaCl, 15-25 mM MgCl 2 10-20 mM Tris-HCl, 0.5-0.75 mM dithiothreitol and 100-200 mug/mL bovine serum albumin, and pH is 7-8.
In some embodiments of the invention, the reaction buffer comprises 20mM NaCl, 15mM MgCl 2 10mM Tris-HCl, 0.5mM dithiothreitol and 100. mu.g/mL bovine serum albumin, pH 7.9.
In a fourth aspect, the present invention provides an amplification system for nucleic acid amplification, the amplification system comprising a primer, RNase H and RPA enzyme; wherein, the primer is used for amplifying nucleic acid in a sample to be detected, the RPA enzyme is used for recombinase polymerase amplification, and the concentration of the RNase H is not higher than 1U/muL.
In the present invention, the RPA enzyme may be an enzyme conventionally used in the art for Recombinase Polymerase Amplification (RPA), including a Recombinase capable of binding a single-stranded nucleic acid primer, a single-stranded DNA binding protein (SSB), and a strand displacement DNA Polymerase.
In some embodiments of the invention, the concentration of the RNase H is 0.1-0.5U/. mu.L.
In some embodiments of the invention, the primers are primers for amplifying SARS-CoV-2 nucleic acid; preferably a primer for amplifying the N gene of SARS-CoV-2.
The primers comprise a forward primer and a reverse primer; the forward primer is preferably selected from one or more of nucleotide sequences shown in SEQ ID NO. 5-8, and the reverse primer is preferably selected from one or more of nucleotide sequences shown in SEQ ID NO. 9-12.
In some embodiments of the invention, the nucleotide sequence of the forward primer is shown as SEQ ID NO. 5, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 9; or the nucleotide sequence of the forward primer is shown as SEQ ID NO. 6, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 10; or the nucleotide sequence of the forward primer is shown as SEQ ID NO. 7, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 11; or, the nucleotide sequence of the forward primer is shown as SEQ ID NO. 8, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 12.
In the present invention, the working concentration of the primer can be conventional in the art, and is preferably 0.2-2. mu.M, such as 0.4. mu.M.
In some embodiments of the invention, the amplification system further comprises a reverse transcriptase; when the nucleic acid to be detected is RNA, the reverse transcriptase is used for reverse transcription of RNA into DNA.
A fifth aspect of the invention provides a lysis system for the lysis of a pathogen, the lysis system comprising a pathogen transfer fluid and a lysis fluid; the pathogen transfer solution is used for maintaining the pathogen activity of a sample to be detected, and the lysis solution is used for exposing pathogen nucleic acid in the sample to be detected; wherein the volume ratio of the pathogen transfer solution to the lysis solution is 1 (0.5-10).
In the invention, the pathogen transfer solution and the lysate are conventional in the field, for example, the pathogen transfer solution is a pathogen transfer solution of Beijing Youkang MT0301, and the lysate is a lysate of Epicentre QE 09050.
In some embodiments of the invention, the volume ratio of the pathogen transfer solution to the lysis solution is 1 (1-5); preferably 1: 1.
In the present invention, the pathogen may be a pathogenic organism conventional in the art, preferably selected from pathogenic fungi, viruses and pathogenic prokaryotes; the pathogenic prokaryotes preferably include bacteria, mycoplasma and chlamydia;
in some embodiments of the invention, the pathogen is a virus, preferably an RNA virus, such as SARS-CoV-2, IAV (influenza A virus) or IBV (influenza B virus).
A sixth aspect of the invention provides a kit for nucleic acid detection comprising a detection system according to the third aspect.
In some embodiments of the invention, the gene editing system of the test system is as described in the first aspect.
In some embodiments of the invention, the detection system further comprises a nucleic acid probe.
In the present invention, the nucleic acid probe is a probe with a nucleic acid sequence as a skeleton, which is conventional in the art, and is preferably a fluorescence-labeled single-stranded dna (ssdna). The fluorescent label is that two ends of the single-stranded DNA are respectively connected with a luminescent group and a quenching group. For example, when the sequence of the single-stranded DNA is CCCCC, the luminophore may be FAM and the quencher may be BHQ 1.
In some embodiments of the invention, the kit further comprises an amplification system comprising an amplification primer and RPA enzyme; wherein, the amplification primer is used for amplifying nucleic acid in a sample to be detected, and the RPA enzyme is used for recombinase polymerase amplification; the amplification system is preferably as described in the fourth aspect.
In other embodiments of the invention, the kit further comprises a lysis system comprising a pathogen transfer fluid and a lysis fluid; the pathogen transfer solution is used for maintaining the pathogen activity of a sample to be detected, and the lysis solution is used for exposing pathogen nucleic acid in the sample to be detected; the lysis system is preferably as described in the fifth aspect.
In some embodiments of the invention, the kit comprises a detection system, an amplification system, and a lysis system, as described above.
The seventh aspect of the present invention provides a crRNA for detecting SARS-CoV-2, which is a crRNA for detecting the N gene of SARS-CoV-2; wherein the crRNA is selected from one or more of nucleotide sequences shown as SEQ ID NO 1-4.
In some embodiments of the invention, the crRNA is a nucleotide sequence shown in any one of SEQ ID NO 1-4.
An eighth aspect of the present invention provides a method of nucleic acid detection, the method comprising: reacting a sample to be tested with the detection system according to the third aspect, and collecting a fluorescent signal generated by the reaction.
In some embodiments of the invention, the reaction time is 5-25 min; for example, 10 to 25min, 10 to 20min or 15 to 20 min.
In some embodiments of the invention, the temperature of the reaction is 35 to 45 ℃; for example, 35 to 42 ℃, 37 to 42 ℃ or 39 to 42 ℃.
In some embodiments of the present invention, the method further comprises amplifying the nucleic acid in the sample to be tested in the amplification system according to the fourth aspect before performing the reaction.
In some embodiments of the present invention, the method further comprises, before performing the reaction, lysing the sample to be tested using the lysis system as described in the fifth aspect to expose the nucleic acid in the sample to be tested, so as to perform a nucleic acid extraction-free detection.
In some embodiments of the present invention, the method further comprises amplifying the nucleic acid in the sample to be tested in the amplification system according to the fourth aspect before performing the reaction; and prior to the amplification reaction, the sample to be tested is subjected to lysis with the lysis system according to the fifth aspect, so as to expose the nucleic acid in the sample to be tested.
In the invention, the reaction volume ratio of the amplification system to the detection system is 1 (1-5); preferably 1 (2-4), for example 1: 3.
In the present invention, the amplification system is pre-stored in a closed container in which the detection reaction occurs.
In some embodiments of the invention, the detection system is added to the closed container after the amplification is completed, preferably by injection, to achieve decap-free detection.
In the present invention, the temperature of the cracking is 37 ℃ to 95 ℃, for example, 65 ℃ to 95 ℃.
A ninth aspect of the present invention provides a use of the gene editing system according to the first aspect, the nucleic acid according to the second aspect, the detection system according to the third aspect, the amplification system according to the fourth aspect, the cleavage system according to the fifth aspect, the kit according to the sixth aspect, or the crRNA according to the seventh aspect in preparing a reagent for detecting a nucleic acid of a pathogen.
In the present invention, the pathogen is as described in the fifth aspect.
On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.
The reagents and starting materials used in the present invention are commercially available.
The positive progress effects of the invention are as follows:
(1) the kit of the invention uses the crRNA of the N gene targeting SARS-CoV-2, can identify and cut the gene (the LOD is reduced from 100copies/mL to 1copy/mL in the preferred embodiment) in RNA virus with high sensitivity and high specificity, and can detect the wild type and each mutant strain of SARS-CoV-2 in a broad spectrum;
(2) the optimized reaction system can improve the detection efficiency in the shortened reaction time and the reduced reaction temperature, reduce the detection limit (up to 1 copies/mu L) and has low false positive rate;
(3) when the kit is used for detecting RNA virus, the sensitivity can reach 95.92%, the highest positive coincidence rate can reach 95.56%, the highest negative coincidence rate can reach 100%, and the kit has excellent detection efficiency.
(4) The kit can realize continuous and closed (uncovering-free) closed tube detection, and realizes the compatibility of each reaction by optimizing a reaction system; and can realize nucleic acid detection without nucleic acid extraction, and has higher application value.
Drawings
FIG. 1 is a diagram showing the result of optimizing the sequence of the crRNA of the N gene of SARS-CoV-2 according to the present invention.
FIG. 2 is a diagram showing the result of detecting SARS-CoV-2 by using the optimized crRNA and primer specificity of SARS-CoV-2N gene according to the present invention.
FIG. 3 is a diagram showing the result of broad-spectrum detection of SARS-CoV-2 wild type and each mutant strain by using optimized crRNA and primers for SARS-CoV-2N gene according to the present invention.
FIG. 4 is a schematic diagram of an optimization of the process of the present invention; in the figure:
a is a schematic diagram of extraction-free and uncovering-free reaction system and flow design;
b is a schematic diagram of the optimization result of the amplification time;
c is a schematic diagram of the reaction time optimization result of detection;
d is a schematic diagram of an RNase H concentration optimization result;
e is a schematic diagram of the optimized result of the reaction buffer solution;
f is a schematic diagram of the detected reaction temperature optimization result;
g is a schematic diagram of the volume ratio optimization result of the amplification system and the detection system;
h is a schematic diagram of the optimization result of the fluorescent probe;
i is a schematic diagram of the result of detecting the lowest copy number of RNA before and after the optimization of a reaction system.
FIG. 5 is a diagram showing the efficiency of detecting clinical specimens (SARS-CoV-2, A flow and B flow) by the optimized reaction system.
FIG. 6 is a schematic diagram showing the optimization results of the reaction system for the nucleic acid extraction exemption; in the figure:
a is a schematic diagram of pathogenic nucleic acid lysis mode screening;
b is a schematic diagram of an optimization result of the proportion of the pathogen transfer solution and the lysis solution;
c is a schematic diagram of an optimization result of the cracking temperature;
d is a schematic representation of the limit of detection (LOD) results using the SARS-CoV-2 pseudovirus assay reaction;
e is a schematic diagram of the detection limit result of detecting the Influenza A (IAV) compared with RT-qPCR.
Fig. 7 is a schematic diagram of the result of the positive coincidence rate between the IAV positive sample (all samples with Ct value less than 36) detected by the CRISPR-Cas12a reaction system without nucleic acid extraction and cover opening and the RT-qPCR technology.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. Experimental procedures without specifying specific conditions in the following examples were selected in accordance with conventional procedures and conditions, or in accordance with commercial instructions.
Example 1
Clinical samples and ethical statement
SARS-CoV-2 samples were collected from Shanghai customs port outpatient clinics. Influenza A Virus (IAV), Influenza B Virus (IBV) and negative samples were collected by rekins hospital. All samples were first used for clinical molecular diagnostics and the excess samples were stored for further study without collecting any personal identity information. The study was approved by the ethical committee of the seiku hospital affiliated with the Shanghai university of science and technology and the Shanghai university of transportation medical school.
Extraction of RNA and RT-qPCR assays from clinical samples
RNA was extracted from Nasopharyngeal (NP) swab Virus Transfer Medium (VTM) (Yocon Biology, Beijing, China) using a TIANAmp Virus DNA/RNA Kit (TIANGEN, Beijing, China) according to the product instructions.
RT-qPCR was performed in a QuantStaudio 6Flex System thermocycler (Applied Biosystems, USA) using the One Step PrimeScript RT-PCR Kit (Takara, China). The primers were designed according to the instructions of the Chinese Center for Disease Control (CDC).
The cycling conditions for the reaction were as follows: reverse transcription was performed at 42 ℃ for 5min, heat activated at 95 ℃ for 10s and 40 cycles at 95 ℃ for 5s denaturation step followed by annealing and extension step at 60 ℃ for 34 s.
Primer design and RT-RPA detection
The inventors designed RT-RPA primers for the N gene of SARS-CoV-2 (as shown in Table 1). The RNA of SARS-CoV-2 was amplified using different forward and reverse primer combinations and the best primer pair was screened for validation by Cas12a fluorescence reaction. The best performing primer pair was then selected for the subsequent RT-RPA reaction.
TABLE 1 RT-RPA primers for the N gene of SARS-CoV-2
Figure RE-GDA0003766931790000071
Figure RE-GDA0003766931790000081
Preparation of crRNA and RNA targets
To generate RNA targets, the target sequences of the IAV-M, IBV-HA and SARS-CoV-2-N genes were cloned into the PUC57 plasmid and then PCR amplified using primers containing the T7 promoter. The PCR product was confirmed by gel electrophoresis and used as an IVT template (in vitro transcription template) after purification by a gel extraction kit (Omega, USA). The IVT templates of crRNA (as shown in Table 2) and target RNA were transcribed overnight at 37 ℃ using the HiScribe T7 Quick High Yield RNA Synthesis Kit (NEB, USA). The DNA template in the transcribed RNA was removed by addition of DNase I (DNase I) and the synthesized RNA was purified by phenol-chloroform extraction followed by ethanol precipitation. The concentration of the purified RNA is quantified by a Nanodrop spectrophotometer, and the copy number calculation formula is as follows: RNA copy number (copy number/. mu.l) [6.02 × 10 ] 23 X RNA concentration (ng/. mu.L). times.10 -9 ]/(full transcript length × 330).
TABLE 2 CrRNA of N Gene of SARS-CoV-2
Figure RE-GDA0003766931790000082
RT-RPA and Cas12 a-based nucleotide detection optimization
Prior to optimization, the detection was performed as described in Wang et al, 2021. The detection comprises two steps: (1) RT-RPA was performed using a commercial KIT (WLRB8207KIT, AmpFature, China) according to the product instructions. Briefly, 25. mu.L of the reaction containing 14.7. mu.L of rehydration buffer, 5. mu.L of the RNA sample to be tested, 0.4. mu.M of each primer, and 14 mM magnesium acetate was incubated at 42 ℃ for 30 minutes. (2) For Cas12a fluorescence detection, 20 μ L of reaction mixture containing 5 μ L RT-RPA product, 2 μ L10 × Buffer 3.1(NEB, B7203S), 100nM crRNA, 50nM LbCas12a, and 1.25 μ M single stranded dna (ssdna) reporter probe was incubated for 30 min at 37 ℃. The fluorescence signal was monitored using a SpectraMax iD3 multimode microplate reader (. lamda.ex: 485 nm; lamda.em: 550 nm).
As shown in fig. 4, we optimized various reaction conditions for this assay based on a contamination-free detection method in a closed-tube reaction. (B) And (C) to optimize reaction time, we added RT-RPA products at different reaction times (5, 10, 15 and 20 min) in Cas12 a-based assays and measured the kinetics of the fluorescence signal. (D) For RT-RPA optimization, isothermal amplification reactions were performed by adding different concentrations of ribonuclease H (RNase H) (0, 0.1, 0.2, 0.5 and 1.0U/. mu.L). (E) To optimize the reaction buffer, Na was added at various concentrations + (0, 5, 10, 20, 50 and 100mM), Mg 2+ Cas12 a-based assays were performed in buffers of (0, 5, 10, 15, 20, and 25mM), Tris-HCl (0, 5, 10, 20, 50, and 100mM), DTT (dithiothreitol) (0, 0.5, 1, 2, 5, and 10mM), or BSA (bovine serum albumin) (0, 50, 100, 200, and 500mM), and the best Buffer (i.e., optimized Buffer in the figure) was selected. (F) For incubation temperature optimization, Cas12 a-mediated fluorescence reactions were incubated at different temperatures (35, 37, 39, 42, and 45 ℃). (G) For RT-RPA product input optimization, the assay was performed at different volume ratios (1:1, 1:2, 1:3, 1:4 and 1:5) of RT-RPA product to Cas12a reaction. (H) For ssDNA reporter probe optimization, Cas12 a-mediated fluorescence reactions were performed using ssDNA reporter probes with various sequences (5A-, 5T-, 5C-, and 5G-FQ reporter genes).
For all optimization experiments, the following reagents were used:
RT-RPA kit, 100nM crRNA, 50nM LbCas12a, and 1.25. mu. MssDNA reporter probe. The optimization was performed in an iterative manner, with only one reagent modified per experiment. The best choice of each reaction condition is based on better fluorescence kinetics or lower LOD as the best detection performance. When an optimal reaction condition is determined, the reaction condition prior to the substitution is integrated into the protocol for the next reaction condition optimization.
After the optimal reaction conditions are screened out, the optimized reaction conditions are as follows: (1) in the RT-RPA reaction step, 0.1U/. mu.L RNase H was added to 25. mu.L reaction mixture, followed by incubation at 42 ℃ for 15 min. (2) Cas12 a-mediated detection procedure was then optimized, with 25. mu.L of RT-RPA product in a 100. mu.L reaction, 1 × optimized reaction buffer (pH 7.9, 10mM Tris-HCl, 20mM NaCl, 15mM MgCl) 2 0.5mM dithiothreitol, 100. mu.g/mL bovine serum albumin, 100nM crRNA, 50nM LbCas12a, and 1.25. mu.M 5C-FQ reporter probe, incubated at 42 ℃ for 10 minutes.
LOD before and after optimization is shown in fig. 4 (I). As can be seen, the above method reduces the detection limit LOD from 100 copies/. mu.L to 1 copy/. mu.L
Pollution-free detection method in closed tube reaction
By pre-loading the reaction solution into the syringe, we developed a closed-tube nucleic acid detection method without having to re-open the cap to avoid amplicon contamination. First, 25. mu.L of RT-RPA reaction reagent containing the template was added to the tube, capped, and incubated at 42 ℃ for 15 minutes. Next, during the RT-RPA reaction, 75 μ Ι _ of Cas12a reaction mixture without amplicon was placed in a syringe. Third, after isothermal amplification, Cas12a reaction solution was injected into the tube from the cap and mixed with RT-RPA product by syringe without re-opening the cap, followed by incubation for 10 min at 42 ℃. Finally, the results can be measured by a fluorescence detection device.
RNA hands-free assay
To screen for optimal lysis methods, lentiviruses containing the N gene fragment of SARS-CoV-2(Beyotime, China) were incorporated into VTM to mimic clinical samples. Mixing the collected sample with a candidate lysis solution: (1) 0.2% Triton X-100; (2)100mM TCEP and 1mM EDTA; (3) equal volume of Quickextract DNA Extraction Solution (Lucigen, USA); heating only (heat only) and nuclease free water (RNase-free ddH) 2 O) as a control. After heating at 95 ℃ for 5 minutes, 5. mu.L of the sample was addedThe product was used as input to the Cas12 a-based closed-tube reaction described above to assess cleavage efficiency. For lysis buffer volume optimization, samples were diluted in optimal lysis buffer at volume ratios of 1:1, 1:5 and 1: 10. For cleavage temperature optimization, the cleavage reaction was carried out at 37, 65 and 95 ℃. The results are shown in FIGS. 6A to E.
Statistical analysis
Data were analyzed using GraphPad Prism 8. The double confidence intervals for sensitivity, specificity, positive predictive value (PPA) and negative predictive value (NPA) were calculated using the cloner-Pearson method. The results of positive coincidence rate with RT-qPCR technique for IAV positive samples (all samples with Ct value less than 36) are shown in FIG. 5 and FIG. 7. As can be seen from FIG. 5, the highest positive match rate can reach 95.56%, and the highest negative match rate is 100%.
SEQUENCE LISTING
<110> Shanghai science and technology university
Ruijin Hospital Affiliated to Medical College of Shanghai Jiaotong University
<120> nucleic acid detection kit based on CRISPR and application thereof
<130> P22012385C
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Claims (11)

1. A gene editing system comprising a nuclease and a guide RNA;
wherein the sequence of the guide RNA is selected from one or more of nucleotide sequences shown as SEQ ID NO 1-4.
2. The gene editing system of claim 1, wherein the guide RNA has a sequence as set forth in any one of SEQ ID NOs 1 to 4; and/or the presence of a gas in the gas,
the nuclease is a Cas protein;
preferably, the Cas protein is Cas12a or Cas13 a;
more preferably, the Cas protein is AsCas12a, BbCas12a, BoCas12a, FnCas12a, HkCas12a, Lb4Cas12a, Lb5Cas12a, LbCas12a, OsCas12a or TsCas12 a.
3. An isolated nucleic acid encoding the gene editing system of claim 1 or 2.
4. A detection system for nucleic acid detection, wherein the detection system comprises a gene editing system and a reaction buffer; wherein the gene editing system comprises nuclease and guide RNA, and the reaction buffer solution comprises 10-50 mM NaCl and 5-50 mM MgCl 2 5-50 mM Tris-HCl, 0.1-1.0 mM dithiothreitol and 50-200 mug/mL bovine serum albumin, and the pH value is 7-8;
preferably, the reaction buffer solution comprises 20-30 mM NaCl, 15-25 mM MgCl 2 10-20 mM Tris-HCl, 0.5-0.75 mM dithiothreitol and 100-200 mug/mL bovine serum albumin, wherein the pH value is 7-8; and/or, the nuclease is a Cas protein; and/or the working concentration of the guide RNA is 50-200 nM, such as 100nM, and the working concentration of the nuclease is 25-100 nMSuch as 50 nM;
more preferably, the reaction buffer comprises 20mM NaCl, 15mM MgCl 2 10mM Tris-HCl, 0.5mM dithiothreitol and 100. mu.g/mL bovine serum albumin, pH 7.9; and/or, the Cas protein is Cas12a or Cas13 a; the Cas protein is preferably AsCas12a, BbCas12a, BoCas12a, FnCas12a, HkCas12a, Lb4Cas12a, Lb5Cas12a, LbCas12a, OsCas12a or TsCas12 a.
5. An amplification system for nucleic acid amplification, comprising a primer, RNase H and RPA enzyme; wherein, the primer is used for amplifying nucleic acid in a sample to be detected, and the RPA enzyme is used for recombinase polymerase amplification; the concentration of the RNase H is not higher than 1U/. mu.L;
preferably, the concentration of the RNase H is 0.1-0.5U/. mu.L; the primer is used for amplifying nucleic acid of SARS-CoV-2; and/or, the amplification system further comprises a reverse transcriptase;
more preferably, the primer is a primer for amplifying the N gene of SARS-CoV-2; the forward primer of the primer is preferably selected from one or more of nucleotide sequences shown as SEQ ID NO. 5-8, and the reverse primer is preferably selected from one or more of nucleotide sequences shown as SEQ ID NO. 9-12; and/or the working concentration of the primer is 0.2-2 μ M, such as 0.4 μ M.
6. A lysis system for the lysis of pathogens, wherein the lysis system comprises a pathogen transfer fluid and a lysis fluid; the pathogen transfer solution is used for maintaining the pathogen activity of a sample to be detected, and the lysis solution is used for exposing pathogen nucleic acid in the sample to be detected; the pathogen transfer solution preferably comprises 0.8% sodium chloride, 0.04% potassium chloride, 0.014% calcium chloride, 0.02% magnesium sulfate (heptahydrate), 0.012% disodium hydrogen phosphate (heptahydrate), 0.006% monopotassium phosphate, 0.035% sodium bicarbonate, 0.1% glucose, 0.002% phenol red sodium salt; the percentage is mass percentage; wherein the volume ratio of the pathogen transfer solution to the lysis solution is 1 (0.5-10);
preferably, the volume ratio of the pathogen transfer solution to the lysis solution is 1 (1-5); and/or, the pathogen is selected from the group consisting of pathogenic fungi, viruses and pathogenic prokaryotes; the pathogenic prokaryotes preferably include bacteria, mycoplasma and chlamydia;
more preferably, the volume ratio of the pathogen transfer solution to the lysis solution is 1: 1; and/or the pathogen is a virus, preferably an RNA virus, such as SARS-CoV-2, IAV or IBV.
7. A kit for nucleic acid detection, comprising the detection system of claim 4;
preferably, the gene editing system is the gene editing system of claim 1 or 2; and/or, the detection system further comprises a nucleic acid probe; and/or the presence of a gas in the gas,
the kit also comprises an amplification system, wherein the amplification system comprises an amplification primer and RPA enzyme; wherein, the amplification primer is used for amplifying nucleic acid in a sample to be detected, and the RPA enzyme is used for recombinase polymerase amplification; and/or the presence of a gas in the gas,
the kit also comprises a cracking system, wherein the cracking system comprises a pathogen transfer solution and a cracking solution; the pathogen transfer solution is used for maintaining the pathogen activity of a sample to be detected, and the lysis solution is used for exposing pathogen nucleic acid in the sample to be detected;
more preferably, the amplification system is the amplification system of claim 5; and/or the lysis system is the lysis system of claim 6; and/or the presence of a gas in the gas,
the nucleic acid probe is fluorescence-labeled single-stranded DNA; the fluorescent label preferably comprises a luminescent group and a quenching group which are respectively positioned at two ends of the single-stranded DNA; the sequence of the single-stranded DNA is preferably CCCCC; for example, the luminescent group is FAM and the quencher group is BHQ 1.
8. A crRNA for detecting SARS-CoV-2, wherein the crRNA is a crRNA for detecting SARS-CoV-2N gene;
wherein the crRNA is selected from one or more of nucleotide sequences shown as SEQ ID NO 1-4.
9. A method of nucleic acid detection, the method comprising: reacting a sample to be tested with the detection system of claim 4, and collecting a fluorescent signal generated by the reaction;
preferably, the reaction time is 5-25 min; and/or the reaction temperature is 35-45 ℃;
more preferably, the reaction time is 10-25 min, preferably 10-20 min, for example 15-20 min; and/or the temperature of the reaction is 35-42 ℃, preferably 37-42 ℃, for example 39-42 ℃.
10. The method of claim 9, further comprising amplifying the nucleic acid in the test sample in the amplification system of claim 5 prior to performing the reaction; and/or, the method further comprises, prior to performing the reaction, lysing the test sample using the lysis system of claim 6 to expose nucleic acids in the test sample;
preferably, the reaction volume ratio of the amplification system to the detection system is 1 (1-5); and/or, the amplification system is pre-stored in a closed container in which the detection reaction occurs;
preferably, the reaction volume ratio of the amplification system to the detection system is 1 (2-4), for example, 1: 3; and/or, said detection system is added to said closed container after said amplification is complete, said addition preferably being by injection; and/or the temperature of the cracking is 37 ℃ to 95 ℃, for example 65 ℃ to 95 ℃.
11. Use of the gene editing system of claim 1 or 2, the nucleic acid of claim 3, the detection system of claim 4, the amplification system of claim 5, the lysis system of claim 6, the kit of claim 7, or the crRNA of claim 8 in the preparation of a reagent for detecting a pathogenic nucleic acid;
preferably, the pathogen is selected from the group consisting of pathogenic fungi, viruses and pathogenic prokaryotes; the pathogenic prokaryotes preferably include bacteria, mycoplasma and chlamydia;
more preferably, the pathogen is a virus, preferably SARS-CoV-2; the pathogenic nucleic acid is preferably the N gene of SARS-CoV-2.
CN202210435996.3A 2022-04-24 2022-04-24 Nucleic acid detection kit based on CRISPR and application thereof Pending CN115029345A (en)

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