CN115992207B - Glycine enhanced one-step CRISPR reaction and application thereof in novel coronavirus detection - Google Patents
Glycine enhanced one-step CRISPR reaction and application thereof in novel coronavirus detection Download PDFInfo
- Publication number
- CN115992207B CN115992207B CN202211412093.XA CN202211412093A CN115992207B CN 115992207 B CN115992207 B CN 115992207B CN 202211412093 A CN202211412093 A CN 202211412093A CN 115992207 B CN115992207 B CN 115992207B
- Authority
- CN
- China
- Prior art keywords
- glycine
- crispr
- reaction
- cas13
- detection
- Prior art date
- Legal status (The legal status 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 status listed.)
- Active
Links
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 title claims abstract description 89
- 238000010354 CRISPR gene editing Methods 0.000 title claims abstract description 55
- 239000004471 Glycine Substances 0.000 title claims abstract description 44
- 238000001514 detection method Methods 0.000 title claims abstract description 35
- 238000006243 chemical reaction Methods 0.000 title abstract description 59
- 241000711573 Coronaviridae Species 0.000 title abstract description 12
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 24
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 24
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 24
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 24
- 108091033409 CRISPR Proteins 0.000 claims abstract description 20
- 239000000523 sample Substances 0.000 claims abstract description 19
- 230000035945 sensitivity Effects 0.000 claims abstract description 11
- 238000002360 preparation method Methods 0.000 claims abstract description 6
- 238000000034 method Methods 0.000 claims description 13
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims description 12
- 102100034343 Integrase Human genes 0.000 claims description 7
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 claims description 5
- 230000003321 amplification Effects 0.000 claims description 5
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 5
- 238000010791 quenching Methods 0.000 claims description 5
- 230000000171 quenching effect Effects 0.000 claims description 5
- 101150098304 cas13a gene Proteins 0.000 claims 1
- 101100385364 Listeria seeligeri serovar 1/2b (strain ATCC 35967 / DSM 20751 / CCM 3970 / CIP 100100 / NCTC 11856 / SLCC 3954 / 1120) cas13 gene Proteins 0.000 abstract description 5
- 108700002099 Coronavirus Nucleocapsid Proteins Proteins 0.000 abstract description 3
- 239000000654 additive Substances 0.000 description 11
- 239000008176 lyophilized powder Substances 0.000 description 9
- 239000000126 substance Substances 0.000 description 8
- 230000000996 additive effect Effects 0.000 description 7
- 201000010099 disease Diseases 0.000 description 7
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 7
- 108020005004 Guide RNA Proteins 0.000 description 6
- 239000013614 RNA sample Substances 0.000 description 6
- 239000000843 powder Substances 0.000 description 6
- 238000013518 transcription Methods 0.000 description 6
- 230000035897 transcription Effects 0.000 description 6
- 238000000338 in vitro Methods 0.000 description 5
- UEGPKNKPLBYCNK-UHFFFAOYSA-L magnesium acetate Chemical compound [Mg+2].CC([O-])=O.CC([O-])=O UEGPKNKPLBYCNK-UHFFFAOYSA-L 0.000 description 5
- 239000011654 magnesium acetate Substances 0.000 description 5
- 229940069446 magnesium acetate Drugs 0.000 description 5
- 235000011285 magnesium acetate Nutrition 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 239000000872 buffer Substances 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 239000003999 initiator Substances 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 239000000725 suspension Substances 0.000 description 4
- 108020004414 DNA Proteins 0.000 description 3
- 235000013305 food Nutrition 0.000 description 3
- 238000004108 freeze drying Methods 0.000 description 3
- 230000001900 immune effect Effects 0.000 description 3
- 239000002773 nucleotide Substances 0.000 description 3
- 125000003729 nucleotide group Chemical group 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 230000004044 response Effects 0.000 description 3
- 238000003757 reverse transcription PCR Methods 0.000 description 3
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- 241001678559 COVID-19 virus Species 0.000 description 2
- 102000053602 DNA Human genes 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 101710203526 Integrase Proteins 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- 241001112090 Pseudovirus Species 0.000 description 2
- 108020004682 Single-Stranded DNA Proteins 0.000 description 2
- 101710137500 T7 RNA polymerase Proteins 0.000 description 2
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 description 2
- 229940098773 bovine serum albumin Drugs 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 238000007710 freezing Methods 0.000 description 2
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 2
- 238000003317 immunochromatography Methods 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 238000012123 point-of-care testing Methods 0.000 description 2
- 239000002096 quantum dot Substances 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 238000011895 specific detection Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- UDGUGZTYGWUUSG-UHFFFAOYSA-N 4-[4-[[2,5-dimethoxy-4-[(4-nitrophenyl)diazenyl]phenyl]diazenyl]-n-methylanilino]butanoic acid Chemical compound COC=1C=C(N=NC=2C=CC(=CC=2)N(C)CCCC(O)=O)C(OC)=CC=1N=NC1=CC=C([N+]([O-])=O)C=C1 UDGUGZTYGWUUSG-UHFFFAOYSA-N 0.000 description 1
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- 208000034657 Convalescence Diseases 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 108020004518 RNA Probes Proteins 0.000 description 1
- 238000010802 RNA extraction kit Methods 0.000 description 1
- 239000003391 RNA probe Substances 0.000 description 1
- 230000006819 RNA synthesis Effects 0.000 description 1
- 108091028733 RNTP Proteins 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 239000000443 aerosol Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 229940125532 enzyme inhibitor Drugs 0.000 description 1
- 239000002532 enzyme inhibitor Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 235000013611 frozen food Nutrition 0.000 description 1
- 230000003862 health status Effects 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 244000000010 microbial pathogen Species 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012806 monitoring device Methods 0.000 description 1
- 238000005580 one pot reaction Methods 0.000 description 1
- 230000001769 paralizing effect Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 125000006853 reporter group Chemical group 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000000241 respiratory effect Effects 0.000 description 1
- 108091092562 ribozyme Proteins 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a glycine enhanced one-step CRISPR reaction and application thereof in novel coronavirus detection. The invention relates to the technical field of biology, in particular to a glycine enhanced one-step CRISPR reaction and application thereof in novel coronavirus detection. The invention applies glycine in the preparation of a product that enhances the sensitivity of a CRISPR/Cas13 reagent to detect nucleic acids, wherein the CRISPR/Cas13 reagent comprises crRNA specific for the nucleic acid to be detected, cas13 protein and a fluorescent reporter probe. In the detection of the novel coronavirus N gene, the sensitivity of the glycine-enhanced one-step CRISPR reaction is improved by a factor of 10 compared to the glycine-free one-step CRISPR reaction, reaching 1 copy/microliter (5 copies/reaction). The invention is expected to provide powerful technical support for the real trend of CRISPR reaction to clinical and field detection applications.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a glycine enhanced one-step CRISPR reaction and application thereof in novel coronavirus detection.
Background
The novel coronavirus (SARS-CoV-2) is a respiratory virus that is highly infectious, has a high incidence of disease, and is easily transmitted from person to person. Therefore, rapid and accurate detection of novel coronaviruses is the most effective way to limit further spread of epidemic.
The main detection methods of the novel coronavirus comprise nucleic acid amplification detection, sequencing and immunological detection. Real-time reverse transcription polymerase chain reaction (real time reverse transcription PCR, rRT-PCR) is regarded as a gold standard for clinical examination, but both RT-PCR and gene sequencing are subject to expensive and sophisticated instrumentation, skilled operators, and long detection times. Immunological detection techniques are also commonly used for detection of pathogenic microorganisms, such as enzyme-linked immunosorbent assay (ELISA), colloidal gold immunochromatography, quantum dot immunochromatography, and the like. However, immunological detection is easily affected by the course of disease, and false negative may occur in early disease due to low antibody content in the patient; patients in convalescence are easy to carry the antibody and have false positives. Therefore, there is a need to develop a rapid, accurate, and highly sensitive method for detecting viral nucleic acids.
The regular intermittent clustered short palindromic repeat (Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR) technology is widely applied to point-of-care testing (POCT) due to its excellent detection mechanism, rapid reaction efficiency, excellent detection sensitivity, simple external reaction conditions, and non-specific cleavage of a single-stranded RNA reporter group labeled with fluorescence quenching while specifically recognizing an RNA sequence by specific paralytic cleavage characteristics, i.e., cas13 protein binding to crRNA synthesized in vitro, thereby realizing accurate and efficient detection of a target. However, the prior two-step CRISPR reaction requires an amplified product transfer operation and involves the risk of aerosol contamination. The prior one-step CRISPR reaction has long time consumption and low sensitivity, and can not meet the actual application requirements. Based on the background, development of a rapid and high-sensitivity one-step CRISPR reaction is needed, and a powerful technical support is provided for the CRISPR reaction to truly go to clinical and field detection applications.
Disclosure of Invention
The technical problem to be solved by the invention is that the existing one-step CRISPR reaction has the practical problems of long reaction time, low reaction efficiency, limited sensitivity and the like, and cannot meet the practical detection application requirements.
The invention provides application of glycine in preparation of a product for enhancing sensitivity of detecting nucleic acid by using a CRISPR/Cas13 reagent, wherein the CRISPR/Cas13 reagent comprises crRNA specific to the nucleic acid to be detected, cas13 protein and a fluorescence report probe, and the fluorescence report probe is single-stranded RNA with one end connected with a fluorescent group and the other end connected with a quenching group.
In the above application, the detecting nucleic acid is performed by a one-step method that does not include a step of transferring the nucleic acid amplification product.
In the above application, the detection sample of the CRISPR/Cas13 reagent is derived from an RNA-containing organism, and the CRISPR/Cas13 reagent contains a reverse transcriptase.
The nucleotide sequence of the single-stranded RNA of the fluorescent reporter probe is shown in Table 3.
In the application, the CRISPR/Cas13 reagent contains glycine, wherein the ratio of crRNA to glycine in the CRISPR/Cas13 reagent is 50nM crRNA to 0.1g/mL glycine.
The CRISPR/Cas13 reagent may consist of glycine alone, crRNA specific for the nucleic acid to be detected, cas13 protein and fluorescent reporter probe, and may also contain at least one of the following: cas13 protein, reverse transcriptase, T7 RNA polymerase, ribonuclease H, RNA enzyme inhibitor, rtp, RNA reporter probe, guide RNA, primer, glycine, RPA reagent, magnesium acetate, buffer.
(1) Specifically, the Cas13 protein is an lwaca 13a protein at a concentration of 10-200nM, preferably 50nM.
(2) In particular, the reverse transcriptase is preferably, but not limited to Superscript IV Reverse Transcriptase (Thermo Fisher); the concentration is 0.5-4U/. Mu.L, preferably 2U/. Mu.L.
(3) Specifically, the T7 RNA polymerase concentration is 0.5-4U/. Mu.L, preferably 2U/. Mu.L.
(4) Specifically, the ribonuclease H concentration is 0.05-0.2U/. Mu.L, preferably 0.1U/. Mu.L.
(5) Specifically, the rNTP concentration is 0.25-4mM, preferably 1mM.
(6) Specifically, the fluorescent group of the RNA report probe can be FAM, ROX, HEX, CY, CY5 and the like, and the quenching group can be BHQ1, BHQ2, BHQ3 and the like; the single-stranded RNA probe is prepared according to a conventional method in the field, and the sequence can be 5'-UUUUU-3', 5'-UUUUUU-3', and the like; the concentration is 0.1-4. Mu.M.
(7) Specifically, the guide RNA is prepared by in vitro transcription or is synthesized directly and chemically, and the concentration is 10-200nM, preferably 25nM.
(8) Specifically, the primer concentration is 50-500nM, preferably 125nM.
(9) Specifically, the glycine concentration is 1% -50%, preferably 10%.
(10) Specifically, the buffer was RPA rehydration buffer (TwitDx TM )。
The invention also provides compositions for nucleic acid detection.
The invention also provides a composition for nucleic acid detection comprising glycine and crRNA specific for the nucleic acid to be detected, a Cas13a protein and a fluorescent reporter probe, wherein the fluorescent reporter probe is a single-stranded RNA with one end connected with a fluorescent group and the other end connected with a quenching group.
The invention also provides application of glycine in preparing a nucleic acid detection reagent, wherein the CRISPR/Cas13 reagent is the CRISPR/Cas13 reagent containing glycine.
The invention also provides a method for detecting biological nucleic acid, which comprises the step of detecting whether a sample to be detected contains target nucleic acid or not by using the CRISPR/Cas13 reagent containing glycine.
The above-described applications or methods are non-disease diagnostic applications or methods. The above applications or methods are not directed to obtaining disease diagnosis results or health status of a living human or animal body. The sample to be tested may be a sample from a non-living human or animal body, such as an environmental sample (e.g. air), a food (e.g. frozen food or fresh food).
The organism may be a microorganism.
The method comprises the step of amplifying the nucleic acid of the sample to be detected to obtain a nucleic acid amplification product, and the method does not comprise the step of transferring the nucleic acid amplification product. That is, the reaction of the method is performed in one reaction system. The method is a one-step assay.
The composition is a powder.
The invention also provides application of the composition for detecting nucleic acid in preparing working fluid of a biosensor or the biosensor.
The invention also provides application of the biosensor in an environment pollution monitoring device, a food safety detection device or a disease diagnosis device.
The disease is a novel coronavirus (SARS-CoV-2) infection.
The invention provides an enhanced one-step CRISPR/Cas13 reaction using a chemical additive glycine, which can obviously improve the detection signal intensity, shorten the detection time and improve the detection sensitivity by simply adjusting a reagent system without using a complex sensor detection means. In the detection of the novel coronavirus N gene, the sensitivity of the glycine-enhanced one-step CRISPR reaction is improved by a factor of 10 compared to the glycine-free one-step CRISPR reaction, reaching 1 copy/microliter (5 copies/reaction). The invention is expected to provide powerful technical support for the real trend of CRISPR reaction to clinical and field detection applications.
Drawings
Figure 1 is a one-step CRISPR/Cas13 comparison of several chemical additives and their different concentrations.
Figure 2 is a graph of one-step CRISPR/Cas13 response real-time fluorescence signal under several different chemical additive conditions.
Fig. 3 is a comparison of sensitivity of glycine enhanced one-step CRISPR/Cas13 response versus glycine free group.
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
The quantitative experiments in the following examples were performed in triplicate unless otherwise indicated.
Example 1 Glycine enhanced one-step CRISPR/Cas13 reaction for novel coronavirus RNA detection
1. Preparation of RNA sample to be tested
In this example, a sample of the novel coronavirus pseudovirus SARS-COV-2-abSMNE was purchased from Fubaiao Inc. of Suzhou (cat# FNRV 2593).
Extracting and purifying by using a Tiangen company virus genome DNA/RNA extraction kit (DP 315) to obtain an RNA sample to be detected, and preserving at-20 to-80 ℃ for standby.
2. In vitro transcription of guide RNA (crRNA)
In this example, the guide RNA (crRNA, nucleotide sequence 3 in the sequence listing) was prepared by in vitro transcription. The single-stranded DNA template to be transcribed (the nucleotide sequence is sequence 4 in the sequence table) corresponding to the guide RNA is synthesized by the division of biological engineering (Shanghai).
The method comprises the following specific steps: to 1. Mu.L of a single-stranded DNA template to be transcribed (sequence 4 in the sequence listing) (100. Mu.M) was added 1. Mu.L of 10X standard Taq buffer (New England)Inc.), 1 μl of the oligonucleotide primer (sequence 5 in the sequence listing) (100 μl) for transcription, and 7 μl of enzyme-free water to make a 10 μl mixed system. The mixture was denatured at high temperature (95 ℃ C./5 min) and gradient annealed (stopping at 0.1 ℃ C./s to 4 ℃ C.) using HiScribe T7 High Yield RNA Synthesis Kit (New England>Inc.), in vitro transcription was performed according to the kit instructions. Transcription products using RNA Clean&After purification of the Concentrator-5Kit (ZYMO RESEARCH), the transcribed guide RNA (crRNA) was quantified by Qubit Flex Fluorometer and stored at-80℃for further use.
3. One-step CRISPR/Cas13 reaction
Specific detection systems for one-step CRISPR/Cas13 reactions are shown in table 1 below. Wherein the FAM-5U-BHQ1 fluorescence report probe is: FAM-UUU-BHQ 1, the nucleotide sequences are shown in Table 3. The upstream primer is as follows: CCTCTAATACGACTCACTATAGGCTAGAATGGCTGGCAATGGCGGTGATGCT (sequence 1 in the sequence table), the downstream primer is: ATTTCTTAGTGACAGTTTGGCCTTGTTGTT (SEQ ID NO: 2 of the sequence Listing) were synthesized by the company of Shanghai, inc. The sequences used in the present invention are specifically shown in Table 3.
TABLE 1 one-step CRISPR/Cas13 reaction System
Note that: the RPA lyophilized powder was added to the 50. Mu.L reaction system.
The 50 μl reaction system was added to RPA lyophilized powder, vortexed thoroughly, and split-packed into 200 μl PCR reaction tubes with 10 μl×5 tubes. Pre-freezing the split charging reagent in a freeze dryer (Labconco) at-60 ℃ for 1-3 hours, then carrying out negative pressure treatment for 8-12 hours to prepare one-step CRISPR/Cas13 reaction freeze-dried powder, and storing the freeze-dried powder at-20 ℃ for later use. In use, RPA heavy suspension (TwitDx is added to the prepared lyophilized powder TM ) The RNA sample to be tested, and magnesium acetate (14 mM) as a reaction initiator, and fluorescent signals were detected on a fluorescent collection device.
4. Chemical additive screening experiments for one-step CRISPR/Cas13
The following screening types and concentrations were selected:
1) Bovine serum albumin was added at a final concentration of 10%, 5%, 2.5%, 1%, respectively;
2) Glycine was added at final concentrations of 10%, 5%, 2.5%, 1%, respectively;
3) Trehalose is added at final concentration of 10%, 5%, 2.5% and 1%;
4) Mannitol was added at final concentrations of 10%, 5%, 2.5%, 1%, respectively.
The 16 additives with different concentrations are added into the one-step CRISPR/Cas13 reaction system in the step 3 to screen the optimal chemical additives and the using concentration.
To the prepared lyophilized powder to which the above additives were added, 1×rpa heavy suspension (twist dx TM ) 1. Mu.L of the RNA sample to be tested, and magnesium acetate (14 mM) as a reaction initiator, and the fluorescent signal was detected on a fluorescent collection device for 30 minutes.
The results in FIG. 1 show that the one-step reaction lyophilization reagents with 1% bovine serum albumin, 5% glycine, 10% glycine, and 10% trehalose provided no decay in the fluorescence signal intensity at the end of the reaction compared to that before lyophilization. Further, the results in fig. 2 show that when 10% glycine is used as a reactive chemical additive, the reaction rate is significantly improved compared to that when several other additive components are added before, after and during lyophilization. Therefore, 10% glycine has optimal detection efficiency when used as a chemical additive. Subsequent experiments may select 10% glycine as the chemical additive for the reaction.
Example 2 application of glycine-enhanced one-step CRISPR/Cas13 reaction system lyophilized powder 1, preparation of glycine-enhanced one-step CRISPR/Cas13 reaction system lyophilized powder
Specific detection systems for the glycine added enhanced one-step CRISPR/Cas13 reactions are shown in table 2 below.
TABLE 2 Glycine enhanced one-step CRISPR/Cas13 reaction System
Note that: the solute of the glycine solution (20%) was glycine and the solvent was enzyme-free water. The glycine content of the glycine solution (20%) was 0.2g/mL.
The 50 μl reaction system was added to RPA lyophilized powder, vortexed thoroughly, and split-packed into 200 μl PCR reaction tubes with 10 μl×5 tubes. Pre-freezing the split charging reagent in a freeze dryer (Labconco) at-60 ℃ for 1-3 hours, then carrying out negative pressure treatment for 8-12 hours to prepare one-step CRISPR/Cas13 reaction freeze-dried powder, and storing the freeze-dried powder at-20 ℃ for later use.
The preparation method of the glycine-enhanced one-step CRISPR/Cas13 reaction system freeze-dried powder comprises the following steps: in use, RPA heavy suspension (TwitDx is added to the prepared lyophilized powder TM ) The RNA sample to be tested, and magnesium acetate (14 mM) as a reaction initiator, and fluorescent signals were detected on a fluorescent collection device.
2. Glycine enhanced one-step CRISPR/Cas13 response Performance assessment
The ability of glycine-enhanced one-step CRISPR/Cas13 reactions to detect novel coronavirus N genes at different concentration gradients with glycine-free one-step CRISPR/Cas13 reactions was evaluated in this example.
Specifically, the RNA concentration gradients of the novel coronavirus pseudovirus extracted nucleic acid samples are 1000, 500, 100, 50, 10, 5, 1 copy/reaction, and negative quality control.
The reaction system and reaction conditions for the glycine-free one-step CRISPR/Cas13 reaction are the same as in step 3 of example 1. Glycine enhanced one-step CRISPR/Cas13 reaction System Table 2, when used, RPA heavy suspension (TwitDx was added to the prepared lyophilized powder TM ) The RNA sample to be tested, and magnesium acetate (14 mM) as a reaction initiator, and fluorescent signals were detected on a fluorescent collection device.
As shown in FIG. 3, the sensitivity of the glycine-enhanced reaction can be increased ten times based on the original glycine-free reaction, from 50 copies/reaction to 5 copies/reaction.
TABLE 3 sequence information used in the present invention
The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.
Claims (2)
1. Use of glycine in the preparation of a product that enhances the sensitivity of a CRISPR/Cas13 reagent to detect a nucleic acid, the CRISPR/Cas13 reagent comprising crRNA specific for the nucleic acid to be detected, cas13a protein, RPA reagent and a fluorescent reporter probe that is a single-stranded RNA having a fluorophore attached at one end and a quenching group attached at the other end; the ratio of crRNA to glycine is 50nM crRNA to 0.1g/mL glycine;
the detection of nucleic acids is performed by a one-step method that does not include a step of transferring the nucleic acid amplification product.
2. The use according to claim 1, characterized in that: the detection sample of the CRISPR/Cas13 reagent is derived from an RNA-containing organism, the CRISPR/Cas13 reagent containing a reverse transcriptase.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211412093.XA CN115992207B (en) | 2022-11-11 | 2022-11-11 | Glycine enhanced one-step CRISPR reaction and application thereof in novel coronavirus detection |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211412093.XA CN115992207B (en) | 2022-11-11 | 2022-11-11 | Glycine enhanced one-step CRISPR reaction and application thereof in novel coronavirus detection |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN115992207A CN115992207A (en) | 2023-04-21 |
| CN115992207B true CN115992207B (en) | 2024-03-22 |
Family
ID=85994494
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211412093.XA Active CN115992207B (en) | 2022-11-11 | 2022-11-11 | Glycine enhanced one-step CRISPR reaction and application thereof in novel coronavirus detection |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115992207B (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111560469A (en) * | 2020-03-30 | 2020-08-21 | 广州和盛医疗科技有限公司 | Primer group for detecting new coronavirus gene, CRISPR (clustered regularly interspaced short palindromic repeats) sequence combination and application of primer group |
| CN112029901A (en) * | 2020-07-21 | 2020-12-04 | 河南省生物工程技术研究中心 | Reagent for improving specificity of nucleic acid amplification reaction, nucleic acid amplification reaction solution and kit |
| WO2021163584A1 (en) * | 2020-02-12 | 2021-08-19 | The Broad Institute, Inc. | Field deployable crispr-cas diagnostics and methods of use thereof |
| WO2021262099A1 (en) * | 2020-06-26 | 2021-12-30 | Nanyang Technological University | Crispr-based methods for the detection of nucleic acids in a sample |
| CN113957164A (en) * | 2021-10-29 | 2022-01-21 | 上海市质量监督检验技术研究院 | CRISPR One dot detection method of Cronobacter in infant formula milk powder and kit thereof |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020028729A1 (en) * | 2018-08-01 | 2020-02-06 | Mammoth Biosciences, Inc. | Programmable nuclease compositions and methods of use thereof |
| US11639523B2 (en) * | 2020-03-23 | 2023-05-02 | The Broad Institute, Inc. | Type V CRISPR-Cas systems and use thereof |
-
2022
- 2022-11-11 CN CN202211412093.XA patent/CN115992207B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021163584A1 (en) * | 2020-02-12 | 2021-08-19 | The Broad Institute, Inc. | Field deployable crispr-cas diagnostics and methods of use thereof |
| CN111560469A (en) * | 2020-03-30 | 2020-08-21 | 广州和盛医疗科技有限公司 | Primer group for detecting new coronavirus gene, CRISPR (clustered regularly interspaced short palindromic repeats) sequence combination and application of primer group |
| WO2021262099A1 (en) * | 2020-06-26 | 2021-12-30 | Nanyang Technological University | Crispr-based methods for the detection of nucleic acids in a sample |
| CN112029901A (en) * | 2020-07-21 | 2020-12-04 | 河南省生物工程技术研究中心 | Reagent for improving specificity of nucleic acid amplification reaction, nucleic acid amplification reaction solution and kit |
| CN113957164A (en) * | 2021-10-29 | 2022-01-21 | 上海市质量监督检验技术研究院 | CRISPR One dot detection method of Cronobacter in infant formula milk powder and kit thereof |
Non-Patent Citations (1)
| Title |
|---|
| Detection of SARS-CoV-2 with SHERLOCK One-Pot Testing;Julia Joung等;The new england journal of medicine;第1-61页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN115992207A (en) | 2023-04-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US10689716B1 (en) | Materials and methods for detecting coronavirus | |
| CN111850097B (en) | Signal amplification magnetic bead technology system for nucleic acid detection based on CRISPR technology and application thereof | |
| EP1330553B1 (en) | A kit for detecting non-pathogenic or pathogenic influenza a subtype h5 virus | |
| EP4198142A1 (en) | Kit and method for detecting nucleic acid and uses thereof | |
| AU2002214936A1 (en) | A kit for detecting non-pathogenic or pathogenic influenza a subtype H5 virus | |
| CN110724769A (en) | PCR primer group, kit and detection method for detecting African swine fever virus MGF360-505R gene | |
| WO2021188935A1 (en) | Materials and methods for detecting coronavirus | |
| CN111334610B (en) | Universal RT-RAA-LFD amplification primer for dengue virus and detection method | |
| CN115992207B (en) | Glycine enhanced one-step CRISPR reaction and application thereof in novel coronavirus detection | |
| CN115747361B (en) | Real-time fluorescence MIRA and MIRA-LFD primer group for detecting streptococcus iniae and detection method | |
| CN108611442B (en) | Fluorescent quantitative RT-PCR primer and probe for detecting swine atypical pestivirus, method and application | |
| CN116121413A (en) | Real-time fluorescent nucleic acid isothermal amplification detection kit for group B streptococcus and special primer and probe thereof | |
| CN112280832B (en) | Extraction-free nucleic acid detection method and kit | |
| CN111378724B (en) | RNA amplification detection method and detection kit | |
| CN113502341A (en) | Real-time fluorescent nucleic acid isothermal amplification detection kit for treponema pallidum 16s RNA, and special primer and probe thereof | |
| CN114807435A (en) | Kit for detecting respiratory syncytial virus and application thereof | |
| CN112575122B (en) | Dual PCR primer set for rapidly detecting duck type 2 adenovirus and duck circovirus, and detection method and application thereof | |
| KR102526656B1 (en) | Method for detecting nucleic acid | |
| CN116515840B (en) | Kit and detection method for detecting bovine viral diarrhea virus type 3 | |
| CN118308540B (en) | Detection kit for Hantavirus HTNV type and SEOV type | |
| EP1308521B1 (en) | A process and a kit for detecting foot-and-mouth disease virus | |
| US20240110252A1 (en) | Compositions and Kits for Rapid Detection of SARS-CoV-2 and Methods of Production and Use Thereof | |
| EP4332237A1 (en) | Isothermal gene amplification method, gene detection method, virus detection method, and kit used therefor | |
| JPH11243995A (en) | Detection of urinary bladder cancer in urine sample | |
| CN115961103A (en) | Non-amplified virus nucleic acid detection method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |