CN115992207A - 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
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- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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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 from a Concentrator-5Kit (ZYMO RESEARCH), the samples were counter-rotated by Qubit Flex FluorometerThe guide RNA (crRNA) thus obtained was quantified 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 (10)
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, a Cas13 protein and a fluorescent reporter probe that is a single-stranded RNA having a fluorescent group attached at one end and a quenching group attached at the other end.
2. The use according to claim 1, characterized in that: 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.
3. Use according to claim 1 or 2, 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.
4. A use according to claim 1, 2 or 3, characterized in that: the CRISPR/Cas13 reagent contains glycine, and the ratio of crRNA to glycine in the CRISPR/Cas13 reagent is 50nM crRNA to 0.1g/mL glycine.
5. A composition for nucleic acid detection, characterized in that the composition comprises glycine and crRNA specific for the nucleic acid to be detected, cas13a protein and a fluorescent reporter probe, which is a single-stranded RNA having a fluorescent group attached at one end and a quenching group attached at the other end.
6. Use of glycine for the preparation of a nucleic acid detection reagent, the CRISPR/Cas13 reagent being the CRISPR/Cas13 reagent of any one of claims 1-3.
7. A method of detecting a biological nucleic acid comprising detecting whether a test sample contains a nucleic acid of interest using the CRISPR/Cas13 reagent of any one of claims 1-3.
8. The method according to claim 7, wherein: 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.
9. Use of the composition of claim 5 for the preparation of a working fluid for a biosensor or a biosensor.
10. Use of the biosensor of claim 9 in an apparatus for environmental pollution monitoring, food safety detection or disease diagnosis.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN202211412093.XA CN115992207B (en) | 2022-11-11 | 2022-11-11 | Glycine enhanced one-step CRISPR reaction and application thereof in novel coronavirus detection |
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Citations (7)
| 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 |
| US20210102242A1 (en) * | 2018-08-01 | 2021-04-08 | Mammoth Biosciences, Inc. | Programmable nuclease compositions and methods of use thereof |
| WO2021163584A1 (en) * | 2020-02-12 | 2021-08-19 | The Broad Institute, Inc. | Field deployable crispr-cas diagnostics and methods of use thereof |
| US20210292721A1 (en) * | 2020-03-23 | 2021-09-23 | Feng Zhang | Novel type v crispr-cas systems and 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 |
-
2022
- 2022-11-11 CN CN202211412093.XA patent/CN115992207B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20210102242A1 (en) * | 2018-08-01 | 2021-04-08 | Mammoth Biosciences, Inc. | Programmable nuclease compositions and methods of use thereof |
| WO2021163584A1 (en) * | 2020-02-12 | 2021-08-19 | The Broad Institute, Inc. | Field deployable crispr-cas diagnostics and methods of use thereof |
| US20210292721A1 (en) * | 2020-03-23 | 2021-09-23 | Feng Zhang | Novel type v crispr-cas systems and 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 |
|---|
| JULIA JOUNG等: "Detection of SARS-CoV-2 with SHERLOCK One-Pot Testing", THE NEW ENGLAND JOURNAL OF MEDICINE, pages 1 - 61 * |
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