CN116814627B - SgRNA composition, kit for detecting novel coronavirus SARS-CoV-2 and detection method - Google Patents
SgRNA composition, kit for detecting novel coronavirus SARS-CoV-2 and detection method Download PDFInfo
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Abstract
The application provides an sgRNA composition for detecting novel coronavirus SARS-CoV-2, which comprises a first sgRNA, a second sgRNA and a third sgRNA, wherein the sequence of the first sgRNA comprises a nucleotide sequence shown as SEQ ID NO. 1, the sequence of the second sgRNA comprises a nucleotide sequence shown as SEQ ID NO. 2, and the sequence of the third sgRNA comprises a nucleotide sequence shown as SEQ ID NO. 3. The application also provides a kit for detecting the novel coronavirus SARS-CoV-2 and a detection method of the novel coronavirus SARS-CoV-2.
Description
Technical Field
The application relates to the field of medical biology, in particular to an sgRNA composition, a kit for detecting novel coronavirus SARS-CoV-2 and a detection method.
Background
Therefore, the screening of the novel coronavirus SARS-CoV-2 is particularly important. At present, virus nucleic acid detection can be carried out through reverse transcription polymerase chain reaction (RT-PCR), and the detection method has high sensitivity and strong specificity, and can find patients at early infection or at incubation period; however, the method involves the steps of nucleic acid inactivation, reverse transcription, amplification and the like, needs to rely on professional amplification instruments and experimental environments, has long detection time, and is not beneficial to rapid screening of viruses.
Disclosure of Invention
In view of the above, the application provides a sgRNA composition, a kit for detecting novel coronavirus SARS-CoV-2 and a method for detecting novel coronavirus SARS-CoV-2, which can be used for rapidly and effectively detecting novel coronavirus SARS-CoV-2, and has the advantages of low detection limit, high detection sensitivity and specificity, no need of specialized instruments and detection environments, simpler detection conditions, and capability of rapidly detecting novel coronavirus SARS-CoV-2.
In a first aspect, the application provides a sgRNA composition for detecting novel coronaviruses SARS-CoV-2, comprising a first sgRNA, a second sgRNA and a third sgRNA, wherein the sequence of the first sgRNA comprises the nucleotide sequence shown as SEQ ID NO. 1, the sequence of the second sgRNA comprises the nucleotide sequence shown as SEQ ID NO. 2, and the sequence of the third sgRNA comprises the nucleotide sequence shown as SEQ ID NO. 3.
In a second aspect, the application provides the use of the sgRNA composition of the first aspect in a kit for detecting novel coronavirus SARS-CoV-2.
In a third aspect, the application provides a kit for detecting novel coronavirus SARS-CoV-2, comprising the sgRNA composition of the first aspect.
Optionally, the kit further comprises a Cas13a protein and a reporter gene.
Optionally, the kit further comprises at least one of a dilution buffer and an rnase inhibitor.
Further, the pH value of the dilution buffer is 6.5-6.9.
Further, the dilution buffer comprises 18mM-21mM of 4-hydroxyethyl piperazine ethane sulfonic acid sodium salt, 45mM-58mM of potassium chloride, 4.5mM-6.3mM of magnesium chloride, and 4.5% v/v-5.5% v/v of glycerol.
Alternatively, the minimum detection limit of the kit for detecting the novel coronavirus SARS-CoV-2 is 150copies/mL.
In a fourth aspect, the present application provides a method for detecting SARS-CoV-2, which comprises: the sgRNA composition of the first aspect or the kit for detecting the novel coronavirus SARS-CoV-2 of the second aspect is used for detecting a sample to be detected.
Optionally, mixing and incubating the first sgRNA, the second sgRNA, the third sgRNA with the Cas13a protein to form an RNP complex; and mixing the sample to be tested with the RNP complex and the reporter gene to form a reaction system, and obtaining a fluorescent signal value and analyzing a result after the reaction.
Further, when the first, second, and third sgrnas are mixed with Cas13a protein, the molar ratio of the Cas13a protein, the first, second, and third sgrnas is (3-4): 1:1:1.
Further, the incubation includes treatment at 35-42 ℃ for 10-15 min.
Further, the concentration of the RNP complex in the reaction system is 80nM to 110nM.
Further, the concentration of the reporter gene in the reaction system is 300nM-400nM.
Further, the reaction includes treatment at 30-60 ℃ for 10-15 min.
The sgRNA composition provided by the application can be used for rapidly and effectively detecting the novel coronavirus SARS-CoV-2, has low detection limit and high detection sensitivity and specificity, does not need to rely on professional instruments and detection environments, can reach the same detection level of RT-PCR, has more convenient detection conditions and detection time, can realize the specific rapid detection of the novel coronavirus SARS-CoV-2, and is beneficial to disease treatment and control.
Drawings
Fig. 1 is a graph of fluorescent signals of a first set of samples 4.
FIG. 2 is a schematic representation of a first set of different samples under fluorescence.
Fig. 3 is a graph of fluorescent signals of a first set of samples 4.
Detailed Description
The following description is of exemplary embodiments of the application, and it should be noted that modifications and variations can be made by persons skilled in the art without departing from the principles of the application, and these modifications and variations are also considered to be within the scope of the application.
In the related art, in order to accurately detect the novel coronavirus SARS-CoV-2, detection needs to be performed through PCR and other ways, and special equipment and instruments are needed, and the time consumption is long, such as expensive instruments, special technicians and reaction processes with long reaction time and different reaction temperatures and reaction time periods are needed in RT-PCR; and other detection methods, such as test paper detection, are adopted, so that the detection time is greatly reduced, but the detection accuracy is also reduced, false negative is caused, and the accurate judgment and control of infection are not facilitated.
The application provides an sgRNA composition for detecting novel coronavirus SARS-CoV-2, comprising a first sgRNA, a second sgRNA and a third sgRNA, wherein the sequence of the first sgRNA comprises a nucleotide sequence (GACCACCCCAAAAAUGAAGGGGACUAAAACAGGGUCCACCAAACGUAAUGCGG) shown in SEQ ID NO. 1, the sequence of the second sgRNA comprises a nucleotide sequence (GACCACCCCAAAAAUGAAGGGGACUAAAACUUUGGUGUAUUCAAGGCUCCCU) shown in SEQ ID NO. 2, and the sequence of the third sgRNA comprises a nucleotide sequence (GACCACCCCAAAAAUGAAGGGGACUAAAACAAGCGCAGUAAGGAUGGCUAGU) shown in SEQ ID NO. 3. Wherein sgRNA (single guide RNA) is an important component in a CRISPR knockout system, which can be matched with a target gene, and is helpful for realizing the identification and detection of the target gene. The sgRNA composition provided by the application contains the first sgRNA, the second sgRNA and the third sgRNA, and the first sgRNA, the second sgRNA and the third sgRNA can specifically and efficiently target the novel coronavirus SARS-CoV-2 gene sequence, so that the novel coronavirus SARS-CoV-2 can be accurately positioned and identified, and the detection of the novel coronavirus SARS-CoV-2 is facilitated. The inventor researches and discovers that the first sgRNA, the second sgRNA or the third sgRNA can not effectively locate and identify the novel coronavirus SARS-CoV-2 when being singly used, but when the first sgRNA, the second sgRNA and the third sgRNA are jointly used, the novel coronavirus SARS-CoV-2 can be effectively located and identified, non-target genes (such as influenza virus, MERS, SARS and the like) can not be identified, erroneous judgment is prevented, the detection limit of the novel coronavirus SARS-CoV-2 is low, and the accurate detection of the novel coronavirus SARS-CoV-2 is facilitated.
The application provides the application of the sgRNA composition in any of the above embodiments in a kit for detecting novel coronavirus SARS-CoV-2. The sgRNA composition provided by the application can effectively locate and identify the novel coronavirus SARS-CoV-2, so that the sgRNA composition can be used in a kit for detecting the novel coronavirus SARS-CoV-2, and the detection sensitivity and accuracy are improved.
The application provides a kit for detecting novel coronavirus SARS-CoV-2, comprising the sgRNA composition in any of the above embodiments.
In one embodiment of the application, the kit further comprises a Cas13a protein and a reporter gene. Cas13a protein (C2C 2 protein) is one of proteins with RNA enzyme digestion activity in a Cas protein family of CRISPR effect, and is matched with a target gene through sgRNA, so that the cleavage activity of non-target genes is started, and a reporter gene can be continuously cleaved under the condition of not damaging a substrate to generate a stable signal, thereby achieving the purpose of specifically detecting low-copy-number novel coronavirus SARS-CoV-2.
In the present application, the Cas13a protein may be from at least one of oral ciliated (Leptotrichia buccalis, lbu), wei De ciliated (Leptotrichia wadei, lwa), and sandy ciliated (Leptotrichia shahii, lsh). The application is not limited to the source and sequence of Cas13a protein.
In the present application, the nucleotide sequence of the reporter gene may be selected as desired. In one embodiment of the application, the nucleotide sequence of the reporter gene may be UUUUU.
In one embodiment of the application, the reporter gene is linked to a marker which is capable of generating a signal during the detection process, thereby allowing the detection of the novel coronavirus SARS-CoV-2. In one embodiment of the application, the label includes a fluorescent group and a quenching group. In one embodiment, the reporter gene is linked at the 5 'end to a fluorophore and at the 3' end to a quencher. The fluorescent group and the quenching group may be selected as desired. Specifically, the fluorescent group may include at least one of AMCA, APC, FAM, FITC, cy, cy5, cy7, HEX, and ROX. In an embodiment, the fluorescent group may be AMCA or APC, which is advantageous for obtaining a strong fluorescent signal and for facilitating the judgment of the result. Specifically, the quenching group may include at least one of IABkFQ, BHQ1, BHQ2, BHQ3, TAMRA, DABCYL and ECLIPSE. In one embodiment of the application, the marker may be biotin; that is, biotin is attached to the reporter group. In one embodiment, the kit further comprises an enzyme-labeled avidin and a chromogenic substrate. Specifically, the enzyme-labeled avidin may be horseradish peroxidase-labeled avidin, and the chromogenic substrate may be 3,3', 5' -tetramethylbenzidine or o-phenylenediamine.
In one embodiment of the application, the kit further comprises at least one of a dilution buffer and an rnase inhibitor. The dilution buffer can dissolve components to be dissolved in the kit, can be used in a subsequent reaction system, and can also serve as a negative control in the detection of the kit. In the application, the dilution buffer does not react with each component in the kit, and can dissolve the components to be dissolved in the kit, and the specific composition of the dilution buffer can be selected according to the needs. In one embodiment of the application, the pH of the dilution buffer is between 6.5 and 6.9. Not only can dissolve the components in the kit, but also can not destroy the activity of the components. In particular, the pH of the dilution buffer may be, but is not limited to, 6.5, 6.6, 6.7, 6.8 or 6.9, etc. In one embodiment of the application, the dilution buffer comprises sodium 4-hydroxyethylpiperazine ethanesulfonate (HEPES-Na), potassium chloride (KCl), magnesium chloride (MgCl 2) and glycerol (C 3H8O3). The solvent of the dilution buffer may be water, such as ultrapure water, deionized water, double distilled water, or the like. In one embodiment, the dilution buffer comprises 18mM-21mM (e.g., 18mM, 19mM, 20mM, 21mM, etc.) sodium salt of 4-hydroxyethylpiperazine ethanesulfonate, 45mM-58mM (e.g., 46mM, 47mM, 49mM, 50mM, 52mM, 53mM, 55mM, 56mM, etc.), 4.5mM-6.3mM (e.g., 4.5mM, 4.8mM, 5mM, 5.1mM, 5.5mM, 5.8mM, 6mM, 6.2mM, etc.), and 4.5% v/v-5.5% v/v (e.g., 4.6% v/v, 4.8% v/v, 5% v/v, 5.1% v/v, 5.2% v/v, or 5.5% v/v, etc.) magnesium chloride. It is understood that glycerol in the range of 4.5% v/v to 5.5% v/v means that the volume fraction of glycerol in the dilution buffer is 4.5% to 5.5%. The diluted buffer solution is split-packed according to the requirement and stored at a low temperature (e.g. 4 ℃) for standby.
In one embodiment of the application, the minimum detection limit of the kit for detecting the novel coronavirus SARS-CoV-2 is 150copies/mL. The kit provided by the application has low detection limit on the novel coronavirus SARS-CoV-2, can realize accurate, efficient and specific detection, and is beneficial to infection prevention and control.
In one embodiment of the application, the sgRNA composition or kit further comprises a fourth sgRNA, the sequence of which comprises the nucleotide sequence shown in SEQ ID NO. 4 (GACCACCCCAAAAAUGAAGGGGACUAAAACGUUGGUGGUGCAGGAGGCAUUG). The fourth sgRNA can identify human conserved gene GAPDH (glyceraldehyde-3-phosphate dehydrogenase), thereby ensuring that human samples are collected in samples to be detected in the detection process, preventing false negative, ensuring that the samples to be detected have no problem and improving the detection accuracy.
The application provides a novel method for detecting coronavirus SARS-CoV-2, which comprises the following steps: the sgRNA composition in any of the above embodiments or the kit for detecting novel coronavirus SARS-CoV-2 in any of the above embodiments is used to detect a sample to be detected. In one embodiment of the present application, the present application provides a method for detecting SARS-CoV-2, a novel coronavirus for the purpose of non-disease diagnosis. For example, the method can detect whether the novel coronavirus SARS-CoV-2 exists in the external environment, such as the surface of an object, and the method can be applied to the fields of customs detection, logistics detection and the like.
In one embodiment of the application, the first sgRNA, the second sgRNA, the third sgRNA, and the Cas13a protein are mixed and incubated to form an RNP complex; and mixing the sample to be tested with RNP (ribonucleoprotein) complex and a reporter gene to form a reaction system, reacting to obtain a fluorescent signal value and analyzing the result.
In one embodiment of the application, cas13a protein is solubilized with a dilution buffer to form a Cas13a protein solution to facilitate incubation. In one embodiment, the concentration of Cas13a protein solution may be 500pmol/L to 1000pmol/L. Specifically, the concentration of the Cas13a protein solution may be, but is not limited to, 600pmol/L, 700pmol/L, 750pmol/L, 800pmol/L, 900pmol/L, 950pmol/L, etc. Cas13a protein solution can be stored under-zero (e.g., -20 ℃) and thawed for later use at low temperature (e.g., -4 ℃).
In one embodiment of the application, the sgRNA composition can be stored after dissolution with water for use. The first sgRNA, the second sgRNA and the third sgRNA may be dissolved in water after being mixed, or the first sgRNA, the second sgRNA and the third sgRNA may be dissolved in water respectively; the above solution can be stored at ultralow temperature (such as-80deg.C). In one embodiment of the application, the first sgRNA is dissolved in water to form a first sgRNA stock solution. Specifically, the concentration of the first sgRNA stock solution may be 50. Mu.M-100. Mu.M (e.g., 60. Mu.M, 70. Mu.M, 75. Mu.M, 80. Mu.M, 90. Mu.M, etc.). In one embodiment of the application, the second sgRNA is dissolved in water to form a second sgRNA stock solution. Specifically, the concentration of the second sgRNA stock solution may be 50. Mu.M-100. Mu.M (e.g., 60. Mu.M, 70. Mu.M, 75. Mu.M, 80. Mu.M, 90. Mu.M, etc.). In one embodiment of the application, the third sgRNA is dissolved in water to form a third sgRNA stock solution. Specifically, the concentration of the third sgRNA stock solution may be 50. Mu.M-100. Mu.M (e.g., 60. Mu.M, 70. Mu.M, 75. Mu.M, 80. Mu.M, 90. Mu.M, etc.).
In one embodiment of the application, mixing the first, second, and third sgrnas with the Cas13a protein comprises mixing the first, second, and third sgRNA stock solutions with the Cas13a protein solution. In another embodiment of the present application, the first sgRNA stock solution, the second sgRNA stock solution, and the third sgRNA stock solution can be diluted with water to form a first sgRNA solution, a second sgRNA solution, and a third sgRNA solution, respectively, and then mixed with the Cas13a protein solution. In one embodiment of the application, the molar ratio of Cas13a protein, first sgRNA, second sgRNA, and third sgRNA when mixed is (3-4): 1:1:1, thereby ensuring binding of the first, second, third sgRNA to Cas13a protein.
In one embodiment of the application, mixing and incubating the first, second, and third sgrnas with the Cas13a protein forms an RNP complex, including mixing and incubating the first, second, and third sgRNA solutions with the Cas13a protein solution forms a solution containing the RNP complex.
In one embodiment of the application, the incubation comprises treatment at 35℃to 42℃for 10min to 15min. Specifically, the incubation temperature may be, but not limited to, 35 ℃, 36 ℃, 37 ℃, 38 ℃, 39 ℃, 40 ℃, 41 ℃ or 42 ℃ and the like, and the incubation time may be, but not limited to, 10min, 11min, 12min, 13min, 14min or 15min and the like. The incubation temperature is low, the RNP compound can be formed without special heating, for example, the container for containing liquid can be directly covered by a palm, the incubation can be realized, and the operation is simple and convenient.
In one embodiment of the present application, the reporter gene may be dissolved in water and stored for later use. In one embodiment of the application, the reporter is dissolved in water to form a reporter solution. Specifically, the concentration of the reporter solution may be 300nM to 400nM (e.g., 320nM, 330nM, 340nM, 350nM, 375nM, 380nM, 390nM, etc.).
In one embodiment of the application, the concentration of RNP complex in the reaction system is 80nM to 110nM, thereby facilitating rapid and efficient detection. Specifically, the concentration of RNP complex in the reaction system may be, but not limited to, 80nM, 85nM, 90nM, 95nM, 100nM, 105nM or 110nM, etc.
In one embodiment of the application, the concentration of the reporter gene in the reaction system is 300nM-400nM, thereby facilitating rapid and efficient detection. Specifically, the concentration of the reporter gene in the reaction system may be, but is not limited to, 310nM, 320nM, 330nM, 340nM, 350nM, 360nM, 370nM, 380nM or 390nM, etc.
In one embodiment of the application, the reaction system further comprises an RNase inhibitor for preventing degradation of the RNA sequence. Specifically, the concentration of the RNase inhibitor in the reaction system may be 0.05U/. Mu.L to 1U/. Mu.L, such as 0.05U/. Mu.L, 0.08U/. Mu.L, 0.1U/. Mu.L, 0.3U/. Mu.L, 0.5U/. Mu.L, 0.7U/. Mu.L, 0.8U/. Mu.L, or 1U/. Mu.L, etc.
In one embodiment of the application, the reaction comprises a treatment at 30℃to 60℃for 10min to 15min. Specifically, the reaction temperature may be, but not limited to, 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃ or the like, and the reaction time may be, but not limited to, 10min, 11min, 12min, 13min, 14min, 15min or the like. The reaction temperature is not high, the reaction is only needed to be carried out at normal temperature or simply heated, for example, a container for containing liquid can be directly covered by a palm, the reaction can be realized, and the operation is simple and convenient. In one embodiment, the reaction comprises a treatment at 30℃to 40℃for 10min to 15min.
In the application, the fluorescence detection is carried out on the reacted liquid, so as to obtain a fluorescence signal value, and whether the sample to be detected has the novel coronavirus SARS-CoV-2 is judged according to the fluorescence signal value. It can be understood that the reaction system without the sample to be detected has a blank fluorescence value, i.e. a background fluorescence value, and the reaction system with the sample to be detected has a post-reaction fluorescence value after the reaction, and the difference between the post-reaction fluorescence value and the blank fluorescence value is an actual fluorescence value, i.e. a fluorescence signal value. In the application, if the fluorescence signal value is more than 50, the sample to be detected is positive, namely the novel coronavirus SARS-CoV-2 is provided.
In one embodiment of the application, when a fourth sgRNA is included in the sgRNA composition or kit, the fourth sgRNA can be mixed and incubated with Cas13a protein to form a fourth RNP-containing complex; and mixing the sample to be tested with the fourth RNP complex and the reporter gene to form a reaction system, reacting to obtain a fluorescent signal value and analyzing the result. After detection, the fluorescence signal is strong (for example, the fluorescence signal value is greater than 50), which indicates that the human body sample is collected in the sample to be detected, and false negative is avoided.
Compared with the detection time of RT-PCR (generally more than 60 min), the detection method provided by the application has the advantages that the detection time is short (less than 30 min), the detection process only needs a fluorescent detection device, the price is low, the operation is simple, expensive instruments (such as an RT-PCR instrument and the like) are not needed, the detection limit is low, the sensitivity reaches the level of RT-PCR, the application prospect is better, and the detection method can be used in the aspects of laboratory detection, consumer self-screening, external environment detection and the like. Therefore, the sgRNA composition, the kit for detecting the novel coronavirus SARS-CoV-2 and the method for detecting the novel coronavirus SARS-CoV-2 can realize high-accuracy, high-sensitivity, high-specificity and rapid detection and screening of the novel coronavirus SARS-CoV-2, and can meet the daily detection requirement.
Example 1
A kit for detecting novel coronavirus SARS-CoV-2 comprises a first sgRNA (SEQ ID NO: 1), a second sgRNA (SEQ ID NO: 2) and a third sgRNA (SEQ ID NO: 3).
Example 2
A kit for detecting novel coronavirus SARS-CoV-2 comprises a first sgRNA (SEQ ID NO: 1), a second sgRNA (SEQ ID NO: 2), a third sgRNA (SEQ ID NO: 3), a Cas13a protein and a reporter gene (UUUUU).
Example 3
A kit for detecting novel coronavirus SARS-CoV-2 comprises a first sgRNA (SEQ ID NO: 1), a second sgRNA (SEQ ID NO: 2), a third sgRNA (SEQ ID NO: 3), a Cas13a protein, a reporter gene (UUUUU), a dilution buffer and an RNase inhibitor.
Effect example 1
The kit for detecting the novel coronavirus SARS-CoV-2 in the embodiment 3 is adopted, wherein 4-hydroxyethyl piperazine ethane sulfonic acid sodium salt, potassium chloride, magnesium chloride and glycerol are dissolved in double distilled water to form a dilution buffer solution, the dilution buffer solution is preserved for standby at the temperature of 4 ℃, the concentration of the 4-hydroxyethyl piperazine ethane sulfonic acid sodium salt in the dilution buffer solution is 20mM, the concentration of the potassium chloride is 50mM, the concentration of the magnesium chloride is 5mM, and the volume ratio of the glycerol is 5%; the preparation method comprises the steps of dissolving Lbu-derived Cas13a protein (purchased from the Achillea Hui Cheng organism) with a dilution buffer to prepare 500pmol/L Cas13a protein solution, preserving at-20 ℃, and thawing at 4 ℃ for later use; the first sgRNA (SEQ ID NO: 1), the second sgRNA (SEQ ID NO: 2) and the third sgRNA (SEQ ID NO: 3) were synthesized and dissolved with ultrapure water, respectively, to form a first sgRNA mother liquor, a second sgRNA mother liquor and a third sgRNA mother liquor at a concentration of 100. Mu.M. mu.L of the first, second and third stock solutions of sgRNA were mixed with 7. Mu.L of ultrapure water to form a first, second and third solution of sgRNA having a concentration of 12.5. Mu.M. The reporter gene (sequence UUUUU) is synthesized, and the 5 'end of the sequence is connected with fluorescent molecule AMCA, and the 3' end is connected with quenching molecule IABkFQ. The reporter gene was diluted with ultrapure water to a reporter gene solution having a concentration of 400 nM.
Mixing the Cas13a protein solution, the first sgRNA solution, the second sgRNA solution and the third sgRNA solution (the molar ratio of Cas13a protein to first sgRNA to second sgRNA to third sgRNA is 3:1:1), and incubating at 37 ℃ for 10min to form a solution containing RNP complex.
The detection samples comprise COVID-19 pseudovirus whole genome quality control (GW-CRBM 002, cyanine good gene), nasopharyngeal swab samples during positive infection period, infection recovery period and nasopharyngeal swab samples after infection recovery and negative samples (dilution buffer solution, a first stream sample and a second stream sample), wherein COVID-19 pseudovirus whole genome quality control is diluted into detection samples with different concentrations by deionized water (including 1.5X10 3 copies/ml sample 1, 1.5X10 2 copies/ml sample 2,1.5X 10copies/ml sample 3), nasopharyngeal swab samples during positive infection period of patients are samples 4, nasopharyngeal swab samples during positive infection recovery period of patients (samples 2-3 days after positive) are samples 5, nasopharyngeal swab samples after infection recovery of patients are samples 6, dilution buffer solution is used as sample 7 (negative control), a first stream sample detected by RT-PCR is used as sample 8, and a second stream sample detected by RT-PCR is used as sample 9.
Mixing the detection sample with RNP complex-containing solution, reporter gene solution and RNase inhibitor to form a reaction system, wherein the reaction system is shown in Table 1, reacting at 37deg.C for 15min, and reading fluorescence value after reaction at 365nm and 610nm wavelength of excitation light; wherein, the system formed by the RNP complex-containing solution, the reporter gene solution and the RNase inhibitor mixed solution in each group of reaction systems reads blank fluorescence values under the wavelength of 365nm of excitation light and 610nm of emitted light, the results are shown in Table 2, and the fluorescence values after reaction are shown in Table 3; the actual fluorescence values were obtained by subtracting the blank fluorescence values from the corresponding post-reaction fluorescence values of each group, and the results are shown in Table 4 (five sets of parallel samples were carried out for each sample). Wherein, the reaction products are placed in Syngene G, BOX F3 fluorescence imaging equipment, FIG. 1 is a fluorescent signal diagram of a first group of samples 4, the fluorescent signal can be obviously seen, FIG. 2 is a schematic diagram of a first group of different samples under fluorescence, the obvious fluorescence of samples 1,2,4 and 5 can be seen, and the samples in the rest EP tubes are not fluorescent.
TABLE 1 reaction system
| Name of the name | Volume (mu L) | Final concentration |
| Solutions containing RNP complexes | 0.6 | 100nM |
| Reporter gene solution | 1 | 400nM |
| Detecting a sample | 1 | - |
| RNase inhibitors | 1 | 2.5nM |
| Dilution buffer | 16.4 | - |
TABLE 2 blank fluorescence values
| First group of | Second group of | Third group of | Fourth group | Fifth group of | |
| Sample 1 | 2269 | 2269 | 2280 | 2276 | 2268 |
| Sample 2 | 2284 | 2274 | 2271 | 2281 | 2280 |
| Sample 3 | 2287 | 2260 | 2261 | 2269 | 2256 |
| Sample 4 | 2223 | 2223 | 2208 | 2222 | 2229 |
| Sample 5 | 2241 | 2254 | 2255 | 2236 | 2251 |
| Sample 6 | 2231 | 2239 | 2235 | 2230 | 2233 |
| Sample 7 | 2219 | 2215 | 2216 | 2230 | 2219 |
| Sample 8 | 2231 | 2234 | 2235 | 2238 | 2240 |
| Sample 9 | 2238 | 2229 | 2245 | 2232 | 2235 |
TABLE 3 fluorescence values after reaction
| First group of | Second group of | Third group of | Fourth group | Fifth group of | |
| Sample 1 | 2500 | 2501 | 2499 | 2475 | 2488 |
| Sample 2 | 2397 | 2384 | 2390 | 2387 | 2397 |
| Sample 3 | 2297 | 2264 | 2275 | 2283 | 2273 |
| Sample 4 | 2349 | 2337 | 2356 | 2353 | 2332 |
| Sample 5 | 2333 | 2351 | 2343 | 2343 | 2328 |
| Sample 6 | 2247 | 2245 | 2237 | 2239 | 2253 |
| Sample 7 | 2230 | 2233 | 2226 | 2238 | 2228 |
| Sample 8 | 2244 | 2244 | 2240 | 2257 | 2250 |
| Sample 9 | 2246 | 2238 | 2258 | 2244 | 2248 |
TABLE 4 actual fluorescence values
Meanwhile, the detection samples are detected by using a commercial kit (novel coronavirus 2019-nCoV nucleic acid detection kit, DA0990-DA0997, DA gene) (five groups of samples are subjected to detection) and the Ct value of the detection results is shown in Table 5, wherein the Ct value is positive under 35.
Table 5 commercial kit detection results Ct value
| First group of | Second group of | Third group of | Fourth group | Fifth group of | Average value of | |
| Sample 2 | 32.1 | 31.5 | 31.7 | 31.9 | 31.2 | 31.68 |
| Sample 3 | 35.1 | 34.8 | 35.6 | 35.2 | 35.3 | 35.2 |
| Sample 4 | 28.5 | 28.9 | 27.3 | 26.5 | 25.6 | 27.36 |
| Sample 5 | 32.6 | 33.6 | 32.4 | 31.8 | 33.5 | 32.78 |
| Sample 6 | 37.6 | 38.2 | 36.9 | 37.2 | 36.8 | 37.34 |
To avoid false negatives, samples 4, 5, and 6 were tested while sample 7 was used as a negative control. A fourth sgRNA (SEQ ID NO: 4) was synthesized and dissolved with ultrapure water, respectively, to form a fourth sgRNA solution having a concentration of 12.5. Mu.M. The Cas13a protein solution and the fourth sgRNA solution were mixed (molar ratio of Cas13a protein to fourth sgRNA 1:1), incubated for 10min at 37 ℃ to form a solution containing RNP complexes. Synthesizing a reporter gene (the sequence is UUUUU), wherein the 5 'end of the sequence is connected with a fluorescent molecule APC, and the 3' end of the sequence is connected with a quenching molecule IABkFQ; the reporter gene was diluted with ultrapure water to a reporter gene solution having a concentration of 400 nM. Mixing a solution containing RNP complex, a reporter gene solution, a detection sample and an RNase inhibitor according to a reaction system shown in Table 1, reacting at 37 ℃ for 15min, and reading a fluorescence value after reaction at 365nm of excitation light and 610nm of emitted light; wherein, the system formed by the RNP complex-containing solution, the reporter gene solution and the RNase inhibitor mixed solution in the reaction system reads blank fluorescence values at the wavelength of 365nm of excitation light and 610nm of emission light, the results are shown in Table 6, and the fluorescence values after the reaction are shown in Table 7; the actual fluorescence values were obtained by subtracting the blank fluorescence values from the corresponding post-reaction fluorescence values of each group, and the results are shown in Table 8 (five sets of parallel samples were carried out for each sample). Wherein, fig. 3 is a graph of the fluorescence signal of the first group of samples 4, the fluorescence signal can be clearly seen.
TABLE 6 blank fluorescence values
| First group of | Second group of | Third group of | Fourth group | Fifth group of | |
| Sample 4 | 2221 | 2225 | 2219 | 2232 | 2225 |
| Sample 5 | 2240 | 2229 | 2235 | 2237 | 2236 |
| Sample 6 | 2234 | 2231 | 2236 | 2228 | 2229 |
| Sample 7 | 2225 | 2221 | 2226 | 2219 | 2227 |
TABLE 7 fluorescence values after reaction
TABLE 8 actual fluorescence values
As can be seen from Table 8, the fluorescence value of sample 7 of the negative control is low, and the fluorescence value of sample 4-6 is much higher than that of sample 7, indicating that sample 4-6 is a valid sample, avoiding the occurrence of false negatives. As can be seen from Table 4, the fluorescence values of samples 7-9 of the negative control are low, the fluorescence values of samples 1-2 and 4-5 are far higher than the fluorescence value of the negative control group and the actual fluorescence value is larger than 50, which indicates that samples 1-2 and 4-5 are positive, and the fluorescence value of sample 3 is equivalent to the fluorescence value of the negative control group, which indicates that the novel coronavirus SARS-CoV-2 cannot be effectively detected at the concentration of 15copies/ml, and the fluorescence value of sample 6 is equivalent to the fluorescence value of the negative control group, which indicates that sample 6 is negative and is not infected with the novel coronavirus SARS-CoV-2. It can be seen that the kit and the detection method provided by the application can effectively detect the novel coronavirus SARS-CoV-2, the minimum detection limit is 150copies/ml, and other viruses (such as influenza viruses) can not be misjudged, so that the novel coronavirus SARS-CoV-2 can be efficiently and specifically detected.
As can be seen from Table 5, the detection results obtained by the detection using the currently commercial kit are almost equivalent to those obtained by the detection method provided by the present application, for example, the novel coronavirus SARS-CoV-2 in sample 4 can be detected, but the novel coronavirus SARS-CoV-2 in sample 6 can not be detected, and it can be seen that the detection kit and the detection method provided by the present application can approach the existing RT-PCR detection level.
Effect example 2
The kit for detecting the novel coronavirus SARS-CoV-2 in the embodiment 3 is adopted, wherein 4-hydroxyethyl piperazine ethane sulfonic acid sodium salt, potassium chloride, magnesium chloride and glycerol are dissolved in double distilled water to form a dilution buffer solution, the dilution buffer solution is preserved for standby at the temperature of 4 ℃, the concentration of the 4-hydroxyethyl piperazine ethane sulfonic acid sodium salt in the dilution buffer solution is 20mM, the concentration of the potassium chloride is 50mM, the concentration of the magnesium chloride is 5mM, and the volume ratio of the glycerol is 5%; the preparation method comprises the steps of dissolving Lbu-derived Cas13a protein (purchased from the Achillea Hui Cheng organism) with a dilution buffer to prepare 500pmol/L Cas13a protein solution, preserving at-20 ℃, and thawing at 4 ℃ for later use; the first sgRNA (SEQ ID NO: 1), the second sgRNA (SEQ ID NO: 2) and the third sgRNA (SEQ ID NO: 3) were synthesized and dissolved with ultrapure water, respectively, to form a first sgRNA mother liquor, a second sgRNA mother liquor and a third sgRNA mother liquor at a concentration of 100. Mu.M. mu.L of the first, second and third stock solutions of sgRNA were mixed with 7. Mu.L of ultrapure water to form a first, second and third solution of sgRNA having a concentration of 12.5. Mu.M. The reporter gene (sequence UUUUU) is synthesized, and the 5 'end of the sequence is connected with fluorescent molecule AMCA, and the 3' end is connected with quenching molecule IABkFQ. The reporter gene was diluted with ultrapure water to a reporter gene solution having a concentration of 400 nM.
Cas13a protein solution, first sgRNA solution, second sgRNA solution, third sgRNA solution were mixed (molar ratio of Cas13a protein, first sgRNA, second sgRNA, and third sgRNA was 3:1:1), incubated at 37 ℃ for 10min, forming a solution containing RNP complex. The Cas13a protein solution was incubated with the first sgRNA solution (molar ratio of Cas13a protein to first sgRNA 1:1) for 10min at 37 ℃ to form a solution containing the first RNP complex. The Cas13a protein solution was incubated with the second sgRNA solution (molar ratio of Cas13a protein to second sgRNA 1:1) for 10min at 37 ℃ to form a solution containing the second RNP complex. The Cas13a protein solution was incubated with the third sgRNA solution (molar ratio of Cas13a protein to third sgRNA 1:1) for 10min at 37 ℃ to form a solution containing a third RNP complex.
Mixing a solution containing a first RNP complex with the sample 1, the reporter solution and the RNase inhibitor to form a reaction system (experiment 1), mixing a solution containing a second RNP complex with the sample 1, the reporter solution and the RNase inhibitor to form a reaction system (experiment 2), mixing a solution containing a third RNP complex with the sample 1, the reporter solution and the RNase inhibitor to form a reaction system (experiment 3), mixing a solution containing an RNP complex with the sample 1, the reporter solution and the RNase inhibitor to form a reaction system (experiment 4), reacting the reaction systems at 37 ℃ for 15min, and reading fluorescence values after reaction at 365nm of excitation light and 610nm of emitted light; wherein, the system formed by the mixed solution of the solution containing RNP complex/the solution containing first RNP complex/the solution containing second RNP complex/the solution containing third RNP complex, the reporter gene solution and the RNase inhibitor in each group of reaction systems reads blank fluorescence values at the wavelength of 365nm of excitation light and 610nm of emission light, the results are shown in Table 10, and the fluorescence values after reaction are shown in Table 11; the actual fluorescence values were obtained by subtracting the blank fluorescence values from the corresponding post-reaction fluorescence values of each group, and the results are shown in Table 12 (five groups of parallel samples were carried out for each experiment).
TABLE 9 reaction system
TABLE 10 blank fluorescence values
| First group of | Second group of | Third group of | Fourth group | Fifth group of | |
| Experiment 1 | 2272 | 2269 | 2276 | 2271 | 2275 |
| Experiment 2 | 2280 | 2269 | 2271 | 2276 | 2277 |
| Experiment 3 | 2277 | 2269 | 2273 | 2272 | 2279 |
| Experiment 4 | 2273 | 2271 | 2281 | 2269 | 2275 |
TABLE 11 fluorescence values after reaction
| First group of | Second group of | Third group of | Fourth group | Fifth group of | |
| Experiment 1 | 2280 | 2271 | 2289 | 2275 | 2288 |
| Experiment 2 | 2297 | 2284 | 2290 | 2287 | 2287 |
| Experiment 3 | 2286 | 2273 | 2282 | 2288 | 2289 |
| Experiment 4 | 2520 | 2501 | 2496 | 2495 | 2498 |
TABLE 12 actual fluorescence values
It can be seen that when the first sgRNA, the second sgRNA or the third sgRNA is used alone for detection, the sample cannot be effectively detected, but the method of combining the first sgRNA, the second sgRNA and the third sgRNA can effectively detect the novel coronavirus SARS-CoV-2, and has the advantages of high specificity, low detection limit, simple operation of the whole detection process, easy reaction conditions and short reaction time, thereby realizing the efficient and specific rapid detection of the novel coronavirus SARS-CoV-2; the kit and the detection method provided by the application have wide application prospects.
The foregoing examples illustrate only a few embodiments of the application and are described in detail herein without thereby limiting the scope of the application. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the application, which are all within the scope of the application. Accordingly, the scope of protection of the present application is to be determined by the appended claims.
Claims (5)
1. The application of the sgRNA composition in preparing a kit for detecting novel coronavirus SARS-CoV-2 is characterized in that the sgRNA composition consists of a first sgRNA, a second sgRNA and a third sgRNA, wherein the sequence of the first sgRNA is SEQ ID NO. 1, the sequence of the second sgRNA is SEQ ID NO. 2, and the sequence of the third sgRNA is SEQ ID NO. 3.
2. The kit for detecting the novel coronavirus SARS-CoV-2 is characterized by comprising an sgRNA composition, lbu Cas a protein and a reporter gene, wherein the sgRNA composition consists of a first sgRNA, a second sgRNA and a third sgRNA, the sequence of the first sgRNA is SEQ ID NO. 1, the sequence of the second sgRNA is SEQ ID NO. 2, the sequence of the third sgRNA is SEQ ID NO. 3, the sequence of the reporter gene is UUUUU, the 5 'end of the sequence of the reporter gene is connected with a fluorescent molecule, and the 3' end of the sequence of the reporter gene is connected with a quenching molecule.
3. The kit for detecting novel coronavirus SARS-CoV-2 as recited in claim 2, further comprising at least one of a dilution buffer and an rnase inhibitor;
The pH value of the dilution buffer is 6.5-6.9;
The dilution buffer comprises 18mM-21mM of 4-hydroxyethyl piperazine ethane sulfonic acid sodium salt, 45mM-58mM of potassium chloride, 4.5mM-6.3mM of magnesium chloride and 4.5% v/v-5.5% v/v of glycerol.
4. A method for detecting SARS-CoV-2, a novel coronavirus for the purpose of non-disease diagnosis, comprising:
Mixing and incubating a first sgRNA, a second sgRNA, a third sgRNA with a Lbu Cas a protein to form an RNP complex, the first sgRNA having the sequence of SEQ ID No. 1, the second sgRNA having the sequence of SEQ ID No. 2, the third sgRNA having the sequence of SEQ ID No. 3, the first, second, third sgrnas being mixed with the Lbu Cas a protein, the molar ratio of the Lbu Cas a protein, the first, second and third sgrnas being (3-4): 1:1:1, the incubating comprising treating at 35 ℃ to 42 ℃ for 10min to 15min;
Mixing a sample to be detected with the RNP complex and a reporter gene to form a reaction system, wherein the sequence of the reporter gene is UUUUU, the 5 'end of the sequence of the reporter gene is connected with a fluorescent molecule, the 3' end of the sequence of the reporter gene is connected with a quenching molecule, a fluorescent signal value is obtained after reaction, whether the sample to be detected contains novel coronavirus SARS-CoV-2 is judged by analyzing the fluorescent signal value, wherein the reaction system without the sample to be detected has a blank fluorescent value, the reaction system with the sample to be detected has a post-reaction fluorescent value after reaction, the difference between the post-reaction fluorescent value and the blank fluorescent value is the fluorescent signal value, and if the fluorescent signal value is more than 50, the fluorescent signal value indicates that the sample to be detected contains novel coronavirus SARS-CoV-2, and the reaction comprises treating for 10min-15min at 30-60 ℃.
5. The assay of claim 4, wherein the concentration of said RNP complex in said reaction system is from 80nM to 110nM;
the concentration of the reporter gene in the reaction system is 300nM-400nM.
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