Cas13a / Cas12 combined RNA virus nucleic acid detection kit and detection method
Through the RNA virus nucleic acid detection kit used in combination with Cas13a/Cas12, the cascade amplification mechanism of LbuCas13a and LbCas12 proteins is used to solve the problems of cumbersome detection and insufficient sensitivity in the prior art, and achieve high specificity and high sensitivity RNA viruses Detection, suitable for rapid detection of various RNA viruses.
Patent Information
- Application Number
- CN202411430997.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-14
- Publication Date
- 2024-12-27
AI Technical Summary
The existing RNA virus detection methods have problems such as high instrumentation and operation requirements, cumbersome operation, non-specific amplification leading to false positive results, and low detection sensitivity, and are particularly difficult to deal with micro detection.
Using the RNA virus nucleic acid detection kit used in Cas13a/Cas12, it contains the double-labeled DNA probe of LbuCas13a protein, LbCas12 protein, primer crRNA-13, primer crRNA-12, fluorophores and fluorophore quenching groups, through the virus-free The cascade of nucleic acid amplification amplifies the output signal to improve detection specificity and sensitivity.
Isothermal detection without the need for transcriptional process is achieved, false positive errors are reduced, detection specificity is high, sensitivity reaches 10fM, and there is almost no positive reaction to non-target viruses.
Smart Images
Figure CN119193926A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of virus detection technology, and specifically to an RNA virus nucleic acid detection kit and a detection method for a combination of Cas13a / Cas12. Background Art
[0002] RNA viruses are a type of virus that uses RNA as its genetic material. Many RNA viruses can infect humans and cause diseases, such as coronaviruses (such as the new coronavirus, SARS-CoV-2), flaviviruses (such as Zika virus, ZIKV; dengue virus, DENV), filoviruses (such as Ebola virus, EBOV) and influenza virus. Diseases caused by RNA viruses seriously threaten human life and health, and hinder people's normal life and socioeconomic development. Currently, there are three main detection methods for them: molecular detection (PCR nucleic acid detection), rapid antigen detection and antibody detection.
[0003] After reverse transcription, RNA can be quantitatively detected by combining PCR with real-time fluorescence detection. However, this method has high requirements for instruments and operators, cumbersome operation process, non-specific amplification, and prone to aerosol contamination and false positive results. The use of CRISPR-based detection methods further improves the specificity of RNA virus detection. In order to facilitate detection, RNA / DNA probes usually need to be labeled (such as fluorophores and quenchers), which may reduce the stability of the detection and significantly increase the cost of detection. In the CRISPR / Cas system, Cas13a was developed as a highly specific gene detection and editing tool, but its sensitivity is not high, and the detection limit can only reach the pM level, which is difficult to cope with trace detection. Summary of the invention
[0004] In order to solve the problems in the background technology, the present invention provides a Cas13a / Cas12-combined RNA virus nucleic acid detection kit and a detection method. The kit detects viral nucleic acid without amplifying the viral nucleic acid and improves the detection limit.
[0005] To achieve the above object, the first technical solution adopted by the present invention is: The Cas13a / Cas12 combined RNA virus nucleic acid detection kit comprises: LbuCas13a protein, LbCas12 protein, primer crRNA-13, primer crRNA-12, primer Target-12, and a DNA probe double-labeled with a fluorescent group and a fluorescent quenching group; Among them, the primer crRNA-13 is the guide RNA corresponding to the LbuCas13a protein, and the primer crRNA-13 sequence includes a fixed sequence and a binding sequence, wherein the binding sequence is a 20nt fragment that can be complementary to the RNA of the RNA virus, and the binding sequence is located at the 3' end of the fixed sequence; Primer crRNA-12 is the guide RNA corresponding to the LbCas12 protein, and its sequence is shown in SEQ ID NO.2; Primer Target-12 is the target substrate corresponding to the LbCas12 protein, and its sequence is shown in SEQ ID NO.3.
[0006] Preferably, the RNA virus comprises any one of a coronavirus, a flavivirus, a filovirus and an influenza virus.
[0007] Preferably, when the RNA virus is a new coronavirus, the primer crRNA-13 sequence is as shown in SEQ ID NO.1.
[0008] Preferably, the molar ratio of LbuCas13 protein, LbCas12a protein, primer crRNA-13, primer crRNA-12, RNA to be tested, primer Target-12, and DNA probe is 1:1:1:1:(10 -7 ~0.1):1:(2~10).
[0009] Preferably, the sequence of the DNA probe is shown as SEQ ID NO.4.
[0010] Preferably, it also comprises 10×LbuCas13a buffer, consisting of 200 mM HEPES, 2 M KCl, 50% glycerol, and 10 mM DTT at pH = 7.0.
[0011] The second technical solution adopted by the present invention is: The RNA virus nucleic acid detection method of Cas13a / Cas12 combined for non-diagnostic purposes, using the RNA virus nucleic acid detection kit combined with Cas13a / Cas12 described in any of the first technical solutions for detection, comprises the following steps: Provide a DNA probe double-labeled with LbuCas13a protein, LbCas12 protein, primer crRNA-13, primer crRNA-12, primer Target-12, a fluorescent group and a fluorescent quenching group; The LbuCas13a protein, primer crRNA-13, and primer crRNA-12 were mixed, incubated at room temperature for 15 minutes, and then the RNA to be tested was added, and incubated at 37° C. for 20 to 60 minutes to obtain a first mixed solution; The LbCas12 protein, the primer Target-12, the DNA probe double-labeled with the fluorescent group and the fluorescent quenching group are mixed evenly with the first mixed solution, incubated at 37°C for 10-90 minutes, and then incubated at 70°C for 10-60 minutes to obtain a second mixed solution; Determining the peak fluorescence intensity of the second mixed solution; The presence of RNA virus and the content of RNA virus when RNA virus exists are determined according to the peak value of the fluorescence intensity of the second mixed solution.
[0012] Preferably, the preparation method of primer crRNA-13 / crRNA-12 is as follows: 4 μL 10 μM crRNA-13 DNA template chain / crRNA-12 DNA template chain is mixed with 4 μL 10 μM promoter and 4 μL 10X phi 29 buffer, and then added to 10 μL ultrapure water. After incubation at 90°C for 5 minutes, incubate at 37°C for 30 minutes, continue to add 0.5 μL phi 29 pol and 2 μL dNTP and incubate at 37°C for 30 minutes, then add 8 μL 5X Trans Buffer, 4 μL rNTP, and 1 μL 20 U / μL T7 pol and incubate at 37°C overnight for 12 to 18 hours; after the incubation is completed, 8 μL 5X DNase I buffer and 4 μL DNase I pol are added to the system and incubated at 37°C for 0.5 to 4 hours, and finally incubated at 75°C for 10 minutes.
[0013] Preferably, the DNA template chain of crRNA-12 is MB-12, and the sequence is shown in SEQ ID NO.6.
[0014] When the RNA virus is the new coronavirus, the DNA template chain of crRNA-13 is MB-13, and the sequence is shown in SEQ ID NO.5.
[0015] Preferably, the preparation method of primer Target-12 is: the sense strand and antisense strand of Target-12 are mixed evenly in a molar ratio of 1:1, hybridized at 90° C. for 10 min, and then cooled at room temperature.
[0016] Preferably, the positive strand of Target-12 is MB-Z, and the sequence is shown in SEQ ID NO.7; the positive strand of Target-12 is MB-F, and the sequence is shown in SEQ ID NO.8.
[0017] Compared with the prior art, the present invention has the following beneficial effects: 1) The kit of the present invention does not require a transcription process during the detection process, and can perform isothermal detection.
[0018] 2) During the detection process of the kit of the present invention, there is no need to amplify the viral nucleic acid, which effectively reduces false positive errors and outputs signals through the cascade amplification of LbuCas13-LbCas12.
[0019] 3) The detection method of the present invention has high specificity and is designed to detect viral RNA, and will hardly produce a positive reaction to other non-target viruses.
[0020] 4) The detection method of the present invention has high sensitivity, and the minimum detection limit can be as low as 10fM. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Figure 1 It is a schematic diagram of the principle of the method for detecting RNA virus nucleic acid provided by the present invention; the green irregular ellipse represents LbuCas13a; the blue irregular ellipse represents LbCas12; FAM, fluorescent dye group; BHQ1, quenching group; Figure 2 This is a schematic diagram of the principle of detecting RNA virus nucleic acid in Comparative Example 2; Figure 3 is the standard curve drawn in Example 4; Figure 4 This is the result of detecting the novel coronavirus pseudovirus on food packaging in Example 5; Figure 5 This is the result of the selective test on other coronavirus pseudovirus RNA in Example 6. DETAILED DESCRIPTION
[0022] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with specific embodiments.It should be understood that these descriptions are exemplary only, and are not intended to limit the scope of the present invention.In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present invention.Unspecified conditions in the embodiments are carried out according to the conditions of normal conditions or manufacturer recommendations.Reagents used or instruments that do not specify manufacturers are conventional products that can be purchased and obtained commercially.
[0023] The principle of the present invention for detecting viral nucleic acid is: crRNA-13 that can be complementary to it is designed according to the specific RNA sequence of viral nucleic acid, crRNA-13 can be combined with LbuCas13a, and when the target virus specific RNA exists, it is accurately identified and combined by crRNA-13, and the conformation of LbuCas13a protein is changed, and the "side cutting" activity of indiscriminately cutting RNA is activated. Due to the influence of "side cutting", the crRNA-12 in the detection system is cracked, and it is impossible to combine with LbCas12 and identify Target-12, and the "side cutting" activity of LbCas12 will not be opened, and the DNA probes of fluorescent groups and quenching groups are modified at both ends, i.e. TG-Reporter cannot be cut, resulting in a decrease in fluorescence intensity; on the contrary, when there is no target virus specific RNA, TG-Reporter is cut by activated LbCas12, and high fluorescence is shown.
[0024] It should be noted that the RNA of the target RNA virus to be detected is also referred to as Target-13 below, and its sequence can be artificially designed according to different virus detection needs. The following specific embodiments only take the new coronavirus as an example. The Target-13 sequence used in the following specific embodiments is 5'-CGCATTACGTTTGGTGGACC-3', which is synthesized by Shanghai Sangon Biotechnology Co., Ltd. and transcribed.
[0025] The detection kit and the detection method of the present invention can be used to detect relevant RNA viruses in food, and can also be used for relevant RNA virus detection in clinic.
[0026] The first embodiment of the present invention provides a Cas13a / Cas12 combined RNA virus detection kit, comprising: LbuCas13a protein, LbCas12 protein, primer crRNA-13, primer crRNA-12, primer Target-12, a DNA probe double-labeled with a fluorescent group and a fluorescent quenching group; Among them, the primer crRNA-13 is the guide RNA corresponding to the LbuCas13a protein, and the primer crRNA-13 sequence includes a fixed sequence and a binding sequence, wherein the binding sequence is a 20nt fragment that can be complementary to the RNA of the RNA virus, and the binding sequence is located at the 3' end of the fixed sequence; Primer crRNA-12 is the guide RNA corresponding to the LbCas12 protein, and its sequence is shown in SEQ ID NO.2; Primer Target-12 is the target substrate corresponding to the LbCas12 protein, which is a double-stranded DNA, and its sequence is shown in SEQ ID NO.3.
[0027] Understandably, the sequence of primer crRNA-13 varies depending on the RNA virus to be detected, and different RNA viruses contain specific RNA fragments with different genetic information, and the binding site of primer crRNA-13 is designed according to these specific RNA fragments. The sequence of primer crRNA-13 is expressed as: 5'-fixed sequence-binding sequence-3'.
[0028] The detection kit of the present invention is suitable for all RNA virus detection, such as coronavirus (such as new coronavirus, SARS-CoV-2), flavivirus (such as Zika virus, ZIKV; dengue virus, DENV), filovirus (such as Ebola virus, EBOV) and influenza virus (Influenza virus), etc. When the RNA virus is new coronavirus (SARS-CoV-2), the primer crRNA-13 sequence is shown in SEQ ID NO.1.
[0029] crRNA is transcribed from a DNA template, and Target-12 is hybridized by two complementary DNA chains, which can reverse complement the binding end of crRNA-12. After crRNA activates the corresponding LbuCas13a and / or LbCas12, the activated LbuCas13a and / or LbCas12 can "side-cut" the corresponding intermediates and TG-Reporter.
[0030] It should be noted that the fluorescent group and quenching group in the double-labeled DNA probe (TG-Reporter) of the fluorescent group and the fluorescent quenching group can be any common group that can be matched and modified on the nucleic acid molecule, and the excitation and emission wavelengths are adaptively adjusted by the technicians in this field according to the different groups. For example, the ROX red fluorescent group can be matched with the BHQ2 quenching group, with an excitation wavelength of 585nM and an emission wavelength of 605nM; the fluorescent group FAM can be matched with the BHQ1 quenching group, with an excitation wavelength of 450~500nM and an emission wavelength of 500~600nM.
[0031] A second embodiment of the present invention provides a method for detecting RNA viruses using a combination of Cas13a / Cas12 for non-diagnostic purposes, using the RNA virus detection kit combined with Cas13a / Cas12 described in any of the first embodiments for detection, comprising the following steps: Provide a DNA probe double-labeled with LbuCas13a protein, LbCas12 protein, primer crRNA-13, primer crRNA-12, primer Target-12, a fluorescent group and a fluorescent quenching group; The LbuCas13a protein, primer crRNA-13, and primer crRNA-12 were mixed, incubated at room temperature for 15 minutes, and then the RNA to be tested was added, and incubated at 37° C. for 20 to 60 minutes to obtain a first mixed solution; The LbCas12 protein, the primer Target-12, the DNA probe double-labeled with the fluorescent group and the fluorescent quenching group are mixed evenly with the first mixed solution, incubated at 37°C for 10-90 minutes, and then incubated at 70°C for 10-60 minutes to obtain a second mixed solution; Determining the peak fluorescence intensity of the second mixed solution; The presence of RNA virus and the content of RNA virus when RNA virus exists are determined according to the peak value of the fluorescence intensity of the second mixed solution.
[0032] In some specific embodiments, taking the novel coronavirus as an example, the RNA virus detection method using Cas13a / Cas12 in combination comprises the following steps: 1) 4 μL of primers MB-12 (10 μM) and MB-13 (10 μM) were mixed with 4 μL of primer promoter (10 μM) and 4 μL of 10X phi 29 buffer, respectively, and added to 10 μL of ultrapure water. After incubation at 90°C for 5 min, incubation at 37°C for 30 min, 0.5 μL of phi 29 pol and 2 μL of dNTP were added and incubated at 37°C for 30 min. Then, 8 μL of 5X TransBuffer, 4 μL of rNTP, and 1 μL of T7 pol (20 U / μL) were added and incubated at 37°C overnight for 12-18 h. After the incubation is completed, 8 μL 5X DNase I buffer and 4 μL DNase I pol are added to the system and incubated at 37°C for 0.5-4 h to remove the DNA template. Finally, the system is incubated at 75°C for 10 min to inactivate the enzyme in the system to obtain the corresponding crRNA-13 and crRNA-12.
[0033] 2) LbuCas13a, crRNA-13, and crRNA-12 were mixed in a 1:1:1 molar ratio in 10×LbuCas13a buffer with a pH value of 7.5±0.5 and 24.4uL ultrapure water. After incubation at room temperature for 15 minutes, the mixture was incubated in a 1:1:1 ratio of LbuCas13:crRNA-13:crRNA-12:new coronavirus RNA. -7 ~0.1) and incubate at 37°C for 20-60 min to obtain a first mixed solution.
[0034] 3) MB-Z and MB-F were mixed evenly in a 1:1 molar ratio, hybridized at 90°C for 10 min, and then cooled at room temperature to obtain Target-12. LbCas12: DNA probe: Target-12 = 1: (2-10): 1 molar ratio was mixed in 10×LbuCas13a buffer at pH 7.5±0.5, mixed with the first mixed solution, incubated at 37°C for 10-90 min, and then incubated at 70°C for 10-60 min to obtain a second mixed solution.
[0035] 4) Take 20~50μL of the incubation product in step 3) into a black 384-well plate and read the fluorescence.
[0036] A standard curve can be obtained by adding different amounts of SARS-CoV-2 RNA standard (i.e., Target-13) and reading the fluorescence energy. The fluorescence peak value can be substituted into the standard curve using the actual sample to test the fluorescence peak value using this method and then calculated to calculate the corresponding mass concentration of SARS-CoV-2, thus providing a reference for the detection of SARS-CoV-2.
[0037] In the above steps, MB-Z and MB-F are the sense strand and antisense strand constituting Target-12, respectively, Promoter is the promoter, MB-13 is the DNA template strand of crRNA-13, and MB-12 is the DNA template strand of crRNA-12.
[0038] Preferably, the overnight incubation time in step 1) is 16 hours, and the enzyme inactivation incubation time is 2 hours.
[0039] Preferably, the pH value of the LbuCas13a buffer in step 2) is 7.0, and the composition is 20mM HEPES, 200mM KCl, 5% glycerol and 1mM DTT. Specifically, the final concentration of LbuCas13a, LbCas12a, crRNA-13 and crRNA-12 is 100nM.
[0040] Preferably, the molar ratio of LbCas12, DNA probe, and Target-12 in step 3) is 1:10:1, and the incubation is 30 min at 37°C and 10 min at 70°C. Specifically, the final concentrations of TG-Reporter and Target-12 are 1 μM and 200 nM, respectively.
[0041] In order to make the technical solution of the present invention clearer, the novel coronavirus detection kit, detection method and detection effect of the combined use of Cas13a / Cas12 are described in detail through multiple specific embodiments below.
[0042] Some of the sequences involved in the following examples are shown in Table 1, where FAM represents a modified fluorescent group and BHQ1 represents a modified quenching group. The nucleotide sequences in Table 1 were commissioned to a professional synthesis company, Shanghai Shenggong Biotechnology Co., Ltd.
[0043] Table 1 Related sequences .
[0044] Example 1 Preparation of crRNA-13 In this embodiment, the crRNA-13 solution is prepared in the following steps: Add 4 μL of 10 μM promoter stock solution, 4 μL of 10 μM crRNA-13 DNA template chain solution (MB-13) and 4 μL of 10X phi 29 buffer to 10 μL ultrapure water, shake well, anneal at 90°C for 5 minutes, and then place at 37°C for 30 minutes, add 0.5 μL phi 29 pol and 2 μL dNTP, shake well, incubate at 30°C for 30 minutes, add 1 μL T7 pol (20 U / μL), 8 μL 5X Trans Buffer and 4 μL rNTP mixture, mix well, incubate at 37°C for 16 hours, add 8 μL 5X DNase I buffer and 4 μL DNase I pol was incubated at 37°C for 2 h to remove the DNA template, and finally incubated at 75°C for 10 min to inactivate the enzyme in the system to obtain a crRNA-13 solution. The sequence of crRNA-13 is 5'-TAGACCACCCCAAAAATGAAGGGGACTAAAACGGTCCACCAAACGTAATGCG-3', as shown in SEQ ID NO.1.
[0045] Example 2 Preparation of crRNA-12 In this embodiment, the crRNA-12 solution is prepared in the following steps: Add 4 μL of 10 μM promoter stock solution, 4 μL of 10 μM crRNA-12 DNA template chain solution (MB-12) and 4 μL of 10X phi 29 buffer to 10 μL of ultrapure water, shake well, anneal at 90°C for 5 minutes, and then place at 37°C for 30 min, add 0.5 μL of phi 29 pol and 2 μL of dNTP, shake well, incubate at 30°C for 30 min, add 1 μL of T7 pol (20 U / μL), 8 μL of 5X Trans Buffer and 4 μL of rNTP mixture, mix well, incubate at 37°C for 16 h, add 8 μL of 5X DNase I buffer and 4 μL of DNase I pol was incubated at 37°C for 2 h to remove the DNA template, and finally incubated at 75°C for 10 min to inactivate the enzyme in the system to obtain a crRNA-12 solution. The sequence of crRNA-12 was 5'-TAATTTCTACTAAGTGTAGATCGAGAATGGCTGCGCCATAC-3', as shown in SEQ ID NO.2.
[0046] Example 3 Preparation of target dsDNA Target-12 In this embodiment, the target dsDNA solution is prepared in the following steps: 20 μL of 10 μM MB-Z solution and 20 μL of 10 μM MB-F solution were mixed, shaken, incubated at 90°C for 10 min, and naturally cooled to room temperature to obtain a dsDNA solution, i.e., Target-12 solution. The sequence of Target-12 is 5'-GTATGGCGCAGCCATTCTCG-3', as shown in SEQ ID NO.3.
[0047] Example 4 Drawing a standard curve In this embodiment, the standard curve solution is drawn, and the steps are as follows: 1) Prepare the novel coronavirus pseudovirus standard solution Prepare 2 μL of new coronavirus pseudovirus standard solutions with RNA contents of 0, 10fM, 100fM, 1pM, 10pM, 100pM, 1nM, 10nM, and 100nM respectively.
[0048] 2) Draw a standard curve ① Take 2 μL of the 2uM crRNA-13 solution prepared in Example 1, the 2uM crRNA-12 solution prepared in Example 2, and the new coronavirus pseudovirus standard solution, mix them, add 0.4 μL of 10 μM LbuCas13a, 4 μL of 10×LbuCas13a buffer, pH 7.5, and 24.4 μL of ultrapure water, mix them, and incubate at room temperature for 15 min to obtain a first mixed solution.
[0049] ② Take 0.8 μL of Target-12 solution with a concentration of 5 uM in Example 4, 0.4 μL of LbCas12 solution with a concentration of 10 μM, and 4 μL of TG-Reporter solution with a concentration of 10 μM, and mix them with the first mixed solution in 10×LbuCas13a buffer at pH 7.5±0.5, shake well, incubate at 37°C for 30 min, and then incubate at 70°C for 60 min to obtain a second mixed solution. Take 40 uL of the second mixed solution, measure its fluorescence intensity, and record the peak fluorescence intensity corresponding to the new coronavirus pseudovirus standard solution.
[0050] ③ Use the new coronavirus pseudovirus standard solutions of 0, 10fM, 100fM, 1pM, 10pM, 100pM, 1nM, and 10nM respectively to replace the total new coronavirus pseudovirus standard solution in step ①, repeat the operations of steps ① and ②, and obtain a series of fluorescence intensity peaks of the new coronavirus pseudovirus standard solutions. The molar concentration of the new coronavirus pseudovirus standard solution is used as the abscissa, and the difference between the fluorescence intensity peak of the new coronavirus pseudovirus standard solution and the fluorescence intensity of group 0 is used as the ordinate to draw a standard curve. The regression equation of the fitted standard curve is y = 3416.03x + 50177.10, R 2 = 0.992, where y represents the difference in fluorescence intensity and x represents the molar concentration of the new coronavirus pseudovirus. The standard curve is as follows Figure 3 shown.
[0051] Based on the standard curve, the detection limit of this method was 10 fM.
[0052] Example 5 Detection of novel coronavirus pseudovirus on food packaging In this embodiment, the pseudo-COVID-19 virus is added to the outer packaging of food and tested, and the steps are as follows: 1) Take several pieces of food packaging, apply 0, 10fM, 100fM, and 100pM of the new coronavirus pseudovirus solution respectively, rinse with saline, mix well, and use a viral DNA / RNA extraction kit to extract RNA, quantify it to 2μL with saline, and record them as 1# to 4# sample solutions respectively.
[0053] 2) Detection of novel coronavirus pseudovirus sample solution ① Mix 2 μL of 1# sample solution with 2 μL of 2uM crRNA-13 solution prepared in Example 1 and 2 μL of 2uM crRNA-12 solution prepared in Example 2, add 0.4 μL of 10 μM LbuCas13a, 4 μL of 10×LbuCas13a buffer, pH 7.5, and 24.4 μL of ultrapure water, mix, and incubate at room temperature for 15 min to obtain a first mixed solution.
[0054] ② Take 0.8 μL of Target-12 solution with a concentration of 5 uM in Example 4, 0.4 μL of LbCas12 solution with a concentration of 10 μM, and 4 μL of TG-Reporter solution with a concentration of 10 μM, mix with the first mixed solution in 10×LbuCas13a buffer at pH 7.5±0.5, shake well, incubate at 37°C for 30 min, and incubate at 70°C for 60 min to obtain a second mixed solution. Take 40 uL of the second mixed solution to measure its fluorescence intensity, and record the peak fluorescence intensity corresponding to the new coronavirus pseudovirus standard solution.
[0055] ③ Use 2#~4# sample solutions to replace 1# sample solution in step ①, repeat steps ① and ②, obtain the peak fluorescence intensity of 2#~4# sample solutions and record them, subtract the peak fluorescence intensity of 2#~4# sample solutions from the peak fluorescence intensity of 1# sample solution, substitute the difference data into the standard curve, and calculate the content of new coronavirus in 2#~4# sample solutions. The results are as follows Figure 4 shown. Figure 4 The "recovery rate" on the vertical axis refers to the ratio of the new coronavirus content calculated by the standard curve to the actual content of the added standard new coronavirus. The recovery rate reflects the degree of conformity between the test results of the embodiment and the actual situation. The closer the recovery rate is to 100%, the more consistent the test results are with the actual situation. If it is >100%, it means that the test results are higher than the actual situation, otherwise it is lower.
[0056] Example 6 Specificity of the method of the present invention In this example, the selectivity of the detection method provided by the present invention for other coronaviruses was investigated, and the steps were as follows: (1) Use ultrapure water to prepare 2 μL of 1 nM solutions of SARS-CoV-2, SARS-CoV, and MERS-CoV.
[0057] (2) 2 μL of the above-mentioned different types of pseudovirus solutions were mixed with 2 μL of the 2 uM crRNA-13 solution prepared in Example 1 and 2 μL of the 2 uM crRNA-12 solution prepared in Example 2, and 0.4 μL of 10 μM LbuCas13a, 4 μL of 10×LbuCas13a buffer and 24.4 μL of ultrapure water were added thereto, mixed, and incubated at 37° C. for 30 min to obtain a first mixed solution.
[0058] (3) Take 0.8 μL of the Target-12 solution prepared in Example 4, 0.4 μL of a 10 μM LbCas12 solution and 4 μL of a 10 μM TG-Reporter solution and mix them with the first mixed solution, shake well, incubate at 37°C for 30 min, and incubate at 70°C for 60 min to obtain a second mixed solution. Take 40 uL of the second mixed solution, measure its fluorescence intensity, and record the fluorescence intensity peaks corresponding to the new coronavirus pseudovirus (SARS-CoV-2) standard solution, the atypical pneumonia pseudovirus (SARS-CoV) standard solution, and the Middle East respiratory syndrome coronavirus (MERS-CoV) standard solution.
[0059] Depend on Figure 5 It can be seen that the peak fluorescence intensity of the SARS pseudovirus and Middle East respiratory syndrome pseudovirus groups is equivalent to that of the blank control group, while the peak fluorescence intensity of the new coronavirus pseudovirus group is significantly higher than the fluorescence intensity of the SARS pseudovirus group, the Middle East respiratory syndrome pseudovirus and the blank control group (Blank), indicating that the detection method provided by the present invention is highly specific and selective for the new coronavirus.
[0060] Example 7 Signal-to-Noise Ratio Determination In this example, the signal-to-noise ratio of the method for detecting the novel coronavirus provided by the present invention was examined, and the steps were as follows: (1) Mix 2 μL of 500 pM novel coronavirus pseudovirus standard solution with 2 μL of crRNA-13 solution prepared in Example 1, crRNA-12 solution prepared in Example 2, and novel coronavirus pseudovirus standard solution, add 0.4 μL of 10 μM LbuCas13a, 4 μL of 10×LbuCas13a buffer, and 24.4 μL of ultrapure water, mix, and incubate at 37°C for 30 min to obtain a first mixed solution. Take 0.8 μL of Target-12 solution configured in Example 4, 0.4 μL of 10 μM LbCas12 solution, and 4 μL of 10 μM TG-Reporter solution and mix with the solution in the premix, shake well, and incubate at 37°C for 30 min to obtain a second mixed solution. Take 40uL of the second mixed solution, and use a fluorescence spectrometer to measure its fluorescence intensity under the conditions of an excitation wavelength of 480nm and an emission wavelength range of 510-650nm, and record the peak fluorescence intensity corresponding to the new coronavirus pseudovirus standard solution.
[0061] (2) Replace the SARS-CoV-2 pseudovirus solution in step (1) with an equal amount of ultrapure water, repeat the operation in step (1), and record the peak fluorescence intensity.
[0062] (3) Calculate the ratio of the peak fluorescence intensity measured in step (2) to that measured in step (1), that is, the signal-to-noise ratio. The results are shown in Table 2.
[0063] Comparative Example 1 In this comparative example, the crRNA-13 solution prepared in Example 1, the crRNA-12 solution prepared in Example 2, and the dsDNA solution prepared in Example 4 were used for novel coronavirus pseudovirus detection. The LbuCas13a enzyme cleavage time was 15 min, and the signal-to-noise ratio was investigated.
[0064] The experimental steps are as follows: (1) Take 2 μL of crRNA-13 solution, crRNA-12 solution and 2 μL of SARS-CoV-2 pseudovirus standard solution, mix them, add 0.4 μL of 10 μM LbuCas13a, 4 μL of 10×LbuCas13a buffer and 24.4 μL of ultrapure water, mix them, and incubate at 37°C for 15 min.
[0065] (2) Take 0.8 μL of the Target-12 solution prepared in Example 4, 0.4 μL of a 10 μM LbCas12 solution and 4 μL of a 10 μM TG-Reporter solution, mix them with the solution in step (1), shake well, incubate at 37°C for 30 min, then measure the fluorescence intensity of the reaction mixture and record the peak fluorescence intensity corresponding to the new coronavirus pseudovirus standard solution.
[0066] (3) Replace the COVID-19 pseudovirus standard solution in step (1) with an equal amount of ultrapure water, repeat steps (1) and (2), and record the peak fluorescence intensity.
[0067] (4) Calculate the ratio of the peak fluorescence intensity measured in step (3) to that measured in step (2), that is, the signal-to-noise ratio. The results are shown in Table 2.
[0068] Comparative Example 2 In this comparative example, the crRNA-13 solution prepared in Example 2 was used to perform a cascade-free, single LbuCas13a detection experiment on the new coronavirus pseudovirus to examine its signal-to-noise ratio. Figure 2 shown.
[0069] The experimental steps are as follows: (1) Take 2 μL of 20 μM crRNA-13 solution, add 0.4 μL of 10 μM LbuCas13a, 4 μL of 10×LbuCas13a buffer, 4 μL of 10 μM U5-Reporter solution and 25.6 μL of ultrapure water, mix well, and incubate at 37°C for 10 min.
[0070] (2) Take 2 μL of the new coronavirus pseudovirus standard solution and mix it with the solution in step (1), shake well, incubate at 37°C for 30 min, then measure the fluorescence intensity of the reaction mixture and record the peak fluorescence intensity corresponding to the new coronavirus pseudovirus standard solution.
[0071] (3) Replace the COVID-19 pseudovirus standard solution in step (2) with an equal amount of ultrapure water, repeat steps (1) and (2), and record the peak fluorescence intensity.
[0072] (4) Calculate the ratio of the peak fluorescence intensity measured in step (3) to that measured in step (2), that is, the signal-to-noise ratio. The results are shown in Table 2.
[0073] Table 2 Signal-to-noise ratio of Example 7 and Comparative Examples 1 and 2 .
[0074] As can be seen from Table 2, the signal-to-noise ratio of the detection method provided by the present invention is significantly higher than that of the comparative example, indicating that the detection method of the present invention has high performance such as sensitivity.
[0075] In summary, the RNA virus detection method of the present invention has high sensitivity and specificity, can be used to detect all RNA viruses, and is expected to be used for clinical and food-related RNA virus detection, with good application prospects.
[0076] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.
Claims
1. A Cas13a / Cas12-combined RNA virus nucleic acid detection kit, characterized in that: Comprising: LbuCas13a protein, LbCas12 protein, primer crRNA-13, primer crRNA-12, primer Target-12, a DNA probe double-labeled with a fluorescent group and a fluorescent quenching group; Among them, the primer crRNA-13 is the guide RNA corresponding to the LbuCas13a protein, and the primer crRNA-13 sequence includes a fixed sequence and a binding sequence, wherein the binding sequence is a 20nt fragment that can be complementary to the RNA of the RNA virus, and the binding sequence is located at the 3' end of the fixed sequence; Primer crRNA-12 is the guide RNA corresponding to the LbCas12 protein, and its sequence is shown in SEQ ID NO.2; Primer Target-12 is the target substrate corresponding to the LbCas12 protein, which is a double-stranded DNA, and its sequence is shown in SEQ ID NO.
3.
2. The RNA virus nucleic acid detection kit combined with Cas13a / Cas12 as claimed in claim 1, characterized in that, The molar ratio of LbuCas13 protein, LbCas12a protein, primer crRNA-13, primer crRNA-12, RNA to be tested, primer Target-12, and DNA probe is 1:1:1:1:(10 -7 ~0.1):1:(2~10).
3. The RNA virus nucleic acid detection kit combined with Cas13a / Cas12 as claimed in claim 1, characterized in that, The sequence of the DNA probe is shown in SEQ ID NO.
4.
4. The RNA virus nucleic acid detection kit combined with Cas13a / Cas12 as claimed in claim 1, characterized in that, Also included is 10×LbuCas13a buffer consisting of 200 mM HEPES, 2 M KCl, 50% glycerol, 10 mM DTT at pH = 7.
0.
5. The RNA virus nucleic acid detection kit combined with Cas13a / Cas12 as claimed in claim 1, characterized in that, The RNA virus includes any one of a coronavirus, a flavivirus, a filovirus and an influenza virus.
6. The RNA virus nucleic acid detection kit combined with Cas13a / Cas12 as claimed in claim 5, characterized in that, When the RNA virus is a new coronavirus, the primer crRNA-13 sequence is as shown in SEQ ID NO.
1.
7. A method for detecting RNA virus nucleic acid using a combination of Cas13a / Cas12 for non-diagnostic purposes, characterized in that: Detection is performed using a Cas13a / Cas12 combined viral nucleic acid detection kit as described in any one of claims 1 to 5, comprising the following steps: Provide a DNA probe double-labeled with LbuCas13a protein, LbCas12 protein, primer crRNA-13, primer crRNA-12, primer Target-12, a fluorescent group and a fluorescent quenching group; The LbuCas13a protein, primer crRNA-13, and primer crRNA-12 were mixed, incubated at room temperature for 15 minutes, and then the RNA to be tested was added, and incubated at 37° C. for 20 to 60 minutes to obtain a first mixed solution; The LbCas12 protein, the primer Target-12, the DNA probe double-labeled with the fluorescent group and the fluorescent quenching group are mixed evenly with the first mixed solution, incubated at 37°C for 10-90 minutes, and then incubated at 70°C for 10-60 minutes to obtain a second mixed solution; Determining the peak fluorescence intensity of the second mixed solution; The presence of the target RNA virus and the content of the target RNA virus when the target RNA virus exists are determined according to the peak value of the fluorescence intensity of the second mixed solution.
8. The RNA virus nucleic acid detection method for the non-diagnostic purpose of Cas13a / Cas12 combination as claimed in claim 7, characterized in that, The preparation method of primer crRNA-13 / crRNA-12 is as follows: 4 μL 10 μM crRNA-13 DNA template chain / crRNA-12 DNA template chain is mixed with 4 μL 10 μM promoter and 4 μL 10X phi 29 buffer, and then added to 10 μL ultrapure water. After incubation at 90°C for 5 minutes, incubation at 37°C for 30 minutes, 0.5 μL phi 29 pol and 2 μL dNTP are added and incubated at 37°C for 30 minutes, and then 8 μL 5X Trans Buffer, 4 μL rNTP, and 1 μL 20 U / μL T7 pol are added and incubated at 37°C overnight for 12~18 hours; after the incubation is completed, 8 μL 5X DNase I buffer and 4 μL DNase I pol are added to the system and incubated at 37°C for 0.5~4 hours, and finally incubated at 75°C for 10 minutes.
9. The RNA virus nucleic acid detection method for the non-diagnostic purpose of Cas13a / Cas12 combination as claimed in claim 8, characterized in that, The DNA template chain of crRNA-12 is MB-12, and the sequence is shown in SEQ ID NO.
6.
10. The RNA virus nucleic acid detection method for the non-diagnostic purpose of Cas13a / Cas12 combination as claimed in claim 7, characterized in that, The preparation method of primer Target-12 is as follows: the sense strand and antisense strand of Target-12 are mixed evenly in a molar ratio of 1:1, hybridized at 90° C. for 10 min, and then cooled at room temperature.