Probe group for detecting SARS-CoV-2 coronavirus nucleic acid and application

Abstract

The invention discloses a probe group for detecting SARS-CoV-2 coronavirus nucleic acid and application of the probe group, and belongs to the field of biological detection. The crRNA sequence for detecting the SARS-CoV-2 coronavirus nucleic acid is any one of the following sequences: LbucrRNA1, LbucrRNA2, LbucrRNA3 and LbucrRNA4 aiming at an S gene, and N1crRNA and N2crRNA aiming at an N gene of a mutant strain EG.5.1. 1, and the sequence is shown in a sequence table. Based on the capability of cutting free RNA and targeting a specific RNA sequence of the LbuCas13a protein, a colloidal carbon immunochromatography test strip is used as a visual detection mode, an immunochromatography Cas13a nucleic acid detection method is established, rapid detection of the SARS-CoV-2 coronavirus can be completed within 90 min through the method, a novel simple, convenient and rapid detection method is provided for disease prevention and control, and the method is suitable for popularization and application. And the method is simple to operate and does not need assistance of large-scale instruments and equipment.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biology detection, and specifically relates to a probe group for detecting SARS-CoV-2 coronavirus nucleic acid and an application thereof. Background Art

[0002] SARS-CoV-2 coronavirus is a new type of respiratory RNA virus with high transmission ability and mutation. Effective and accurate detection of the virus is an effective means of disease prevention and control. RT-qPCR is the gold standard for detecting SARS-CoV-2 coronavirus. This method is accurate and reliable, but it usually takes a long time and is complex to operate and depends on instruments and equipment. Therefore, the development of a low-cost, convenient and efficient method for detecting SARS-CoV-2 coronavirus variants is of vital importance to controlling respiratory diseases caused by the virus.

[0003] In terms of specificity and operability, compared with traditional PCR methods, the technology based on clustered regularly interspaced short palindromic repeats (CRISPR) has been shown to have unique advantages in molecular diagnosis. Due to the high specificity of Cas / guide-RNA in recognizing targets, CRISPR targeted detection methods have been used to identify point mutations of SARS-CoV-2 coronavirus. As a key effector in the CRISPR system, Cas13a protein has unique ribonuclease activity. Cas13a protein can accurately cut the target RNA under the guidance of CRISPR RNA (crRNA), and at the same time stimulate the non-specific cutting activity of Cas13a protein to cut RNA on the surface of Cas13a protein. LbuCas13a protein shows excellent cutting efficiency among homologous Cas13a proteins. By using LbuCas13a protein, sensitive and specific cutting of nucleic acids can be achieved. At the same time, it can be combined with immunochromatographic test strips to achieve the conversion and amplification of cutting signals, and the test results can be presented quickly and observably. It has great potential in the rapid and low-cost detection of SARS-CoV-2 coronavirus.

[0004] The interpretation method of immunochromatographic test strips for Cas13a detection of RNA viruses at home and abroad is mainly the "line method", that is, there is a band on the "T" line of the detection line, and the nucleic acid test result is judged to be positive. This type of interpretation method is easy to read the test result intuitively, that is, when the target RNA is present, the Cas13a protein plays a cutting role. When the probe is not completely cut, the complete probe will still be intercepted by the quality control line C line, and the cut free probe containing the marker will be captured and bound by the specific antibody at the detection line, so that the bands at the C line and the T line are both visible, and the result is positive. However, when the probe is completely cut and broken, there is no colloidal gold enrichment at the quality control (C) line, and there is only a band at the detection (T) line, and there is no band at the quality control line (C) line. This method does not meet the rigor of in vitro diagnosis.

[0005] To be more in line with the rigor of in vitro diagnosis, if the "line elimination method" detection mode is adopted, that is, there is no band at the "T" line of the detection line, and the nucleic acid test result is judged to be positive, the opposite thinking should be adopted when laying the specific antibody corresponding to the probe on the test strip. When the target RNA is not present, Cas13a cannot play a cutting role, and the probe is intact and can be captured by the "T line" of the test strip. Excessive antibodies labeled with colloidal carbon are captured at the C (quality control) line, so that both the C line and the T line have bands, and the result is judged to be negative; when the target RNA is present, Cas13a plays a cutting role, the probe is completely cut, and the probe containing one end of the marker is not captured by the antibody at the "T line", and is freed to the C line and captured by the C antibody, so that there is no band on the T line and there is a band on the C line, and the result is judged to be positive. Summary of the invention

[0006] The object of the present invention is to provide a probe set and application for detecting SARS-CoV-2 coronavirus nucleic acid. Based on the ability of LbuCas13a protein to cut free RNA and target specific RNA sequences, the present invention uses colloidal carbon immunochromatography test strips as a visual detection method to establish a method for immunochromatography Cas13a nucleic acid detection.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0008] The present invention first provides a probe group for detecting SARS-CoV-2 coronavirus nucleic acid, which is a crRNA sequence for detecting SARS-CoV-2 coronavirus nucleic acid, which is any one of the following: LbucrRNA1, LbucrRNA2, LbucrRNA3, LbucrRNA4 for the S gene, and N1crRNA and N2crRNA for the N gene of the mutant strain EG.5.1.1, and the sequence is as follows:

[0009]

[0010] The present invention also provides a kit for detecting SARS-CoV-2 coronavirus nucleic acid, which comprises the probe set for detecting SARS-CoV-2 coronavirus nucleic acid described in the present invention.

[0011] Furthermore, the kit for detecting SARS-CoV-2 coronavirus nucleic acid comprises a colloidal carbon immunochromatographic test strip by elimination method and a CRISPR reaction system, wherein the CRISPR reaction system comprises the probe set for detecting SARS-CoV-2 coronavirus nucleic acid of the present invention, and the immunochromatographic test strip comprises, in order of sample flow direction, a sample pad, a binding pad containing colloidal carbon-labeled rabbit anti-biotin antibody, an NC membrane containing T lines and C lines, and absorbent paper;

[0012] When the probe is FAB-polyU-11nt, the T line is formed by rabbit anti-6-FAM polyclonal antibody, and the C line is formed by goat anti-rabbit IgG;

[0013] When the probe is DB-polyU-11nt, the T line is formed by Anti-Digoxigenin antibody, and the C line is formed by goat anti-rabbit IgG;

[0014] When the probe is TB-polyU-11nt, the T line is formed by Anti-TAMRA antibody [5G5], and the C line is formed by goat anti-rabbit IgG;

[0015] When the probe is CB-polyU-11nt, the T line is formed by Anti-Cy5 antibody [CY5-15], and the C line is formed by goat anti-rabbit IgG;

[0016] The sequences of each probe are as follows:

[0017]

[0018] Furthermore, the CRISPR reaction system is as follows:

[0019] Targeting the S gene system

[0020]

[0021] Targeting N gene system

[0022]

[0023] After system 1 is configured, add it to a clean RNase-Free PCR tube, and then place it in a PCR instrument at 37°C for 10 minutes as step 1 system. After the reaction, the Cas13a / crRNA mixture is added to system 2 for step 2.

[0024] Targeting S gene or N gene system II

[0025]

[0026]

[0027] After the reaction system is configured, react at 37°C in a PCR instrument for 90 minutes, then terminate the reaction at 4°C for 2 minutes, insert the prepared colloidal carbon immunochromatography test strip, and read the test results within 5 to 10 minutes.

[0028] The present invention also provides the use of the kit in detecting SARS-CoV-2 coronavirus nucleic acid, and the use is not for the purpose of diagnosing or treating the disease.

[0029] The present invention adopts the above technical scheme, uses LbuCas13a protein as the detection effector protein, combines CRISPR-Cas13a detection with immunochromatographic test paper interpretation, and adopts the detection presentation method of "line elimination method", that is, no band at the "T" line of the test strip detection line is interpreted as positive, thereby constructing an immunochromatographic-LbuCas13a detection method.

[0030] Beneficial effects of the present invention:

[0031] 1. The colloidal carbon immunochromatographic test strip was successfully constructed and combined with the Cas13a reaction to construct the immunochromatographic-LbuCas13a detection method. Five inactivated SARS-CoV-2 coronavirus positive samples and four negative samples screened by fluorescent PCR and colloidal gold test strip methods were tested. The results showed that both positive and negative samples were successfully detected, with 100% consistency with the fluorescent PCR method and the colloidal gold method, proving the accuracy of the method.

[0032] 2. Analysis of qPCR quantification and digital PCR results determined that the minimum detection limit of the immunochromatographic LbuCas13a detection method was 381.75 copies / μL.

[0033] 3. The detection mode of the "line elimination method" combined with Cas13a adopted in the present invention does not require amplification of the target, effectively avoiding aerosol contamination generated during amplification.

[0034] 4. The sensitivity and specificity of the SARS-CoV-2 coronavirus variants were tested using the method of the present invention, demonstrating that the method can quickly, sensitively and accurately detect the SARS-CoV-2 coronavirus variants. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1It is the electrophoresis diagram of crRNA in vitro transcription template PCR product; A: M: Takara 20bp DNA ladder Marker; Lanes 1-4 represent: LwcrRNA1 in vitro transcription template, LwcrRNA2 in vitro transcription template, LwcrRNA3 in vitro transcription template, LwcrRNA4 in vitro transcription template; B: M: Takara 20bp DNA Marker; Lanes 1-3 represent: LbucrRNA1 in vitro transcription template, LbucrRNA2 in vitro transcription template, LbucrRNA3 in vitro transcription template; Lanes 4 and 5 represent the forward and reverse primer fragments without PCR, respectively; Lane 6 represents a mixture of forward and reverse primer fragments without PCR.

[0036] Figure 2 It is a diagram for detecting the activity of Cas13a protein; A: commercial LwacrRNA protein activity detection; B: LbuCas13a protein activity detection of the present invention.

[0037] Figure 3 For LbuCas13a protein activity detection; 1: negative control group; 2: experimental group containing target RNA.

[0038] Figure 4 Schematic diagram of the detection principle of single-plex CRISPR test strips.

[0039] Figure 5 For immunochromatography LbuCas13a specific detection; 0: water + probe. 1: water + probe + lbuCas13a protein; 2: water + probe + lbuCas13a protein + crRNA; 3: water + probe + lbuCas13a protein + crRNA + ssRNA.

[0040] Figure 6 This is a graph of the SARS-CoV-2 coronavirus fluorescence PCR test results and some test strip test results.

[0041] Figure 7 Laboratory samples for immunochromatographic Cas13a detection; 0: negative control; 2-6: inactivated SARS-CoV-2 coronavirus positive RNA samples determined by fluorescence PCR method No. 002-006; 1: negative sample No. 001; 7: negative sample No. 007; 8: negative sample No. 008; 9: negative sample No. 009.

[0042] Figure 8 qPCR standard curve results; A: qPCR amplification curve; B: qPCR standard curve.

[0043] Fig. 9 This is a one-dimensional result diagram of digital PCR.

[0044] Fig.10 This is the result graph of immunochromatographic Cas13a detection sensitivity; 0: negative control; inactivated SARS-CoV-2 coronavirus RNA sample; 1: 6108 copies / μL sample; 2: 3054 copies / μL sample; 3: 1527 copies / μL sample; 4: 763.5 copies / μL sample; 5: 381.75 copies / μL sample; 6: 190 copies / μL sample; 7: 9595 copies / μL sample.

[0045] Fig.11 This is a graph of the test results of inactivated SARS-CoV-2 coronavirus and influenza virus samples; the T1 line corresponds to the probe TARMA test group (the test sample is SARS-CoV-2 coronavirus); the T2 line corresponds to the Dig group test line (the test sample is influenza), 0: negative control; 1-8: SARS-CoV-2 coronavirus and influenza virus samples. DETAILED DESCRIPTION

[0046] The present invention is further described below in conjunction with the accompanying drawings and embodiments. The experimental methods without specific conditions are based on conventional experimental conditions in the art or conditions recommended by the manufacturer.

[0047] Goat anti-rabbit IgG and rabbit anti-6-FAM polyclonal antibodies were purchased from Sangon Biotechnology Co., Ltd., and Anti-Digoxigenin antibody, Anti-TAMRA antibody [5G5], and Anti-Cy5 antibody [CY5-15] were all purchased from abcam.

[0048] The LwaCas13a protein is a purchased commercial protein, and the LbuCas13a protein was obtained by self-expression and purification through the Escherichia coli cloning expression system with reference to the following literature.

[0049] [1]Liu L, Li

[0050] [2] Adler BA, Hessler T, Cress BF, Lahiri A, Mutalik VK, Barrangou R, Banfield J, Doudna JA. Broad-spectrum CRISPR-Cas13a enables efficient phage genome editing. Nat Microbiol. 2022: 7(12): 1967-1979. doi: 10.1038 / s41564-022-01258-x. Example 1 Preparation of crRNA and extraction of target RNA

[0051] (1) To verify the activity of LbuCas13a protein, the corresponding crRNA was designed based on the SARS-CoV-2 coronavirus (S gene) pseudovirus provided by our research group (by inserting the partial genome of the bacteriophage and the SARS-CoV-2 coronavirus (S gene) into the pSE380 plasmid and expressing it in Escherichia coli Er2566 to obtain pseudovirus particles). The crRNA preparation method first synthesized the RNA corresponding to the DNA Oligo, formed double-stranded DNA by PCR, and then prepared crRNA by in vitro transcription. The DNA template fragment corresponding to the RNA was synthesized by Anhui General Biotechnology Co., Ltd. The DNA template sequence is shown in Table 1.

[0052] Table 1 Synthetic DNA template sequences

[0053]

[0054] The synthesized DNA fragments were used as templates and PCR amplified (the 5' end of the upstream primer had a T3 promoter sequence) to obtain DNA fragments with a T3 promoter sequence. The primers used for amplification were synthesized by Anhui General Biological Co., Ltd., and the primer sequences are shown in Table 2.

[0055] Table 2 Primers for amplification of synthetic DNA fragments

[0056]

[0057] The PCR amplification system was configured with reference to Table 3 and Table 4 and PCR amplification was performed. The amplification product was detected by agarose gel electrophoresis.

[0058] Table 3 In vitro transcription template crRNA double-strand amplification system

[0059]

[0060] Table 4 In vitro transcription template crRNA double-strand amplification program

[0061]

[0062] (2) Extraction of SARS-CoV-2 coronavirus (S gene) pseudovirus RNA using Bio-Tech RNAeasy TM The virus RNA was extracted using a viral RNA extraction kit, the concentration was determined using Nanodrop 2000, and the cells were stored at -80°C.

[0063] (3) Purification of double-stranded DNA products from in vitro transcription templates

[0064] ① Take 150 μL of 1×TE (pH 8.0) and mix it with the above double-stranded PCR amplification product.

[0065] ② Add 5 μL of nucleic acid precipitation aid to 195 μL of the system and mix well with a pipette.

[0066] ③ Add 20 μL of 5 M sodium chloride to the mixed system and mix well by pipetting.

[0067] ④ Add 2 volumes of anhydrous ethanol to the above mixed system, mix well with a pipette, and place in a -80℃ refrigerator for 40 min.

[0068] ⑤ Take out from -80℃ refrigerator, centrifuge at 12,000×g for 10 min to collect nucleic acid, and remove supernatant with a pipette in a clean bench.

[0069] ⑥ Add 1 mL of 70% ethanol to the precipitate, gently invert, and centrifuge at 12,000×g for 1 min. Carefully discard the supernatant and open the lid to dry for 5 min to remove residual alcohol.

[0070] ⑦ Dissolve the nucleic acid precipitate with 10 μL of DEPC Treated Water solvent and let it stand for 5 minutes to obtain the purified in vitro transcription template. Use Nanodrop 2000 to measure the concentration.

[0071] Example 2 In vitro transcription of crRNA

[0072] (1) Take the purified in vitro transcription template crRNA double strand and prepare the RNA transcription reaction system. The transcription reaction system is shown in Table 5.

[0073] Table 5 RNA transcription reaction system

[0074]

[0075] The prepared mixture was placed in a 37°C incubator for 16 h.

[0076] (2) Purification of in vitro transcription products

[0077] ① Add 4 μL DNase I (100 mg / mL) to the in vitro transcription system and place it at 37°C for 15 min to remove the template DNA.

[0078] ② Add 135 μL DEPC Treated Water and 15 μL 3M sodium acetate solution (pH 5.2) to the reaction system and mix well by pipetting.

[0079] ③ Add 200 μL of phenol-chloroform (25:24:1 = phenol: chloroform: isoamyl alcohol) to the reaction system and mix well.

[0080] ④ Centrifuge the reaction mixture at 12,000×g for 10 min until upper and lower layers appear. Take the upper aqueous phase and transfer it to another clean 1.5mL centrifuge tube.

[0081] ⑤ Add 2 volumes of anhydrous ethanol to the supernatant, place in a -80°C refrigerator for 40 min, centrifuge at 12,000×g for 20 min, remove the supernatant, and collect the nucleic acid precipitate.

[0082] ⑥ Wash the nucleic acid precipitate with 1 mL of pre-cooled 70% ethanol, centrifuge at 12,000×g for 1 min, and discard the supernatant.

[0083] ⑦Resuspend the RNA precipitate in 20 μL DEPC Treated Water, measure the concentration using Nanodrop 2000, and store at -80°C.

[0084] (3) Results: crRNA was synthesized by in vitro transcription. First, the DNA sequence corresponding to crRNA was amplified by PCR to obtain a double-stranded template product containing T3 promoter. Figure 1 As shown in the figure, the theoretical value of the target product fragment is 78bp. The results show that compared with the fragment without PCR, with 20bp DNA ladder marker as a reference, the size of the target fragment product meets the theoretical value and can be used for subsequent in vitro transcription. The in vitro transcribed crRNA was purified by phenol chloroform extraction, with a concentration of about 500ng / μL, and stored at -80℃ for subsequent protein activity verification experiments.

[0085] Example 3LbuCas13a protein activity detection

[0086] (1) Verify the accuracy of crRNA preparation and reaction system. Take commercial LwaCas13a protein, S gene RNA and LwcrRNA, and configure Cas13a protein reaction system together with fluorescent reporter molecules. The sequence of fluorescent reporter molecules is shown in Table 6, and the reaction system is shown in Tables 7 and 8.

[0087] Table 6 Fluorescent reporter molecule sequences

[0088]

[0089]

[0090] Table 7 LwaCas13a protein reaction system 1

[0091]

[0092] Place the above reaction system in a PCR instrument and set the reaction program: 37°C, 10 min. This is the step 1 system. After the reaction is complete, add the reaction system according to the table below to proceed to step 2.

[0093] Table 8 LwaCas13a protein reaction system II

[0094]

[0095] In the blank control group, an equal amount of RNase-Free H2O was added to the reaction system instead of LwcrRNA, ssRNA and LwaCas13a protein. The probe used in the system was FQ-polyU-11nt. After all the reaction systems were configured, they were added to a clean black ELISA plate, pipetted and mixed, centrifuged briefly, and bubbles were removed. A fluorescence ELISA reader was used, with the excitation wavelength set to 492nm and the emission wavelength set to 518nm. The reaction procedure was: 37°C, 2h, and fluorescence was detected every 5min.

[0096] The fluorescence change results are shown in Figure 2 As shown in A. It can be seen that when the target RNA is present, the commercial LwaCas13a protein can achieve normal probe cleavage and the fluorescence signal is significantly enhanced compared with the blank control group. The above results prove that the crRNA preparation and detection system are correct and can complete the detection and signal capture of Cas13a protein.

[0097] (2) Verify the activity of LbuCas13a protein.

[0098] ① Take the purified LbuCas13a protein, S gene RNA and LbucrRNA, and configure the Cas13a protein reaction system (microplate reader detection) together with the fluorescent reporter molecule. The reaction system is consistent with Tables 7 and 8.

[0099] ② Take the purified LbuCas13a protein, S gene RNA and LbucrRNA, and configure the Cas13a protein reaction system (test strip detection) together with the fluorescent reporter molecule. The reaction system is shown in Tables 9 and 10.

[0100] Table 9 LbuCas13a protein reaction system 1

[0101]

[0102]

[0103] Place the above reaction system in a PCR instrument and set the reaction program: 37°C, 10 min. This is the step 1 system. After the reaction is complete, add the reaction system according to the table below to proceed to step 2.

[0104] Table 10 LbuCas13a protein reaction system II

[0105]

[0106] In the blank control group, an equal amount of RNase-Free H2O was added to the reaction system instead of LbucrRNA, ssRNA and LbuCas13a protein. The probe used in the system was FB-polyU-11nt. After all the reaction systems were prepared, they were added to a clean PCR tube. The above reaction system was placed in a constant temperature incubator and reacted at 37°C for 2 hours. After the reaction was completed, The Cas12 / Cas13-specific nucleic acid detection test strip is inserted into the PCR tube containing the reaction solution for detection, and the test results are read within 5 to 10 minutes.

[0107] Results Figure 2 As shown in B, the enzyme-labeled assay showed that compared with the blank control group, the fluorescence intensity of the experimental group gradually increased with the extension of the detection time, proving that the purified LbuCas13a protein had the expected protein activity.

[0108] In addition, Tiosbio's Cas12 / 13 nucleic acid test strips were used for testing. The interpretation method was as follows: positive means that both the quality control line (C line) and the test line (T line) have red strips or the test strip quality control line (C line) does not show color, and the test line (T line) shows color; negative means that a red strip appears on the quality control line (C line) and the test line (T line) does not show color; invalid means that neither the quality control line (C line) nor the test line (T line) has strips. Figure 3 As shown, compared with the negative control group, the experimental group containing target RNA showed T line color development and C line color development, which was positive according to the test strip judgment method, proving the expected activity of LbuCas13a protein.

[0109] Example 4 Preparation and Assembly of Immunochromatographic Test Strips

[0110] 1. Colloidal carbon labeled antibodies

[0111] Rabbit anti-biotin antibody was selected as the labeled antibody, and colloidal carbon was used to couple the antibody. The colloidal carbon labeled antibody kit of Beijing Nanogene Biotechnology Co., Ltd. was used in this experiment. For detailed operation steps, please refer to the kit instruction manual).

[0112] 2. Preparation of immunochromatographic test strips

[0113] The immunochromatographic test strip comprises, in order according to the sample flow direction, a sample pad, a binding pad containing colloidal carbon-labeled rabbit anti-biotin antibody, an NC membrane containing T lines and C lines, and absorbent paper;

[0114] When the probe is FAB-polyU-11nt, the T line is formed by rabbit anti-6-FAM polyclonal antibody, and the C line is formed by goat anti-rabbit IgG;

[0115] When the probe is DB-polyU-11nt, the T line is formed by Anti-Digoxigenin antibody, and the C line is formed by goat anti-rabbit IgG;

[0116] When the probe is TB-polyU-11nt, the T line is formed by Anti-TAMRA antibody [5G5], and the C line is formed by goat anti-rabbit IgG;

[0117] When the probe is CB-polyU-11nt, the T line is formed by Anti-Cy5 antibody [CY5-15], and the C line is formed by goat anti-rabbit IgG;

[0118] The specific method for preparing the immunochromatographic test strips is based on Kang Jingru's colloidal carbon immunochromatographic experimental method, with slight modifications:

[0119] (1) Sample pad treatment

[0120] Select the sample pad material, cut the sample pad material into 16mm×10cm, place the sample pad in a clean slot, and evenly add about 20mL of the pre-prepared sample pad treatment solution to the sample pad to ensure that the sample pad is fully soaked. Soak for about 10 minutes and turn it over once in the middle. Then put the sample pad in an oven to dry, set the temperature to 37℃, dry for 3 hours, and temporarily store it in a drying oven for subsequent test strip assembly.

[0121] (2) Test strip marking

[0122] The bottom plate and NC membrane were cut into 10 cm lengths, the absorbent paper was cut into 2.2 cm × 10 cm, the bottom plate protective glue was torn off, the NC membrane and absorbent paper were pasted, and the plate was placed on a slicer. The corresponding antibody or secondary antibody was diluted to an appropriate concentration with coating buffer (0.032 g Na2CO3, 0.059 g NaHCO3, 0.878 g NaCl, 5 g trehalose, 0.05 g NaN3 were weighed, 80 mL ddH2O was added to dissolve, the volume was fixed to 100 mL, and 10 mL methanol was added) and a line was drawn on the NC membrane. The drawn test strips were dried at 37 ° C for 3 h and temporarily stored in a drying oven for subsequent test strip assembly.

[0123] (3) Carbon spraying on bonding pad

[0124] Select the conjugate pad material, cut the conjugate pad material into 8mm×10cm, spray the prepared colloidal carbon-coated antibody evenly on the conjugate pad with a gun tip, and then dry the conjugate pad at 37℃ for 3h. Store in a constant temperature drying oven.

[0125] (4) Test strip assembly and chromatography

[0126] After the test strips with T and C lines are dried, they are assembled with the conjugate pad (8mm×10cm) sprayed with colloidal carbon labeled antibodies and the treated sample pad (16mm×10cm). The assembled test strips are then cut into 3.4mm×6cm using a slicer. They are then placed in an aluminum foil bag filled with a desiccant and sealed for storage.

[0127] 3. Principle of the Invention

[0128] The present invention adopts the detection mode of line elimination method, i.e., no band is detected as a positive result. The principle of nucleic acid detection of the present invention is based on Cas13a protein cutting ability, and Cas13a cutting can be characterized by free RNA. Therefore, a suitable probe can be used as the main detection object of test strip detection. When the detection target does not exist, i.e., the probe is not cut, the probe can be completely captured at the T (detection) line, thereby causing the enrichment of colloidal carbon here, thereby showing a band, i.e., proving that the target is not detected, and judging that the result detection target is negative. When the detection target exists, the probe is cut, and one end with colloidal carbon labeling is free to the C (quality control) line, and there is no colloidal carbon enrichment at the T (detection) line, and the result is judged to be positive.

[0129] In this embodiment and Figure 4 For example, the two ends of the probe marker are FAM and Biotin respectively, the liquid sample to be tested is the mobile phase, and chromatography starts from the sample pad.

[0130] At the binding pad, the Biotin at one end of the probe will bind to the colloidal carbon-labeled rabbit anti-biotin antibody; when the detection target is present, based on the detection principle of Cas13a, the probe will be cut off, and the colloidal carbon-labeled rabbit anti-biotin antibody will be freed to the C (quality control) line and captured by the goat anti-rabbit secondary antibody, forming a black color visible to the naked eye. The Anti-FAM antibody at the T (detection) line captures the other end of the broken FAM-labeled probe, but there is no colloidal carbon labeling, so there is no band.

[0131] When there is no detection target, the intact probe (containing a FAM label at one end) labeled with colloidal carbon will be captured by the Anti-FAM antibody at the T line, thereby forming a black color visible to the naked eye at the T (detection) line.

[0132] Regardless of whether the probe in the sample is cut or not, the excess colloidal carbon-labeled rabbit anti-Biotin antibody will continue to chromatograph with the sample, and the goat anti-rabbit secondary antibody on the C (quality control) line will unconditionally bind to the colloidal carbon-labeled rabbit anti-Biotin antibody to form a black line visible to the naked eye.

[0133] Example 5 LbuCas13a detection accuracy verification

[0134] (1) First, based on the Cas13a detection system in Example 3, the concentration of RNase Inbitior in the system and the concentration of RNA fluorescence reporter analysis were optimized, and the detection reaction time was optimized. After optimization, the final concentration of RNase Inbitior in the reaction system was 1U / μL, the final concentration of RNA fluorescence reporter molecule was 4nM, and the reaction time was ≥45min. The optimized LbuCas13a detection system is shown in Tables 11 and 12.

[0135] (2) Configure the Cas13a protein reaction system, set up two groups of negative controls and one group of blank controls. In the first group, an equal amount of RNase-Free H2O was added to the reaction system instead of LbucrRNA and S gene RNA. In the second group, an equal amount of RNase-Free H2O was added to the reaction system instead of ssRNA. In the blank control group, an equal amount of RNase-Free H2O was added to the reaction system instead of LwcrRNA, S gene RNA and LwaCas13a protein. After all the reaction systems are configured, add them to a clean RNase-free PCR tube and react at 37°C for 90 minutes. Insert it into the prepared test strip and read the test results within 5 to 10 minutes.

[0136] Test results are shown in Figure 5 As shown, only when LbuCas13a protein, LbucrRNA and S gene RNA are present, there is no band at the detection line, and the result is determined to be positive. When any one component is missing, there is a band at the detection line, that is, the result is determined to be negative. This result further proves the specific cleavage activity of LbuCas13a protein.

[0137] (3) Detection of inactivated samples of SARS-CoV-2 coronavirus

[0138] To ensure the accuracy of the immunochromatographic Cas13a protein detection effect, the inactivated SARS-CoV-2 coronavirus nucleic acid samples were tested using Daan Gene's SARS-CoV-2 Coronavirus 2019-nCoV Nucleic Acid Detection Kit (Fluorescence PCR Method) and Shanghai Bojie SARS-CoV-2 Coronavirus (2019-nCoV) Antigen Detection Kit (Colloidal Gold Method) to screen out positive samples, and then the immunochromatographic LbuCas13a protein detection system was configured for RNA samples according to Tables 11 and 12.

[0139] Table 11 Optimized LbuCas13a detection system 1

[0140]

[0141] After the above reaction system is prepared, add it to a clean RNase-Free PCR tube, and then place it in a PCR instrument at 37℃ for 10 minutes as step 1 system. After the reaction is completed, add the reaction system according to the table below to proceed to step 2.

[0142] Table 12 Optimized LbuCas13a detection system II

[0143]

[0144] The probe used in the system is TB-polyU-11nt (see Table 6). After the above reaction system is configured, it is placed in a constant temperature incubator and reacted at 37°C for 90 minutes. The prepared single-target colloidal carbon test strip is inserted and the test results are read within 5 to 10 minutes.

[0145] Conclusion: (1) The SARS-CoV-2 coronavirus kit (qPCR method) and the SARS-CoV-2 coronavirus (test strip method) were used to perform qPCR detection on inactivated SARS-CoV-2 coronavirus samples. The kit set up 3 groups of genes for amplification, see Figure 6 , red is the human internal reference gene, green is the new crown N gene and blue is the ORF1 gene. The qPCR test results show that the Ct values ​​of the internal reference genes are all <35, indicating that the test samples are correct. The N gene and ORF1 gene have curves running out, and the Ct values ​​are all <35. The results are interpreted as positive test results. There are bands at the T line detection and the C line detection of the test strip test method, and the results are judged as positive, otherwise negative. The result analysis showed that there were 5 positive cases and 4 negative cases in the nucleic acid test.

[0146] (2) Based on the above test results, the test samples were analyzed using the immunochromatographic Cas13a detection method constructed by the present invention. The results are shown in Figure 7As shown, the test results showed 100% consistency with the fluorescence PCR method and the test strip method, and the positive samples could be correctly detected, proving the accuracy and specificity of the immunochromatographic Cas13a detection method.

[0147] Example 6 Immunochromatography LbuCas13a Detection Sensitivity Determination

[0148] (1) qPCR quantitative experiment

[0149] In vitro transcribed RNA was used as the qPCR standard, and SARS-CoV-2 coronavirus positive sample RNA was used as the detection object for qPCR quantitative experiments. The test results are as follows Figure 8 As shown, the standard curve was successfully run, the test samples were within the range of the standard curve, and the established standard curve correlation coefficient was 0.99869, proving that the credibility was high. The average Ct value of the SARS-CoV-2 coronavirus positive sample RNA was 21.32.

[0150] (2) Analysis of digital PCR droplet results

[0151] To determine the copy number of qPCR standards, a digital PCR experiment was performed, and the results of the ROX channel detection in one dimension are as follows Fig. 9 As shown in the figure, the middle line is the threshold line, the positive droplets are distributed above the threshold line, and the negative droplets are distributed below the threshold line. The ordinate represents the fluorescence intensity, and the abscissa represents the sample well number. The test result analysis shows that the target sequence exists, the positive droplets are distributed evenly, the peak value is high, and the results of four repetitions are consistent with small errors. According to the number of positive samples, the copy number of the qPCR standard determined by software analysis and calculation is 1.219×10 8 copies / μL.

[0152] (3) Immunochromatographic Cas13a detection sensitivity

[0153] Based on the copy number of the qPCR standard sample measured above, the copy number of the test sample can be calculated to be 6108 copies / μL after conversion by the Ct value of the qPCR standard curve. This sample was diluted in a gradient manner and the sensitivity was determined by the immunochromatographic Cas13a detection method. The test results are shown in Fig.10 shown.

[0154] The results showed that when the copy number of the sample concentration dropped to 190 copies / μL, colloidal carbon was enriched at the immunochromatographic Cas13a detection line, and the result was negative. Therefore, it was inferred that the minimum detection limit of the immunochromatographic Cas13a detection method was 381.75 copies / μL.

[0155] Example 7 Detection of SARS-CoV-2 coronavirus inactivated samples

[0156] crRNA was designed for the N gene of the SARS-CoV-2 coronavirus mutant strain EG.5.1.1 and prepared by referring to the in vitro transcription method of Example 1. The primer sequences are shown in Table 13 and were synthesized by Anhui General Biotechnology Co., Ltd. The preparation of the double-stranded product and the detailed steps of in vitro transcription were the same as those in Example 2.

[0157] Table 13 SARS-CoV-2 coronavirus sample crRNA

[0158]

[0159]

[0160] Take the prepared SARS-CoV-2 coronavirus crRNA and LbuCas13a protein and configure the reaction system with reference to Tables 14 and 15.

[0161] Table 14 Immunochromatographic Cas13a SARS-CoV-2 coronavirus detection system 1

[0162]

[0163] After the above reaction system is configured, add it to a clean RNase-Free PCR tube, and then place it in a PCR instrument at 37°C for 10 minutes as the step 1 system.

[0164] Table 15 Immunochromatographic SARS-CoV-2 Coronavirus Detection System II

[0165]

[0166] The probe used in the system was TB-polyU-11nt (see Table 6). After the reaction system was configured, the reaction was carried out at 37°C for 90 minutes in a PCR instrument, and then the reaction was terminated at 4°C for 2 minutes.

[0167] After the above sample detection reaction is completed, take 50 μL of the reaction system and insert it into the prepared dual-target immunochromatographic test strip, and read the test results within 5 to 10 minutes.

[0168] Eight inactivated SARS-CoV-2 coronavirus positive RNA samples and eight inactivated influenza A(H3N2) positive RNA samples were tested with dual-target test strip immunochromatographic Cas13a. The test results are as follows: Fig.11As shown (the construction of multi-target immunochromatographic test strips can be based on the detection system probes, and the corresponding capture antibodies can be selected for laying. One probe can correspond to one detection target, and multiple detection T lines can be laid on the prepared colloidal carbon test strips to construct multi-target detection immunochromatographic test strips; one probe corresponds to one detection target, and different probes are captured by different antibodies. Then, during the experiment, a multi-tube detection system is configured, and samples are added separately to react. After the reaction, each system is mixed and the detection results are presented on the same test strip, thereby realizing multi-target detection).

[0169] The results showed that the influenza sample in test strip 1 was not positive, and the SARS-CoV-2 coronavirus was not positive in test strip 7, which may be due to the low sample concentration. The remaining numbered positive samples were successfully detected. The specificity and feasibility of multi-target immunochromatographic Cas13a detection were verified.

Claims

1. A probe set for detecting SARS-CoV-2 coronavirus nucleic acid, characterized in that: The crRNA sequence for detecting SARS-CoV-2 coronavirus nucleic acid is any one of the following: LbucrRNA1, LbucrRNA2, LbucrRNA3, LbucrRNA4 for the S gene, N1crRNA and N2crRNA for the N gene of the mutant strain EG.5.1.1, the sequence is as follows:

2. A kit for detecting SARS-CoV-2 coronavirus nucleic acid, characterized in that: It includes the probe set for detecting SARS-CoV-2 coronavirus nucleic acid as described in claim 1.

3. A kit for detecting SARS-CoV-2 coronavirus nucleic acid, characterized in that: The invention comprises a colloidal carbon immunochromatographic test strip of line elimination method and a CRISPR reaction system, wherein the CRISPR reaction system comprises the probe group for detecting SARS-CoV-2 coronavirus nucleic acid as claimed in claim 1; the immunochromatographic test strip of line elimination method comprises, in order according to the sample flow direction, a sample pad, a binding pad containing colloidal carbon-labeled rabbit anti-biotin antibodies, an NC membrane containing T lines and C lines, and absorbent paper.

4. A kit for detecting SARS-CoV-2 coronavirus nucleic acid according to claim 3, characterized in that: When the probe is FAB-polyU-11nt, the T line is formed by rabbit anti-6-FAM polyclonal antibody, and the C line is formed by goat anti-rabbit IgG; When the probe is DB-polyU-11nt, the T line is formed by Anti-Digoxigenin antibody, and the C line is formed by goat anti-rabbit IgG; When the probe is TB-polyU-11nt, the T line is formed by Anti-TAMRA antibody [5G5], and the C line is formed by goat anti-rabbit IgG; When the probe is CB-polyU-11nt, the T line is formed by Anti-Cy5 antibody [CY5-15], and the C line is formed by goat anti-rabbit IgG; The sequences of each probe are as follows:

5. A kit for detecting SARS-CoV-2 coronavirus nucleic acid according to claim 3, characterized in that: The CRISPR reaction system is as follows: Targeting the S gene system Targeting N gene system After system 1 is configured, add it to a clean RNase-Free PCR tube, and then place it in a PCR instrument at 37°C for 10 minutes as step 1 system. After the reaction, the Cas13a / crRNA mixture is added to system 2 for step 2. Targeting S gene or N gene system II After the reaction system is configured, react at 37°C in a PCR instrument for 90 minutes, then terminate the reaction at 4°C for 2 minutes, insert the prepared colloidal carbon immunochromatography test strip, and read the test results within 5 to 10 minutes.

6. Use of the kit as claimed in any one of claims 2 to 5 in detecting SARS-CoV-2 coronavirus nucleic acid, wherein the use is not for the purpose of diagnosing or treating the disease.

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