A RT-LAMP primer-probe composition for identifying Pansacoronavirus and its application

Abstract

The present invention discloses a primer-probe composition and application of the RT-LAMP method for identifying pan-Sabei coronavirus, which belongs to the technical field of virus molecular detection. The primer-probe composition includes outer primers F3 and B3, inner primers FIP and BIP, loop primers LF and LB, and the nucleotide sequences are shown in SEQ ID NO.1-6, respectively. The primer-probe composition provided by the present invention can realize the specific detection of a broad-spectrum Sabei coronavirus sample. The detection device provided by the present invention has high detection sensitivity, and the detection limit is as low as 200 copies / mL; the detection efficiency is high, and a single sample only takes about 35 minutes from collection to completion of detection, which can significantly improve the efficiency of nucleic acid detection in response to the epidemic; the operation is simple, the result interpretation is intuitive, the device cost is low, and the conditions for industrial promotion are met.

Description

Technical Field

[0001] The invention belongs to the technical field of virus molecular detection, and specifically relates to an RT-LAMP primer-probe composition, a kit and a visual integrated detection device for identifying Pan-Sabe coronavirus, which are suitable for qualitative detection of Pan-Sabe coronavirus. Background Art

[0002] Since the outbreak of the novel coronavirus (SARS-CoV-2), its variants have not only changed in virulence, but also shown a strong ability to spread, which can cause large-scale infection in a short period of time. SARS-CoV-2 belongs to the β-CoV-B lineage, also known as Sarbecoviruses. Other subgenus Sarbecoviruses include the SARS coronavirus (SARS-CoV) that caused the severe acute respiratory syndrome (SARS) epidemic in 2003, and the SARS-related coronavirus (SARSr-CoV) that exists in bats, pangolins and many other wild animals.

[0003] Sarbecoviruses have many host animals. As early as 20 years ago, people had discovered bat-derived Sarbecoviruses. In 2005, the first viral relative of SARS-CoV was found in rhinoceros bats, proving that wild animals can be the natural hosts of Sarbecoviruses. Existing studies have shown that bats are most likely the natural host of SARS-CoV-2, which adapts and evolves through intermediate hosts and then infects humans. Sarbecoviruses are prone to gene recombination and mutation during evolution due to their unique genome replication and RNA synthesis mechanisms, such as jumping polymerase reactions, discontinuous template extension and selection, which results in the continuous emergence of new highly pathogenic members during genetic evolution, posing a more extensive threat of zoonotic diseases. Since the 21st century, highly pathogenic Sarbecoviruses have repeatedly crossed species barriers to invade humans, leading to a continuous increase in serious diseases and causing immeasurable damage to human life safety and global public health.

[0004] Therefore, establishing a new broad-spectrum detection method for the Sarbecoviruses subgroup can not only cope with the persistent threat of SARS-CoV-2 variants, but also monitor the transmission dynamics of SARS-CoV and SARSr-CoV in real time, prevent the recurrence of SARS and future large-scale epidemics caused by cross-species transmission of potentially highly pathogenic SARSr-CoV, which has positive and far-reaching significance for public health and economic development in countries around the world.

[0005] At present, the detection methods for Pan-Sab coronavirus infection mainly include antigen detection, antibody detection and nucleic acid detection, but most of them are specifically developed for SARS-CoV-2. Although the detection time of antigen and antibody detection is short, they are not ideal in terms of sensitivity and specificity, and are prone to false positives and false negatives. They are generally used as a supplementary method for nucleic acid detection. Real-time reverse transcription polymerase chain reaction (RT-qPCR) is the gold standard for the diagnosis of SARS-CoV and SARS-CoV-2, but this method requires the configuration of expensive and sophisticated experimental equipment and has high technical requirements for operators. It is not suitable for home self-examination or use in grassroots testing institutions in rural areas, remote mountainous areas and other difficult conditions.

[0006] Therefore, developing a molecular diagnostic method that is simple to operate, has integrated equipment, high specificity and sensitivity, and can quickly identify Sarbecoviruses is a technical challenge that professionals in this field urgently need to solve. Summary of the invention

[0007] The purpose of the present invention is to provide a primer-probe combination for universal detection of Pan-Sabe coronavirus, a kit including the primer-probe combination, and a visual integrated device for detection using the kit, and to provide a detection method that is efficient, simple, and more suitable for emergency detection and bedside self-testing under harsh conditions.

[0008] To achieve the above object, the present invention adopts the following technical solution:

[0009] The present invention designs the target gene in the 3' non-coding region (3'UTR for short). The 3'UTR region of the virus is not only involved in transcriptional regulation, but also in the stability and transport process of the transcript. For example, some RNA binding proteins of the virus can regulate the stability and transport of RNA by binding to the characteristic sequence in the 3'UTR. Sabae coronavirus continues to evolve and mutate in response to various environmental changes, but the 3'UTR region has better sequence conservation and less prone to base mutation than other coding genes such as S, N, ORF-1b, RdRp, E, etc. due to its basic functions.

[0010] The present invention downloads the SARS-CoV-2 Wuhan strain and various variant strains (including subtypes such as Alpha, Beta, Gamma, Delta, Zeta, Lota, Lambda, Mu, XBB.1.5, EG.5.1, etc.) from NCBI-GenBank and GISAID databases, and the full gene sequences of SARS-CoV and bat-source and pangolin-source SARSr-CoV in different regions in 2003, totaling about 1000 strains. Through gene homology comparison analysis, a highly conservative and universal gene region of all pan-sabelian coronaviruses is found in 3'UTR. Then, according to the primer design principle of reverse transcription loop-mediated isothermal amplification technology (RT-LAMP), a primer sequence with moderate Tm value and GC content, no hairpin structure, and less dimer is selected, and after verification by bioinformatics analysis and molecular biology experiments, a set of RT-LAMP primers and probe combinations suitable for broad-spectrum sabelian coronavirus detection are screened out.

[0011] In summary, the present invention provides an RT-LAMP primer-probe composition for identifying Pan-Sabe coronavirus, wherein the primer-probe composition comprises outer primers F3 and B3, inner primers FIP and BIP, and loop primers LF and LB. Specifically, the primer sequences are as follows:

[0012] Forward outer primer F3:

[0013] 5'-CTCGARTARTAMAYAGCACAAGTAG-3' (SEQ ID NO. 1);

[0014] Reverse outer primer B3:

[0015] 5'-AATCACATGGGGATAGCACT-3' (SEQ ID NO. 2);

[0016] Forward inner primer FIP:

[0017] 5'-ATGTGGGTGGCTCTTTCAAGTCCTCCTKTAGTTAACTTTAATCT CACATAGCA-3' (SEQ IDNO.3);

[0018] Reverse inner primer BiP:

[0019] 5'-ACGATCGAGKGTACAGTGAAYAATGCCTAAAATTAATTTTACACATTAGGGC-3' (SEQ IDNO.4);

[0020] Forward loop primer LF:

[0021] 5'-TAATGTTACAYTGATTAAAGA-3' (SEQ ID NO. 5);

[0022] Reverse loop primer LB:

[0023] 5'-AGGGAGAGCTGCCTATATGGAAG-3' (SEQ ID NO. 6);

[0024] Among them, R, Y, M, and K are substituted bases, R represents A or G base, Y represents C or T base, M represents A or C base, and K represents G or T base.

[0025] Further, the 5' end of the loop primer LF is labeled with 6-carboxyfluorescein (6-FAM) or fluorescein isothiocyanate (FITC), and the 5' end of the loop primer LB is labeled with biotin. The labeled primer enters the amplification product along with the isothermal amplification reaction, so that the amplification product carries 6-FAM / FITC and biotin labels, and then the chromatographic double antibody sandwich method can be used to quickly detect the nucleic acid amplification product. The labels that can be used in the present invention are not limited to this.

[0026] In the present invention, the primer-probe combination is used to construct an isothermal amplification reaction system, and an isothermal amplification reaction is performed. The RNA of the sample to be tested is reverse transcribed into cDNA by reverse transcriptase, and the cDNA undergoes a strand displacement amplification reaction under the action of Bst polymerase, and whether a specific amplification product is generated is detected to determine whether the sample to be tested is Pan-Sabe coronavirus.

[0027] In the present invention, the pan-Sabe coronavirus includes: SARS-CoV-2 and its variants, SARS-CoV, animal-derived SARSr-CoV and potential new SARSr-CoV.

[0028] The present invention provides application of the RT-LAMP primer-probe combination in preparing a kit for detecting Pan-Sabe coronavirus.

[0029] Furthermore, the kit includes a primer combination solution, in which the concentration of the outer primers F3 and B3 is 0.2-0.6 μM, the concentration of the inner primers FIP and BIP is 0.8-2.4 μM, and the concentration of the loop primers LF and LB is 0.4-1.2 μM.

[0030] Furthermore, the kit also includes an isothermal amplification reaction solution, which contains a buffer, dNTPs, AMV reverse transcriptase, betaine, Mg 2+ , Bst polymerase or a mixture of two or more.

[0031] The present invention establishes a detection method for pan-Sabe coronavirus nucleic acid loop-mediated isothermal amplification, and completes the visualization of the detection results through nucleic acid detection test strips. The detection method comprises the following steps:

[0032] (1) After collecting a sample using a throat swab or a nasal swab, immerse the swab in a sample tube containing a nucleic acid lysis solution to obtain a sample to be tested;

[0033] (2) adding the sample to be tested to the isothermal amplification reaction solution containing the RT-LAMP primer probe combination, placing the reaction system on a heating assembly, and heating at 58-60° C. for 30 min;

[0034] (3) The amplified product is added dropwise to the sample well of the nucleic acid gold-labeled test strip detection device, and the result is read after 5 minutes. When the test line of the test strip is positive (i.e., both the C line and the T line are colored), it indicates that the sample contains Sabae coronavirus nucleic acid. If the quality control line does not show color, it means that the device is invalid. Regardless of whether the test line is colored or not, the result is invalid and needs to be retested.

[0035] The nucleic acid detection test strip is a colloidal gold test strip, which is sequentially provided with a sample pad, a colored particle conjugate pad and a water absorbent pad, and the joints of the above parts partially overlap. A detection line (T line) and a quality control line (C line) are sequentially provided along the liquid flow direction, wherein the colored particles on the colored particle conjugate pad are coated with streptavidin, the detection line (T line) is coated with anti-FAM / FITC antibodies, and the quality control line (C line) is coated with anti-streptavidin antibodies, and the colored particles are selected as colloidal gold particles.

[0036] The present invention uses the colloidal gold test strip method as a result interpretation method. The amplification product of the RT-LAMP reaction of the positive sample carries FAM / FITC and biotin labels, and after being passed through a colored particle conjugate pad, it is combined with streptavidin-colloidal gold particles, and can be combined with the anti-FAM / FITC antibody on the test line of the test strip and the anti-streptavidin antibody on the quality control line respectively. The test result is that the test line and the quality control line are colored at the same time. Negative samples cannot be amplified due to the lack of target genes, so there is no specific amplification product. The test result is that only the quality control line is combined with streptavidin-colloidal gold particles to develop color. In this way, the visual interpretation of the amplification results is achieved, and positive and negative results are distinguished.

[0037] Furthermore, the present invention provides a visual integrated detection device for Pan-Sabe coronavirus based on microfluidics combined with colloidal gold technology, the device comprising a shell and a heating component, a colloidal gold test strip is arranged in the shell, a judgment result display window is provided on the shell surface corresponding to the test strip detection area, an RT-LAMP reaction well is provided on the shell near one end of the test strip sample pad, and the upper end of the reaction well wall is connected to the test strip sample pad through a microfluidic channel; an isothermal amplification reaction reagent including the RT-LAMP primer probe combination is pre-embedded in the reaction well; and the reaction well is connected to the heating component.

[0038] Microfluidics technology uses microchannels to achieve precise control of liquid flow, thereby integrating components such as sample addition, amplification reaction, and visual interpretation of results, thereby simplifying operations.

[0039] Furthermore, the components of the isothermal amplification reaction reagent include: 1×isothermal amplification buffer, FIP / BIP 0.8-2.4 μM, F3 / B3 0.2-0.6 μM, LF / LB 0.4-1.2 μM, dNTPs 2.5 mM, AMV reverse transcriptase 10 U, betaine 5 M, MgSO4 6 mM, and Bst polymerase 8 U.

[0040] Furthermore, the heating component maintains a temperature of 58-60°C.

[0041] The present invention provides a method for detecting Pan-Sabe coronavirus using the visual integrated detection device, comprising the following steps:

[0042] (1) After collecting the sample using a throat swab or a nasal swab, immerse the swab in a sample tube containing a nucleic acid lysis solution to quickly lyse and extract nucleic acid;

[0043] (2) Add the rapidly extracted nucleic acid from the sample to be tested to the RT-LAMP reaction well containing the isothermal amplification reaction reagent, turn on the heating component, and react at 58-60° C. for 30 minutes;

[0044] (3) Add buffer to the reaction wells where amplification has been completed, so that the amplified product flows into the microchannel connected to the test strip along with the buffer and contacts the colloidal gold test strip. The result is read after 5 minutes.

[0045] The above method is simple to operate. The entire amplification and detection process only requires one sample injection, and there is no need to open the cover and add reagents, thus avoiding the chance of contamination of the amplification product. It is suitable for clinical emergency diagnosis and bedside self-examination of suspected patients.

[0046] The present invention realizes the detection of Pan-Sabe coronavirus based on RT-LAMP technology. The method requires simple equipment and not only has high sensitivity, specificity and repeatability similar to RT-qPCR, but also has the advantages of simple operation, short detection time and intuitive results. The whole process can complete visual detection within 35 minutes, which can meet the requirements of rapid detection.

[0047] Compared with the prior art, the present invention has the following beneficial effects:

[0048] (1) The present invention uses the highly conserved region of the 3' non-coding region of Pan-Sabe coronavirus as the detection target to design an RT-LAMP primer-probe combination for identifying Pan-Sabe coronavirus. The combination has good broad spectrum and high specificity, and has no cross-reaction with common respiratory viruses such as influenza A and B viruses, parainfluenza virus, 229E coronavirus, OC43 coronavirus, HKU1 coronavirus, human rhinovirus, measles virus, human bocavirus, human metapneumovirus, respiratory syncytial virus, mumps virus, and rubella virus.

[0049] (2) The present invention uses the conservative region of the 3' non-coding region of the pan-Sabe coronavirus as the detection target. Compared with the existing commonly used target genes for fluorescent RT-qPCR, such as S, N, ORF-1b, RdRp, E, etc., the 3' non-coding region selected by the present invention is more conservative and universal, and can achieve broad-spectrum detection of Sabella coronavirus, can detect various SARS-CoV-2 variants, can also detect SARS-CoV, can also detect animal-derived SARS coronaviruses and potential new SARSr-CoV, can cope with the current and future epidemic detection of new coronavirus variants, and at the same time prevent possible future recurrence of SARS-CoV and cross-species transmission of SARSr-CoV.

[0050] (3) The detection device provided by the present invention has high detection sensitivity and a detection limit as low as 2.0×10 2 copies / mL.

[0051] (4) The detection device provided by the present invention has high detection efficiency. It only takes about 35 minutes from the collection to the completion of the detection of a single sample, which can significantly improve the efficiency of nucleic acid detection in response to the epidemic.

[0052] (5) The detection device provided by the present invention has intuitive results and low device cost, and does not rely on any precision instruments. It is suitable for use in mountainous areas, rural areas, communities and other grassroots areas with harsh on-site detection environments, and has the conditions for industrial promotion. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 This is the result of optimizing the reaction time of Pan-Sabe coronavirus RT-LAMP method. In the figure, ①-⑦ are respectively SARS-CoV-2 nucleic acid 2.0×106 copies / mL, 2.0×10 5 copies / mL, 2.0×10 4 copies / mL, 2.0×10 3 copies / mL, 2.0×10 2 copies / mL, 2.0×10 1 copies / mL and negative control.

[0054] Figure 2 This is the result of the optimization of the reaction temperature of the Pan-Sabe coronavirus RT-LAMP method. In the figure, ①-⑦ are respectively SARS-CoV-2 nucleic acid 2.0×10 6 copies / mL, 2.0×10 5 copies / mL, 2.0×10 4 copies / mL, 2.0×10 3 copies / mL, 2.0×10 2 copies / mL, 2.0×10 1 copies / mL and negative control.

[0055] Figure 3 This is a graph of the specific detection results of the pan-Sabe coronavirus RT-LAMP method. In the figure, 1-20 are respectively 1: SARS-CoV-2 Omicron strain, 2: SARS-CoV, 3: bat-origin SARSr-CoV, 4: 229E coronavirus, 5: OC43 coronavirus, 6: HKU1 coronavirus, 7: NL63 coronavirus, 8: human rhinovirus, 9: measles virus, 10: human bocavirus, 11: human metapneumovirus, 12: respiratory syncytial virus, 13: parainfluenza virus types 1-4, 14: influenza A virus H1N1, 15: influenza A virus H3N2, 16: influenza B virus Victoria type, 17: influenza B virus Yamagata type, 18: mumps virus, 19: rubella virus, 20: nucleic acid detection results of negative control.

[0056] Figure 4 This is the result of the sensitivity test of Pan-Sabe coronavirus RT-LAMP method. In the figure, ①-⑦ are SARS-CoV-2 nucleic acid 2.0×10 6 copies / mL, 2.0×10 5 copies / mL, 2.0×10 4 copies / mL, 2.0×10 3 copies / mL, 2.0×10 2 copies / mL, 2.0×10 1copies / mL and negative control.

[0057] Figure 5 This is a flow chart of the use of the visual integrated detection device provided by the present invention.

[0058] Figure 6 This is a detection example diagram of the visual integrated detection device provided by the present invention. DETAILED DESCRIPTION

[0059] The present invention will be further described below in conjunction with specific embodiments. The following embodiments are only used to illustrate the present invention and are not intended to limit the scope of application of the present invention. Without departing from the spirit and essence of the present invention, modifications or replacements made to the inventive method, steps or conditions all belong to the scope of the present invention.

[0060] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0061] The Bst polymerase, constant temperature amplification buffer, and 100 mmol / LMgSO4 used in the following examples were purchased from NEB; 10 mmol / L dNTPs and AMV reverse transcriptase (10 U / μL) were purchased from Sangon Biotechnology (Shanghai) Co., Ltd.; betaine was purchased from Shanghai Biotechnology Co., Ltd.; primers were commissioned to Shanghai Biotechnology Co., Ltd. for synthesis; closed colloidal gold test strip detection device was purchased from Hangzhou Ustar Biotechnology Co., Ltd.; Premix Ex TaqTM (PerfectReal Time) was purchased from TaKaRa, batch number: AK2404; the nucleic acids involved in the examples were provided by Zhejiang Provincial Center for Disease Control and Prevention.

[0062] Example 1: Design of universal RT-LAMP primer probe for Pan-Sabe coronavirus

[0063] 1. Obtain the gene sequence of Sabei coronavirus:

[0064] The complete gene sequence of SARS-CoV-2 (MN908947.3) and the complete gene sequences of SARS-CoV-2 variants (EPI_ISL_581117, EPI_ISL_1314210.2, EPI_ISL_660190, EPI_ISL_2261510, EPI_ISL_3218258, EPI_ISL_1111137, EPI_ISL_3473618, EPI_ISL_1315070, EPI_ISL_677211, EPI_ISL_3578149, EPI_ISL_765494, EPI_ISL_ 3568746, EPI_ISL_1111128, EPI_ISL_3569078, EPI_ISL_1908878, EPI_ISL_2310477), the complete gene sequences of SARS-CoV from different regions in 2003 (AY274119, AY291451, AY395003, AY559096, AY278488, AY278741), the complete gene sequences of SARS-like coronaviruses from bats (MN996532, MG772934, MW251308), and the complete gene sequences of SARS-like coronaviruses from pangolins (EPI_ISL_410721, EPI_ISL_410539).

[0065] 2. Design of universal primers and probe sequences:

[0066] The gene sequences obtained in step 1 of homology comparison were screened out using DNAMAN software to identify conservative gene fragments. Sequence analysis showed that the sequence divergence in the 3'UTR region was smaller than that in coding genes such as S, N, ORF-1b, RdRp, and E. Therefore, through homology comparison, highly conservative and universal gene regions were found in the 3'UTR as targets for RT-LAMP detection.

[0067] According to the design principles of primers and probes of the RT-LAMP method, the design of primers and probes was carried out in the conservative gene fragments screened out; the complementarity of the upstream and downstream primers was analyzed, and the gene sequences with moderate Tm value and GC content, no hairpin structure, and fewer complementary bases were selected, and then a set of primer probe sequences was designed. Including 1 pair of external primers F3 and B3, internal primers FIP and BIP, and 1 pair of single-label detection loop primers LF and LB. The designed specific primers and probes are as follows (Table 1):

[0068] Table 1. Specific primers and probes

[0069]

[0070] Among them, Biotin is a biotin labeling group, FITC is a fluorescein isothiocyanate labeling group, and the labeling group can also be replaced by a 6-carboxyfluorescein labeling group (FAM). The bases are merged, R represents A or G bases, Y represents C or T bases, M represents A or C bases, and K represents G or T bases.

[0071] Example 2: Determination of a universal RT-LAMP reaction detection method for Pan-Sabe coronavirus

[0072] 1. Test samples

[0073] The quantified SARS-CoV-2 nucleic acid was diluted 10-fold to prepare 2.0×10 6 copies / mL, 2.0×10 5 copies / mL, 2.0×10 4 copies / mL, 2.0×10 3 copies / mL, 2.0×10 2 copies / mL, 2.0×10 1 There were 6 reaction systems with different concentrations of 10 copies / mL, and sterile double-distilled water was set as the negative control.

[0074] 2. Optimize the RT-LAMP reaction system

[0075] Orthogonal method was used to optimize the reaction time, Mg 2+ The results showed that the amount of amplified product tended to be saturated when the reaction time was 30 min. Figure 1 Mg 2+ The amplification reaction efficiency reached the maximum when the concentration was 6mmol / μL; the nucleic acid amplification efficiency was optimal when the annealing temperature was 58℃ and 60℃ ( Figure 2 ).

[0076] Therefore, preferably, the RT-LAMP reaction system is as shown in Table 2, and the reaction conditions are: reaction at 60° C. for 30 min. After the reaction is completed, the reaction product is added dropwise to the colloidal gold test strip, and the result can be read after the test strip develops color.

[0077] Table 2. RT-LAMP reaction system

[0078]

[0079] 3. Determine the testing process

[0080] (1) Rapid cleavage of RNA from suspected patients;

[0081] Use a cotton swab to collect a throat swab or nasal swab and immerse the swab in a sample tube containing nucleic acid lysis solution.

[0082] (2) Nucleic Acid Amplification

[0083] The sample to be tested is added to the reaction solution containing the above reaction system, and a buffer solution is added. The sample is heated and reacted at 60°C for 30 minutes. The sample is then contacted with a colloidal gold test strip and the result can be read after the test strip develops color.

[0084] 4. Interpretation of results

[0085] The structure of the colloidal gold test strip is as follows: a sample pad, a colored particle conjugate pad and a water absorbent pad are arranged in sequence, and the joints of the parts partially overlap. A detection line (T line) and a quality control line (C line) are arranged in sequence along the flow direction of the liquid, wherein the colored particles on the colored particle conjugate pad are coated with streptavidin, the detection line (T line) is coated with anti-FITC antibodies, and the quality control line (C line) is coated with anti-streptavidin antibodies, and the colored particles are selected as colloidal gold particles.

[0086] When the test line of the test strip is positive (i.e. both the C line and the T line are red), it means that the sample contains Sabae coronavirus nucleic acid. If the quality control line does not show red, the result is invalid. The interpretation method is as follows:

[0087] Negative (-): only a red band appears in the quality control area (C line);

[0088] Positive (+): A red band appears in the quality control area (C line) and the test area (T line);

[0089] Invalid: There is no red strip in the quality control area (line C), indicating that the test strip is invalid.

[0090] Example 3: Pan-Sabe coronavirus visual integrated RT-LAMP method detection performance test

[0091] 1. Specificity detection

[0092] The detection method determined in Example 2 was used to detect the nucleic acids of common human coronaviruses (HCoV-229E, HCoV-OC43, HCoV-HKU1 and HCoV-NL63), human rhinovirus (HRV), measles virus (MV), human bocavirus (HBoV), human metapneumovirus (HMPV), respiratory syncytial virus (RSV), parainfluenza virus (PIV1-4), influenza A virus (H1N1 and H3N2), influenza B virus (Victoria and Yamagata subtypes), mumps virus (MUV), and rubella virus (RV).

[0093] The results show that Figure 3), the nucleic acids of SARS-CoV-2Omicron strain, SARS-CoV, and bat-origin SARSr-CoV were positive on the test line (both T line and C line were red), while the remaining 16 pathogens had no specific reaction (only C line was red), indicating that the method of the present invention has strong specificity in detecting pan-Sabe coronavirus.

[0094] 2. Sensitivity detection

[0095] The quantified SARS-CoV-2 nucleic acid was diluted 10-fold to prepare 2.0×10 6 copies / mL, 2.0×10 5 copies / mL, 2.0×10 4 copies / mL, 2.0×10 3 copies / mL, 2.0×10 2 copies / mL, 2.0×10 1 The reaction system was divided into 6 reaction systems with different concentrations of 100 copies / mL, and sterile double distilled water was set as a negative control. The method described in Example 2 was used for nucleic acid amplification. The results showed that ( Figure 4 ), minimum detection limit 2.0×10 2 copies / mL, indicating that this method has high detection sensitivity and can meet the needs of detecting low-load viruses carried by infectious sources and related vector hosts in epidemic areas, playing a role in monitoring and early warning.

[0096] 3. Accuracy detection

[0097] Nucleic acids were extracted from 208 clinical samples suspected of coronavirus infection, and then nucleic acid amplification was performed using a commercial RT-qPCR kit and the RT-LAMP method described in Example 2, respectively.

[0098] The results showed (Table 3) that 17 cases were confirmed as positive and 191 cases were confirmed as negative by RT-qPCR. The results of RT-LAMP amplification after 30 minutes were consistent with the results of fluorescence RT-qPCR.

[0099] Table 3. Clinical specimen test results

[0100]

[0101] The above results prove that the method of the present invention has the same accuracy as the RT-qPCR method, but at the same concentration, the response time of the method of the present invention is shorter, the instrumentation is simpler, and it is more suitable for emergency detection and bedside self-testing under harsh conditions.

[0102] Example 4: Pan-Sabe coronavirus RT-LAMP method visual integrated device for detection of clinical and environmental samples

[0103] The present embodiment provides a visual integrated RT-LAMP detection device based on microfluidics combined with colloidal gold paper strip technology, the device includes a shell and a heating component, a colloidal gold test strip is arranged in the shell, a display window is provided on the shell surface corresponding to the test strip detection area, an RT-LAMP reaction hole is provided on the shell near one end of the test strip sample pad, and the upper end of the reaction hole wall is connected to the test strip sample pad through a microfluidic channel; the reaction hole is connected to the heating component.

[0104] according to Figure 5 According to the process shown, use a cotton swab to collect environmental samples or nasal swabs, immerse the swab in a sample tube containing a nucleic acid lysis solution, then add the sample to be tested to the above device containing the reaction system shown in Table 2, add buffer, turn on the heating component, and react at 60°C for 30 minutes. After the reaction is completed, add the second tube of buffer, so that the amplification product flows into the microchannel connected to the test strip with the buffer, and contacts with the colloidal gold test strip. The result can be read after the test strip develops color.

[0105] In this example, three clinical samples and two environmental samples were taken and nucleic acid tests were performed according to the above process. The results are as follows: Figure 6 As shown, the test results of the three clinical samples were positive (both the T line and the C line were red), the test result of environmental sample 1 was positive (both the T line and the C line were red), and the test result of environmental sample 2 was negative (only the C line was red).

[0106] The above-described embodiments are only preferred solutions of the present invention and are not intended to limit the present invention. A person skilled in the relevant technical field may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, any technical solution obtained by equivalent replacement or equivalent transformation falls within the protection scope of the present invention.

Claims

1. An RT-LAMP primer-probe composition for identifying Pan-Sabe coronavirus, characterized in that, The pan-Sabe coronavirus includes: SARS-CoV-2 and its variants, SARS-CoV, and animal-derived SARSr-CoV; the primer probe composition is designed for the conserved region of the 3' non-coding region of the pan-Sabe coronavirus, including outer primers F3 and B3, inner primers FIP and BIP, loop primers LF and LB, the nucleotide sequence of F3 is shown in SEQ ID NO.1, the nucleotide sequence of B3 is shown in SEQ ID NO.2, the nucleotide sequence of FIP is shown in SEQ ID NO.3, the nucleotide sequence of BIP is shown in SEQ ID NO.4, the nucleotide sequence of LF is shown in SEQ ID NO.5, and the nucleotide sequence of LB is shown in SEQ ID NO.

6.

2. The RT-LAMP primer-probe composition for identifying Pan-Sabe coronavirus according to claim 1, characterized in that The 5' end of the loop primer LF is labeled with 6-carboxyfluorescein or fluorescein isothiocyanate, and the 5' end of the loop primer LB is labeled with biotin.

3. The use of the RT-LAMP primer-probe combination as claimed in claim 1 or 2 in the preparation of a kit for detecting Pan-Sabe coronavirus, characterized in that: The pan-Sabe coronavirus includes: SARS-CoV-2 and its variants, SARS-CoV, and animal-derived SARSr-CoV.

4. The use according to claim 3, characterized in that The kit comprises a primer combination liquid, in which the concentrations of the outer primers F3 and B3 are 0.2-0.6 μM, the concentrations of the inner primers FIP and BIP are 0.8-2.4 μM, and the concentrations of the loop primers LF and LB are 0.4-1.2 μM.

5. The use according to claim 3, characterized in that The kit also includes an isothermal amplification reaction solution, which contains a buffer, dNTPs, AMV reverse transcriptase, betaine, Mg 2+ , Bst enzyme or a mixture of two or more.

6. The use according to claim 3, characterized in that The pan-Sabe coronavirus includes: SARS-CoV-2Omicron strain, SARS-CoV, and bat-origin SARSr-CoV.

7. A visual integrated detection device for Pan-Sabe coronavirus, characterized in that: The pan-Sabe coronavirus includes: SARS-CoV-2 and its variants, SARS-CoV, and animal-derived SARSr-CoV; the device includes a shell and a heating component, a colloidal gold test strip is arranged in the shell, a result judgment display window is arranged on the shell surface corresponding to the test strip detection area, an RT-LAMP reaction well is arranged on the shell near one end of the test strip sample pad, and the upper end of the reaction well wall is connected to the test strip sample pad through a microfluidic channel; an isothermal amplification reaction reagent including the RT-LAMP primer-probe combination as described in claim 1 or 2 is pre-embedded in the reaction well; the reaction well is connected to the heating component.

8. The visual integrated detection device for Pan-Sabe coronavirus according to claim 7, characterized in that: The components of the isothermal amplification reaction reagent include: 1×isothermal amplification buffer, FIP / BIP 0.8-2.4 μM, F3 / B3 0.2-0.6 μM, LF / LB 0.4-1.2 μM, dNTPs 2.5 mM, AMV reverse transcriptase 10 U, betaine 5 M, MgSO4 6 mM, and Bst polymerase 8 U.

9. The visual integrated detection device for Pan-Sabe coronavirus according to claim 7, wherein the heating component maintains a temperature of 58-60°C.

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