Nucleic acid sequence, product, method and application for novel coronavirus detection

Abstract

The invention provides a nucleic acid sequence, a product, a method and application for novel coronavirus detection, and relates to the technical field of biological detection, and in order to detect novel coronaviruses, different fluorescent markers are designed for amplification primers of ORF1ab genes and N genes of the novel coronaviruses, and a multi-cross isothermal amplification reaction is adopted, so that the novel coronavirus detection sensitivity is improved. Therefore, amplification products of the ORF1ab gene and the N gene can be obtained only under one temperature condition, and the ORF1ab gene and the N gene of the genome of a virus sample to be detected are simultaneously detected by using the transverse flow measurement biosensor strip. By adopting the amplification primer sequence provided by the invention, the novel coronavirus can be visually detected, the detection speed is high, the operation is simple, the time and cost are saved, and the method is suitable for being popularized to clinical detection of the novel coronavirus.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to a nucleic acid sequence, product, method and application for detecting a novel coronavirus. Background Art

[0002] Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), also known as the new coronavirus infection (COVID-19), has caused extremely high morbidity and millions of deaths worldwide, posing a serious threat to human health and public safety. Although humans continue to develop new vaccines to respond to the outbreak, the continued weakening of specific antibody activity in the human body and the immune escape of the constantly mutating virus strains have not completely curbed the spread of SARS-CoV-2 in humans. As the SARS-CoV-2 virus strains continue to mutate, they are characterized by lower virulence and stronger transmission ability.

[0003] At present, real-time reverse transcription polymerase chain reaction (rRT-PCR) has been the gold standard for detecting SARS-CoV-2 due to its advantages of high sensitivity and specificity. However, the rRT-PCR method has a cumbersome operation process, requires expensive instruments (rRT-PCR instrument), and the reaction procedure is complicated and time-consuming. Therefore, this method is not suitable for detection in some areas with backward laboratory conditions, as well as emergency bedside detection. In addition, antigen detection is also one of the commonly used clinical detection methods. Although this method is easy to operate and time-saving, it has low sensitivity. In the early stage of infection, when the amount of viral vector in the body is low, false negative results are prone to occur. Therefore, there is an urgent need to develop a simple, cost-saving, rapid and more sensitive detection technology to diagnose COVID-19.

[0004] Based on this, the present invention provides a new strategy for detecting the new coronavirus, so as to detect the new coronavirus conveniently and quickly. Summary of the invention

[0005] The present invention provides a nucleic acid sequence, product, method and application for detecting the novel coronavirus, so as to detect the novel coronavirus more conveniently and quickly.

[0006] In a first aspect of an embodiment of the present invention, a nucleic acid sequence for novel coronavirus detection is provided, wherein the nucleic acid sequence has a multiple cross-displacement amplification reaction primer sequence shown in SEQ ID No. 1 to SEQ ID No. 20;

[0007] Among them, SEQ ID No.1 to SEQ ID No.10 are used to detect the ORF1ab gene of the new coronavirus SARS-CoV-2, and SEQ ID No.11 to SEQ ID No.20 are used to detect the N gene of the new coronavirus SARS-CoV-2.

[0008] In an optional embodiment of the present invention, the 5' end of the primer sequence shown in SEQ ID No.4 is connected to fluorescein isothiocyanate; the 5' end of the primer sequence shown in SEQ ID No.14 is connected to digoxigenin; the 5' end of the primer sequences shown in SEQ ID No.3 and SEQ ID No.13 are connected to biotin.

[0009] In a second aspect of an embodiment of the present invention, a kit for detecting a novel coronavirus is provided, the kit comprising a nucleic acid amplification reagent, the nucleic acid amplification reagent comprising a multiple cross-displacement amplification reaction primer sequence shown in SEQ ID No. 1 to SEQ ID No. 20;

[0010] Among them, SEQ ID No.1 to SEQ ID No.10 are used to detect the ORF1ab gene of the new coronavirus SARS-CoV-2, and SEQ ID No.11 to SEQ ID No.20 are used to detect the N gene of the new coronavirus SARS-CoV-2.

[0011] In an optional embodiment of the present invention, the 5' end of the primer sequence shown in SEQ ID No.4 is connected to fluorescein isothiocyanate; the 5' end of the primer sequence shown in SEQ ID No.14 is connected to digoxigenin; the 5' end of the primer sequences shown in SEQ ID No.3 and SEQ ID No.13 are connected to biotin.

[0012] In an optional embodiment of the present invention, the kit further comprises: 2×Bst DNA polymerase buffer, DNA polymerase, avian fibroblast virus reverse transcriptase, RNA template, and visual detection reagent. Avian fibroblast virus reverse transcriptase (AMV) is added to the multiple cross-continuous temperature amplification (MCDA) system to achieve reverse transcription and gene amplification in the same system.

[0013] In a third aspect of an embodiment of the present invention, a detection system for detecting a novel coronavirus is provided, the detection system comprising: a test kit and a lateral flow biosensor strip; the test kit comprises a nucleic acid amplification reagent, the nucleic acid amplification reagent comprises a multiple cross-displacement amplification reaction primer sequence shown in SEQ ID No. 1 to SEQ ID No. 20;

[0014] Among them, SEQ ID No.1 to SEQ ID No.10 are used to detect the ORF1ab gene of the new coronavirus SARS-CoV-2, and SEQ ID No.11 to SEQ ID No.20 are used to detect the N gene of the new coronavirus SARS-CoV-2.

[0015] In an optional embodiment of the present invention, the lateral flow measurement biosensor strip comprises a sample pad, a gold label pad, a fiber membrane, a water absorbent pad and a back plate, wherein the sample pad, the gold label pad, the fiber membrane and the water absorbent pad are sequentially assembled on the back plate, and the fiber membrane is provided with: a detection line 1, a detection line 2 and a quality control line, wherein the gold label pad is coated with gold nanoparticle-coupled streptavidin, the detection line 1 and the detection line 2 are respectively coated with anti-fluorescein isothiocyanate antibodies and anti-digoxigenin antibodies, and the quality control line is coated with biotin-coupled bovine serum albumin.

[0016] In a fourth aspect of an embodiment of the present invention, a non-diagnostic detection method for novel coronavirus detection is provided, the method comprising:

[0017] Extracting the genome of the virus sample to be tested;

[0018] Based on the nucleic acid sequence having multiple cross-displacement amplification reaction primer sequences shown in SEQ ID No. 1 to SEQ ID No. 20, a multiple cross-displacement isothermal amplification reaction is performed using the genome of the virus sample to be detected as a template to obtain an amplification product;

[0019] The amplification product is detected using a lateral flow biosensor strip.

[0020] In an optional embodiment of the present invention, the primer concentrations in the reaction system of the multiple cross-isothermal amplification reaction are: the concentrations of cross primers CP1 and CP2 are both 1.6 μM, the concentrations of replacement primers F1 and F2 are both 0.4 μM, and the concentrations of amplification primers R1, R2, D1*, D2, C1* and C2 are all 0.8 μM.

[0021] In an optional embodiment of the present invention, the nucleic acid sequence according to any one of the first aspects above is used for non-diagnostic purposes in the detection of new coronavirus.

[0022] The present invention includes the following advantages: The present invention provides a nucleic acid sequence, product, method and application for the detection of the new coronavirus, and MCDA amplification primers are designed for the ORF1ab gene and N gene of the new coronavirus SARS-CoV-2, respectively, so that the ORF1ab gene and N gene of the new coronavirus can be detected at the same time. In the embodiment of the present invention, different fluorescent markers are designed for the amplification primers for the ORF1ab gene and the N gene, and a multi-cross constant temperature amplification reaction is adopted, so that the amplification products of the ORF1ab gene and the N gene can be obtained only under one temperature condition, and the genomic ORF1ab gene and the N gene of the virus sample to be detected are detected simultaneously using a lateral flow measurement biosensor strip. The nucleic acid sequence provided in the embodiment of the present invention is used to detect the new coronavirus, which has a fast detection speed, does not require a variable temperature device, saves the cost of the laboratory to purchase a PCR amplifier, and has excellent specificity, and can accurately identify the new coronavirus. The detection scheme is convenient, fast, sensitive, and specific, and is suitable for promotion to the clinical detection of the new coronavirus. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for use in the description of the embodiments of the present application will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0024] Figure 1 This is a diagram showing the optimal reaction temperature results of a single-plex RT-MCDA assay provided by an embodiment of the present invention;

[0025] Figure 2 This is a result diagram of the dual-channel gold nanometer flow measurement biosensor provided by an embodiment of the present invention;

[0026] Figure 3 It is a sensitivity result diagram of the single-plex RT-MCDA detection provided by an embodiment of the present invention;

[0027] Figure 4 is a sensitivity result diagram of SARS-CoV-2-mRT-MCDA-LFB detection provided by an embodiment of the present invention;

[0028] Figure 5 It is a specific result diagram of the SARS-CoV-2-mRT-MCDA-LFB detection method provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0029] The following will be combined with the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application. The reagents and instruments used in the embodiments of the present invention are as follows:

[0030] The SARS-CoV-2 virus gene template was an in-house quality control product produced by Shanghai Zhijiang Biotechnology Co., Ltd. Anti-fluorescein isothiocyanate antibody (anti-FITC), anti-digoxigenin antibody (anti-DIG), gold nanoparticle-conjugated streptavidin (SA-G), and biotin-conjugated bovine serum albumin (B-BSA) were purchased from Resenbio. The backing plate, sample pad, gold label pad, fiber membrane, and absorbent pad were purchased from Jie-Yi Company. The MCDA kit and visual detection reagent (VDR) were from Tianjin Huidexin Technology Development Co., Ltd. Avian fibroblast virus reverse transcriptase (AMV) was purchased from BBI Life Sciences Co., Ltd. DL2000 DNAMarker was purchased from Bao Biotechnology (Dalian) Co., Ltd. Primer synthesis was completed by Qingke Biotechnology Co., Ltd. (Beijing, China). The fully automatic magnetic bead-based nucleic acid extraction instrument was purchased from Shanghai Zhijiang Biotechnology Co., Ltd. The PCR instrument was Sensoquest Labcycler, a product of Sensoquest of Germany; the electrophoresis equipment and gel imaging system (Gel Dox XR) were both products of Bio-Rad of the United States. Constant temperature amplification was achieved using a PCR instrument.

[0031] Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), also known as the novel coronavirus infection (COVID-19), has been found to infect humans since 2019 and has caused a pandemic of acute respiratory diseases. It has now caused extremely high morbidity and millions of deaths worldwide, seriously threatening human health and public safety. Initially, COVID-19 causes severe "flu"-like symptoms, with the most common symptoms being fever, cough and difficulty breathing. In severe cases, it can progress to acute respiratory distress syndrome (ARDS), pneumonia, pulmonary fibrosis, renal failure, myocarditis, and even death. Although humans continue to develop new vaccines to respond to the outbreak, the spread of SARS-CoV-2 in humans has not been completely curbed as the activity of specific antibodies in the human body continues to weaken and the constantly mutating virus strains have immune escape. As the SARS-CoV-2 virus strains continue to mutate, they are characterized by lower virulence and stronger transmission ability. At present, most people only show symptoms such as fever, sore throat, headache, and gastrointestinal discomfort after infection, but there is still a serious threat to the life safety of children, the elderly, people with low immunity, and people with underlying diseases.

[0032] At present, real-time reverse transcription polymerase chain reaction (rRT-PCR) has been the gold standard for detecting SARS-CoV-2 due to its high sensitivity and specificity. However, the rRT-PCR method has a cumbersome operation process, requires expensive instruments (rRT-PCR instrument), and the reaction procedure is complicated and time-consuming. Therefore, this method is not suitable for detection in some areas with backward laboratory conditions, as well as emergency bedside detection. In addition, antigen detection is also one of the commonly used clinical detection methods. Although this method is easy to operate and time-saving, it has low sensitivity. In the early stage of infection, when the amount of viral vector in the body is low, false negative results are prone to occur. Therefore, there is an urgent need to develop a simple, cost-saving, rapid and more sensitive detection technology to diagnose COVID-19.

[0033] Multi-cross displacement amplification (MCDA) technology is a new gene amplification technology established in recent years. This technology can identify the target sequence at a constant temperature through ten primers. It is characterized by simple operation, no need for complex instruments and working environment, short amplification time, and high specificity and sensitivity. The development of MCDA technology has been relatively mature, and detection of various strains including Acinetobacter baumannii, Listeria monocytogenes, Klebsiella pneumoniae, Candida albicans, etc. has been achieved. However, according to current research, the method of combining MCDA and reverse transcription to detect RNA viruses is rare. In the present invention, the inventor established mRT-MCDA-LFB technology, that is, reverse transcription and MCDA amplification are carried out simultaneously in the same system, so as to realize the identification of two groups of RNA gene sequences at a constant temperature, and double amplification, and then through the method of dual-channel flow measurement biosensor, the results are judged by naked eyes. The present invention successfully developed a set of rapid and effective SARS-CoV-2-mRT-MCDA-LFB detection methods, which provides a convenient and effective means for clinical diagnosis of COVID-19.

[0034] Based on MCDA technology, the inventors have developed a solution for rapid detection of the new coronavirus based on MCDA technology, targeting the currently common new coronavirus model SARS-CoV-2.

[0035] In a first aspect of an embodiment of the present invention, a nucleic acid sequence for detecting a novel coronavirus is provided, wherein the nucleic acid sequence has a multiple cross-displacement amplification reaction primer sequence shown in SEQ ID No.1 to SEQ ID No.20; wherein SEQ ID No.1 to SEQ ID No.10 are used to detect the ORF1ab gene of the novel coronavirus SARS-CoV-2, and SEQ ID No.11 to SEQ ID No.20 are used to detect the N gene of the novel coronavirus SARS-CoV-2.

[0036] In the embodiment of the present invention, MCDA amplification primers were designed for two specific genes on the new coronavirus SARS-CoV-2 virus: ORF1ab (open reading frame 1a / b) gene and N (nucleocapsid protein) gene, so as to simultaneously detect the ORF1ab gene and N gene of the new coronavirus.

[0037] In the present embodiment, the primers that can be labeled include CP1, CP2, D1, C1, R1, D2, the 5' end of C2 and R2.

[0038] The labeled primers are all marked with asterisks to distinguish them. In the embodiments of the present invention, the specific primer sequences and modifications are shown in Table 1 below:

[0039] Table 1

[0040]

[0041]

[0042] The 5' end of the primer sequence shown in SEQ ID No.4 is connected to fluorescein isothiocyanate; the 5' end of the primer sequence shown in SEQ ID No.14 is connected to digoxigenin; the 5' end of the primer sequences shown in SEQ ID No.3 and SEQ ID No.13 are connected to biotin.

[0043] In the embodiment of the present invention, the MCDA reaction system contains 10 primers that recognize 10 regions of the target sequence, including: 2 cross primers: CP1 and CP2 (Cross Primer, CP), CP1 contains Cls (complementary sequence of C1 region) and P1, i.e. 5′-Cls-P1, CP2 contains C2s (complementary sequence of C2 region) and P2, i.e. 5′-C2s-P2, the two cross primers CP1 and CP2 are the main primers mediating MCDA amplification; 2 replacement primers: F1 and F2, which play a replacement role in the MCDA reaction and replace the cross primers CP1 and CP2; 6 amplification primers: D1, C1, R1, D2, C2 and R2 can accelerate the MCDA reaction and increase the amount of MCDA product.

[0044] In a second aspect of an embodiment of the present invention, a kit for detecting the new coronavirus is provided, the kit comprising a nucleic acid amplification reagent, the nucleic acid amplification reagent containing multiple cross-displacement amplification reaction primer sequences shown in SEQ ID No. 1 to SEQ ID No. 20; wherein SEQ ID No. 1 to SEQ ID No. 10 are used to detect the ORF1ab gene of the new coronavirus SARS-CoV-2, and SEQ ID No. 11 to SEQ ID No. 20 are used to detect the N gene of the new coronavirus SARS-CoV-2.

[0045] Specifically, the kit also includes: 2×Bst DNA polymerase buffer, DNA polymerase, avian fibroblast virus reverse transcriptase, RNA template, and visual detection reagent.

[0046] In a third aspect of an embodiment of the present invention, a detection system for detecting a novel coronavirus is provided, the detection system comprising: a test kit and a lateral flow biosensor strip; the test kit comprises a nucleic acid amplification reagent, the nucleic acid amplification reagent comprises a multiple cross-displacement amplification reaction primer sequence shown in SEQ ID No.1 to SEQ ID No.20; wherein, SEQ ID No.1 to SEQ ID No.10 are used to detect the ORF1ab gene of the novel coronavirus SARS-CoV-2, and SEQ ID No.11 to SEQ ID No.20 are used to detect the N gene of the novel coronavirus SARS-CoV-2.

[0047] Specifically, the lateral flow measurement biosensor strip comprises a sample pad, a gold label pad, a fiber membrane, a water absorbent pad and a back plate, wherein the sample pad, the gold label pad, the fiber membrane and the water absorbent pad are sequentially assembled on the back plate, and the fiber membrane is provided with: a detection line 1, a detection line 2 and a quality control line, wherein the gold label pad is coated with streptavidin coupled with gold nanoparticles, the detection line 1 and the detection line 2 are coated with anti-fluorescein isothiocyanate antibodies and anti-digoxigenin antibodies respectively, and the quality control line is coated with biotin-coupled bovine serum albumin.

[0048] In an embodiment of the present invention, the lateral flow measurement biosensor strip is specifically: a dual-channel biodetector (LFB). Specifically, the LFB comprises five parts, a sample pad, a gold label pad, a fiber membrane, a water-absorbing pad and a back plate. First, the sample pad, the gold label pad, the fiber membrane and the water-absorbing pad are assembled on the back plate in sequence. Then, SA-G (streptavidin coupled to gold nanoparticles) is coated at the gold label pad position, anti-FITC (anti-fluorescein isothiocyanate antibody) and anti-DIG (anti-digoxigenin antibody) are coated at the detection line 1 and the detection line 2 positions respectively, and B-BSA (biotin-coupled bovine serum albumin) is coated at the control line (CL) position. After drying, it is packaged in a plastic shell and can be used.

[0049] In a fourth aspect of an embodiment of the present invention, a non-diagnostic detection method for novel coronavirus detection is provided, the method comprising the following steps:

[0050] Step 1, extracting the genome of the virus sample to be detected;

[0051] Step 2, based on the nucleic acid sequence having multiple cross-displacement amplification reaction primer sequences shown in SEQ ID No. 1 to SEQ ID No. 20, using the genome of the virus sample to be detected as a template to perform a multiple cross-displacement isothermal amplification reaction to obtain an amplification product;

[0052] Step 3: Detect the amplification product using a lateral flow biosensor strip.

[0053] Among them, when conducting a multiple cross-isothermal amplification reaction, the two sets of amplification primers of the ORF1ab gene and the N gene were mixed in the same reaction system, and the primer concentrations were: the concentration of the replacement primers F1 and F2 was 0.4 μM, the concentration of the amplification primers R1, R2, D1*, D2, C1* and C2 was 0.8 μM each, and the concentration of the cross primers CP1 and CP2 was 1.6 μM.

[0054] Specifically, the reaction system of the multi-cross isothermal amplification reaction also includes: 12.5 μL of 2×Bst DNA polymerase buffer, 1.25 μL of DNA polymerase (Bst 2.0), 1 μL of avian fibroblast virus reverse transcriptase, 1 μL of RNA template (genome of the virus sample to be detected), 1 μL of visual detection reagent (VDR), and deionized water is added to 25 μL. The reaction conditions of the multi-cross isothermal amplification reaction are: the amplification reaction is carried out at a constant temperature of 65° C. for 40 minutes to obtain the amplified product.

[0055] In the embodiment of the present invention, the inventor analyzed the MCDA amplification product by nucleic acid amplification indicator and 2.0% agarose gel electrophoresis for the amplification results. For positive results, the reaction system is still sky blue and appears as a ladder band in the agarose gel. For negative results, the reaction system changes from blue to colorless and transparent, and there is no ladder band in the agarose gel. In order to determine the optimal detection temperature, the inventor conducted amplification experiments at 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C and 70°C for each group of single-plex amplification systems (the reaction template was 100pg per reaction). The experimental results showed that when the temperature was between 63-68°C, the reaction system showed a clear sky blue color, indicating that when the temperature was between 63-68°C, a good amplification effect could be achieved, while the color of the reaction system below 63°C and above 68°C was not obvious, indicating that the amplification effects below 63°C and above 68°C were poor (see Figure 1 , Figure 1 Parts A and B in 1 are the amplification results of ORF1ab-RT-MCDA at different temperatures, and parts C and D in 1 are the amplification results of N-RT-MCDA at different temperatures). Therefore, considering the stability of the experiment, the inventors adopted 65°C as the optimal detection temperature for the RT-MCDA experiment.

[0056] In the embodiment of the present invention, a multiple cross-displacement amplification reaction primer sequence (a nucleic acid sequence having a multiple cross-displacement amplification reaction primer sequence shown in SEQ ID No. 1 to SEQ ID No. 20) is used, and a multiple cross-isothermal amplification reaction is performed using the genome of the virus sample to be detected as a template to obtain a corresponding amplification product. The amplification product is then detected using a lateral flow biosensor, thereby realizing the detection and typing identification of the new coronavirus.

[0057] Specifically, the operation in step 3 includes: dripping a total of 10 μL of MCDA product in the sample pad area of ​​the LFB, then adding 100 μL of detection buffer in the same area, siphoning the MCDA product and moving it from bottom to top (moving from the sample pad to the absorption pad). When the MCDA product reaches the gold label pad, one end of the double-labeled product (biotin-labeled end) reacts with streptavidin (SA-G) coupled to gold nanoparticles. As the product moves forward, the other end of the double-labeled product (FITC-labeled end or Dig-labeled end) will attach to the antibody in the detection line 1 or detection line 2 area, thereby fixing the double-labeled product on the detection line. When the product aggregates in the detection line area, the SA-G at the other end will undergo a color reaction. If the MCDA product contains two types of labeled products, both detection line 1 and detection line 2 can be colored, thereby achieving dual-channel visual detection of the MCDA product. In addition, any excess SA-G will directly react with the biotin-coupled bovine serum albumin in the quality control line (CL) area, resulting in a color reaction, and the function of the LFB can be judged by the result of the above color reaction. Through the above detection method, the new coronavirus can be detected, getting rid of expensive instruments and complex reaction conditions, and the detection can be completed quickly within an hour through 40 minutes of gene amplification and 5 minutes of lateral flow biosensor reading results.

[0058] Based on the same inventive concept as above, in an embodiment of the present invention, non-diagnostic applications of the above nucleic acid sequences in the detection of new coronaviruses are also provided.

[0059] The present invention is for detecting the novel coronavirus, and different fluorescent markers are designed for the amplification primers of the ORF1ab gene and the N gene of the novel coronavirus, and a multi-cross constant temperature amplification reaction is adopted, so that the amplification products of the ORF1ab gene and the N gene can be obtained under only one temperature condition, and the ORF1ab gene and the N gene of the genome of the virus sample to be detected are detected simultaneously by using a lateral flow measurement biosensor strip. The amplification primer sequence provided by the present invention can be used for visual detection of the novel coronavirus, with a fast detection speed, high specificity and high sensitivity, and its operation is simple, time and cost saving, and it is suitable for promotion to the clinical detection of the novel coronavirus.

[0060] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0061] Example 1: Feasibility verification of dual-channel visual detection of mRT-MCDA products

[0062] In order to realize the mRT-MCDA method to detect ORF1ab and N genes simultaneously, the inventors added the primer sets corresponding to ORF1ab and N genes into the same reaction system and amplified them at a constant temperature of 65°C for 40 minutes. Although nucleic acid amplification indicators and agarose gel electrophoresis cannot accurately interpret mRT-MCDA products, the detection results can be displayed on the dual-channel LFB. For details, please refer to Figure 2 The two red lines marked with CL and TL1 indicate that the ORF1ab gene was successfully amplified (see Figure 2 In the first block LFB, the two red lines marked with CL and TL1 indicate ORD1ab positivity), while the two red lines marked with CL and TL2 indicate successful amplification of the N gene (see Figure 2 In block II LFB, the two red lines CL and TL2 indicate N positive). If TL1, TL2 and CL all appear in the detection area, the test result is positive (see Figure 2 In the LFB of panel III, the three red lines CL, TL1 and TL2 indicate that ORD1ab and N are both positive. If the LFB shows only a single CL band, the test result is negative (see Figure 2 In the fourth block of LFB, CL has only one red line indicating negative). It can be seen from the results that the inventors have successfully designed the mRT-MCDA-LFB technology to detect the new coronavirus SARS-CoV-2.

[0063] Example 2: Sensitivity verification of single-plex RT-MCDA method for amplification of ORF1ab and N genes

[0064] The SARS-CoV-2 gene template was diluted to 100ng, 10ng, 1ng, 100pg, 10pg, 1pg, 100fg, 10fg, and 1fg per reaction, and the ORF1ab and N genes were amplified at 65°C. The experimental results are shown in Figure 3 ( Figure 3 Parts A and B are the sensitivity results of ORF1ab-RT-MCDA. Figure 3 Parts C and D are the sensitivity results of N-RT-MCDA, where DW is an equal volume of distilled water added as a negative control). It can be seen that the lowest detection limit of ORF1ab-RT-MCDA and N-RT-MCDA is 100 fg.

[0065] Example 3: Sensitivity verification of SARS-CoV-2-mRT-MCDA-LFB detection method

[0066] The serially diluted novel coronavirus genome was subjected to standard MCDA amplification reaction, and two detection methods were used to judge the novel coronavirus MCDA amplification results. The judgment results were as follows: Figure 4 shown.

[0067] Specifically, the gene template of SARS-CoV-2 was serially diluted (1 ng / μL, 100 pg / μL, 10 pg / μL, 1 pg / μL, 100 fg / μL, 10 fg / μL and 1 fg / μL) to determine the sensitivity of the SARS-CoV-2-mRT-MCDA-LFB detection method (the mRT-MCDA-LFB detection method for SARS-CoV-2 proposed in the embodiment of the present invention), and an equal volume of distilled water (DW) was added to the negative control group, and the results were interpreted simultaneously by the visual dye method and the dual-channel LFB method ( Figure 4 1-7 represent the amount of template in each reaction of 1ng, 100pg, 10pg, 1pg, 100fg, 10fg and 1fg, respectively. Number 8 is used as a negative control by adding the same volume of distilled water). If the reaction solution is blue after amplification, it is judged as a positive reaction, and colorless is a negative reaction. The product is detected by LFB. The appearance of red lines in the TL1, TL2 and CL regions indicates a positive result. When the SARS-CoV-2 genome content in the reaction system is lower than its detection limit, or the added template is double distilled water (DW), LFB only appears a red line in the CL region, indicating a negative result. For this result, refer to Figure 4 The lowest detection limit of the SARS-CoV-2-mRT-MCDA-LFB detection method was 100 pg per reaction. Compared with the single-plex RT-MCDA method, multiple amplification had no effect on its sensitivity.

[0068] Example 5: Specificity verification of SARS-CoV-2-mRT-MCDA-LFB detection method

[0069] The specificity of the SARS-CoV-2-mRT-MCDA-LFB detection method was evaluated using common viruses, mycoplasmas, chlamydia, and bacteria as templates (see Table 2 for strain information). Figure 5 The results of the detection of SARS-CoV-2 gene templates (LFB-1) and non-SARS-CoV-2 templates such as influenza A virus, influenza B virus, and respiratory syncytial virus (LFB-2-29) are shown. In addition, LFB-30 shows that an equal volume of distilled water was added as a negative control. Figure 5 As shown, the SARS-CoV-2-mRT-MCDA-LFB method detected SARS-CoV-2 as positive, while the results of other samples were negative. The above results show that the SARS-CoV-2-mRT-MCDA-LFB detection method has extremely high specificity in detecting SARS-CoV-2.

[0070] Table 2 Strain information and specific results of MCDA-LFB detection method:

[0071]

[0072]

[0073] In the present invention, a nucleic acid sequence, product, method and application for the detection of the new coronavirus have been successfully developed. The nucleic acid sequence can simultaneously amplify the specific genes ORF1ab (open reading frame 1a / b) and N (nucleocapsid protein) of the new coronavirus in a reaction system at a constant temperature (65°C). The detection kit prepared using the above nucleic acid sequence is used to detect the new coronavirus, which gets rid of expensive instruments and complex reaction conditions. The results are read by 40 minutes of gene amplification and 5 minutes of lateral flow biosensor, and the detection can be completed quickly within an hour. And the nucleic acid sequence has very high sensitivity and specificity. Therefore, the nucleic acid sequence, product and method for the detection of the new coronavirus based on MCDA proposed in the present invention have effective practical value in clinical diagnosis and response to the outbreak of the new crown epidemic.

[0074] The nucleic acid sequence, product, method and application for the detection of the new coronavirus provided by the present invention are introduced in detail above. Specific examples are used herein to illustrate the principle and implementation mode of the present invention. The description of the above embodiments is only used to help understand the method of the present invention and its core idea. At the same time, for those skilled in the art, according to the idea of ​​the present invention, there will be changes in the specific implementation mode and application scope. In summary, the content of this specification should not be understood as limiting the present invention.

Claims

1. A nucleic acid sequence for detecting a novel coronavirus, characterized in that: The nucleic acid sequence has a multiple cross-displacement amplification reaction primer sequence shown in SEQ ID No. 1 to SEQ ID No. 20; Among them, SEQ ID No.1 to SEQ ID No.10 are used to detect the ORF1ab gene of the new coronavirus SARS-CoV-2, and SEQ ID No.11 to SEQ ID No.20 are used to detect the N gene of the new coronavirus SARS-CoV-2.

2. The nucleic acid sequence for detecting novel coronavirus according to claim 1, characterized in that: The 5' end of the primer sequence shown in SEQ ID No. 4 is connected to fluorescein isothiocyanate; the 5' end of the primer sequence shown in SEQ ID No. 14 is connected to digoxigenin; the 5' end of the primer sequences shown in SEQ ID No. 3 and SEQ ID No. 13 are connected to biotin.

3. A kit for detecting a novel coronavirus, characterized in that: The kit includes a nucleic acid amplification reagent, wherein the nucleic acid amplification reagent contains multiple cross-displacement amplification reaction primer sequences shown in SEQ ID No. 1 to SEQ ID No. 20; Among them, SEQ ID No.1 to SEQ ID No.10 are used to detect the ORF1ab gene of the new coronavirus SARS-CoV-2, and SEQ ID No.11 to SEQ ID No.20 are used to detect the N gene of the new coronavirus SARS-CoV-2.

4. The kit for detecting novel coronavirus according to claim 3, characterized in that: The 5' end of the primer sequence shown in SEQ ID No.4 is connected to fluorescein isothiocyanate; the 5' end of the primer sequence shown in SEQ ID No.14 is connected to digoxigenin; the 5' end of the primer sequences shown in SEQ ID No.3 and SEQ ID No.13 are connected to biotin.

5. The kit for detecting novel coronavirus according to claim 3, characterized in that: The kit also includes: 2×Bst DNA polymerase buffer, DNA polymerase, avian fibroblast virus reverse transcriptase, RNA template, and visual detection reagent.

6. A detection system for novel coronavirus detection, characterized in that: The detection system comprises: a test kit and a lateral flow biosensor strip; the test kit comprises a nucleic acid amplification reagent, and the nucleic acid amplification reagent comprises a multiple cross-displacement amplification reaction primer sequence shown in SEQ ID No. 1 to SEQ ID No. 20; Among them, SEQ ID No.1 to SEQ ID No.10 are used to detect the ORF1ab gene of the new coronavirus SARS-CoV-2, and SEQ ID No.11 to SEQ ID No.20 are used to detect the N gene of the new coronavirus SARS-CoV-2.

7. The detection system for detecting the novel coronavirus according to claim 6, characterized in that: The transverse flow measurement biosensor strip comprises a sample pad, a gold label pad, a fiber membrane, a water absorbent pad and a back plate, wherein the sample pad, the gold label pad, the fiber membrane and the water absorbent pad are sequentially assembled on the back plate, and the fiber membrane is provided with: a detection line 1, a detection line 2 and a quality control line, wherein the gold label pad is coated with streptavidin coupled with gold nanoparticles, the detection line 1 and the detection line 2 are respectively coated with anti-fluorescein isothiocyanate antibody and anti-digoxigenin antibody, and the quality control line is coated with biotin-coupled bovine serum albumin.

8. A non-diagnostic detection method for novel coronavirus detection, characterized in that: The method comprises: Extracting the genome of the virus sample to be tested; Based on the nucleic acid sequence having multiple cross-displacement amplification reaction primer sequences shown in SEQ ID No. 1 to SEQ ID No. 20, a multiple cross-displacement isothermal amplification reaction is performed using the genome of the virus sample to be detected as a template to obtain an amplification product; The amplification product is detected using a lateral flow biosensor strip.

9. The non-diagnostic detection method for novel coronavirus detection according to claim 8, characterized in that: The primer concentrations in the reaction system of the multiple cross isothermal amplification reaction are: the concentrations of the cross primers CP1 and CP2 are both 1.6 μM, the concentrations of the displacement primers F1 and F2 are both 0.4 μM, and the concentrations of the amplification primers R1, R2, D1*, D2, C1* and C2 are all 0.8 μM.

10. Use of the nucleic acid sequence according to claim 1 or claim 2 for non-diagnostic purposes in the detection of new coronavirus.

Images