A rapid detection method for novel coronavirus and a rapid identification method for variant strains

Abstract

The application discloses a rapid detection and variation strain rapid identification method of a novel coronavirus, and comprises the following steps: S1, taking PCR amplification freeze-dried microspheres; S2, novel coronavirus detection; S3, novel coronavirus variation strain identification, which comprises the following steps: S31, mixing the amplification product of the sample with a positive novel coronavirus detection result with hybridization liquid, adding the novel coronavirus variation strain rapid identification chip, centrifuging the mixed liquid to flow into the reaction pool, and heating the constant-temperature reaction until the reaction liquid completely flows into the identification necessary pool outside the reaction pool; S32, scanning the reaction pool by using a scanner, and the fluorescence signal with flocculation is the corresponding mutation type, and the fluorescence signal without flocculation indicates that the corresponding mutation type is not possessed. The application can rapidly identify and determine which variation strain.

Description

Technical Field

[0001] This invention relates to public health and epidemic prevention, and in particular to a method for rapid detection of novel coronavirus and rapid identification of variant strains, as well as a chip for rapid identification of novel coronavirus variant strains. Background Technology

[0002] Most commercially available COVID-19 nucleic acid tests can be completed within 1-3 hours, determining whether a result is positive or negative, and even the concentration of the virus. However, COVID-19 is a highly mutating virus. Some mutant strains, such as Alpha, Belta, Delta, and Omicron, especially Omicron, have significantly increased infectivity, requiring greater attention from health authorities. This also places new demands on testing technology, requiring it not only to diagnose infection but also to identify the specific mutant strain. Current methods for identifying the mutant strain involve gene sequencing and comparison, a complex and time-consuming process. Summary of the Invention

[0003] To address the aforementioned deficiencies, this invention provides a method for rapid detection of the novel coronavirus and rapid identification of variant strains, which can quickly identify and determine which variant strain it is.

[0004] A rapid detection method for novel coronavirus and rapid identification of variant strains, comprising the following steps:

[0005] S1, Take PCR amplification lyophilized microspheres;

[0006] S2, Novel Coronavirus Testing, this step includes;

[0007] S20, extract nucleic acid samples;

[0008] S21, add the nucleic acid sample extracted in S20 to the PCR amplification lyophilized microspheres to reconstitute the PCR amplification lyophilized microspheres, seal, shake thoroughly to dissolve the PCR amplification lyophilized microspheres, and then centrifuge briefly.

[0009] S22, placed in a real-time quantitative PCR instrument for amplification;

[0010] S23, obtain the test result. If the test result is positive, proceed to S3.

[0011] S3, Identification of novel coronavirus variants, this step includes:

[0012] S31, after mixing the amplification product of the sample that tested positive for the novel coronavirus with the hybridization solution, add it to the novel coronavirus variant rapid identification chip, centrifuge the mixture and let it flow into the reaction cell, heat it to a constant temperature until the liquid after the reaction completely flows into the identification cell outside the reaction cell.

[0013] S32. Scan the reaction cell with a scanner. Clusters of fluorescent signals indicate the corresponding mutation type, while the absence of clusters of fluorescent signals indicates the absence of the corresponding mutation type.

[0014] Preferably, the PCR amplification lyophilized microspheres are prepared by the following method and then stored or transported:

[0015] S11, Preparation: Mix primers, probes, enzymes and excipients in the proportions required for the novel coronavirus to form PCR amplification reagents;

[0016] S12, spotting: The PCR amplification reagent formed in S11 is spotted onto a pre-cooled lyophilized plate using a pipette or an automatic bead dispenser to form PCR amplification reagent microspheres.

[0017] S13, freeze-drying: The plate prepared by S12 is placed in a vacuum freeze-drying oven for dehydration to form freeze-dried microspheres.

[0018] S14, Filling: Remove the PCR amplification lyophilized microspheres from the lyophilization tooling plate and load them into tubes, one PCR amplification lyophilized microsphere per tube. Load the tubes into the wells of the plate, seal them under vacuum, and the PCR amplification lyophilized microsphere kit is formed.

[0019] S15, Packaging.

[0020] Preferably, the dehydration process in the vacuum freeze-drying oven includes the following two steps:

[0021] S131, sublimation drying;

[0022] S132, desorption and drying.

[0023] Preferably, the freeze-drying tooling plate is a tooling plate that has been pre-cooled to -65°C.

[0024] Preferably, the novel coronavirus variant rapid identification chip includes a chip substrate, on which at least one variant rapid identification unit is disposed. Each variant rapid identification unit includes a sample pool, a first reaction pool, a second reaction pool, a third reaction pool, a fourth reaction pool, and a fifth reaction pool. The variant rapid identification unit also includes a connecting channel and a separating channel. One end of the connecting channel is connected to the sample pool, and the other end is connected to the separating channel. The first reaction pool, the second reaction pool, the third reaction pool, the fourth reaction pool, and the fifth reaction pool are each connected to the separating channel. The first reaction pool, the second reaction pool, the third reaction pool, the fourth reaction pool, and the fifth reaction pool are used to pre-load the spotting solution for novel coronavirus variant identification.

[0025] Preferably, the rapid identification unit for mutant strains is further provided with an identification pool, a first siphon channel, a second siphon channel, a third siphon channel, a fourth siphon channel, and a fifth siphon channel. The first reaction pool is connected to the identification pool through the first siphon channel, the second reaction pool is connected to the identification pool through the second siphon channel, the third reaction pool is connected to the identification pool through the third siphon channel, the fourth reaction pool is connected to the identification pool through the fourth siphon channel, and the fifth reaction pool is connected to the identification pool through the fifth siphon channel.

[0026] Preferably, the rapid identification unit for mutant strains is also equipped with a collection pool, which is connected to the separation channel.

[0027] Preferably, the novel coronavirus variant rapid identification chip also includes a chip cover that covers the chip substrate.

[0028] Preferably, the chip cover is also provided with a first exhaust hole and a second exhaust hole, and the mutant strain rapid identification unit is also provided with a first exhaust channel and a second exhaust channel. One end of the first exhaust channel is connected to the collection pool and the other end is connected to the first exhaust hole. One end of the second exhaust channel is connected to the identification pool and the other end is connected to the second exhaust hole.

[0029] Preferably, the chip cover is provided with a sample dispensing port, which is connected to the sample cell. Attached Figure Description

[0030] Figure 1 This is a flowchart illustrating the preparation process of the freeze-dried microspheres for novel coronavirus 2019-nCoV nucleic acid detection according to the present invention.

[0031] Figure 2 This is a schematic diagram of the freeze-dried microsphere tooling plate of the present invention;

[0032] Figure 3 This invention relates to a PCR amplification lyophilized microsphere kit.

[0033] Figure 4 This is a schematic diagram of the rapid identification chip for novel coronavirus variant strains in Embodiment 2 of the present invention;

[0034] Figure 5 This is a schematic diagram of the rapid identification chip for novel coronavirus variant strains in Embodiment 3 of the present invention;

[0035] Figure 6 This is a process flow diagram of the rapid novel coronavirus detection method of the present invention;

[0036] Figure 7 This is a flowchart of the rapid identification process for novel coronavirus variant strains according to the present invention;

[0037] Figure 8This is a flowchart illustrating the rapid detection and rapid identification process for novel coronavirus strains according to the present invention.

[0038] Figure 9 This is a schematic diagram of a comparative chip of the present invention. Detailed Implementation

[0039] The invention will now be further described with reference to the accompanying drawings.

[0040] In the description of this invention, it should be noted that the terms "upper" and "lower" indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, or the orientation or positional relationship in which the product of this invention is usually placed when in use. They are only used to facilitate the description of this invention and to simplify the description, and are not intended to indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this invention.

[0041] In the description of this invention, it should also be noted that, unless otherwise explicitly specified and limited, the terms "fitting," "connecting," and "connection" should be interpreted broadly. For example, "connection" can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium; it can be a welding connection or an encapsulated connection, etc. For those skilled in the art, the specific meaning of the above terms in this invention can be understood according to the specific circumstances.

[0042] Example 1: Preparation of freeze-dried microspheres for novel coronavirus (2019-nCoV) nucleic acid detection

[0043] Currently, most reagent kits on the market for detecting the novel coronavirus (2019-nCoV) nucleic acid are qPCR kits. These kits contain the reaction solution, primers, probes, enzymes, and other liquid materials required for the amplification reaction. The storage and transportation conditions for these qPCR kits are -20℃±5℃, which are quite stringent and unsuitable for conventional storage and transportation.

[0044] To resolve this technical issue, please refer to [reference needed]. Figure 1 , Figure 1 This invention relates to a preparation process for lyophilized microspheres for the detection of novel coronavirus 2019-nCoV. Figure 1 The preparation process includes the following steps:

[0045] S11, Preparation: Mix primers, probes, enzymes, and excipients (such as trehalose, polyethylene glycol, etc.) in the proportions required for the novel coronavirus to form PCR amplification reagents.

[0046] S12, Spotting: Spot the PCR amplification reagent formed in S11 onto the lyophilized substrate (the lyophilized substrate has been pre-cooled to -65°C before spotting; the purpose of pre-cooling is to make the liquid instantly solidify when the PCR amplification reagent is spotted onto the lyophilized substrate).

[0047] S13, lyophilization: The plate prepared in S12 is placed in a vacuum freeze dryer for dehydration to form PCR amplification reagent (lyophilized microspheres).

[0048] Figure 2 In this context, the lyophilized microsphere fixture includes a lyophilized fixture 1 and a plurality of PCR amplification lyophilized microspheres 2 separated from each other on the upper surface of the lyophilized fixture 1.

[0049] Furthermore, in a number of PCR amplification lyophilized microspheres 2, the distance between each two adjacent PCR amplification lyophilized microspheres 2 is basically equal.

[0050] In this step, the dehydration process in the vacuum freeze-drying oven includes the following two steps:

[0051] S131, sublimation drying, the conditions for sublimation drying are shown in Table 1 below.

[0052] S132, analytical drying, the conditions for analytical drying are shown in Table 1 below.

[0053] Table 1

[0054]

[0055] S14, Filling: Remove the PCR amplification lyophilized microspheres 2 from the lyophilization fixture 1 and load them into tubes, one PCR amplification lyophilized microsphere per tube. Place the tubes into the wells of a 96-well plate, seal the 96-well plate with sealing film, and then vacuum seal it in an aluminum foil bag, forming a... Figure 3 The PCR amplification lyophilized microsphere kit shown is shown.

[0056] It should be noted that in this invention, S12, the spotting is carried out outside the vacuum freeze-drying oven.

[0057] The well plate can be an 8-well plate or a PCR 96-well plate; no particular limitation is made here.

[0058] Figure 3 The PCR amplification lyophilized microsphere kit includes a substrate 3, a number of PCR amplification lyophilized microspheres 2, and a number of tubes 4 equal to the number of PCR amplification lyophilized microspheres 2. Each tube 4 contains one PCR amplification lyophilized microsphere 2. The PCR amplification lyophilized microsphere kit is also provided with a number of housing units 5. Each housing unit 5 forms a cavity 6 for placing one tube 4.

[0059] Furthermore, several receiving units 5 are integrally formed with the substrate 3, with the upper end of each receiving unit 5 being open and flush with the upper surface of the substrate 3, and the lower end extending through the lower surface of the substrate 3.

[0060] Furthermore, the cross-sectional area of ​​each accommodating unit 5 gradually decreases from the top to the bottom.

[0061] S15, Packaging: Package the PCR amplification lyophilized microsphere kit from S4 for easy transport and storage.

[0062] The PCR amplification lyophilized microsphere kit prepared in this embodiment solves the problem of the relatively strict storage and transportation conditions of existing qPCR kits, which require storage and transportation at -20℃±5℃. The PCR amplification lyophilized microsphere kit can be transported and stored at 2℃-8℃. On the other hand, it shortens the time for subsequent nucleic acid detection.

[0063] Example 2: Rapid Identification Chip for Novel Coronavirus Mutant Strains

[0064] Please refer to Figure 4 , Figure 4 The present invention discloses a rapid identification chip 7 for novel coronavirus variant strains, comprising a chip substrate 71, on which a rapid identification unit 72 for variant strains is disposed. The rapid identification unit 72 for variant strains includes a sample pool 721, a first reaction pool 7221, a second reaction pool 7222, a third reaction pool 7223, a fourth reaction pool 7224, and a fifth reaction pool 7225. The rapid identification unit 72 for variant strains also includes a connecting channel 723 and a separating channel 724. One end of the connecting channel 723 is connected to the sample pool 721, and the other end is connected to the separating channel 724. The first reaction pool 7221, the second reaction pool 7222, the third reaction pool 7223, the fourth reaction pool 7224, and the fifth reaction pool 7225 are each connected to the separating channel 724.

[0065] Furthermore, the novel coronavirus variant rapid identification chip 7 also includes a chip cover 73, which covers the chip substrate 71.

[0066] Furthermore, a sample dispensing port 74 is provided on the chip cover 73, which is connected to the sample cell 721.

[0067] Furthermore, the rapid identification unit 72 for mutant strains is also provided with an identification pool 725, a first siphon channel 7261, a second siphon channel 7262, a third siphon channel 7263, a fourth siphon channel 7264, and a fifth siphon channel 7265. The first reaction pool 7221 is connected to the identification pool 725 through the first siphon channel 7261, the second reaction pool 7222 is connected to the identification pool 725 through the second siphon channel 7262, the third reaction pool 7223 is connected to the identification pool 725 through the third siphon channel 7263, the fourth reaction pool 7224 is connected to the identification pool 725 through the fourth siphon channel 7264, and the fifth reaction pool 7225 is connected to the identification pool 725 through the fifth siphon channel 7265.

[0068] Furthermore, the rapid identification unit 72 for mutant strains is also equipped with a collection pool 727, which is connected to the separation channel 724.

[0069] Furthermore, the chip cover 73 is also provided with a first exhaust hole 75 and a second exhaust hole 76, and the mutant strain rapid identification unit 72 is also provided with a first exhaust channel 7281 and a second exhaust channel 7282. One end of the first exhaust channel 7281 is connected to the collection pool 727 and the other end is connected to the first exhaust hole 75. One end of the second exhaust channel 7282 is connected to the identification pool 725 and the other end is connected to the second exhaust hole 76.

[0070] Example 3

[0071] Please refer to Figure 5 In this embodiment, three mutant strain rapid identification units 72 are provided on the chip substrate 71. These three mutant strain rapid identification units 72 are arranged at equal intervals along the circumference with the center of the chip substrate 71 as the center.

[0072] In Example 2 or 3, before the chip cap 73 is closed, different liquids are spotted into each of the five reaction chambers: the first reaction chamber 7221, the second reaction chamber 7222, the third reaction chamber 7223, the fourth reaction chamber 7224, and the fifth reaction chamber 7225. The five reaction chambers contain, respectively, amino-modified mutant strain-specific probe spotting solution, amino-modified positive internal control (IC) probe spotting solution, amino-modified hybridization positive probe spotting solution, and blank control (BC) spotting solution (see Table 2 for details). After spotting, the reaction is allowed to proceed for 16 hours. Then, the chip cap 73 is closed, and the chip is placed in a vacuum bag, vacuum-sealed, and pressurized.

[0073] Table 2 below shows the liquids contained in the first reaction chamber 7221, the second reaction chamber 7222, the third reaction chamber 7223, the fourth reaction chamber 7224, and the fifth reaction chamber 7225 in one embodiment. Of course, these liquids may not be packed according to Table 2. For example, DMSO solution can be packed into the first reaction chamber 7221, while DMSO solution and the S gene N501Y site probe can be packed into the fifth reaction chamber 7225. This invention does not impose any particular limitation.

[0074] Table 2

[0075]

[0076]

[0077] The preparation methods for the S gene N501Y site probe and the S gene D614G site probe are as follows:

[0078] (1) Download the sequence of the novel coronavirus mutant strain. The mutation sites of the novel coronavirus mutant strain S gene include the S gene N501Y site and the S gene D614G site. Based on the mutation sequence, the probe sequence is designed so that the modified group -NH2 (amino) on the probe can bind to the -COOH on the surface of PDMS at a certain temperature. The amplified product can specifically bind to the probe.

[0079] (2) The probe synthesized in (1) is dissolved, quantified, and then mixed with the spotting buffer to form the S gene N501Y site probe and the S gene D614G site probe.

[0080] Example 4

[0081] Based on the PCR amplification of lyophilized microspheres 2 in Example 1, this example provides a rapid method for detecting the novel coronavirus.

[0082] Please refer to Figure 6 , Figure 6 The rapid novel coronavirus detection method of this embodiment includes the following steps:

[0083] S20, start the nucleic acid extractor, open the chamber door, add 200μL of SARS-CoV-2 pseudovirus sample to the well of the binding buffer plate, and place the binding buffer plate, washing buffer 1 plate, washing buffer 2 plate, and elution buffer plate into their corresponding positions on the device from left to right. Install the magnetic sleeve and close the chamber door. Open the fully automated nucleic acid extractor software, select the extraction program, and perform sample extraction.

[0084] S21, take the PCR amplification lyophilized microspheres 2 prepared in Example 1, add 20 μL of nucleic acid sample extracted by LS20 to the PCR amplification lyophilized microspheres 2 to reconstitute the PCR amplification lyophilized microspheres 2, seal, shake thoroughly to dissolve the PCR amplification lyophilized microspheres 2, and then centrifuge briefly (the purpose is to avoid some sample remaining on the PCR membrane, tube wall and tube cap), and wait for the instrument to be used for detection.

[0085] S22, place it in a real-time quantitative PCR instrument and amplify according to the procedure in Table 3. Simultaneously select the ROX, FAM, and VIC channels, and select 60℃ and 30s for the fluorescence acquisition point.

[0086] Table 3

[0087]

[0088] S23, the test results are obtained.

[0089] This embodiment uses the PCR amplification lyophilized microspheres 2 prepared in Example 1. When performing nucleic acid detection, it is only necessary to reconstitute the PCR amplification lyophilized microspheres 2 with the nucleic acid sample, which saves the steps of preparing the amplification reaction solution and makes the operation very convenient and fast.

[0090] Example 5

[0091] Based on Embodiment 2 or 3 above, this embodiment provides a method for rapid identification of novel coronavirus variant strains.

[0092] Please refer to Figure 7 , Figure 7 The rapid identification method for novel coronavirus variants in this embodiment includes the following steps:

[0093] S31, the amplification product of the sample that tested positive for the novel coronavirus is mixed with the hybridization solution (the hybridization solution is existing technology and contains SDS, SSC, Denhardt's Solution, formamide, and hybridization quality control probe), and then added to the novel coronavirus variant strain rapid identification chip of Example 2 or 3. After centrifugation, it is placed in a 50°C water bath for 10 minutes until the liquid completely flows into the identification pool 725 outside the reaction pool.

[0094] S32. Scan the reaction cell with a scanner. Clusters of fluorescent signals indicate the corresponding mutation type, while the absence of clusters of fluorescent signals indicates the absence of the corresponding mutation type.

[0095] In this embodiment, using the novel coronavirus variant rapid identification chip of embodiment 2 or 3, when the nucleic acid test is positive, only a batch of novel coronavirus variant rapid identification chips produced in advance need to be injected to quickly identify whether it is a novel coronavirus variant that matches it, so as to quickly control the epidemic.

[0096] Example 6

[0097] Based on Embodiment 1 above, and on Embodiment 2 or 3 above, this embodiment provides a method for rapid detection of the novel coronavirus and rapid identification of variant strains.

[0098] Please refer to Figure 8 , Figure 8 The rapid detection and rapid identification method for novel coronavirus strains in this embodiment includes the following steps:

[0099] S1, Take 2 lyophilized microspheres from Example 1 for PCR amplification.

[0100] S2, Novel Coronavirus Testing, this step includes:

[0101] S20, start the nucleic acid extractor, open the chamber door, add 200μL of SARS-CoV-2 pseudovirus sample to the well of the binding buffer plate, and place the binding buffer plate, washing buffer 1 plate, washing buffer 2 plate, and elution buffer plate into their corresponding positions on the device from left to right. Install the magnetic sleeve and close the chamber door. Open the fully automated nucleic acid extractor software, select the extraction program, and perform sample extraction.

[0102] S21, add 20 μL of the nucleic acid sample extracted in S20 to the PCR amplification lyophilized microspheres 2 to reconstitute the PCR amplification lyophilized microspheres 2, seal, shake thoroughly to dissolve the PCR amplification lyophilized microspheres 2, then centrifuge briefly (to avoid some sample remaining on the PCR membrane, tube wall and cap), and wait for testing.

[0103] S22, place it in a real-time quantitative PCR instrument and amplify according to the procedure in Table 2. Simultaneously select the ROX, FAM, and VIC channels, and select 60℃ and 30s for the fluorescence acquisition point.

[0104] S23, obtain the test result. If the test result is positive, proceed to S3.

[0105] S3, Identification of novel coronavirus variants, this step includes:

[0106] S31, the amplification product of the sample that tested positive for the novel coronavirus is mixed with the hybridization solution (containing SDS, SSC, Denhardt's Solution, formamide, and hybridization quality control probe), and then added to the novel coronavirus variant strain rapid identification chip of Example 2 or 3. After centrifugation, it is placed in a 50°C water bath for 10 minutes until the liquid after the reaction completely flows into the identification pool 725 outside the reaction pool.

[0107] S32. Scan the reaction cell with a scanner. Clusters of fluorescent signals indicate the corresponding mutation type, while the absence of clusters of fluorescent signals indicates the absence of the corresponding mutation type.

[0108] Comparative Example 1

[0109] Please refer to Figure 9 , Figure 9 As a comparison of the present invention, the microcontroller chip 8 differs from the novel coronavirus variant rapid identification chip 7 of Example 2 in that it lacks the identification pool 725, as well as the first siphon channel 7261, the second siphon channel 7262, the third siphon channel 7263, the fourth siphon channel 7264, the fifth siphon channel 7261, and the second exhaust channel 7282. The rest of the structure is exactly the same.

[0110] In the following Examples 7 and Comparative Examples 2, Sample 1 of Example 7 is the same as Sample 1 of Comparative Examples 2, Sample 2 of Example 7 is the same as Sample 2 of Comparative Examples 2, and Sample 3 of Example 7 is the same as Sample 3 of Comparative Examples 2.

[0111] Example 7

[0112] This embodiment is a specific implementation of the rapid detection method for novel coronavirus and rapid identification of variant strains in Embodiment 6.

[0113] S1, Take 2 lyophilized microspheres from Example 1 for PCR amplification.

[0114] S2, Novel Coronavirus Testing, this step includes:

[0115] S20, start the nucleic acid extractor, open the chamber door, and add 200μL of each of the three pseudovirus samples (sample 1, sample 2, and sample 3) into the wells of the binding buffer plate. Place the binding buffer plate, washing buffer 1 plate, washing buffer 2 plate, and elution buffer plate into their corresponding positions on the device from left to right. Install the magnetic sleeve and close the chamber door. Open the fully automated nucleic acid extractor software, select the extraction program, and begin sample extraction.

[0116] S21, add 20 μL of the nucleic acid sample extracted in S20 to the PCR amplification lyophilized microspheres 2 to reconstitute the PCR amplification lyophilized microspheres 2, seal, shake thoroughly to dissolve the PCR amplification lyophilized microspheres 2, then centrifuge briefly (to avoid some sample remaining on the PCR membrane, tube wall and cap), and wait for testing.

[0117] S22, place it in a real-time quantitative PCR instrument and amplify according to the procedure in Table 2. Simultaneously select the ROX, FAM, and VIC channels, and select 60℃ and 30s for the fluorescence acquisition point.

[0118] S23, the test results were obtained, among which, the test results of samples 1, 2 and 3 were positive for the novel coronavirus 2019-nCoV.

[0119] S3, Identification of novel coronavirus variants, this step includes:

[0120] S31, after each of the samples that tested positive for the novel coronavirus (sample 1, sample 2, sample 3) was mixed with the hybridization solution (containing SDS, SSC, Denhardt's Solution, formamide, and hybridization quality control probe), it was added to the novel coronavirus variant strain rapid identification chip 7 of Example 2 or 3 (each sample corresponds to a variant strain rapid identification unit 72), centrifuged, and then placed in a 50°C water bath for 10 minutes until the liquid completely flowed into the identification pool 725 outside the reaction pool.

[0121] S32. Scan the reaction cell with a scanner. Clusters of fluorescent signals indicate the corresponding mutation type; the absence of clusters indicates the absence of the corresponding mutation type. The detection results for samples 1, 2, and 3 are as follows: Sample 1 has a mutation at the N501Y site of the S gene; Sample 2 has a mutation at the D614G site of the S gene; no mutation was detected in Sample 3. Sample 3 represents another mutation type of COVID-19 and requires further identification (using identification chips containing the corresponding viral mutant sequences).

[0122] Comparative Example 2

[0123] The method and steps for detecting the novel coronavirus and identifying variant strains in this comparative embodiment are basically the same as those in Example 7. It is a parallel experiment conducted simultaneously with Example 7, and the fake virus samples (Sample 1, Sample 2, and Sample 3) are also identical to those in Example 7. The difference lies in step S3, where the microcontroller chip 8 of Comparative Example 1 is used, and the scanning is performed after the reaction when the liquid remains in the reaction tank.

[0124] The detection results for samples 1, 2, and 3 are: scan interpretation failed, unable to identify and distinguish mutant types.

[0125] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A rapid detection method for novel coronavirus (for non-diagnostic purposes) and a rapid identification method for variant strains, comprising the following steps: S1, Take PCR amplification lyophilized microspheres; S2, Novel Coronavirus Testing, this step includes; S20, extract nucleic acid samples; S21, add the nucleic acid sample extracted in S20 to the PCR amplification lyophilized microspheres to reconstitute the PCR amplification lyophilized microspheres, seal, shake thoroughly to dissolve the PCR amplification lyophilized microspheres, and then centrifuge briefly. S22, placed in a real-time quantitative PCR instrument for amplification; S23, obtain the test result. If the test result is positive, proceed to S3. S3, Identification of novel coronavirus variant strains, this step includes: S31, after mixing the amplification product of the sample that tested positive for the novel coronavirus with the hybridization solution, add it to the novel coronavirus variant rapid identification chip, centrifuge the mixture and let it flow into the reaction cell, heat it to a constant temperature until the liquid after the reaction completely flows into the identification cell outside the reaction cell. S32. Scan the reaction cell with a scanner. Clusters of fluorescent signals indicate the corresponding mutation type, while the absence of clusters of fluorescent signals indicates the absence of the corresponding mutation type. The PCR amplification lyophilized microspheres were prepared by the following method and then preserved or transported: S11, Preparation: Mix primers, probes, enzymes and excipients in the proportions required for the novel coronavirus to form PCR amplification reagents; S12, spotting: The PCR amplification reagent formed in S11 is spotted onto a pre-cooled lyophilized plate using a pipette or an automatic bead dispenser to form PCR amplification reagent microspheres. S13, freeze-drying: The plate prepared by S12 is placed in a vacuum freeze-drying oven for dehydration to form freeze-dried microspheres. S14, Filling: Remove the PCR amplification lyophilized microspheres from the lyophilization tooling plate and load them into tubes, one PCR amplification lyophilized microsphere per tube. Load the tubes into the wells of the plate, seal them under vacuum, and the PCR amplification lyophilized microsphere kit is formed. S15, Packaging; The novel coronavirus variant rapid identification chip includes a chip substrate, on which at least one variant rapid identification unit is disposed. Each variant rapid identification unit includes a sample pool, a first reaction pool, a second reaction pool, a third reaction pool, a fourth reaction pool, and a fifth reaction pool. The variant rapid identification unit also includes a connecting channel and a separation channel. One end of the connecting channel is connected to the sample pool, and the other end is connected to the separation channel. The first reaction pool, the second reaction pool, the third reaction pool, the fourth reaction pool, and the fifth reaction pool are each connected to the separation channel. The first reaction pool, the second reaction pool, the third reaction pool, the fourth reaction pool, and the fifth reaction pool are used to pre-load the spotting solution for novel coronavirus variant identification. The rapid identification unit for the mutant strain is also equipped with an identification pool, a first siphon channel, a second siphon channel, a third siphon channel, a fourth siphon channel, and a fifth siphon channel. The first reaction pool is connected to the identification pool through the first siphon channel, the second reaction pool is connected to the identification pool through the second siphon channel, the third reaction pool is connected to the identification pool through the third siphon channel, the fourth reaction pool is connected to the identification pool through the fourth siphon channel, and the fifth reaction pool is connected to the identification pool through the fifth siphon channel. The novel coronavirus mutant strain rapid identification chip also includes a chip cover, which covers the chip substrate; the chip cover is also provided with a first exhaust hole and a second exhaust hole, and the mutant strain rapid identification unit is also provided with a first exhaust channel and a second exhaust channel, one end of the first exhaust channel is connected to the collection pool and the other end is connected to the first exhaust hole, and one end of the second exhaust channel is connected to the identification pool and the other end is connected to the second exhaust hole.

2. The method for rapid detection of novel coronavirus for non-diagnostic purposes and rapid identification of variant strains according to claim 1, characterized in that, The vacuum freeze-drying oven dehydration process includes the following two steps: S131, sublimation drying; S132, desorption and drying.

3. The method for rapid detection of novel coronavirus for non-diagnostic purposes and rapid identification of variant strains according to claim 1, characterized in that, The freeze-drying tooling plate is a tooling plate that has been pre-cooled to -65°C.

4. The method for rapid detection of novel coronavirus for non-diagnostic purposes and rapid identification of variant strains according to claim 1, characterized in that, The rapid identification unit for mutant strains is also equipped with a collection pool, which is connected to the separation channel.

5. The method for rapid detection of novel coronavirus for non-diagnostic purposes and rapid identification of variant strains according to claim 1, characterized in that, The chip cover is provided with a sample dispensing port, which is connected to the sample cell.

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