Household visual detection kit, detection method and system for simple diseases

Abstract

The invention discloses a household visual detection kit for simple diseases as well as a detection method and system. The household visual detection kit comprises an RPA detection kit, freeze-dried powder is packaged in a reagent tube A, freeze-dried chromogenic dye is packaged in a reagent tube B, and ionic liquid is packaged in a reagent tube C; according to the LAMP detection kit, ionic liquid containing solid paraffin particles is packaged in a reaction tube, and freeze-dried powder is arranged in a tube cover; the kit disclosed by the invention is simple in operation steps, can be transported at room temperature, can be directly used without preparing a reagent after a package is opened on a detection site, is judged to be visual color development judgment by naked eyes, realizes immediate instrument-free detection, avoids aerosol pollution, saves time and labor, and is high in detection efficiency. Ordinary people can also rapidly, accurately and conveniently carry out nucleic acid detection on pathogens (target genes) at home, and powerful guidance can be provided for reasonable drug use of families.

Description

Technical Field

[0001] The present invention relates to the technical field of detection kits, and in particular to a simple disease home visual detection kit, a detection method and a system. Background Art

[0002] Commonly used nucleic acid tests in hospitals, such as the fluorescent quantitative PCR method, have the advantages of high sensitivity, early detection of pathogen infection, high accuracy, and low false positive and false negative rates, but require professional laboratories, professionals, and expensive special instruments, and the detection time is long, so it is not suitable for real-time home detection; the detection of gold-labeled test strips is used for antigen detection, such as the new coronavirus. This method is easy to use and fast, and can be used for real-time home detection, but the detection sensitivity is low, it is not easy to detect early pathogen infection, the accuracy is low, and the false positive and false negative rates are high, and positive samples still need to be further tested and confirmed by fluorescent quantitative PCR. Therefore, the present invention proposes a simple disease home visual detection kit and a detection method and system to solve the problems existing in the prior art. Summary of the Invention

[0003] In response to the above problems, the purpose of the present invention is to propose a simple disease home visual detection kit and detection method and system. The simple disease home visual detection kit and detection method and system kit have simple operation steps and can be transported at room temperature. After opening the package at the detection site, it can be used directly without preparing reagents. The result is determined by visual color development with the naked eye, realizing instant instrument-free detection, avoiding aerosol contamination, saving time and effort, and ordinary people can also perform nucleic acid detection of pathogens (target genes) at home quickly, accurately and conveniently.

[0004] To achieve the purpose of the present invention, the present invention is implemented by the following technical solutions: a simple disease home visual detection kit, including an RPA detection kit and a LAMP detection kit;

[0005] The RPA detection kit includes reagent tube A, reagent tube B, and reagent tube C. The reagent tubes A and B are double-tube structures, and the tube cap of the reagent tube C is a tube cap structure connected to a dripper. The reagent tube A is encapsulated with lyophilized powder, the reagent tube B is encapsulated with lyophilized color dye, and the reagent tube C is encapsulated with ionic liquid;

[0006] The LAMP detection kit includes a reaction tube and a tube cover. The reaction tube is encapsulated with an ionic liquid containing solid paraffin particles, and the tube cover is provided with freeze-dried powder. The reaction tube and the tube cover are an adapted eight-tube and tube cover structure.

[0007] Further, the freeze-dried powder in the A reagent tube is made by mixing enzymes, primers, dNTPmix, protective agent glycerol and trehalose, and then freeze-drying; the freeze-dried powder in the tube cap is made by mixing enzymes, primers, dNTPmix, protective agent glycerol, trehalose and color developing dye, and then freeze-drying; the ionic liquid is made by mixing buffer, ddH2O and Mg 2+ .

[0008] Further, the detection method of the RPA detection kit comprises the following steps:

[0009] Step one, insert the sampling swab with sample into the sampling tube for sample dissolution, then take out the sampling swab and cover the tube cap with the dropper, and then perform lysis, pathogen inactivation and nucleic acid release;

[0010] Step two, after lysis of the sample, shake the sampling tube, then remove the tube cap on the dropper, drop one drop of supernatant in the dropper on the tube cap into the A reagent tube as a template, then remove the tube cap of the C reagent tube, drop 4 drops of ionic liquid in the C reagent tube into the A reagent tube to obtain a reaction solution, and then cover the double-tube cap on the A reagent tube and the B reagent tube;

[0011] Step three, hold the A reagent tube and the B reagent tube of the double-tube cap in the palm for 5-10 min, then invert the A reagent tube and the B reagent tube, tilt the B reagent tube on one side, pour the reaction solution in the A reagent tube into the B reagent tube, place the double-tube cap vertically, fully dissolve the freeze-dried color developing dye in the B reagent tube, and finally judge the result according to the color contrast of color development.

[0012] Further, in step one, insert the sampling swab into the sampling tube, and agitate for 30-40 times within 30-40 s for sample dissolution; after covering the tube cap with the dropper, stand still for 1-10 min, then repeatedly pinch the lower part of the sampling tube for 5-8 times or heat in a constant temperature metal bath for 1-10 min, to fully lyse, inactivate pathogens and release nucleic acids.

[0013] Further, in step two, the amount of droplet in the dropper on the tube cap is 10 μL, after dropping the ionic liquid, pinch the lower part of the A reagent tube for 5-8 times to mix the ionic liquid reaction solution uniformly; in step three, after pouring the reaction solution into the B reagent tube, place the double-tube cap vertically and pinch the lower part of the B reagent tube for 5-8 times to dissolve and mix the freeze-dried color developing dye uniformly for color development.

[0014] Further, the detection method of the LAMP detection kit comprises the following steps:

[0015] Step one, insert the sampling swab with sample into the sampling tube for sample dissolution, then take out the sampling swab and cover the tube cap with the dropper, and then perform lysis, pathogen inactivation and nucleic acid release;

[0016] Second step, after sample lysis, shake the sampling tube, then pull off the tube cap on the dropper, drop a drop of supernatant in the tube cap on the dropper into the reaction tube as a template, then cover the tube cap containing the freeze-dried powder and shake it hard, dissolve the freeze-dried powder on the tube cap and mix with the template, invert the reaction tube and stand for 1-2 min, then stand the reaction tube, shake the solution into the bottom of the tube and mix evenly, and put it into the amplification instrument for nucleic acid amplification;

[0017] Third step, after amplification, the reaction tube is subjected to light treatment, then the color development is observed and the results are compared and judged by color.

[0018] Further, the light treatment in the third step includes one of using a flashlight of a specific wavelength to irradiate, irradiating under sunlight, and using a portable color development instrument to develop color.

[0019] A simple home visual detection system for diseases includes a detection acquisition module, an AI judgment module, and a symptomatic drug dispensing module. The detection acquisition module performs real-time detection at home based on RPA and LAMP detection reagent kits and obtains detection result pictures or instant diagnosis or self-use of a family medicine kit for real-time medication. The AI judgment module performs intelligent judgment on uploaded detection result pictures and inquiry information based on an AI health consultation platform and gives real-time judgment results. The symptomatic drug dispensing module intelligently dispenses drugs in real time according to judgment results and user needs and choices, and sends drugs in real time for users to use at home according to usage and dosage.

[0020] Further, the AI judgment module includes an AI filing module and an inquiry module. The AI filing module establishes a personal health record based on user registration information on the AI health consultation platform. The inquiry module performs real-time intelligent judgment and result output based on AI and user inquiry information, and manages user chronic diseases.

[0021] Further, the symptomatic drug dispensing module includes a drug recommendation module, a drug purchase selection module, and an intelligent positioning module. The drug recommendation module gives the name of the drug, the indication, and the usage and dosage information based on real-time judgment results for users to use a family medicine kit for real-time medication or use a drug purchase program. The drug purchase selection module is used for online real-time drug purchase according to user choices. The intelligent positioning module is used for searching the nearest contracted pharmacy according to the user's location range and giving real-time symptomatic drug dispensing and real-time drug delivery for users to use at home according to usage and dosage.

[0022] The beneficial effects of the present invention are as follows: the reagent kit of the present invention is pre-packaged, freeze-dried, and packaged, and can be transported at room temperature, avoiding the need for cold chain transportation. The operation steps are simple, and the kit can be directly used after opening the package at the detection site without the need to prepare reagents. The result is determined by visual color development with the naked eye, realizing instant instrument-free detection, avoiding aerosol pollution, saving time and effort, and ordinary people can also perform nucleic acid detection of pathogens (target genes) at home quickly, accurately, and conveniently, which can provide powerful guidance for the rational use of medicines at home. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a flow chart of the detection system of Example 1 of the present invention.

[0024] Figure 2 This is the RPA home testing flowchart.

[0025] Figure 3 This is a specific experimental diagram of the RT-RPA detection method for the new coronavirus in Example 2 of the present invention.

[0026] Figure 4 This is a sensitivity experiment diagram of the RT-RPA detection method for the new coronavirus in Example 2 of the present invention.

[0027] Figure 5 This is the LAMP home testing flow chart.

[0028] Figure 6 This is a specific experimental diagram of the RT-LAMP detection method for the new coronavirus in Example 3 of the present invention.

[0029] Figure 7 This is a sensitivity experiment diagram of the RT-LAMP detection method for the new coronavirus in Example 3 of the present invention. DETAILED DESCRIPTION

[0030] In order to deepen the understanding of the present invention, the present invention will be further described in detail below with reference to the examples. The examples are only used to explain the present invention and do not constitute a limitation on the scope of protection of the present invention.

[0031] Example 1

[0032] according to Figure 1As shown, the present embodiment provides a simple disease home visual detection system, which includes a detection acquisition module, an AI judgment module, and a symptomatic drug dispensing module. The detection acquisition module performs real-time detection at home based on RPA and LAMP detection kits and acquires detection result pictures or performs immediate diagnosis or uses a family medicine kit for real-time medication. The AI judgment module intelligently judges the uploaded detection result pictures and disease inquiry information based on an AI health consultation platform and gives real-time judgment results. The symptomatic drug dispensing module intelligently dispenses drugs in real time according to real-time judgment results and user needs and choices, and sends the drugs in real time for the user to use at home according to the usage and dosage, and to perform real-time symptomatic treatment at home.

[0033] The AI judgment module includes an AI filing module and an inquiry module. The AI filing module establishes a personal health record based on the user's registration information on the AI health consultation platform. The inquiry module intelligently judges and outputs results in real time based on AI and user inquiry information, and manages the user's chronic diseases.

[0034] For chronic disease management, the user can use the home heart rate, blood pressure, blood lipids, blood glucose, blood oxygen, urine test, blood indicators, biochemical indicators, height, weight, waist circumference, and other chronic disease data collected by the home intelligent terminal collection system through the Internet of Things AI data transmission to the health consultation center. The user's health record, AI doctor, and artificial doctor in the health consultation center are used for chronic disease management. Then, the AI sends the user the personalized prevention and treatment methods, medication guidance, online drug dispensing, and disease warning in real time according to the user's chronic disease dynamic real-time management analysis results, such as the risk of cerebral infarction, myocardial infarction, cancer, and other chronic diseases, such as hypertension, type 2 diabetes, metabolic syndrome, obesity, heart disease, stroke, atherosclerosis, fatty liver, gout, chronic renal failure, prostate cancer, cirrhosis, liver cancer, chronic atrophic gastritis, gastric ulcer, gastric cancer, rheumatoid arthritis, type 1 diabetes, hypothyroidism, hyperthyroidism, hyperprolactinemia, prolactinoma, polycystic ovary syndrome, ovarian premature aging, and amenorrhea. The user can also conduct health consultation, follow-up, and human-computer interaction.

[0035] The symptomatic drug dispensing module includes a drug recommendation module, a drug purchase selection module, and an intelligent positioning module. The drug recommendation module gives the user the name of the drug, the indication, and the usage and dosage information for real-time medication or selection of the drug type and purchase program based on the real-time judgment results. The drug purchase selection module is used for online real-time drug purchase according to the real-time judgment results and user choices. The intelligent positioning module is used to search for the nearest contracted pharmacy according to the user's location range and to give real-time symptomatic drug dispensing and real-time drug delivery for the user to use at home according to the usage and dosage, as shown in the attached instructions. Figure 1

[0036] ​The following examples provide a specific implementation process of a simple disease home visual detection kit detection method.

[0037] Example 2

[0038] According to Figure 2-Figure 4 The present embodiment provides a new coronavirus SARS-CoV-2 RT-RPA nucleic acid home instant visual detection method.

[0039] Specifically, a single-tube nucleic acid reaction tube is used to detect the new coronavirus SARS-CoV-2, and the target gene N gene is NCBI Reference Sequence: NC_045512.2.

[0040] New coronavirus RPA primer sequence:

[0041] N-F: 5'-cctcttctcgttcctcatcacgtagtcgc-3';

[0042] N-R: 5'-cctcagcagcagatttcttagtgacagtttggc-3'.

[0043] The following steps are included:

[0044] 1. Reagent pre-packaging, freeze-drying

[0045] The reaction solution on the tube cover in the single-tube nucleic acid reaction tube contains the following components: 2M trehalose 3.3μL, R-mix (containing 50% glycerol) 2.5μL, E-mix (containing 50% glycerol) 5μL, 25mM dNTP mix 2.8μL, primer F / R 100uM 0.18μL, RNase inhibitor (40U / μL) 1μL, ReverTraAce (100U / μL) 1μL, wherein the glycerol content is strictly set according to 22.1% W / M / V. Freeze-drying, packaging with aluminum foil bag, and labeling as A reagent.

[0046] The ion solution at the bottom of the reaction tube main body contains the following components: 2x buffer (Mg-free) 25μL, ddH2O 9.5μL, 280mM Mg(OAc)2 2.5μL, concentrated hydrochloric acid (HCl 37.5%) 3μL. The total volume is 40μL. Since RPA is constant temperature amplification at 37 degrees Celsius, there is no need to add paraffin to seal the tube. A separate tube cover is placed on the main body of the reaction tube, packaged with an aluminum foil bag, and labeled as B reagent.

[0047] 2. Template direct extension lysis extraction

[0048] Gently rotate a disposable nasal swab 3-5 times deep within the nasal cavity. Insert the swab into a sampling tube containing 0.5 ml of lysis solution (components: 0.5 M NaOH and 10 mM Na2EDTA, 3 μL of RNase inhibitor (40 U / μL)), labeled Reagent C. Agitate the swab 30-40 times per second, pressing the tip against the tube wall 5-8 times. Discard the swab. Cap the tube, press the bottom of the tube 5-8 times, and let it sit at room temperature for 1 minute.

[0049] 3. Reaction process

[0050] When in use, first remove the A reagent tube cap of the reaction tube body, turn over the sampling tube C reagent, design the aperture of the sampling tube dropper, and drop exactly 10μL. Add a filter element in the sampling tube dropper to filter out the residue of the sample liquid, add 1 drop 10μL of the sample template prepared in the sampling tube to the tube cap, discard the independent tube cap covering the ionic liquid B reagent, cover the tube cap containing the freeze-dried powder reagent and sample template on the centrifuge tube, invert for 2 minutes, mix with the ionic liquid, dissolve the freeze-dried powder, turn upside down 5-8 times to mix, shake the liquid into the bottom of the tube with your hand, put it in the palm of your hand, clench your fist for 5 minutes, and perform DNA amplification.

[0051] 4. Identification of RPA amplification products

[0052] After the RPA detection reaction is completed, add 3 μL of 50×SYBR Green I dye to the nucleic acid amplification product, stir the tube 5-8 times with your fingers to mix, and observe the color reaction directly under sunlight or fluorescent light with the naked eye. If the solution turns green after color development, the sample is a positive sample; if the solution turns orange after color development, the sample is a negative sample. Figure 2 shown.

[0053] 5. Specificity Experiment

[0054] For the novel coronavirus pseudovirus (N gene), human coronavirus OC43, human coronavirus 229E, porcine epidemic diarrhea virus (coronavirus), influenza B virus, Japanese encephalitis virus, rhinovirus and adenovirus, the sampling tube C reagent template was used for direct amplification. The extraction lysate components were: 0.5M NaOH and 10mM Na2EDTA, 3μL of RNase inhibitor (40U / μL), and the sample: lysate = 1:10. Genomic RNA was extracted and crude RNA samples were prepared as RNA templates for RT-RPA specificity tests. The experimental results are shown in the attached instructions. Figure 2As shown in the figure (in the figure, 1 represents the new coronavirus pseudovirus (N gene); 2 represents human coronavirus OC43; 3 represents human coronavirus 229E; 4 represents porcine epidemic diarrhea virus (coronavirus); 5 represents influenza B virus; 6 represents Japanese encephalitis virus; 7 represents rhinovirus; 8 represents adenovirus; N represents negative control genomic RNA), it can be seen from the figure that only 1 new coronavirus pseudovirus is positive green, and the rest of the tubes are orange-red, which confirms that the pre-packaged and freeze-dried RPA reaction system has a strong specificity for the new coronavirus, as shown in the attached instruction manual. Figure 3 shown.

[0055] 6. Sensitivity test

[0056] VP / mL (number of virus particles / ml). Dilute the novel coronavirus pseudovirus (VP / μL (number of virus particles / μL)) to 10 5 VP / μL, 10 4 VP / μL, 10 3 VP / μL, 10 2 VP / μL, 10 1 VP / μL, 10 0 VP / μL, the diluted novel coronavirus pseudovirus solution was extracted using a template direct amplification, the lysis buffer composition: 0.5M NaOH and 10mM Na2EDTA, RNase inhibitor (40U / μL) 3μL, according to the sample: lysis buffer = 1:10, the genomic RNA was extracted and used as a template for RT-RPA amplification test, and its sensitivity was measured. The experimental results are shown in the attached instructions. Figure 2 As shown (in the figure, M represents Marker; 1 represents 10 5 VP / μL; 2 means 10 4 VP / μL; 3 means 10 3 VP / μL; 4 means 10 2 VP / μL; 5 means 10 1 VP / μL; 6 means 10 0 VP / μL; N represents negative control (human negative genomic RNA). 1 There are still positive bands at VP / μL; 1-5 in the 0.2ml centrifuge tube are all green, 6 and N are orange, indicating that the detection limit of the new coronavirus RT-RPA test is 10 1 VP / μL, indicating that RT-RPA has high sensitivity in detecting new coronavirus, as shown in the attached instructions. Figure 4 shown.

[0057] 7. Repeatability test

[0058] 10×10 3 , 10×102 A 2019-nCoV positive reference sample was tested using four batches of reagents. Inter-batch precision was measured 40 times, and intra-batch precision was repeated 40 times within the same batch. The inter-batch and intra-batch coefficients of variation (CV) were analyzed to verify reproducibility. The inter-batch and intra-batch CVs were less than 3%, indicating that the established 2019-nCoV RT-RPA detection system has high reproducibility.

[0059] 8. Stability test

[0060] The 10×10 3 , 10×10 2 The positive reference sample was tested 20 times to verify its stability. The reagent stored at room temperature for 6 months was used to detect 10×10 3 , 10×10 2 The results of the 20 replicates of the 2000 copies / ml SARS-CoV-2 positive reference sample were all positive, indicating that the established RT-RPA detection system for SARS-CoV-2 has good stability.

[0061] Example 2

[0062] according to Figure 5-Figure 7 As shown, this embodiment provides a method for real-time visualization detection of the new coronavirus SARS-CoV-2 RT-LAMP.

[0063] Specifically, the eight-tube nucleic acid reaction tube RT-LAMP detection method was used to detect the new coronavirus, and the target gene was ORF1ab (targeting the RdRp region), NCBI Reference Sequence: NC_045512.2.

[0064] SARS-CoV-2 LAMP primer sequences:

[0065] ORF1ab-F3: 5'-ACCAACTGGAGTTCATGC-3'

[0066] ORF1ab-B3: 5'-CGATTGAGAAACCACCTGTC-3'

[0067] ORF1ab-FIP:

[0068] 5'-GCTTGTGCTGTTTGCCTGTCTTTTTGGCACAGACTTAGAAGG-3'

[0069] ORF1ab-BIP:

[0070] 5'-AGCTGGTACGGACACAACTATTTTTCCATTTATAACAGCAGCGTA-3'

[0071] The following steps are involved:

[0072] 1. Reagent freeze-drying

[0073] The reaction solution lyophilized in the cap of each tube of an eight-tube strip contained the following components: 0.2 μL of 100 μM FIP / BIP, 0.05 μL of 100 μM F3 / B3, 1 μL of CFI dye (Shanghai Boman Biotechnology Co., Ltd.), 1.5 μL of 2 M trehalose, 1.4 μL of 25 mM dNTP mix, 2 μL of betaine (5 M), 1 μL of 8 U / μL Bst enzyme, 1 μL of RNase inhibitor (40 U / μL), 1 μL of ReverTraAce (100 U / μL), 0.2 μL of 50% glycerol, and a total glycerol content (including glycerol in the enzyme) of 22.1% (W / V). Pglycerol = 1.2613 g / cm 3 , where the glycerol content is strictly set at 22.1% W / M / V, mixed and lyophilized in a 0.2 ml tube cap, sealed in an aluminum foil bag, and labeled as reagent A. The ionic liquid contains the following components: ddH2O 7.75 μL, 10× buffer (Mg-free) 2.5 μL, 100 mM Mg 2+ 1.75μL, concentrated hydrochloric acid (HCl37.5%) 3μL, mix well and put into a 0.2ml nucleic acid reaction tube, add 10μL melted paraffin, let the nucleic acid reaction tube stand at room temperature for 10 minutes, then seal it in an aluminum foil bag and mark it as reagent B.

[0074] 2. Template direct expansion cracking and extraction

[0075] Gently rotate a disposable nasal swab 3-5 times deep within the nasal cavity. Insert the swab into a sampling tube containing 0.5 ml of lysis solution (components: 0.5 M NaOH and 10 mM Na2EDTA, 3 μL of RNase inhibitor (40 U / μL)), labeled Reagent C. Agitate the swab 30-40 times per second, pressing the tip against the tube wall 5-8 times. Discard the swab. Cap the tube, press the bottom of the tube 5-8 times, and let it sit at room temperature for 1 minute.

[0076] 3. Reaction process

[0077] In use, first take out the A reagent tube cover of the reaction tube main body, invert the sampling tube C reagent, design the aperture of the sampling tube dropper head, drop one drop of 10 μL, add a filter core in the sampling tube dropper head to filter out the residue of the sample liquid, add one drop of 10 μL of the sample template prepared by the sampling tube on the tube cover, discard the independent tube cover on the ionic liquid B reagent, cover the tube cover containing the freeze-dried powder reagent and the sample template on the centrifugal tube, invert for 2 min, mix with the ionic liquid, dissolve the freeze-dried powder, invert up and down for 5-8 times, shake the liquid into the tube bottom with hands, and then put into a constant temperature amplification instrument for 60℃, 30 min for DNA amplification.

[0078] 4. LAMP amplification product identification

[0079] In the LAMP reaction system, one component of CFI is added before the reaction, and after the reaction is completed, the color change of the negative and positive reaction systems is observed; according to the color, under normal circumstances, the positive sample is sky blue, and the negative sample is violet. The fluorescent dye in CFI, under normal circumstances, the positive sample is green, and the negative sample is orange. CFI is a double-color system, as shown in the drawings of the specification. Figure 5

[0080] 5. Specificity experiment

[0081] The new coronavirus pseudovirus (N gene), human coronavirus OC43, human coronavirus 229E, porcine epidemic diarrhea virus (coronavirus), influenza B virus and Japanese encephalitis virus were respectively amplified with the sampling tube C reagent template, and the extraction lysis liquid components were: 0.5M NaOH and 10mM Na2EDTA, RNAase inhibitor (40U / μL) 3μL, according to the sample: lysis liquid = 1:10, extract the genomic RNA, and prepare the crude RNA sample as the RNA template for the RT-RPA specificity test. The portable fluorescence color developing instrument is used for color development, first put the sample into the sample hole, cover the light shielding cover, adjust the side button of the color developing instrument to change the intensity of the light, put the mobile phone camera directly against the observation hole above the instrument, and take a photo with the mobile phone to record the experimental results. The experimental results are shown in the drawings of the specification (in the figure, 1 represents human coronavirus OC43; 2 represents human coronavirus 229E; 3 represents porcine epidemic diarrhea virus (coronavirus); 4 represents influenza B virus; 5 represents Japanese encephalitis virus; 7-8 represents new coronavirus pseudovirus (ORF1ab gene) 9 represents ddH2O; 10 represents negative control genomic RNA), under blue light, the positive sample is green and the negative sample is orange. Figure 3

[0082] ​​Under sunlight or light, HNB color development showed that the color of 7-8 new coronavirus pseudoviruses was sky blue, indicating positive, and the rest were violet, indicating negative. The fluorescent dye color development was positive green only in 7-8 new coronavirus pseudoviruses, and the rest were orange-red negative; it was confirmed that the RPA reaction system with pre-packaged and freeze-dried reagents had strong specificity for the new coronavirus, as shown in the attached instructions. Figure 6 shown.

[0083] 6. Sensitivity test

[0084] VP / mL (number of virus particles / ml). Dilute the novel coronavirus pseudovirus (VP / μL (number of virus particles / μL)) to 10 5 VP / μL, 10 4 VP / μL, 10 3 VP / μL, 10 2 VP / μL, 10 1 VP / μL, 10 0 VP / μL, the diluted novel coronavirus pseudovirus solution was extracted using template direct amplification, the lysis buffer reagent C components: 0.5MNaOH and 10mMNa2EDTA, RNase inhibitor (40U / μL) 3μL, according to the sample: lysis buffer = 1:10, the genomic RNA was extracted and used as a template for RT-LAMP amplification test to determine its sensitivity. The experimental results are shown in the attached instructions. Figure 4 As shown (1 in the figure represents 10 5 VP / μL; 2 means 10 4 VP / μL; 3 means 10 3 VP / μL; 4 means 10 2 VP / μL; 5 means 10 1 VP / μL; 6 means 10 0 VP / μL; 7 represents positive plasmid control; blank represents negative control (human negative genomic RNA). 1 There are still positive bands when VP / μL; 1-5 in 0.2ml centrifuge tube all show green, 10 5 VP / μL, 10 4 VP / μL, 10 3 VP / μL, 10 2 VP / μL, 10 1 VP / μL were all positive, 7, the positive control plasmid was green, positive, 1VP / μL, blank showed orange, negative, indicating that the detection limit of the new coronavirus RT-LAMP test was 10 1 VP / μL, indicating that RT-LAMP has high sensitivity in detecting new coronavirus, as shown in the attached instructions. Figure 7 shown.

[0085] 7. Repeatability test

[0086] Detection of 10×10 by RT-LAMP method of novel coronavirus 3 , 10×10 2 A 200-copy / ml SARS-CoV-2 positive reference sample was tested using four batches of reagents. Inter-batch precision was measured 40 times, and intra-batch precision was repeated 40 times within the same batch. The inter-batch and intra-batch coefficients of variation (CV) were analyzed to verify reproducibility. The inter-batch and intra-batch CVs were <3%, indicating that the established SARS-CoV-2 RT-LAMP assay has high reproducibility.

[0087] 8) Stability test

[0088] The 10×10 3 , 10×10 2 The positive reference was tested 20 times to verify its stability. The reagent stored at room temperature for 6 months was used to detect 10×10 3 , 10×10 2 The results of the 20 replicates of the 2000 copies / ml SARS-CoV-2 positive reference sample were all positive, indicating that the established RT-LAMP detection system for SARS-CoV-2 is stable.

[0089] Example 3

[0090] This example provides a compliance experiment for detecting the novel coronavirus using the RT-RPA detection kit and RT-LAMP detection kit of the present application and detecting the novel coronavirus using the commercially available novel coronavirus fluorescence quantitative PCR detection kit and novel coronavirus antigen detection kit (colloidal gold method).

[0091] The selection of sample sources fully considered the influence of factors such as different geographical regions, different time, different infection stages and physiological states. A total of 109 samples were verified for nucleic acid and antigen testing, including 59 new coronavirus nasopharyngeal nucleic acid-positive samples and 45 nasopharyngeal nucleic acid-negative samples. The RT-RPA detection kit and RT-LAMP detection kit of this application were used together with a certain brand of listed new coronavirus fluorescence quantitative PCR detection kit and a certain brand of new coronavirus antigen detection kit (colloidal gold method) for testing. The nucleic acid results of nasopharyngeal nucleic acid samples were compared with the new coronavirus negative / positive results of nasal samples of the RT-RPA detection kit, RT-LAMP detection kit of this application, the listed new coronavirus fluorescence quantitative PCR detection kit and the new coronavirus antigen detection kit (colloidal gold method). The positive and negative compliance rates of the four new coronavirus detection kits were calculated respectively. The calculation method is shown in Table 1 and the formula below.

[0092] Table 1 Calculation of nasopharyngeal sample detection using the research kit and the control kit

[0093]

[0094] Positive coincidence rate = A / (A+C)×100%

[0095] Negative coincidence rate = D / (B+D)×100%

[0096] Total compliance rate = (A+D) / (A+B+C+D)×100%

[0097] A total of 109 samples were tested for nucleic acid and antigen verification, including 59 nasopharyngeal nucleic acid-positive samples of the new coronavirus and 50 nasopharyngeal nucleic acid-negative samples for nucleic acid and antigen testing. According to the nucleic acid results of the nasopharyngeal nucleic acid samples, the RT-RPA detection kit and RT-LAMP detection kit of this application were compared with the negative / positive results of the marketed new coronavirus fluorescence quantitative PCR detection kit. The total positive coincidence rate was 100% (59 / 59), the total negative coincidence rate was 100% (50 / 50), and the total coincidence rate was 100% (109 / 109); compared with the negative / positive results of the antigen detection kit of the nasal antigen samples, the total positive coincidence rate was 69% (41 / 59), the total negative coincidence rate was 74% (37 / 50), and the total coincidence rate was 71.56% (78 / 101).

[0098] Comparing the negative / positive results of the RT-RPA detection kit and RT-LAMP detection kit of this application with the marketed novel coronavirus fluorescence quantitative PCR detection kit, the total positive compliance rate is 100%; the total compliance rate of the novel coronavirus antigen detection kit with the RT-RPA detection kit and RT-LAMP detection kit of this application is 71.56% (78 / 101), indicating that the novel coronavirus antigen detection kit has high false positive and false negative rates and low accuracy.

[0099] The results of the comparison of the RT-RPA detection kit and RT-LAMP detection kit of the present application with the commercially available novel coronavirus fluorescence quantitative PCR detection kit and novel coronavirus antigen detection test strips (colloidal gold method) are shown in Table 2 below.

[0100]

[0101]

[0102] As can be seen from Table 2 above, the RPA detection kit and LAMP detection kit of the present application have reagent pre-packaging, freeze-drying, and packaging, with simple operation steps, avoiding the need for cold chain transportation. Through room temperature transportation, the test results are determined by visual color determination with the naked eye, realizing instant instrument-free detection, avoiding aerosol contamination, saving time and effort, and ordinary people can also perform nucleic acid detection of pathogens (target genes) quickly, accurately, and conveniently at home.

[0103] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the foregoing embodiments. The foregoing embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims and their equivalents.

Claims

1. A simple disease home visual detection kit, characterized by: Including RPA detection kit and LAMP detection kit; The RPA detection kit includes reagent tube A, reagent tube B, and reagent tube C. The reagent tubes A and B are double-tube structures, and the tube cap of the reagent tube C is a tube cap structure connected to a dripper. The reagent tube A is encapsulated with lyophilized powder, the reagent tube B is encapsulated with lyophilized color dye, and the reagent tube C is encapsulated with ionic liquid; The LAMP detection kit includes a reaction tube and a tube cover. The reaction tube is encapsulated with an ionic liquid containing solid paraffin particles, and the tube cover is provided with freeze-dried powder. The reaction tube and the tube cover are an adapted eight-tube and tube cover structure.

2. A simple disease home visual detection kit according to claim 1, characterized in that: The freeze-dried powder in the A reagent tube is prepared by mixing enzyme, primer, dNTPmix, protective agent glycerol and trehalose and then freeze-drying. The freeze-dried powder in the tube cap is prepared by mixing enzyme, primer, dNTPmix, protective agent glycerol, trehalose and color dye and then freeze-drying. The ionic liquid is prepared by mixing buffer, ddH2O and Mg 2+ Made by mixing.

3. The detection method of a simple disease home visual detection kit according to claim 1, characterized in that: The detection method of the RPA detection kit comprises the following steps: Step 1: insert the sampling swab with the sample into the sampling tube to dissolve the sample, then remove the sampling swab and cover the tube with a cap connected to a dropper to lyse, inactivate pathogens and release nucleic acids; Step 2: After the sample is lysed, shake the sampling tube, then remove the tube cap on the dropper, and drip the supernatant in the dropper cap on the dropper into reagent tube A as a template. Then remove the tube cap of reagent tube C, and drip 4 drops of the ionic liquid in reagent tube C into reagent tube A to react to obtain a reaction solution. Cover the double tube cap on reagent tubes A and B; Step 3. Hold the double-tube reagent tube A and reagent tube B in the palm of your hand and keep them warm for 5-10 minutes. Then invert the A and B reagent tubes and tilt them toward the side of the B reagent tube. Pour the reaction solution in the A reagent tube into the B reagent tube. Place the double-tube upright to fully dissolve the freeze-dried color development dye in the B reagent tube and finally judge the result based on the color comparison.

4. The detection method of a simple disease home visual detection kit according to claim 3, characterized in that: In step 1, the sampling swab is inserted into the sampling tube and stirred 30-40 times within 30-40 seconds to dissolve the sample; after covering the tube with a dropper, the tube is left to stand for 1-10 minutes, and then the bottom of the sampling tube is repeatedly pinched 5-8 times or heated in a constant temperature metal bath for 1-10 minutes to fully lyse and inactivate the pathogen and release the nucleic acid.

5. The detection method of a simple disease home visual detection kit according to claim 3, characterized in that: In the step 2, the dripping amount of the dropper on the tube cover is 10 μL. After the ionic liquid is dripped in, the lower part of the A reagent tube is pinched 5-8 times to allow the ionic liquid to react and mix evenly to obtain a reaction liquid. In the step 3, the reaction liquid is poured into the B reagent tube, and the double tube is placed upright and the lower part of the B reagent tube is pinched 5-8 times to dissolve and mix evenly the freeze-dried color developing dye.

6. The detection method of a simple disease home visual detection kit according to claim 1, characterized in that: The LAMP detection kit detection method comprises the following steps: The first step is to insert the sampling swab with the sample into the sampling tube to dissolve the sample, then remove the sampling swab and cover the tube with a cap connected to a dropper to lyse, inactivate pathogens and release nucleic acids; Step 2: After the sample is lysed, shake the sampling tube, then remove the tube cap on the dropper, drop the supernatant in the dropper cap on the reaction tube into the reaction tube as a template, then cover the tube cap containing the lyophilized powder and shake it vigorously to dissolve the lyophilized powder on the tube cap and mix it with the template, invert the reaction tube and let it stand for 1-2 minutes, then turn the reaction tube upright, shake the solution into the bottom of the tube and mix it evenly, and put it into the amplification instrument for nucleic acid amplification; The third step is to expose the reaction tube to light after the amplification is completed, and then observe the color development and compare the results.

7. A simple disease home visual detection kit, detection method and detection system according to claim 6, characterized in that: The light treatment in the third step includes one of irradiating with a flashlight of a specific wavelength, irradiating under sunlight, and irradiating with a portable colorimeter for color development.

8. A simple disease home visual detection system, characterized by: It includes a detection acquisition module, an AI judgment module and a symptomatic medication delivery module. The detection acquisition module performs real-time home detection based on the RPA detection kit and the LAMP detection kit and obtains the detection result picture or makes an immediate diagnosis or uses the home medicine box in real time. The AI ​​judgment module makes an intelligent judgment on the uploaded detection result picture and consultation information based on the AI ​​health consultation platform and gives a real-time judgment result. The symptomatic medication delivery module performs intelligent real-time symptomatic medication according to the judgment result and user needs and choices, and delivers medicine in real time for users to use at home according to usage and dosage.

9. A simple home visual disease detection system according to claim 8, characterized in that: The AI ​​judgment module includes an AI file creation module and a consultation module. The AI ​​file creation module establishes a personal health file based on the user's registration information on the AI ​​health consultation platform. The consultation module is based on AI and combines the user's consultation information to perform real-time intelligent judgment and result output as well as user chronic disease management.

10. A simple home visual disease detection system according to claim 8, characterized in that: The symptomatic medication dispensing and delivery module includes a medication recommendation module, a medication purchase selection module, and an intelligent positioning module. The medication recommendation module provides the symptomatic medication name, main symptoms, and usage and dosage information based on real-time judgment results for users to use their home medicine box for real-time medication or use a medication purchase program. The medication purchase selection module is used to purchase medications online in real time according to the user's selection. The intelligent positioning module is used to search for the nearest contracted pharmacy based on the user's location range and provide real-time symptomatic medication and real-time delivery for users to use at home in real time according to usage and dosage.

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