CN112646929A - Novel coronavirus nucleic acid chromatography detection kit and application - Google Patents
Novel coronavirus nucleic acid chromatography detection kit and application Download PDFInfo
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Abstract
The invention relates to the technical field of gene detection, and relates to a novel coronavirus nucleic acid chromatography detection kit and application thereof. Aiming at the technical problems that the detection of the novel coronavirus detection kit in the prior art is long in time consumption, special equipment is needed, DNA is not easy to degrade and pollute, the sensitivity is low and the specificity is not strong, the application provides the novel coronavirus nucleic acid chromatography detection kit and the application, the RNA extraction process is avoided, a PCR instrument is not needed, RNA isothermal amplification is based on, an amplification product is RNA, the degradation is easy, and the aerosol pollution possibility is extremely low; the specially designed primer and probe have the advantages of high sensitivity, strong specificity and simple operation.
Description
Technical Field
The invention relates to the technical field of gene detection, in particular to a novel coronavirus nucleic acid chromatography detection kit and application thereof.
Background
The novel coronavirus belongs to a coronavirus of beta genus, and has an envelope, wherein particles are circular or elliptical, and the diameter of the particles is 60-140 nm. The clinical manifestations are fever, dry cough and hypodynamia, and severe diseases such as dyspnea and/or hypoxemia gradually appear in severe patients; the critically ill rapidly progresses to acute respiratory distress syndrome, septic shock, and difficult to correct metabolic acidosis and coagulation dysfunction. The novel coronavirus is RNA positive strand virus, and has sequence similarity of 79.6% with SARS coronavirus. On day 11/2/2020, the International Committee for Classification of virology tentatively named the novel coronavirus SARS-CoV-2.
The method for accurately and quickly screening SARS-CoV-2 infectors and isolating them is the most effective means for epidemic prevention and control at present. Currently, the detection of SARS-CoV-2 is mainly based on both nucleic acid and antibody detection, and the real-time fluorescence RT-PCR method based on nucleic acid detection is the main method for SARS-CoV-2 detection. Nucleic acid detection reagents which are marketed or published in patents are liquid detection reagents, need to be prepared for use firstly, and have higher requirements on experiments and skills; the kit requires cold chain transportation, which increases the detection cost. And the detection sensitivity of the colloidal gold detection reagent on the market is relatively low, and certain risks of false negative and false positive exist. Other published techniques for detecting nucleic acids based on novel coronaviruses, such as the Crispr technique and the RT-LAMP technique. Although isothermal detection can be achieved, there is a certain risk of contamination because the amplification product is DNA. On the basis of RNA isothermal amplification technology, the method combines colloidal gold chromatography technology to establish a 2019-nCoV diagnosis method which is simple and rapid to operate and low in price, and has very important significance.
The invention discloses a Chinese patent application publication No. CN111187860A, named as 'novel coronavirus multiple PCR rapid detection kit', and discloses a novel coronavirus multiple PCR rapid detection kit, which comprises: the PCR reaction solution freeze-dried powder comprises a PCR reaction solution freeze-dried powder, a primer pool freeze-dried powder and a buffer solution, wherein the primer pool freeze-dried powder comprises a primer pair and a fluorescent probe listed in a sequence table. The scheme optimizes a primer system for detecting SARS-CoV-2 by aiming at the multiplex PCR, designs a brand new primer and a probe pool, and can achieve the aim of sensitively and specifically meeting the early-stage rapid detection of the new coronavirus clinically. However, the scheme needs to extract the viral RNA by a magnetic bead method or a column method, needs special instruments, is easy to pollute in actual detection, cannot detect the viral RNA in real time, and cannot detect the viral RNA in basic medical units.
In view of the above demand for rapid detection of pathogens by epidemic prevention and control, a novel coronavirus detection method which can be used for instant detection, is not easy to pollute, has high sensitivity and strong specificity, and can be used for detection in primary medical units is needed.
Disclosure of Invention
1. Problems to be solved
Aiming at the technical problems that the detection of the novel coronavirus detection kit in the prior art is long in time consumption, special equipment is needed, DNA is not easy to degrade and pollute, the sensitivity is low and the specificity is not strong, the application provides the novel coronavirus nucleic acid chromatography detection kit and the application, the RNA extraction process is avoided, a PCR instrument is not needed, RNA isothermal amplification is based on, an amplification product is RNA, the degradation is easy, and the aerosol pollution possibility is extremely low; the specially designed primer and probe have the advantages of high sensitivity, strong specificity and simple operation.
2. Technical scheme
In order to achieve the purpose, the technical scheme is as follows:
the novel coronavirus amplification reagent comprises an ORF1ab gene and/or N gene amplification reagent; the ORF1ab gene amplification reagent contains an ORF1ab gene amplification primer, and the ORF1ab gene amplification primer sequence is shown as SEQ ID NO 1-2; the N gene detection reagent contains N gene amplification primers, and the sequence of the N gene amplification primers is shown in SEQ ID NO. 3-4.
A novel coronavirus nucleic acid chromatography detection kit comprises the novel coronavirus amplification reagent.
Further, the kit also comprises a detection reagent; the detection reagent comprises an ORF1ab gene detection probe, wherein the ORF1ab gene detection probe has a sequence shown in SEQ ID NO. 5-9; the detection reagent comprises an N gene detection probe, and the sequence of the N gene detection probe is shown in SEQ ID NO. 10-14. .
Further, the amplification reagent also contains an ACTB internal control gene amplification primer, and the sequence of the ACTB internal control gene amplification primer is shown as SEQ ID NO. 15-16; the detection reagent also comprises an ACTB internal control gene detection probe, and the sequence of the ACTB internal control gene detection probe is shown in SEQ ID NO. 17-21.
Further, the detection reagent also contains a colloidal gold probe, the 5' end of the colloidal gold probe is modified by sulfhydrylation, and the probe sequence is shown in SEQ ID NO. 22.
Further, the kit also comprises a nitrocellulose membrane coated by the coating probe; the coated probe includes:
ORF1ab coats the probe, the sequence of the probe is shown in SEQ ID NO. 23;
n gene coating probe with the sequence as shown in SEQ ID No. 24;
the internal control ACTB coats a probe, and the sequence of the probe is shown as SEQ ID NO. 25;
the quality control is coated with a probe, and the sequence of the probe is shown as SEQ ID NO. 26.
A colloidal gold probe: the 5 ' end of the colloidal gold probe is modified by sulfhydrylation, and a sulfhydryl group can form a covalent bond with the colloidal gold particles, is marked on the colloidal gold particles and can be combined with the 3 ' end of the detection probe and the 3 ' end of the quality control probe;
detecting a probe: the 5 'end can be specifically combined with an amplified RNA product, the 3' end can be combined with a thiolation probe marked on a gold nanoparticle to play a role in enriching the color development of colloidal gold at a detection line, and the two parts are linked by 4-5T;
and (3) capturing a probe: the 5 ' end can be specifically combined with an amplified RNA product, the 3 ' end can be combined with the 5 ' end of a coating probe coated on a corresponding detection line on an NC membrane to play a role in fixing the amplified product RNA, and the two parts are linked by 4-5T;
coating the probe: the coated probe is coated on an NC membrane, 1-10T basic groups are arranged at the 5 'end of the coated probe, the purpose is to increase the hybridization space, the 5' end biotin (digoxin) modification can be combined with streptavidin (anti-digoxin antibody) and coated on the NC membrane, the 3 'end can be combined with the 5' end of the capture probe, and a nanogold-detection probe-RNA-capture probe-coated probe compound is formed and detected;
coating a probe with a quality control line: the 3 'end of the probe can be combined with a nanogold probe, and the 5' end of the probe modified by biotin (digoxin) can be combined with streptavidin (anti-digoxin antibody) and coated on an NC membrane. During chromatography, the C-coated probe can form a 'C-coated probe-nanogold probe' complex in the presence or absence of an RNA amplification product, and the complex forms a macroscopic strip during chromatography. The probe can control the quality of the test paper strip and the detection liquid, and the chromatography process is error-free.
Furthermore, the 5 'end of the coating probe contains 1-10 polyT bases, and the 5' end is marked with biotin or digoxin.
Furthermore, the kit also comprises a detection test strip, and the detection test strip is formed by sequentially fixing absorbent paper, a coated nitrocellulose membrane and a sample pad on a PVC base plate from top to bottom.
The test strip is provided with a detection line and a quality control line, wherein the detection line comprises an N-T line, an ORF1ab-T line and an internal reference-T line, the internal reference-T line is an internal reference detection line, a coated internal reference coated probe can be specifically combined with one end of a capture probe, and an N coated probe coated at the N-T line can be specifically combined with one end of a novel coronavirus N gene capture probe; the ORF1ab coated probe at ORF1ab-T line was able to specifically bind to one end of the novel coronavirus ORF1ab gene capture probe. The C-shaped coated probe coated on the quality control line can be specifically combined with the nano-gold probe. And (3) hybridizing the detection probe, the capture probe, the nanogold probe and the specific amplification product of the nucleic acid to be detected, and then dripping the hybridized products on a test strip for chromatography, wherein the color development of the detection line indicates that the nucleic acid to be detected exists, and the color development of the quality control line indicates that the detection is effective.
A method for using a novel coronavirus nucleic acid chromatography detection kit, which comprises the following steps:
(1) releasing viral nucleic acid using a sample release agent comprising sodium lauroyl sarcosinate, DTT, Tris-HCl, EDTA and SDS;
samples such as pharyngeal swabs and alveolar lavage fluid of patients suspected of having the novel coronavirus are collected, and the viral RNA molecules are released by using a sample releasing agent.
Preferably, the sample releasing agent comprises 1-5% of sodium lauroyl sarcosinate, 1-20 mM DTT, 1-100 mM Tris-HCl, 1-20 mM EDTA and 0.1-0.5% SDS; further, the sample-releasing agent consisted of 2.5% sodium lauroyl sarcosinate, 5mM DTT, 10mM Tris-HCl, 2mM EDTA and 0.1% SDS;
(2) amplifying the virus nucleic acid by using an amplification reagent to obtain an RNA amplification product, wherein the amplification reagent comprises a TMA amplification reaction solution and TMA amplification enzyme; the amplification reaction solution comprises polymerase buffer solution, reverse transcriptase buffer solution, DMSO, dNTPs, NTP and an amplification primer; the amplification primers comprise the ORF1ab gene amplification primer and an N gene amplification primer;
preferably, the amplification reaction solution consists of RNA polymerase buffer solution with a final concentration of 1 XT 7, 1 XMAV reverse transcriptase buffer solution, 10% DMSO, 1.25mM dNTPs, 2.5mM NTP and 400nM amplification primer; wherein the amplification primers comprise 3 groups: a pair of amplification primers of ORF1ab gene, N gene and ACTB internal control gene;
preferably, the reverse transcriptase is AMV or M-MLV; the TMA amplification enzyme system mainly comprises 5U-20U/25 mul reaction reverse transcriptase, 20U-50U/25 mul reaction T7RNA polymerase and 0.1-1U/25 mul reaction RnaseH; further TMA amplification enzymes mainly comprise 8U/25 mu l reaction AMV, 36U/25 mu l reaction T7RNA polymerase and 0.5U/25 mu l reaction RnaseH;
preferably, 2. mu.L of the nucleic acid extract is added to 17. mu.L of the TMA reaction solution, heated at 95 ℃ for two minutes, preheated at 42 ℃ for 2 minutes, added with 1. mu.L of the amplification enzyme, and amplified at 42 ℃ for 30 minutes at constant temperature. If the sample to be detected contains the nucleic acid of the novel coronavirus, a large amount of RNA molecules are amplified and enriched during amplification.
(3) And mixing the RNA amplification product with a detection solution, and placing the mixture on a detection test strip for chromatography, wherein the detection solution comprises an ORF1ab gene detection probe, an N gene detection probe and a colloidal gold probe.
Pre-hybridization:
the RNA isothermal amplification product is mixed with a novel coronavirus detection solution (comprising a detection probe, a colloidal gold probe and a capture probe), and prehybridization is carried out for 10 minutes at 42 ℃. The amplified RNA molecule is combined with the detection probe and the capture probe in a complementary pairing way, and when an amplification product exists, a colloidal gold-detection probe-RNA molecule-capture probe compound can be formed.
Chromatography detection:
dropping the prehybridization product at a sample pad of the test strip, carrying out chromatography on the prehybridization solution along the NC membrane to the direction of absorbent paper, and when an RNA amplification product to be detected exists, forming a 'colloidal gold-detection probe-RNA molecule-capture probe complex', and intercepting the complex by a coating probe coated on the NC membrane during chromatography to form a strip which is seen by naked eyes and is positive. If the RNA product to be detected is not amplified, the colloidal gold-detection probe-RNA molecule-capture probe compound cannot be formed, and the colloidal gold particles cannot be gathered at the T line, so that a macroscopic strip cannot be formed, and the result is negative. No matter whether the RNA product to be detected is amplified or not, the nanogold probe flows forwards along the NC membrane during chromatography, and is combined with a sequence coated at the quality control line when reaching the quality control line, so that the nanogold probe is retained at the quality control line to form a macroscopic colored strip, which is effective in experimental results.
TABLE 1 specific primer and Probe sequences in the kits of the invention
3. Advantageous effects
Compared with the prior art, the technical scheme provided by the invention has the following beneficial effects:
(1) according to the novel coronavirus (2019-nCoV) detection kit, three indexes, namely, a novel coronavirus ORF1ab gene, a novel coronavirus N gene and an internal reference ACTB gene, can be simultaneously amplified in the same tube by an RNA constant-temperature amplification method, the amplified nucleic acid product is RNA, the RNA is easy to degrade in a natural environment, and the effect of preventing pollution is easier to achieve compared with the effect of amplifying DNA by a PCR method.
(2) The novel coronavirus (2019-nCoV) detection kit disclosed by the invention is low in equipment requirement, RNA constant-temperature amplification is carried out in a 42 ℃ environment, amplification reaction can be realized even in a water bath kettle, the requirement of an experimental instrument is reduced to the maximum extent, and the monitoring of new crown epidemic situation of a primary medical team can be met by combining colloidal gold detection.
(3) The novel coronavirus (2019-nCoV) detection kit disclosed by the invention is high in sensitivity, and the minimum detection limit of the kit on RNA copies of ORF1ab and E genes of the novel coronavirus is 100 copies/mL.
(4) The novel coronavirus (2019-nCoV) detection kit disclosed by the invention adopts an RNA constant-temperature amplification technology and a test strip chromatography technology, detects nucleic acid through the test strip, and can judge the result only within about 10 min. The operation is very simple, the technical requirement on experimenters is low, and special instruments and equipment are not needed.
Detailed Description
For a further understanding of the contents of the present invention, reference will now be made in detail to the following examples.
Example 1
Preparation of nucleic acid detection test strip
The main raw materials required for preparing the nucleic acid detection test strip are as follows: nitrocellulose membranes (NC membranes), sample pads, absorbent paper, PVC base plates, and the like.
1. Film spraying:
detecting a line N-T: 5' -end biotinylation modified N gene coated probe sequence, (20. mu.M), film spraying amount: 2-3 muL/cm;
detection line ORF1ab-T line: 5' -end biotinylation modified ORF1ab gene-coated probe sequence, (20. mu.M), film spraying amount: 2-3 muL/cm;
detecting an internal reference-T line: ACTB gene-coated probe sequence modified with biotinylation at the 5' end, (20 μ M), membrane-spraying amount: 2-3 mu L/cm;
quality control line (line C): 5' end biotinylation modification, nano-gold probe capture, (20 mu M), film spraying amount: 2-3 μ L/cm.
And after the film spraying is finished, drying the film in a clean constant temperature box at 37 ℃ for 2 hours, and storing the film in a dry environment for later use.
2. Test strip assembly
And respectively cutting 2cm long absorbent paper, the coated NC film and the sample pad, and sequentially fixing the absorbent paper, the coated NC film and the sample pad on a PVC base plate from top to bottom to obtain the detection test strip.
Example 2
Sensitivity test comparison test
The sensitivity comparison test is carried out by adopting the developing method and the colloidal gold prepared according to the method published by the Chinese patent application CN 111455099A. Selecting pseudo viruses with known concentration and containing 2019-nCoV target genes, carrying out concentration gradient dilution by 10 times, repeating 3 times for each gradient, taking the lowest dilution concentration with 100% positive detection rate as an estimated detection limit, after the estimated detection limit is determined, diluting the 2019-nCoV pseudo viruses to be close to the estimated detection limit concentration value, carrying out detection by using a kit, and carrying out detection for 20 times for each concentration so as to further accurately determine the lowest detection limit concentration (selecting the dilution with the positive rate of more than 95% as the detection limit sensitivity of the kit).
TABLE 22019 determination of detection limits for nCoV
From the data, the lowest detection limit of the kit is as follows: 1X 102copies/mL. The lowest detection limit of the comparison patent is 800 copies/mL.
Example 3
Specificity verification
Other pathogens that are similar or cause symptoms similar to the 2019 novel coronavirus species, such as seasonal influenza A H1N1, novel influenza A H1N1(2009) influenza, influenza A H3N2, H7N9, influenza B Yamagata, influenza B Victoria, respiratory syncytial virus A, respiratory syncytial virus B, parainfluenza I, parainfluenza II, parainfluenza III, rhinovirus A, B, C sets, adenovirus 1, 7, enterovirus A, B, C, D, rotavirus A, norovirus GI, Mycoplasma pneumoniae, Chlamydia pneumoniae, Haemophilus influenzae, Staphylococcus aureus, Streptococcus pneumoniae, coronaviruses (HKU1, OC43, NL63, 229E), MERS coronavirus pseudoviruses, cross-reaction tests were performed to verify the specificity of the kit assays and indicate that there is no cross reaction between the kit and other microorganisms, the specificity of detecting the pathogen by the embodying kit is strong.
Example 4
Validation of clinical samples
1000 samples are tested by applying the reagent and the reference reagent in a clinical unit together, wherein 940 samples of throat swabs and 60 samples of sputum are tested according to the kit and the reference reagent, and the test results are as follows:
TABLE 4 test results of inventive and reference reagents (four-grid table)
Positive compliance rate/clinical sensitivity: 54/54-100%;
negative compliance rate/clinical specificity: 945/946 ═ 99.89%;
the total coincidence rate is as follows: (54+945)/1000 ═ 99.9%;
the value of the coefficient of conformity Kappa (k) was calculated to be 0.99, and in this test, the value of Kappa was 0.99 and more than 0.75, and it was considered that the two were in good conformity. (Kappa is more than or equal to 0.75 to indicate that the consistency of the two is good, 0.75 to 0.4 to indicate that the consistency is general, and Kappa <0.4 to indicate that the consistency of the two is poor).
The above examples are merely representative of preferred embodiments of the present invention, and the description thereof is more specific and detailed, but not to be construed as limiting the scope of the present invention. It should be noted that, for those skilled in the art, various changes, modifications and substitutions can be made without departing from the spirit of the present invention, and these are all within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
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Claims (9)
1. A novel coronavirus amplification reagent, characterized in that: comprises ORF1ab gene and/or N gene amplification reagent; the ORF1ab gene amplification reagent contains an ORF1ab gene amplification primer, and the ORF1ab gene amplification primer sequence is shown as SEQ ID NO 1-2; the N gene detection reagent contains N gene amplification primers, and the sequence of the N gene amplification primers is shown in SEQ ID NO. 3-4.
2. A novel coronavirus nucleic acid chromatography detection kit is characterized in that: comprising the novel coronavirus amplification reagent of claim 1.
3. The novel nucleic acid chromatography detection kit for coronavirus according to claim 2, wherein: the kit further comprises a detection reagent; the detection reagent comprises an ORF1ab gene detection probe, wherein the ORF1ab gene detection probe has a sequence shown in SEQ ID NO. 5-9; the detection reagent comprises an N gene detection probe, and the sequence of the N gene detection probe is shown in SEQ ID NO. 10-14.
4. The novel nucleic acid chromatography detection kit for coronavirus according to claim 3, wherein: the amplification reagent also contains an ACTB internal control gene amplification primer, and the sequence of the ACTB internal control gene amplification primer is shown as SEQ ID NO. 15-16; the detection reagent also comprises an ACTB internal control gene detection probe, and the sequence of the ACTB internal control gene detection probe is shown in SEQ ID NO. 17-21.
5. The novel nucleic acid chromatography detection kit for coronavirus according to claim 4, wherein: the detection reagent also contains a colloidal gold probe, the 5' end of the colloidal gold probe is modified by sulfhydrylation, and the probe sequence is shown in SEQ ID NO. 22.
6. The novel nucleic acid chromatography detection kit for coronavirus according to claim 5, wherein: the kit also comprises a nitrocellulose membrane coated by the coating probe; the coated probe includes:
ORF1ab coats the probe, the sequence of the probe is shown in SEQ ID NO. 23;
n gene coating probe with the sequence as shown in SEQ ID No. 24;
the internal control ACTB coats a probe, and the sequence of the probe is shown as SEQ ID NO. 25;
the quality control is coated with a probe, and the sequence of the probe is shown as SEQ ID NO. 26.
7. The novel nucleic acid chromatography detection kit for coronavirus according to claim 6, wherein: the 5 'end of the coating probe contains 1-10 polyT bases, and the 5' end is marked with biotin or digoxin.
8. The novel nucleic acid chromatography detection kit for coronavirus according to claim 7, wherein: the kit also comprises a detection test strip; the detection test strip is formed by sequentially fixing absorbent paper, a coated nitrocellulose membrane and a sample pad on a PVC base plate from top to bottom.
9. The use of the novel coronavirus nucleic acid chromatography detection kit according to any one of claims 2-8, wherein: the method comprises the following steps:
(1) releasing viral nucleic acid using a sample release agent comprising sodium lauroyl sarcosinate, DTT, Tris-HCl, EDTA and SDS;
(2) amplifying the virus nucleic acid by using an amplification reagent to obtain an RNA amplification product, wherein the amplification reagent comprises a TMA amplification reaction solution and TMA amplification enzyme; the amplification reaction solution comprises polymerase buffer solution, reverse transcriptase buffer solution, DMSO, dNTPs, NTP and an amplification primer;
(3) and mixing the RNA amplification product with a detection solution, and placing the mixture on a detection test strip for chromatography, wherein the detection solution comprises an ORF1ab gene detection probe, an N gene detection probe and a colloidal gold probe.
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| CN113156124A (en) * | 2021-05-28 | 2021-07-23 | 安徽医科大学第二附属医院 | Gene editing-based method for detecting new coronavirus and colloidal gold test strip |
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| CN116024302A (en) * | 2022-10-26 | 2023-04-28 | 江苏迅睿生物技术有限公司 | Duplex nucleic acid detection chromatographic test paper, primer, probe set and kit |
| CN116024302B (en) * | 2022-10-26 | 2023-08-01 | 江苏迅睿生物技术有限公司 | Duplex nucleic acid detection chromatographic test paper, primer, probe set and kit |
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Application publication date: 20210413 |