WO2022236891A1 - Non-invasive detection kit for detecting novel coronavirus antigen, and detection method thereof - Google Patents

Abstract

A non-invasive detection kit for detecting a novel coronavirus antigen, and a detection method thereof. By detecting whether a mouthwash sample contains a novel coronavirus antigen, the detection kit rapidly determines whether a subject is infected with a novel coronavirus. The detection kit comprises a test card and mouthwash; the test card consists of a test strip and a plastic box; according to the passing order of a sample to be tested, the structures in the test card sequentially are a sample pad, a colloidal gold bonding pad, a nitrocellulose membrane, and an absorbent pad, wherein the nitrocellulose membrane comprises a detection line coated with an SARS-CoV-2 recombinant antibody and a quality control line coated with a goat anti-rabbit polyclonal antibody, and the colloidal gold bonding pad comprises the SARS-CoV-2 recombinant antibody and a rabbit IgG antibody. According to the detection method, sampling is performed in a non-invasive sampling mode, it is conveniently and quickly detected whether the subject is infected, non-invasive sampling does not cause any pain, the possibility of secondary infection is eradicated, a result can be quickly obtained within ten minutes, an operation method is simple, and no assistance is needed.

Description

[Title of the invention formulated by ISA under Rule 37.2] Non-invasive detection kit for detecting novel coronavirus antigen and its detection method technical field

The invention relates to a non-invasive detection kit for detecting novel coronavirus antigens and a detection method thereof, belonging to the field of detection of pathogenic microorganisms.

Background technique

People infected with the new coronavirus have fever and weakness as the main clinical symptoms, dry cough as the main respiratory symptoms, and gradually develop dyspnea. Severe patients present with acute respiratory distress syndrome, septic shock, metabolic acidosis, and coagulation disorders, which are difficult to correct. Although the symptoms were similar to those of viral pneumonia, a new type of coronavirus was found in the lower respiratory tract of infected people, which was later named 2019 novel coronavirus (2019-nCoV) or SARS-CoV-2, COVID-19. Novel coronavirus pneumonia is an acute respiratory infectious disease, and the population is generally susceptible. The source of infection seen so far is mainly patients infected with the new coronavirus, and asymptomatic infections may also become the source of infection. Based on the current epidemiological investigation, the incubation period is 1-14 days, mostly 3-7 days. The main manifestations are fever, dry cough, and fatigue. A small number of patients have symptoms such as nasal congestion, runny nose, sore throat, myalgia, and diarrhea.

Coronaviruses are viruses that can infect both humans and animals, and are often the source of infection in areas where humans have frequent contact with wild animals. A large number of scientific studies have confirmed that the SARS-CoV-2 virus originated in nature, and the new coronavirus belongs to the genus β coronavirus. However, the infection mechanism has not been clarified so far, including transmission speed, incubation period, and human-to-human transmission of highly pathogenic viruses. etc., resulting in a large number of infected people in many countries around the world, and the death rate is gradually increasing. At present, the novel coronavirus has become a pandemic, which has a huge impact on the stability of human society and the global economy.

In the face of the severe pandemic situation of the 2019 novel coronavirus disease (COVID-19) spreading globally, rapid identification of patients infected with the novel coronavirus (SARS-CoV-2) is the top priority of prevention and control. The gold standard for SARS-CoV-2 detection is nucleic acid detection, but in clinical work, differences in specimen type, specimen quality, patient condition and other factors may lead to false negative results in nucleic acid detection. In addition, nucleic acid detection relies on laboratory testing instruments, which has the problem of long detection time and the detection cannot be adapted to large-scale screening at the point of care. Therefore, how to accurately and rapidly identify viral infections is an important challenge. For this reason, serum-specific antibodies, as an important immune response product for the body to resist viral infection, are rapidly applied to laboratory detection of SARS-CoV-2 and combined with nucleic acid detection for rapid diagnosis and screening of SARS-CoV-2 patients. check.

New coronavirus pneumonia nucleic acid detection can take patients' nasopharyngeal swabs, sputum and other lower respiratory secretions, blood, stool and other specimens to detect new coronavirus nucleic acid. If the nucleic acid of the specimen is positive, the virus infection can be confirmed. Novel coronavirus infection mainly invades bronchial epithelial cells and alveolar epithelial cells. Lower respiratory tract specimens, such as sputum and airway extracts, should be used as much as possible to reflect the virus infection more accurately.

More and more studies have shown that the positive rate of nasal swabs is higher than that of throat swabs. The Guangdong Center for Disease Control and Prevention and the Sun Yat-Sen University team analyzed the time relationship between the viral load in the nose and throat of 17 patients with new coronary pneumonia and the course of the disease, and found that patients with new coronary pneumonia can detect a higher viral load in the upper respiratory tract shortly after onset , while the viral load in the nasal cavity was higher than that in the throat after nasal and throat swabs were sampled using the same cotton swab. Liu Yanbin and others took throat swabs and nasal swabs from 100 cases of new coronary pneumonia in Wuhan Red Cross Hospital, and compared the positive rate of nucleic acid detection of samples after two methods of sampling. The positive detection rate of CoV-2 viral nucleic acid was 54%, while the positive rate of viral nucleic acid in nasal swab samples was 89%. The positive rate of viral nucleic acid in nasal swab samples was higher than that in throat swab samples, and the difference was statistically significant (χ ² =3.8504, P=0.0497). The positive detection rate of nasal swabs for nucleic acid detection in patients with new coronary pneumonia has obvious advantages. collected a total of 866 respiratory specimens from 213 patients, including 205 throat swabs, 490 nasal swabs, 142 sputum and 29 bronchoalveolar lavage fluid (BALF). At 14 days after the onset, except for BALF, the positive rate of sputum was the highest (74.4%-88.9%), followed by nasal swabs (53.6%-73.3%) in severe and mild case samples. Among the samples collected ≥15 days ago, the positive rate of sputum and nasal swabs was still high, ranging from 42.9% to 61.1%. The positive rate of throat swabs collected ≥8 days is very low, especially in mild cases.

The diagnostic model based on colloidal gold rapid immunochromatography has been paid more and more attention. Rapid antigen detection can make up for the time window and sensitivity of nucleic acid detection, and provide relevant information for infection control measures. Antigen testing is especially important when diagnosing new and emerging human coronaviruses. In this case, especially in the early stages of the disease, it is not very feasible to test positive for the viral DNA of an infected person, but it is possible to demonstrate retrospectively that an immune response has been developed. In the identification of SARS-CoV-2, especially in early screening, rapid antigen detection can make up for the sensitivity and time window problems of nucleic acid detection, and provide relevant information for infection control. Laboratory diagnostics are critical to any attempt at a SARS-CoV-2 pandemic. Studies have shown that self-collected mouthwash/mouthwash samples are not inferior to nasopharyngeal flocked swabs collected by healthcare workers in detecting SARS-CoV-2, and are significantly more acceptable to patients. Mouthwash/gargle samples, in particular, showed the highest sensitivity and were the first choice of those who were sampled. These data should be considered by all planning laboratory testing strategies and algorithms; self-collection of gargle/mouthwash samples avoids the deployment of large numbers of medical personnel trained in sample collection and the consumption of large quantities of personal protective equipment and swabs. It should be emphasized that as many children and adolescents return to school and as many other respiratory viruses become increasingly prevalent and respiratory symptoms become more common, testing volumes will likely be higher by the end of 2021. Self-collected mouthwash/mouthwash samples may also result in substantial cost savings as no PPE or trained healthcare personnel are required to collect the samples. The utility may be higher in low- and middle-income countries or in remote areas, where access to testing clinics would be another barrier to obtaining samples. The stability of RNA recovery in this sample type is stored at room temperature for at least two days, making subsequent sample detection difficult and the risk of missed or false detections.

In the study, saliva samples were significantly less sensitive and acceptable than mouthwash/gargle samples. However, it is important to note that a substantial proportion of SARS-CoV-2 positive participants had negative matched saliva samples, samples were taken within 7 days from initial diagnosis, and most patients with high detection rates of saliva samples were hospitalized patients . Less amenable to high-throughput processing when viral load levels tend to be higher and/or additional artificial saliva processing steps are used. During the evaluation, it was intermittently observed that processing of saliva samples was hampered by variable sample viscosity; additional manual processing of two saliva samples was required due to the presence of excess mucus. For these reasons, rinse/mouthwash samples appear to be more feasible and uniquely advantageous over swab-free and transport medium options.

Given the high user acceptance, no need for swabs and/or transport media, and high diagnostic yield, gargle/gargle samples can be considered a promising sample type for detection of suspected SARS- Those who are initially infected with CoV-2 are especially suitable for large-scale resident self-examination or community-distributed general inspection.

Contents of the invention

In view of the above-mentioned problems existing in the prior art, the object of the present invention is to obtain a non-invasive detection kit and detection method for detecting novel coronavirus antigens for mouthwash samples or samples.

In order to achieve the above-mentioned purpose of the invention, the technical scheme of the non-invasive detection kit for detecting the novel coronavirus antigen used in the present invention is as follows:

The detection kit quickly determines whether the subject is infected with the new coronavirus by detecting whether the mouthwash sample or the sample contains the new crown antigen. The kit is a colloidal gold immunochromatography detection kit, including a test card, a gargle Saliva, the test card is composed of test strips and plastic boxes, the test strips are composed of nitrocellulose membranes, sample pads, colloidal gold bonded pads, absorbent pads, absorbent paper, PVC boards and other supports, and each structure in the test card is in accordance with The passing order of the sample to be tested is: sample pad, colloidal gold binding pad, nitrocellulose membrane and absorption pad, wherein the nitrocellulose membrane area includes the detection line and the quality control line, and the detection line is coated near the colloidal gold conjugate. One end, the quality control line is coated near the end of the absorbent pad, the nitrocellulose membrane includes the detection line coated with SARS-CoV-2 recombinant antibody and the quality control line is coated with goat anti-rabbit polyclonal antibody, and the binding pad includes SARS-CoV-2 Recombinant antibody, rabbit IgG antibody.

Preferably, the test card is provided with a sample hole, and the sample to be tested is dripped into the sample pad through the sample hole. The sample pad, colloidal gold binding pad, nitrocellulose membrane and absorbent pad in the test card are all adsorbed on a support pad. The absorbent pad in the test card has strong water absorption, and the sample to be tested can move from the sample pad to the absorbent pad through capillary action, and then pass through the test line and quality control line on the nitrocellulose membrane in turn, when the new crown of the sample to be tested When the concentration of virus antigen is greater than the lower limit of detection, the new coronavirus antigen in the sample combines with the SARS-COV-2 recombinant antibody, and the detection line to the nitrocellulose membrane combines with the SARS-COV-2 recombinant antibody to form a red band visible to the naked eye.

Preferably, the recombinant antibody is a colloidal gold antibody linked to colloidal gold particles. This kit is optimized and recombined for the RNA sequence fragments of the new coronavirus. The recombined fragments are located in the conserved region, so it can detect all the current new coronaviruses. At present, more than 3,000 mutation sites do not involve these fragments. The detection will not be affected by the mutant strain, and the detection rate is accurate; and the recombinant fragment can effectively avoid the combination of IgA and other sticky proteins in the oral cavity, so the oral samples of this kit have more specificity and more accurate detection results; Several fragments selected by the recombinant fragments have good immunogenicity, relatively high titer for immunomonoclax, and good specificity; although the amino acids of the selected fragments are small, the binding site for immunomonoclax contains the N protein site , can avoid missed detection, and the high-level structure of the protein combined with several fragments has good sensitivity to virus culture.

Preferably, the amino acid sequence of the recombinant antibody is shown in the sequence listing SEQ ID NO.1.

Preferably, the gene sequence for expressing the amino acid sequence of the recombinant antibody is shown in SEQ ID NO.2 in the sequence table.

The way of colloidal gold particles labeling antigens can also be replaced by any other labels known to those skilled in the art, including latex particles, quantum dots, fluorescent nanoparticles and the like. The same or different labels can be selected for the detection line and quality control line, as long as the types of labels can be understood by those skilled in the art.

Preferably, the SARS-COV-2 recombinant antibody is selected from other types of immunoglobulins, including IgA and IgG, preferably non-human IgG, such as mouse IgG.

Preferably, whether there is a red band on the quality control line can be used as a test of whether the liquid flow in the test card meets the preset requirements. If there is no red band on the quality control line, there is an obstacle or error in the flow of the liquid, and the test result of the test line is It should not be used as a reference standard.

Preferably, the nitrocellulose membrane in the test card is adsorbed on a support pad, and the support pad is made of glass fiber, polyester fiber film or non-woven fabric. The SARS-CoV-2 antibody coating protein content on the nitrocellulose membrane should not be less than 2.0mg/mL; the purity should be ≥ 90%, and the SARS-CoV-2 antibody-labeled protein content should not be less than 2.0mg/mL; the purity Should be ≥90%, the protein content of goat anti-rabbit IgG polyclonal antibody should not be less than 3.0mg/mL; the purity should be ≥90%, the rabbit IgG protein content should not be less than 3.0mg/mL; the purity should be ≥90%.

Preferably, the plastic box of the test card is provided with a sampling hole, and the sample to be tested is dripped into the sample pad through the sampling hole; the detection kit also includes a disposable cup and a dropper. Mouthwash is 0.8-0.9% sodium chloride aqueous solution.

Another object of the present invention is to provide a detection method using the above-mentioned non-invasive detection kit for detecting novel coronavirus antigens, characterized in that the detection method comprises the following steps:

a) adding the sample to be tested into the sample hole of the sample pad, so that the sample to be tested flows from the sample pad to the colloidal gold binding pad;

b) The sample to be tested flows through the test line and the quality control line sequentially through capillary action. When the similarity of the sequence to be tested is greater than 90%, the antigen to be tested can be captured by the recombinant antigen, forming a red band;

c) Visually observe whether there is a red band on the test line and the quality control line to determine whether the source of the sample to be tested is infected with the new coronavirus.

Preferably, the detection sample is a mouthwash rinse sample without secondary treatment. When collecting mouthwash, hold 10-15mL of mouthwash in the mouth, tilt the patient's head back 70 degrees, let the mouthwash deep in the throat (remember not to stay at the front of the mouth), and rinse the deep throat repeatedly for 15-20 seconds, Make the mouthwash wash out the virus deep in the throat; after the samples are collected, perform the rapid detection reagent card for the new coronavirus antigen. Since some mouthwash samples are too viscous, the sample volume should be sufficient.

Based on the above features, the detection kit and detection method of the present invention can be used in clinics, homes, and various places and purposes that require rapid detection of novel coronavirus.

In the present invention, the terms "polypeptide" and "protein" are used interchangeably and refer to a polymer of amino acid residues, and are not limited by length. Polypeptides, including antibodies and antibody chains and other polypeptides, such as linkers, may include amino acid residues, including natural and/or unnatural amino acid residues. These terms also include post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, and the like. In certain aspects, polypeptides may contain modifications relative to the native or natural sequence so long as the protein retains the desired activity. These modifications may be deliberate, such as through site-directed mutagenesis, or accidental, such as through host mutations producing proteins or errors due to PCR amplification.

As described herein, the use of "percentage (%) amino acid sequence" and "percentage (%)" and "%" sequence when used for an amino acid sequence (reference polypeptide sequence) is defined as the proportion of amino acid residues in the candidate sequence (e.g. , protein or fragment) consensus, the amino acid residue sequence of the reference polypeptide, the sequence and gaps introduced after adjustment, if necessary, to achieve a maximum percent sequence identity, and disregard any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved by various techniques, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for sequence alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

After testing, the kit of the present application does not cross-react with the following positive samples: Influenza A H1N1, Influenza A H3N2, Influenza A H5N1, Influenza B Yamagata, Influenza B Victoria, Respiratory Syncytial Virus Type A, Respiratory Tract Syncytial virus type B, rhinovirus A, rhinovirus B, adenovirus type 1, adenovirus type 2, adenovirus type 3, adenovirus type 4, adenovirus type 5, adenovirus type 7, parainfluenza virus, Mycobacterium tuberculosis , Haemophilus influenzae, pertussis, Epstein-Barr virus, measles virus, human cytomegalovirus, enterovirus 71, rotavirus, norovirus, mumps virus, varicella-zoster virus, mycoplasma pneumoniae, endemic Human coronavirus HKU1, OC43, NL63, 229E and MERS-Cov antigen positive samples did not cross-react. Blood, saliva, histamine hydrochloride, alpha-interferon, zanamivir, ribavirin, oseltamivir, peramivir, lopinavir, ritonavir, arbidu, levofloxacin, albino Ozithromycin, ceftriaxone, meropenem, tobramycin, etc. do not affect the test results of this product.

The present invention also discloses the application of the above-mentioned immunochromatographic non-invasive detection kit for detecting novel coronavirus antigen, and the detection kit is used for rapid detection of non-invasive mouthwash samples or samples of novel coronavirus.

Compared with the prior art, the present invention uses a non-invasive sampling method to sample, conveniently, quickly and reliably detects whether the subject is infected with the new coronavirus SARS-CoV-2, non-invasive sampling does not cause any pain, and the source of the sample is non-invasive That is to say, the possibility of secondary infection and cross-infection is eliminated, and the risk of virus transmission is reduced; the detection is fast and the result is visible, and the result can be obtained quickly within 10 minutes; the detection result can avoid the hook effect or the front zone effect, and the detection result The accuracy rate is high, the sensitivity of clinical detection can reach more than 95%, and the total coincidence rate reaches 98.32%; the operation method is simple, without any equipment or device assistance, suitable for staff without medical or other training, and the cost of using the detection kit is low , the environment and place of use are not limited, it has strong promotional significance, and is more conducive to the monitoring of high-risk groups. The invention can also prevent asymptomatic individuals from spreading the source of infection, and is an important detection means to save the consumption of medical manpower and material resources. The present invention adopts the principle of colloidal gold immunochromatography sandwich method, uses colloidal gold as a marker, and can quickly and qualitatively detect the presence of novel coronavirus antigens in mouthwash samples, and is suitable for patients with contact history but no symptoms and symptoms of new crown disease The group, mainly for the screening of samples at the early stage of symptoms (0 to 7 days) after infection.

Description of drawings

Fig. 1 is the electrophoresis diagram of the novel coronavirus protein and recombinant protein of the present application;

Fig. 2 is the electrophoresis diagram of the novel coronavirus protein antibody and recombinant protein antibody of the present application;

Fig. 3 is a schematic diagram of the operation of the kit provided by the present invention during and after sampling;

Fig. 4 is a schematic diagram of the expected detection results of the kit of the present invention.

Detailed ways

The non-invasive detection kit for detecting novel coronavirus antigen and its detection method provided by the present invention will be further described in detail and completely below in conjunction with the examples. The embodiments described below are exemplary only for explaining the present invention and should not be construed as limiting the present invention.

The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples were purchased from the market unless otherwise specified.

The kit of this example adopts the principle of colloidal gold immunochromatography sandwich method, uses colloidal gold as a marker, and qualitatively detects the SARS-CoV-2 novel coronavirus antigen in human mouthwash. When the concentration of the corresponding analyte in the sample is not less than the minimum detection limit, it will react with the colloidal gold-labeled antibody to form a complex. Under the action of chromatography, the reaction complex moves forward along the nitrocellulose membrane to the detection line (T ), react with the pre-coated antibody on the nitrocellulose membrane to form an antibody-antigen-antibody complex, and finally form a red reaction line on the detection line, and the result is positive at this time. When the sample does not contain the corresponding analyte or the concentration of the analyte is lower than the minimum detection limit, there is no red reaction line in the detection line, and it is negative at this time. Regardless of whether the sample contains the corresponding analyte, the quality control line (C) will form a red reaction line.

This kit is optimized and recombined for the RNA sequence fragments of the new coronavirus. The recombined fragments are located in the conserved region, so it can detect all the current new coronaviruses. At present, more than 3,000 mutation sites do not involve these fragments. The detection will not be affected by the mutant strain, and the detection rate is accurate; and the recombinant fragment can effectively avoid the combination of IgA and other sticky proteins in the oral cavity, so the oral samples of this kit have more specificity and more accurate detection results; Several fragments selected by the recombinant fragments have good immunogenicity, relatively high titer for immunomonoclax, and good specificity; although the amino acids of the selected fragments are small, the binding site for immunomonoclax contains the N protein site , can avoid missed detection, and the high-level structure of the protein combined with several fragments has good sensitivity to virus culture. The recombined sequence is as follows:

The amino acid sequence is shown in Sequence Table 1, and the gene sequence is shown in Sequence Table 2.

The electrophoresis results of the novel coronavirus protein and recombinant protein are shown in Figure 1, and the electrophoresis results of the novel coronavirus recombinant protein and recombinant protein antibody are shown in Figure 2. As can be seen from the figure, the size of the recombinant protein and the recombinant protein antibody is consistent with the prediction.

This kit includes a test card, mouthwash, disposable cup and dropper. The test card is composed of a test strip and a plastic box. The test strip includes a colloidal gold particle conjugate area and a nitrocellulose membrane area from the invasion area. , Absorbent pad, the detection line is coated at the end close to the colloidal gold conjugate, and the quality control line is coated at the end close to the absorbent pad. The structures in the test card are in order according to the order of the samples to be tested: sample pad, colloidal gold conjugated pad , nitrocellulose membrane and absorbent pad, wherein the nitrocellulose membrane area includes a detection line and a quality control line, wherein the nitrocellulose membrane includes a detection line coated with SARS-CoV-2 recombinant antibody and a quality control line coated with goat anti-rabbit polysaccharide Cloned antibodies, binding pads include SARS-CoV-2 recombinant antibodies, rabbit IgG antibodies.

The SARS-CoV-2 antibody coating protein content on the nitrocellulose membrane should not be less than 2.0mg/mL; the purity should be ≥ 90%, and the SARS-CoV-2 antibody-labeled protein content should not be less than 2.0mg/mL; the purity Should be ≥90%, the protein content of goat anti-rabbit IgG polyclonal antibody should not be less than 3.0mg/mL; the purity should be ≥90%, the rabbit IgG protein content should not be less than 3.0mg/mL; the purity should be ≥90%.

The mouthwash in the kit is 0.8-0.9% sodium chloride aqueous solution.

When sampling, the sampler should not eat, drink, smoke or chew gum and apply lipstick 30 minutes before the collection, and try not to take samples when there are severe oral ulcers, periodontal inflammation, respiratory tract infection or other diseases, Party B will affect the collection results. The samples to be tested, testing reagents and other testing materials need to be placed at room temperature (10-30°C), and the test should be carried out at room temperature (10-30°C).

1. Mouthwash collection process

1. Open the mouthwash;

2. With 10-15ml of mouthwash in the mouth, tilt the patient's head back 70 degrees, let the mouthwash deep in the throat (remember not to stay at the front of the mouth), and rinse the deep throat repeatedly for 15-20 seconds, making the mouthwash Flush out the virus deep in the throat;

3. After the samples are collected, the new coronavirus antigen rapid detection reagent card will be tested. Since some mouthwash samples are too viscous, the sample volume should be sufficient.

Second, the detection program (as shown in Figure 3)

1. Open along the tear opening of the aluminum foil bag, take out the test card and lay it flat;

2. Add about 100 μL (about 3 to 4 drops) of mouthwash sample dropwise into the sample hole of the test card;

3. Observe the displayed results within 15 to 20 minutes, and the displayed results after 30 minutes have no clinical significance.

3. Description of test results (as shown in Figure 4)

Positive: Two red lines, that is, one red reaction line appears on the test line (T) and the quality control line (C).

Negative: a red line, that is, only a red reaction line appears on the quality control line (C).

Invalid: When there is no red line on the quality control line (C), the test is invalid. It is recommended to use a new test card to retest at this time, especially pay attention to whether the sample volume is sufficient.

4. Kit performance test

1. Comparison of detection results of different mouthwash samples after PCR amplification

It can be seen from the comparison that the mouthwash sample has better performance in terms of sensitivity, specificity and accuracy than the saliva sample.

2. Minimum detection limit

The present invention uses mouthwash samples to determine the minimum detection concentration of SARS-CoV-2, and adds heat-inactivated SARS-CoV-2 virus (the stock concentration is 3.5×10 ⁵ TCID ₅₀ /mL) to the negative sample for dilution. Each concentration was diluted 3 times. The detection limit of this reagent is 3.5×10 ² TCID ₅₀ /mL (Table 1).

Table 1 Limit of Detection (LOD) Study Results

concentration	positive/total	positive rate
3.5×10 2 TCID 50 /mL	60/60	100%

3. Clinical sensitivity, specificity and accuracy

Through the collection and detection of 475 clinical samples. Compared with the COVID-19 coronavirus real-time fluorescent quantitative PCR kit (RT-PCR), the clinical (see Table 2) sensitivity of this reagent is 95.00% (95% CI: 90.39%-97.82%), and the specificity is 100% (95% CI: 98.84%-100.00%), the overall coincidence rate was 98.32% (95% CI: 96.71%-99.27%).

Table 2 Test results of clinical samples

Sensitivity: 95.00% (95% CI: 90.39%-97.82%)

Specificity: 100% (95% CI: 98.84%-100.00%)

Overall coincidence rate: 98.32% (95% CI: 96.71%-99.27%).

4. Hook effect

3.5×10 ⁵ TCID ₅₀ /mL SARS-CoV-2 virus was detected after heat inactivation, and there was no Hook effect in the test results of this reagent.

5. Cross reaction

Cross-reactivity with the following organisms was studied. Samples that were positive for the following microorganisms were negative when tested with this reagent.

3.5×10 ³ TCID ₅₀ /mL After heat inactivation of SARS-CoV-2 virus, the following microorganisms (see Table 3) are evaluated at the following concentrations when testing this reagent, and the test results show that they do not affect this reagent Product performance.

Table 3 Microbiological assessment

pathogen type	concentration
Respiratory syncytial virus type A	5.5×10 7 PFU/mL
Respiratory syncytial virus type B	2.8×10 6 TCID 50 /mL
Novel Influenza A H1N1 Virus (2009)	1×10 6 PFU/mL
Seasonal Influenza A (H1N1) Virus	1×10 6 PFU/mL
H3N2 Influenza A virus	1×10 6 PFU/mL
H5N1 Influenza A virus	1×10 6 PFU/mL
Yamagata Influenza B virus	1×10 5 PFU/mL
Victoria Influenza B virus	1×10 6 PFU/mL
rhinovirus	1×10 6 PFU/mL
human adenovirus type 3	5×10 7 TCID 50 /mL
human adenovirus type 7	2.8×10 6 TCID 50 /mL
Enterovirus 71	1×10 6 PFU/mL
Mycobacterium tuberculosis	1×10 5 bacteria/mL
mumps virus	1×10 6 PFU/mL
Human coronavirus 229E strain	1×10 6 PFU/mL
Human coronavirus OC43 strain	1×10 6 PFU/mL

Human coronavirus NL63 strain	1×10 6 PFU/mL
Human coronavirus HKU1 strain	1×10 6 PFU/mL
Parainfluenza virus type 1	7.3×10 6 PFU/mL
Parainfluenza virus type 2	1×10 6 PFU/mL
Parainfluenza virus type 3	5.8×10 6 PFU/mL
Parainfluenza virus type 4	2.6×10 6 PFU/mL
Haemophilus influenzae	5.2×10 6 CFU/mL
Streptococcus pyogenes	3.6×10 6 CFU/mL
Streptococcus pneumoniae	4.2×10 6 CFU/mL
Staphylococcus aureus	3.2×10 8 CFU/mL
Staphylococcus epidermidis	2.1×10 8 CFU/mL
Candida albicans	1×10 7 CFU/mL
Pertussis	1×10 4 bacteria/mL
Mycoplasma pneumoniae	1.2×10 6 CFU/mL
Chlamydia pneumoniae	2.3×10 6 IFU/ml
Legionella pneumophila	1×10 4 bacteria/mL
Mouthwash	N/A

Finally, it is necessary to explain here that: the above examples are only used to further describe the technical solutions of the present invention in detail, and cannot be interpreted as limiting the protection scope of the present invention. Non-essential improvements and adjustments all belong to the protection scope of the present invention.

Claims (10)

1. A non-invasive detection kit for detecting novel coronavirus antigens, the kit is a colloidal gold immunochromatography detection kit, characterized in that the detection kit detects whether the mouthwash sample or the sample contains the new crown antigen In order to quickly determine whether the subject is infected with the new coronavirus, the kit includes a test card and mouthwash, the test card is composed of a test strip and a plastic box, and the test strip includes a nitrocellulose membrane, a sample pad, a colloidal gold binding Pad, absorbent pad, absorbent paper and PVC board, the structures in the test card are in the following order according to the order of the samples to be tested: sample pad, colloidal gold bonded pad, nitrocellulose membrane and absorbent pad, wherein the nitrocellulose membrane includes coated The detection line of SARS-CoV-2 recombinant antibody and the quality control line coated with goat anti-rabbit polyclonal antibody, and the colloidal gold binding pad includes SARS-CoV-2 recombinant antibody and rabbit IgG antibody.
2. The non-invasive detection kit for detecting novel coronavirus antigens according to claim 1, wherein the amino acid sequence of the recombinant antibody is as shown in the sequence table SEQ ID NO.1.
3. The non-invasive detection kit for detecting novel coronavirus antigens according to claim 2, wherein the gene sequence expressing the amino acid sequence of the recombinant antibody is as shown in the sequence table SEQ ID NO.2.
4. The non-invasive detection kit for detecting novel coronavirus antigens according to claim 1, wherein the detection kit also includes a disposable cup and a dropper.
5. The non-invasive detection kit for detecting novel coronavirus antigens according to claim 1, wherein the mouthwash is 0.8-0.9% sodium chloride aqueous solution.
6. The non-invasive detection kit for detecting novel coronavirus antigens according to claim 1, wherein the test card plastic box is provided with a sampling hole, and the sample to be tested is dripped into the sample pad through the sampling hole.
7. The non-invasive detection kit for detecting novel coronavirus antigens according to claim 1, wherein the SARS-CoV-2 antibody coating protein content on the nitrocellulose membrane should not be less than 2.0mg/mL, and the purity Should be ≥90%; SARS-CoV-2 antibody labeled protein content should not be less than 2.0mg/mL, purity should be ≥90%; goat anti-rabbit IgG polyclonal antibody protein content should not be less than 3.0mg/mL, purity should be ≥ 90%; the rabbit IgG protein content should not be less than 3.0mg/mL, and the purity should be ≥90%.
8. A detection method using the non-invasive detection kit for detecting novel coronavirus antigens according to any one of claims 1 to 7, characterized in that the detection method comprises the following steps:

   a) adding the sample to be tested into the sample hole of the sample pad, so that the sample to be tested flows from the sample pad to the colloidal gold binding pad;

   b) The sample to be tested flows through the test line and the quality control line sequentially through capillary action. When the similarity of the sequence to be tested is greater than 90%, the antigen to be tested can be captured by the recombinant antibody, forming a red band;

   c) Visually observe whether there is a red band on the test line and the quality control line to determine whether the source of the sample to be tested is infected with the new coronavirus.
9. The non-invasive detection kit for detecting novel coronavirus antigens according to claim 1, wherein the detection sample is a mouthwash rinse sample without secondary treatment.
10. An application of a non-invasive detection kit for detecting novel coronavirus antigens according to any one of claims 1 to 7, wherein the detection kit is used for rapid detection of mouthwash samples of novel coronavirus or sample.

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