CN111398589A

CN111398589A - Immunochromatography kit for rapidly detecting novel coronavirus N protein and preparation method and application thereof

Info

Publication number: CN111398589A
Application number: CN202010136117.8A
Authority: CN
Inventors: 林斯
Original assignee: Beijing Huaketai Biotechnology Co ltd
Current assignee: Beijing Huaketai Biotechnology Co ltd
Priority date: 2020-02-13
Filing date: 2020-03-02
Publication date: 2020-07-10
Also published as: CN111398588A

Abstract

The invention relates to an immunochromatographic kit for rapidly detecting novel coronavirus N protein, a preparation method and application thereof. The immunochromatographic kit for rapidly detecting the novel coronavirus N protein provided by the invention can be used for detecting the novel coronavirus N protein in a nasopharynx swab, an oropharynx swab, an alveolar lavage fluid, an oral swab or a saliva sample so as to diagnose the novel coronavirus pneumonia.

Description

Immunochromatography kit for rapidly detecting novel coronavirus N protein and preparation method and application thereof

Technical Field

The invention belongs to the field of in-vitro diagnosis, and particularly relates to an immunochromatography kit for rapidly detecting novel coronavirus N protein, and a preparation method and application thereof.

Background

2019 the novel coronavirus (SARS-CoV-2) is a new strain of coronavirus which has never been found in human body before, and after people are infected with coronavirus, the common signs of people include respiratory symptoms, fever, cough, shortness of breath, dyspnea and the like. In more severe cases, the infection can lead to pneumonia, severe acute respiratory syndrome, renal failure, and even death. 1 month and 26 days, the national drug administration (HSA) carries out emergency approval to pass 4 new coronavirus detection products of 4 enterprises such as Huada gene and the like, but the currently approved novel coronavirus nucleic acid detection reagent is based on a nucleic acid detection mode, a single sample needs 3 hours to give a detection result by adopting a nucleic acid fluorescence Polymerase Chain Reaction (PCR) method, and a nucleic acid sequencing method needs 6 hours to give a detection result, more importantly, the detection flux is called as a maximum restriction factor due to extremely high requirements of gene amplification on laboratory environment, the supply capacity of the novel coronavirus rapid detection service is greatly restricted, and the requirement of epidemic situation prevention and control cannot be fully met.

Disclosure of Invention

In view of the above, the main objective of the present invention is to provide an immunochromatographic kit for rapidly detecting a novel coronavirus N protein, and a preparation method and an application thereof.

In order to achieve the purpose, the invention provides the following technical scheme:

an immunochromatographic kit for rapidly detecting novel coronavirus N protein comprises a test strip, wherein the test strip comprises a detection line and a quality control line, the detection line is coated with a monoclonal antibody for resisting the novel coronavirus N protein, and the quality control line is coated with a polyclonal antibody for resisting sheep and rabbit.

In a specific scheme of the invention, the test strip further comprises a PVC plate, a sample pad, a marker pad, a coating pad and a water absorption pad which are sequentially connected are fixed on the PVC plate, a detection line and a quality control line are sequentially arranged on the coating pad, and the sample pad and the marker pad are connected into a whole.

In one embodiment of the invention, one end of the coating pad close to the detection line is connected with a marker pad, and one end close to the quality control line is connected with a water absorption pad.

In a specific embodiment of the invention, the marker pad is coated with another monoclonal antibody against the novel coronavirus N protein and rabbit IgG marked by the marker, and the molar ratio of the monoclonal antibody against the novel coronavirus N protein to the rabbit IgG marked by the marker is 1: 0.2-4.

In a specific embodiment of the present invention, wherein the monoclonal antibody against novel coronavirus N protein is a mouse monoclonal antibody against novel coronavirus N protein or a sheep monoclonal antibody against novel coronavirus N protein, preferably a mouse monoclonal antibody against novel coronavirus N protein.

In one embodiment of the present invention, the label is a fluorescent microsphere, colloidal gold, colloidal selenium, colored latex, or magnetic microsphere.

In a specific embodiment of the present invention, the immunochromatographic kit for rapid detection of a novel coronavirus N protein further comprises a cartridge for mounting a test strip.

In one embodiment of the present invention, wherein the card case comprises:

a bottom trough connected to the PVC sheet;

the upper cover is connected with the bottom groove, and a sample adding hole for adding samples to the sample pad is arranged on the upper cover;

and the observation window is arranged on the upper cover and is used for data acquisition of the detection line and the quality control line.

A preparation method of an immunochromatography kit for rapidly detecting novel coronavirus N protein comprises the following steps:

1) preparation of the coating pad: respectively coating a strain of anti-novel coronavirus N protein antibody and a goat anti-rabbit polyclonal antibody on a nitrocellulose membrane, and drying for later use;

2) preparation of the marker pad: mixing a monoclonal antibody of the novel coronavirus N protein marked by the marker and rabbit IgG marked by the marker, spraying the mixture on a glass cellulose membrane, and drying the mixture for later use;

3) assembling the test strip: bonding a coating pad on a PVC plate, overlapping a water absorption pad at one end close to a quality control line on the coating pad, and overlapping a marker pad and a sample pad connected with the marker pad at one end close to a detection line on the coating pad; then cut into test strips with required width, and then put the test strips into the card shell.

In a specific embodiment of the invention, the molar ratio of the other strain of the mouse anti-coronavirus N protein monoclonal antibody marked by the marker to the rabbit IgG marked by the marker is 1: 0.5-4.

An immunochromatography kit for rapidly detecting a novel coronavirus N protein is used, and a sample used in the method is selected from a nasopharynx swab, an oropharynx swab, an alveolar lavage fluid, an oral swab or saliva.

The nasopharyngeal swab can also be called as a nasal swab, and the nasal swab is used for sampling in the nasal cavity; the oropharyngeal swab can also be called as a pharyngeal swab, and the pharyngeal swab is used for sampling the throat; oropharyngeal swabs were sampled intraorally.

In a specific embodiment of the present invention, wherein the using method comprises the following steps:

the method comprises the following steps:

and (3) pretreating the sample by using a sample preservation solution, dripping the sample to be detected which is treated by 30-100 mu L to the sample adding hole of the kit, standing for 15min, and then inserting into a fluorescence immunoassay analyzer for detection or observing under ultraviolet light to obtain a detection result immediately.

and (3) pretreating the sample by using a sample preservation solution, dripping the sample to be detected which is treated by 60 mu L to a sample adding hole of the kit, standing for 5-20 min, and then inserting into a fluorescence immunoassay analyzer for detection or observing under ultraviolet light to obtain a detection result immediately.

In a specific scheme of the invention, the sample pretreatment mode comprises the steps of adding 0.5m L sample preservation solution into a plastic hose, immersing a cotton swab after sample collection in the sample preservation solution and stirring, extruding the outer side of the plastic hose for a plurality of times by using fingers to enable the sample preservation solution to fully soak the cotton swab, then pulling out the cotton swab, and obtaining the twisted liquid as the sample to be detected.

The kit may also be called a test card.

The invention has the beneficial effects that:

1. the immunochromatographic kit for rapidly detecting the novel coronavirus N protein provided by the invention can detect the novel coronavirus nucleocapsid (N) protein by adopting a double-antibody sandwich method, can detect samples such as nasopharyngeal swabs, oropharyngeal swabs, alveolar lavage fluid, oral swabs or saliva, has high detection speed, can obtain a detection result in 15min, is matched with a fluorescence immunochromatographic instrument for use, is simple to operate, and is suitable for basic medical institutions.

2. The immunochromatographic kit for rapidly detecting the novel coronavirus N protein, provided by the invention, can improve the professional requirements of PCR on instrument operation, personnel, environment and the like, and the defect that a detection antibody cannot be screened in an early stage, can accelerate the screening of first-line suspected cases of epidemic situations, rapidly isolate confirmed persons, and effectively reduce social panic.

3. The immunochromatography kit for rapidly detecting the novel coronavirus N protein provided by the invention can be used for detecting the coronavirus nucleocapsid (N) protein in a nasopharynx swab, an oropharynx swab, an alveolar lavage fluid specimen, an oral swab or a saliva sample so as to be used for diagnosing the novel coronavirus pneumonia.

Drawings

FIG. 1 is a schematic view of an immunochromatographic test strip for rapidly detecting a novel coronavirus N protein provided by the present invention;

FIG. 2A is a schematic diagram of the internal structure of an upper cover of the immunochromatographic assay card for rapid detection of N protein of a novel coronavirus according to the present invention;

FIG. 2B is a schematic diagram of the internal structure of a bottom groove of the immunochromatography detection card for rapidly detecting the N protein of the novel coronavirus provided by the present invention;

the test strip detection device comprises a PVC plate 1, a coating pad 2, a marker pad 3, a water absorption pad 4, a detection line 5, a quality control line 6, a marker joint 7, a sample 8, a sample pad 9, a top cover 11, a bottom groove 12, a sample adding hole 13, an observation window 14, a test strip placement area 15, a positioning column 16, a positioning hole 17, a first limiting part 18, a second limiting part 19 and a third limiting part 20.

Detailed Description

The invention is further described below with reference to the accompanying drawings.

The following materials or reagents, unless otherwise specified, are commercially available.

Example 1:

1. preparation of novel coronavirus N protein immunochromatography detection kit

1) Fluorescent microsphere labeling another strain of mouse anti-novel coronavirus N protein monoclonal antibody

Adding 0.5M L fluorescent microspheres into 1mg carbodiimide (EDC) and 1mg N-hydroxysuccinimide (NHS), stirring at room temperature and 120r/min for 3h, adding 100 mu L of another mouse anti-novel coronavirus N protein monoclonal antibody, stirring at room temperature and 120r/min for 1h, adding 10mg BSA confining liquid, stirring at 120r/min for 1h, centrifuging at 2-8 ℃ and 12000 r/min for 20min, removing supernatant, re-dissolving the solid precipitate obtained after centrifugation into 1M L by using 0.2M phosphate buffer (pH 7.4), adding 1 mu L Proclin300, and storing at 4 ℃ for later use.

2) Labeling rabbit IgG polyclonal antibody by fluorescent microsphere

Adding 1mg of carbodiimide (EDC) and 1mg of N-hydroxysuccinimide (NHS) into 0.5M L fluorescent microspheres, stirring at room temperature and 120r/min for 3h, then adding 100 mu L mg of goat rabbit IgG polyclonal antibody, stirring at room temperature and 120r/min for 1h, then adding 10mg of BSA confining liquid, continuing stirring at 120r/min for 1h, centrifuging at 2-8 ℃ and 11000r/min for 30 min, removing supernatant, finally re-dissolving the solid precipitate to 1M L by using 0.2M phosphate buffer (pH 7.4), then adding 1 mu L Proclin300, and storing at 4 ℃ for later use.

3) Preparation of coating pad

A mouse anti-novel coronavirus N protein monoclonal antibody and a goat anti-rabbit polyclonal antibody are respectively diluted to 1mg/M L by 0.2M phosphate buffer solution (pH 7.4), membrane scribing is carried out on a nitrocellulose membrane (NC membrane) by a membrane scribing and gold spraying instrument, the mouse anti-novel coronavirus N protein monoclonal antibody is used as a detection line (T line) 5, the goat anti-rabbit polyclonal antibody is used as a quality control line (C line) 6, and then the coating pad 2 is prepared by drying for 4 hours at 37 ℃ and under the environment of the humidity of less than 30%.

4) Preparation of marker pad

① the glass cellulose membrane was soaked in 0.2M phosphate buffer (pH 7.4) for 6h and then dried at 35 ℃ for 8 h.

② A mouse anti-novel coronavirus N protein monoclonal antibody marked by fluorescent microspheres with a molar ratio of 1:1 and fluorescent microspheres marked by fluorescent microsphere rabbit IgG are uniformly mixed, sprayed on a glass cellulose membrane at a speed of 10 mu L/cm, and then placed at 45 ℃ for drying for 2 hours to prepare a marker pad 3, and a place, which is coated with the mouse anti-novel coronavirus N protein monoclonal antibody marked by the fluorescent microspheres and the fluorescent microsphere rabbit IgG, on the marker pad 3 is called a marker junction 7.

5) Assembly of immunochromatography detection kit

Firstly, a nitrocellulose membrane 2 is bonded on a PVC plate 1, then a water absorption pad 4 is lapped at one end close to a quality control line 6 on the nitrocellulose membrane 2, a marker pad 3 and a sample pad 9 connected with the marker pad are lapped at one end close to a detection line 5 of the nitrocellulose membrane 2, a strip cutter is used for cutting the test strip (shown in figure 1) with the thickness of 4mm +/-0.1 mm, and the test strip is put into a card shell to prepare the novel coronavirus N protein immunochromatography detection kit.

The kit may also be called a test card.

The card housing is selected from the prior art, for example, the card housing (as shown in fig. 2) may include: a bottom tank 12 connected to the PVC plate 1; an upper cover 11 connected to the bottom tank 12, the upper cover 11 being provided with a sample application hole 13 for applying a sample to the sample pad 9; and the observation window 14 is arranged on the upper cover 11 and is used for data acquisition of the detection line 5 and the quality control line 6.

As shown in fig. 2B, the bottom tank 12 includes: a plurality of positioning holes 17 which are symmetrically distributed and positioned on the inner surface of the test strip, wherein a plurality of first limiting parts 18 used for limiting the test strip to move transversely and second limiting parts 19 used for limiting the test strip to move longitudinally are arranged among the plurality of positioning holes 17; the first limiting part 18 and the second limiting part 19 which are symmetrically arranged enclose a paper strip placing area 15 (a dotted line area) for placing the test paper strip;

as shown in fig. 2A, the upper cover 11 includes: a plurality of positioning posts 16 which cooperate with a plurality of said positioning holes 17, so as to cooperate to fix the upper cover 11 and the bottom slot 12 together; the upper cover 11 further includes a third limiting portion 20 for limiting the up and down movement of the test strip.

An observation window 14 for data acquisition is arranged above the coated pad 2 to expose all the detection lines 5 and the quality control lines 6 for collecting detection results; and the observation window 14 is arranged on the upper cover 11 at a position corresponding to the middle part of the test strip placement area 15. The upper cover 11 is provided with a sample adding hole at a position corresponding to the sample pad 9, so that the sample 8 is dripped on the sample pad 9. The distance between the detection line and the sample adding hole is 15-25 mm.

2. Detection of

Taking a sample to be detected with the thickness of 60 mu L, filling a standard substance or the sample to be detected on a marker pad through a sample adding hole, standing for 15min at room temperature, enabling an antigen to be mixed with the marker for reaction, carrying out chromatography along a nitrocellulose membrane, respectively reacting with a test line and a quality control line, inserting the sample into a fluorescence immunochromatography analyzer for detection, automatically calculating a T/C value of the sample by the analyzer, and judging whether the sample is positive or negative through the range of the sample and the normal value.

The ultraviolet light may also be called ultraviolet light, and an ultraviolet lamp may be specifically used.

3. Evaluation of Performance

1) Precision degree

Two samples with the configuration concentration of (300 +/-75) ng/M L and (100 +/-25) ng/M L are respectively detected by using kits of 3 different batches, the detection is repeated for 10 times for each batch, the average value M and the standard deviation SD of 30 times of concentration value measurement results are calculated, and the detection results of the calculated variation coefficient CV. are shown in the following table 1.

TABLE 1

As can be seen from the data in Table 1, the batch-to-batch variation coefficients are all less than 10.92%, which indicates that the immunochromatography kit for rapidly detecting the novel coronavirus N protein has high precision.

2) Sensitivity of the probe

The polypeptide fragments synthesized by the N protein of the novel coronavirus are configured, the concentrations are 50ng/m L, 100ng/m L and 200ng/m L, and the detection results are shown in the following table 2.

TABLE 2

As can be seen from the detection results in Table 2, the immunochromatographic kit for rapidly detecting the N protein of the novel coronavirus meets the requirement of sensitivity.

Example 2:

nasal/oropharyngeal swab sample detection

The types of samples taken include nasopharyngeal swabs, oropharyngeal swabs.

The method comprises the steps of adding 0.5m L sample preserving fluid into a plastic hose, immersing a cotton stick after the sample is collected in the sample preserving fluid, stirring, extruding the outer side of the plastic hose for a plurality of times by fingers to enable the sample preserving fluid to fully soak the cotton stick, then pulling out the cotton stick, twisting out (namely mixing the sample collected on the cotton stick into the sample preserving fluid) to obtain a sample to be detected, then dropwise adding the sample to be detected after 60 mu L treatment to a sample adding hole of a detection card, standing for 15min, then inserting a fluorescence immunochromatography analyzer for detection, automatically calculating a T/C value of the sample by the analyzer, judging whether the sample is positive or negative according to a normal value range, and when two fluorescence strips are shown under ultraviolet irradiation, obtaining the clinical detection results as shown in the following table 4.

TABLE 3 composition of sample preservation solution

TABLE 4

As can be seen from the clinical test data in table 4, all of the 30 negative samples were detected as negative and consistent with the clinical diagnosis; 8 of 9 positive oropharyngeal swabs detected as positive, 19 of 21 positive oropharyngeal swabs detected as positive, and the total positive coincidence rate was 90%.

Example 3:

oral swab/saliva test

The sample types taken include buccal swabs, saliva.

After the oral swab/saliva sample is collected, the sample is pretreated by a sample preservation solution (the composition is shown in table 5), the treatment mode is that 0.5m L sample preservation solution is added into a plastic hose, a cotton stick after the sample is collected is soaked in the sample preservation solution and stirred, the outer side of the plastic hose is squeezed by fingers for a plurality of times, the cotton stick is fully soaked by the sample preservation solution, then the cotton stick is pulled out, liquid which is obtained by twisting out (namely, the sample collected on the cotton stick is mixed into the sample preservation solution) is a sample to be detected, then the sample to be detected which is well treated by 60 mu L is dripped to a sample adding hole of a detection card, the detection card is placed for 15min in a standing mode, a fluorescence immunochromatography analyzer is inserted for detection, the T/C value of the sample is automatically calculated by the analyzer, the negative and the positive are judged by the range of the normal value, in addition, when two fluorescence strips are shown under the ultraviolet irradiation, the positive.

TABLE 5 sample preservation solution (P.B buffer)

TABLE 6

As can be seen from the clinical test data in table 6, the oral swab sample test results show that 4 positive samples in 8 positive samples are positive, the positive detection rate is 50%, and all negative samples in 12 negative samples are negative, which is consistent with clinical diagnosis; the saliva sample detection result shows that 9 positive samples and 4 positive samples are positive, the positive detection rate is 44.4 percent, and all 11 negative samples are negative, which is consistent with the clinical diagnosis result.

The above-mentioned embodiments are merely examples provided to fully illustrate the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.

Claims

1. The immunochromatographic kit for rapidly detecting the novel coronavirus N protein is characterized by comprising a test strip, wherein the test strip comprises a detection line and a quality control line, the detection line is coated with a monoclonal antibody for resisting the novel coronavirus N protein, and the quality control line is coated with a polyclonal antibody for resisting goat rabbit.

2. The immunochromatographic kit for rapidly detecting a novel coronavirus N-protein according to claim 1, wherein the test strip further comprises a PVC plate, a sample pad, a marker pad, a coating pad and a water absorption pad which are sequentially connected are fixed on the PVC plate, the coating pad is sequentially provided with a detection line and a quality control line, and the sample pad and the marker pad are integrally connected.

3. The immunochromatographic kit for rapidly detecting a novel coronavirus N-protein according to claim 2, wherein the end of the coating pad near the detection line is connected with a marker pad, and the end near the quality control line is connected with a water-absorbing pad.

4. The immunochromatographic kit for rapidly detecting a novel coronavirus N protein according to claim 3, wherein the marker pad is coated with another monoclonal antibody against the novel coronavirus N protein and rabbit IgG labeled with a marker, and the molar ratio of the another monoclonal antibody against the novel coronavirus N protein to the rabbit IgG labeled with the marker is 1: 0.2-4.

5. The immunochromatographic kit for rapidly detecting a novel coronavirus N-protein according to claim 4, wherein the label is a fluorescent microsphere, colloidal gold, colloidal selenium, colored latex, or a magnetic microsphere.

6. The immunochromatographic kit for rapidly detecting a novel coronavirus N-protein of claim 5, which further comprises a card shell for holding a test strip.

7. The immunochromatographic kit for rapidly detecting a novel coronavirus N-protein according to claim 6, wherein the cartridge comprises:

a bottom trough connected to the PVC sheet;

the upper cover is connected with the bottom groove, and a sample adding hole for adding samples to the sample pad is formed in the upper cover;

8. A method for preparing an immunochromatographic kit for rapid detection of a novel coronavirus N protein according to any one of claims 1 to 7, comprising the steps of:

9. A method for using the immunochromatographic kit for rapid detection of a novel coronavirus N-protein according to any one of claims 1 to 7, wherein the sample used in the method is selected from a nasopharyngeal swab, an oropharyngeal swab, an alveolar lavage fluid, an oral swab or saliva.

10. Use according to claim 9, characterized in that it comprises the following steps:

and (3) pretreating the sample by using a sample preservation solution, dripping the sample to be detected which is treated by 30-100 mu L to the sample adding hole of the kit, standing for 15min, and then inserting into a fluorescence immunoassay analyzer for detection, or observing under ultraviolet light, so that a detection result can be obtained immediately.

11. Use according to claim 10,

and (3) pretreating the sample by using a sample preservation solution, dripping the sample to be detected which is treated by 60 mu L to a sample adding hole of the kit, standing for 5-20 min, and then inserting into a fluorescence immunoassay analyzer for detection, or observing under ultraviolet light, so that a detection result can be obtained immediately.

12. The use method of claim 11, wherein the sample is pretreated by adding 0.5m L sample preserving solution into a plastic hose, immersing the cotton swab in the sample preserving solution and stirring, squeezing the outer side of the plastic hose with fingers several times to make the sample preserving solution fully soak the cotton swab, and then pulling out the cotton swab, wherein the stranded liquid is the sample to be tested.