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CN111398593A - Rapid combined detection card and preparation method and application thereof - Google Patents

Rapid combined detection card and preparation method and application thereof Download PDF

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Publication number
CN111398593A
CN111398593A CN202010260203.XA CN202010260203A CN111398593A CN 111398593 A CN111398593 A CN 111398593A CN 202010260203 A CN202010260203 A CN 202010260203A CN 111398593 A CN111398593 A CN 111398593A
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detection
pad
influenza
virus
marker
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林斯
肖燚
胡琛光
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Tianjin Huaketai Biotechnology Co ltd
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Tianjin Huaketai Biotechnology Co ltd
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • G—PHYSICS
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
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    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
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Abstract

The invention relates to a rapid joint detection card and a preparation method and application thereof, wherein the rapid joint detection card comprises a new corona/first flow/second flow detection test strip and a card shell for detecting a sample; the card shell comprises a bottom groove and an upper cover which are mutually clamped, and the new crown/A flow/B flow detection test strip is arranged on the bottom groove of the card shell. The respiratory virus joint detection card provided by the invention can simultaneously detect novel coronavirus N protein, influenza A virus and influenza B virus with joint detection value, so as to screen suspected cases at early stage and rapidly classify diseases of patients, thereby having very important significance for rapid isolation and targeted treatment.

Description

Rapid combined detection card and preparation method and application thereof
Technical Field
The invention belongs to the field of in-vitro diagnosis, and relates to a rapid combined detection card capable of simultaneously detecting novel coronavirus N protein, influenza A virus and influenza B virus, and a preparation method and application thereof.
Background
2019 Novel Coronavirus (NCP), namely "COVID-19", the national Committee for viral Classification of coronavirus research group (CSG) suggests that 2019-nCoV is named "Severe acute respiratory syndrome coronavirus 2", namely "SARS-CoV-2", on the basis of systematic analysis of the relevant coronavirus.
Since the outbreak of pneumonia epidemic situation of new coronavirus infection, a large amount of PCR nucleic acid and IgM/IgG detection reagents appear in the market, and the detection aiming at antigens is almost not available. The single sample can give a detection result in3 hours by adopting a nucleic acid fluorescence PCR method, and can give a detection result in 6 hours by adopting a nucleic acid sequencing method, and more importantly, the detection flux becomes the maximum restriction factor due to the extremely high requirement of gene amplification on the laboratory environment, so that the supply capacity of the novel coronavirus rapid detection service is greatly restricted, and the prevention and control requirements of epidemic situations cannot be fully met. IgM/IgG appears in humans relatively later than the antigen. And most of the current studies are mainly single studies aiming at the detection of pneumonia caused by new coronavirus. And the symptoms of patients caused by other common respiratory viruses such as influenza A virus, influenza B virus and the like are similar to those of patients with new coronavirus pneumonia, so that a plurality of respiratory virus detection reagents are combined to screen suspected cases in the early stage, the diseases of the patients are rapidly classified, and the method has very important significance for rapid isolation and targeted treatment.
Disclosure of Invention
In view of the above, the main objective of the present invention is to provide a rapid combined detection card capable of simultaneously detecting a novel coronavirus N protein, an influenza a virus and an influenza b virus, and a preparation method and an application thereof.
In order to achieve the purpose, the invention provides the following technical scheme:
a rapid combined detection card comprises a new corona/A flow/B flow detection test strip and a card shell for detecting a sample; the card shell comprises a bottom groove and an upper cover which are mutually clamped, and the new crown/A flow/B flow detection test strip is arranged on the bottom groove of the card shell.
In a specific embodiment of the invention, the new corona/first flow/second flow detection test strip comprises a bottom plate, wherein a sample pad, a marker pad, a coating pad and a water absorption pad which are sequentially connected are fixed on the bottom plate; one end of the coating pad, which is close to the detection line, is connected with a marker pad, and one end of the coating pad, which is close to the quality control line, is connected with a water absorption pad; the coating pad is sequentially provided with a first detection line, a second detection line, a third detection line and a quality control line, and the sample pad and the marker pad are connected into a whole.
In a specific embodiment of the present invention, the upper cover is provided with sample adding holes at positions corresponding to the new corona/first flow/second flow detection test strip sample pad, respectively.
In a specific aspect of the present invention, the upper cover is provided with observation windows for data acquisition of the detection line and the quality control line at positions corresponding to the first detection line, the second detection line, the third detection line and the quality control line of the new crown/first flow/second flow detection test strip, respectively.
In a specific embodiment of the present invention, in the new corona/influenza a/b test strip, the first test line, the second test line or the third test line is coated with one of a monoclonal antibody against a novel coronavirus N protein, a monoclonal antibody against influenza a virus and a monoclonal antibody against influenza b virus respectively, and the substances coated on the three test lines are different from each other; the quality control line is coated with a goat anti-rabbit polyclonal antibody.
In a specific embodiment of the invention, the marker pad is coated with another monoclonal antibody against novel coronavirus N protein marked with a marker, another monoclonal antibody against influenza A virus marked with a marker, another monoclonal antibody against influenza B virus marked with a marker, and rabbit IgG marked with a marker.
In a specific embodiment of the invention, the molar ratio of the marker-labeled another anti-novel coronavirus N protein monoclonal antibody, the marker-labeled another anti-influenza A virus monoclonal antibody, the marker-labeled another anti-influenza B virus monoclonal antibody and the marker-labeled rabbit IgG is 1:1:1: (0.1-4).
In one embodiment of the present invention, the label is selected from fluorescent microspheres, colloidal gold, colloidal selenium, colored latex or magnetic microspheres.
In a particular embodiment of the invention, the sample is selected from a nasal swab, a pharyngeal swab, a cavity swab, sputum, saliva, alveolar lavage or blood.
A preparation method of a rapid joint detection card comprises the following steps:
1) uniformly mixing another anti-novel coronavirus N protein monoclonal antibody marked by a marker, another anti-influenza A virus monoclonal antibody marked by the marker, another anti-influenza B virus monoclonal antibody marked by the marker and rabbit IgG marked by the marker, and then coating the obtained mixture on a glass cellulose membrane to prepare a marker pad;
2) respectively and optionally coating a monoclonal antibody against novel coronavirus N protein, a monoclonal antibody against influenza A virus and a monoclonal antibody against influenza B virus on a first detection line, a second detection line and a third detection line, wherein the substances coated on the three detection lines are different, and coating a polyclonal antibody against goat rabbit on a quality control line to prepare a coating pad;
3) the coated pad is adhered to the bottom plate, the water absorbing pad is lapped at one end close to the quality control line of the coated pad, the marker pad and the sample pad connected with the marker pad are lapped at one end close to the detection line of the coated pad, the test strip is cut into test strips by a slitter, and the test strips are put into a card shell to prepare the new crown/A flow/B flow rapid combined detection card.
An application method of a rapid joint detection card comprises the following steps:
treating the collected sample with a sample preservation solution to obtain a sample to be detected, adding 30-200 mu L sample to be detected to a sample adding hole of the new crown/first flow/second flow rapid combined detection card, standing for 5-30 min,
inserting the new corona/A flow/B flow rapid combined detection card into a fluorescence immunochromatographic analyzer for detection, automatically calculating T/C values corresponding to detection lines and quality control lines by the analyzer, and comparing the respective T/C values with the corresponding normal value ranges of the novel coronavirus N protein, the influenza A virus and the influenza B virus to judge whether the corona/A flow/B flow rapid combined detection card is negative or positive;
or directly observing with naked eyes or observing with naked eyes under ultraviolet light, and under the premise that a colored strip or a fluorescent strip appears on the quality control line, when the colored strip or the fluorescent strip appears on any detection line, the detection substance corresponding to the detection line is positive.
The invention has the beneficial effects that:
1. the rapid joint detection card provided by the invention can simultaneously detect novel coronavirus N protein, influenza A virus and influenza B virus with joint detection value, so as to screen suspected cases at early stage and rapidly classify diseases of patients, thereby having very important significance for rapid isolation and targeted treatment.
2. The rapid combined detection card provided by the invention can be used for detecting nasopharynx swabs, oropharynx swabs, alveolar lavage fluid, oral swabs, saliva and other samples, is high in detection speed, can obtain a detection result in 15min, is matched with a fluorescence immunochromatography instrument for use, supports naked eye interpretation or naked eye interpretation under ultraviolet light, is simple to operate, and is suitable for basic medical institutions.
3. The rapid combined detection card 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, and can accelerate the first-line suspected case screening of epidemic situations, thereby rapidly isolating confirmed diagnosis personnel and effectively reducing social panic.
Drawings
FIG. 1 is a schematic structural diagram of the novel test strip for corona/influenza A/influenza B test provided by the present invention;
FIG. 2A is a schematic diagram of an internal structure of an upper cover of a card housing of the rapid joint detection card according to the present invention;
FIG. 2B is a schematic diagram of an internal structure of a bottom slot of a card housing of the rapid joint detection card according to the present invention;
the detection kit comprises a coating pad, a marker pad, a water absorption pad, a sample pad, a bottom groove, a sample placing area, a first limiting part, a second limiting part, a sample placing area, a sample.
Detailed Description
For a further understanding of the invention, reference will now be made to the preferred embodiments of the invention by way of example, and it is to be understood that the description is intended to further illustrate features and advantages of the invention, and not to limit the scope of the claims.
The following materials or reagents, unless otherwise specified, are all commercially available.
Example 1 preparation of a New corona/first flow/second flow test strip
1. Labeling another strain of mouse anti-coronavirus N protein monoclonal antibody by using colloidal gold
Taking 0.5m L colloidal gold solution, adjusting pH to 8.5 with 0.2 mol/L potassium carbonate, standing for 10min, taking 100 mu L of another mouse anti-coronavirus N protein monoclonal antibody, dropwise adding the other mouse anti-coronavirus N protein monoclonal antibody into the colloidal gold solution within 1min under the magnetic stirring of 120r/min, continuously stirring for 30min, adding 0.5m L BSA, uniformly mixing, standing for 30min, centrifuging at the rotating speed of 12000r/min for 10min, removing supernatant, finally, redissolving the solid precipitate obtained after centrifugation to 1m L, adding 1 mu L Proclin300, and preserving at 4 ℃ for later use.
2. Labeling another strain of mouse anti-influenza A virus monoclonal antibody by using colloidal gold
Taking 0.5m L colloidal gold solution, adjusting pH to 8.5 with 0.2 mol/L potassium carbonate, standing for 10min, taking 100 mu L of another mouse anti-influenza A virus monoclonal antibody, dropwise adding the other mouse anti-influenza A virus monoclonal antibody into the colloidal gold solution for 1min under the magnetic stirring of 120r/min, continuously stirring for 30min, adding 0.5m L BSA, uniformly mixing, standing for 30min, centrifuging at the rotating speed of 12000r/min for 10min, removing supernatant, finally, redissolving the solid precipitate obtained after centrifugation to 1m L, adding 1 mu L Proclin300, and preserving at 4 ℃ for later use.
3. Labeling another strain of mouse anti-influenza B virus monoclonal antibody by using colloidal gold
Taking 0.5m L colloidal gold solution, adjusting pH to 8.5 with 0.2 mol/L potassium carbonate, standing for 10min, taking 100 mu L of another mouse anti-influenza B virus monoclonal antibody, dropwise adding the other mouse anti-influenza B virus monoclonal antibody into the colloidal gold solution for 1min under the magnetic stirring of 120r/min, continuously stirring for 30min, adding 0.5m L BSA, uniformly mixing, standing for 30min, centrifuging at the rotating speed of 12000r/min for 10min, removing supernatant, finally, redissolving the solid precipitate obtained after centrifugation to 1m L, adding 1 mu L Proclin300, and storing at 4 ℃ for later use.
4. Goat anti-rabbit IgG polyclonal antibody marked by colloidal gold
Taking 0.5m L colloidal gold solution, adjusting pH to 8.5 with 0.2 mol/L potassium carbonate, standing for 10min, taking 100 mu L goat anti-rabbit IgG polyclonal antibody, dropwise adding the goat anti-rabbit IgG polyclonal antibody into the colloidal gold solution for 1min under the magnetic stirring of 120r/min, continuously stirring for 30min, adding 0.5m L BSA, uniformly mixing, standing for 30min, centrifuging at the rotating speed of 12000r/min for 10min, removing supernatant, finally, redissolving the solid precipitate obtained after centrifugation to 1m L, adding 1 mu L Proclin300, and storing at 4 ℃ for later use.
5. Preparation of coating pad
A mouse anti-novel coronavirus N protein monoclonal antibody, an influenza A virus monoclonal monomer, an influenza B virus 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), and membrane scribing is carried out on a nitrocellulose membrane (NC membrane) by a membrane scribing and gold spraying instrument, wherein the mouse anti-coronavirus N protein monoclonal antibody is used as a first detection line (T1 line) 4, the goat anti-rabbit polyclonal antibody is used as a second detection line (T2 line) 5, the goat anti-rabbit polyclonal antibody is used as a third detection line (T3 line) 6, and the goat anti-rabbit polyclonal antibody is used as a quality control line (C line) 7, and then the coating pad 1 is prepared by drying for 4 hours at 37 ℃ and under the humidity of less than 30%.
6. 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 Another strain of mouse anti-coronavirus N protein monoclonal antibody marked by colloidal gold, another strain of influenza A virus monoclonal monomer marked by colloidal gold, another strain of influenza B virus monoclonal monomer marked by colloidal gold and rabbit IgG marked by colloidal gold are uniformly mixed, then the mixture is sprayed on a glass cellulose membrane according to the speed of 10 mu L/cm, and then the glass cellulose membrane is placed at 45 ℃ for drying for 2h to prepare a marker pad 2, and the marker pad 2 is coated with another strain of mouse anti-coronavirus N protein monoclonal antibody marked by colloidal gold, another strain of influenza A virus monoclonal monomer marked by colloidal gold, another strain of influenza B virus monoclonal monomer marked by colloidal gold and the place of the rabbit IgG marked by colloidal gold is called a marker combining part 8.
The purpose of the marker binding site 8 is to:
when a sample to be detected contains the novel coronavirus N protein, the sample to be detected is dripped on a sample pad 9, the sample to be detected is subjected to forward chromatography under the action of a water absorption pad 3, the novel coronavirus N protein in the sample to be detected at a marker binding part 8 reacts with another strain of mouse anti-novel coronavirus N protein monoclonal antibody coated at the marker binding part, a reacted substance is marked as a first immune complex, then the reacted first immune complex and the colloidal gold-labeled rabbit IgG are subjected to chromatography along with the sample, the reacted first immune complex at a first detection line reacts with a strain of mouse anti-novel coronavirus N protein monoclonal antibody coated on the detection line and stays at the first detection line, the rabbit IgG labeled by fluorescent microspheres at the quality control line reacts with a goat anti-rabbit polyclonal antibody coated on the quality control line and stays at the quality control line, other substances are subjected to chromatography continuously, finally, directly carrying out result interpretation by naked eyes, wherein on the premise that a quality control line displays a color band, when a first detection line displays the color band, the result of the detection of the novel coronavirus N protein in the sample is positive, and when the first detection line does not display the color band, the result of the detection of the novel coronavirus N protein is negative;
when the sample to be detected contains influenza A virus, the sample to be detected is dripped on a sample pad 9, the sample to be detected is subjected to forward chromatography under the action of a water absorption pad 3, the influenza A virus in the sample to be detected at a marker combination part 8 reacts with another strain of influenza A virus monoclonal antibody marked by colloidal gold coated at the marker combination part, the reacted substance is marked as a second immune complex, then the reacted second immune complex and the colloidal gold-marked rabbit IgG are subjected to continuous chromatography along with the sample, the reacted second immune complex at a second detection line reacts with a strain of mouse anti-influenza A virus monoclonal antibody coated on the detection line and stays at the second detection line, the rabbit IgG marked by fluorescent microspheres at a quality control line reacts with a goat anti-rabbit polyclonal antibody coated on the quality control line and stays at the quality control line, other substances are subjected to continuous chromatography, and finally, the result is directly read by naked eyes, on the premise that the quality control line displays a color band, when the second detection line displays the color band, the detection result of the influenza A virus in the sample is positive, and when the second detection line does not display the color band, the detection result of the influenza A virus is negative;
when the sample to be detected contains influenza B virus, the sample to be detected is dripped on a sample pad 9, under the action of a water absorption pad 3, the sample to be detected is subjected to forward chromatography, the influenza B virus in the sample to be detected at a marker combination part 8 reacts with another strain of influenza B virus monoclonal antibody marked by colloidal gold coated at the marker combination part, the reacted substance is marked as a third immune complex, then the reacted third immune complex and the rabbit IgG marked by the colloidal gold are subjected to continuous chromatography along with the sample, the third immune complex reacted at a third detection line reacts with a strain of mouse anti-influenza B virus monoclonal antibody coated on the detection line and stays at the third detection line, the rabbit IgG marked by fluorescent microspheres at a quality control line reacts with a goat anti-rabbit polyclonal antibody coated on the quality control line and stays at the quality control line, other substances are subjected to continuous chromatography, and finally, the result is directly interpreted by naked eyes, on the premise that the quality control line displays a color band, when the third detection line displays the color band, the detection result of the influenza B virus in the sample is positive, and when the third detection line does not display the color band, the detection result of the influenza B virus is negative.
In some embodiments of the present invention, the,the fluorescent microspheres are used as markers, and the detection card added with the sample can be used for respectively collecting fluorescent signals (marked as T) of the first detection line through a fluorescence immunochromatographic instrumenta) The fluorescence signal of the second detection line (denoted as T)b) The fluorescence signal (T) of the third detection linec) And the signal of fluorescence of the control line (denoted as C), calculating Ta/C、Tb/C、 TcValue of/C, will TaComparing the normal value range of the/C and the N protein of the novel coronavirus to judge whether the/C is positive or negative, and comparing the T with the normal value range of the N protein of the novel coronavirusbComparing the/C with the normal value range of influenza A virus to judge whether the virus is positive or negative, and comparing the T with the normal value range of influenza A viruscthe/C is compared with the normal value range of the influenza B virus to judge the negative and positive. Meanwhile, the result can be interpreted under ultraviolet light, on the premise that the quality control line displays the fluorescent strip, when the first detection line displays the fluorescent strip, the detection result of the novel coronavirus N protein in the sample is positive, and when the first detection line does not display the fluorescent strip, the detection result of the novel coronavirus N protein is negative; on the premise that the quality control line displays the fluorescent strip, when the second detection line displays the fluorescent strip, the detection result of the influenza A virus in the sample is positive, and when the second detection line does not display the fluorescent strip, the detection result of the influenza A virus is negative; on the premise that the quality control line displays the fluorescent strip, when the third detection line displays the fluorescent strip, the detection result of the influenza B virus in the sample is positive, and when the third detection line does not display the fluorescent strip, the detection result of the influenza B virus is negative.
7. Preparation of test paper strip
Firstly, a coating pad 1 is adhered on a PVC plate (not shown in the figure), then, one end of a quality control line 7 close to the coating pad 1 is lapped with a water absorption pad 3, one end of a first detection line 4 close to the coating pad 1 is lapped with a marker pad 2 and a sample pad 7 connected with the marker pad, and a strip cutting machine is used for cutting the strip into test strips (shown in the figure 1) with the thickness of 4mm +/-0.1 mm for standby.
EXAMPLE 2 Assembly of Rapid Joint test cards
The new corona/influenza a/influenza b test strip prepared in example 1 was inserted into a card housing to prepare a rapid combination test card.
The card housing (as shown in fig. 2) may include: a bottom groove 10 connected to the bottom plate (specifically, PVC plate); an upper cover 11 connected to the bottom groove 10,
as shown in fig. 2B, the bottom tank 10 includes: a plurality of positioning holes 19 which are symmetrically distributed and are positioned on the inner surface of the test strip, and a plurality of first limiting parts 13 for limiting the transverse movement of the new crown/A flow/B flow test strip and second limiting parts 14 for limiting the longitudinal movement of the test strip are arranged among the plurality of positioning holes 19; the first limiting part 13 and the second limiting part 14, which are symmetrically arranged, enclose a new crown/first flow/second flow test strip placement area 12 (dotted line area) for placing a new crown/first flow/second flow test strip;
as shown in fig. 2A, the upper cover 11 includes: a plurality of positioning posts 18 which are matched with a plurality of positioning holes 19, and are matched with each other to fix the upper cover 11 and the bottom groove 10 together; the upper cover 11 further includes a third limiting portion 15 for limiting the upward and downward movement of the new crown/first flow/second flow test strip.
The upper cover 11 is provided with a sample adding hole 16 for adding a sample to the sample pad 9 of the new crown/first flow/second flow detection test strip;
the observation window 17 is arranged on the upper cover 11 and is used for collecting data of the first detection line 4, the second detection line 5, the third detection line 6 and the quality control line 7 on the coating pad 1 of the new crown/first flow/second flow detection test strip;
the observation window 17 is arranged on the upper cover 11 at a position corresponding to the middle part of the new crown/first-class/second-class test strip placement area 12;
the upper cover 11 is provided with a sample adding hole 16 at a position corresponding to the sample pad 9 of the new crown/first-class/second-class test strip for dropwise adding a sample to be tested on the sample pad 9 of the new crown/first-class/second-class test strip. The distance between the detection line and the sample adding hole is 15-25 mm.
In other embodiments, the sample pad 9 may be omitted, the sample application hole 16 directly corresponds to the label binding site 8 of the label pad 2, and the sample to be tested is directly dropped from the sample application hole 16 to the label binding site for directly performing the reaction.
Example 3 detection
The rapid combined detection card prepared in the embodiments 1 and 2 is used for detecting a sample to be detected, and can be used for detecting a nasal swab, a pharyngeal swab, a cavity swab, sputum, saliva, alveolar lavage fluid or blood.
The detection of pharyngeal swab samples is exemplified.
The method comprises the steps of adding 0.5m L sample preservation liquid into a plastic hose, immersing a cotton swab after the sample is collected into the sample preservation liquid and stirring, extruding the outer side of the plastic hose for a plurality of times by using a finger to fully soak the cotton swab, pulling out the cotton swab, twisting out (namely mixing the sample collected on the cotton swab into the sample preservation liquid) to obtain a sample to be detected, dripping the sample to be detected which is treated by 60 mu L into a sample adding hole 16 corresponding to a sample pad 9 of a new crown/influenza A/B test strip, standing for 15min, and directly reading by naked eyes, wherein when a first test line shows a color band (wine red or purple red), a second test line shows a color band (wine red or purple red), a test result of the N protein of the new coronavirus is positive, when the first test line shows that the N protein of the new coronavirus is negative, when the second test line shows that the second test line shows the N protein of the new coronavirus is negative, and when the second test line shows that the second test line shows the influenza A virus is negative, the second test line shows that the N protein of the new coronavirus is positive, and the second test line shows that the influenza A virus is negative.
Example 4 evaluation of Properties
The following performance evaluation experiments were performed using the combination test cards prepared in examples 1 and 2.
1. Sensitivity of the probe
(1) Sensitivity of novel coronavirus N protein detection result
The polypeptide fragments synthesized by the N protein of the novel coronavirus are respectively prepared into standards with the concentrations of 50ng/m L (L1), 100ng/m L (L2) and 200ng/m L (L3), and the detection results are shown in the following table 1.
TABLE 1
Figure RE-GDA0002506458290000081
(2) Sensitivity of detection results of influenza A virus
Influenza A virus antigen standard solutions were prepared using P.B buffer solutions at concentrations of 200ng/m L (N1), 400ng/m L (N2), and 800ng/m L (N3), respectively, and the test results are shown in Table 2 below.
TABLE 2
Figure RE-GDA0002506458290000091
(3) Sensitivity of influenza B virus detection results
Influenza A virus antigen standard solutions were prepared using P.B buffer solutions at 100ng/m L (P1), 150ng/m L (P2), and 300ng/m L (P3), respectively, and the results of the measurements are shown in Table 3 below.
TABLE 3
Figure RE-GDA0002506458290000092
As can be seen from the detection results in the tables 1, 2 and 3, the rapid combined detection card of the invention can detect the novel coronavirus N protein, the influenza A virus and the influenza B virus, and meets the detection requirement of sensitivity.
2. Specificity of
The standard solutions of adenovirus, syncytial virus, mycoplasma pneumoniae, and chlamydia pneumoniae were used as interfering substances and were dropped 500 μ L to the rapid combination test cards prepared in examples 1 and 2, respectively, for testing, and the test results were observed with the naked eye after 15 minutes, and are shown in table 4.
TABLE 4
Figure RE-GDA0002506458290000093
As shown in the test results in Table 4, the rapid combined test card of the present invention has strong specificity and no cross reaction with respiratory viruses such as other adenoviruses, syncytial viruses, Mycoplasma pneumoniae, Chlamydia pneumoniae, etc.
Example 5 preparation of New corona/first flow/second flow test strip
1. The preparation method of the other strain of mouse anti-coronavirus N protein monoclonal antibody marked by the colloidal gold is the same as that in the example 1;
2. the preparation method of the other strain of mouse anti-influenza A virus monoclonal antibody marked by the colloidal gold is the same as that in the example 1;
3. the preparation method of the other strain of mouse anti-influenza B virus monoclonal antibody marked by the colloidal gold is the same as that in the example 1;
4. the preparation method of the goat anti-rabbit IgG polyclonal antibody marked by the colloidal gold is the same as that of the preparation method in the example 1;
5. the "coated pad" was prepared in the same manner as in example 1;
6. the marker pad was prepared as follows:
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 Another strain of mouse anti-coronavirus N protein monoclonal antibody marked by colloidal gold, another strain of influenza A virus monoclonal monomer marked by colloidal gold, another strain of influenza B virus monoclonal monomer marked by colloidal gold and rabbit IgG marked by colloidal gold are uniformly mixed, then the mixture is sprayed on a glass cellulose membrane according to the speed of 10 mu L/cm, and then the glass cellulose membrane is placed at 45 ℃ for drying for 2h to prepare a marker pad 2, and a place, which is covered by another strain of mouse anti-coronavirus N protein monoclonal antibody marked by colloidal gold, another strain of influenza A virus monoclonal monomer marked by colloidal gold, another strain of influenza B virus monoclonal monomer marked by colloidal gold and rabbit IgG marked by colloidal gold, on the marker pad 2 is called a marker combining part 8.
7. The test strip was prepared in the same manner as in example 1;
8. the "assembly of the rapid combination test card" was prepared in the same manner as in example 2;
9. the "detection" procedure was the same as that in example 3;
example 6 evaluation of Properties
The following performance evaluation experiments were performed using the combination test card prepared in example 5.
1. Sensitivity of the probe
(1) Sensitivity of novel coronavirus N protein detection result
The polypeptide fragments synthesized by the N protein of the novel coronavirus are respectively prepared into standards with the concentrations of 50ng/m L (L1), 100ng/m L (L2) and 200ng/m L (L3), and the detection results are shown in the following table 5.
TABLE 5
Figure RE-GDA0002506458290000101
Figure RE-GDA0002506458290000111
(2) Sensitivity of detection results of influenza A virus
Influenza A virus antigen standard solutions were prepared in PBS at concentrations of 200ng/m L (N1), 400ng/m L (N2), and 800ng/m L (N3), respectively, and the results of the measurements are shown in Table 6 below.
TABLE 6
Figure RE-GDA0002506458290000112
(3) Sensitivity of influenza B virus detection results
Influenza A virus antigen standard solutions were prepared using P.B buffer solutions at 100ng/m L (P1), 150ng/m L (P2), and 300ng/m L (P3), respectively, and the test results are shown in Table 7 below.
TABLE 7
Figure RE-GDA0002506458290000113
As can be seen from the detection results in tables 5, 6 and 7, the rapid combined detection card of the present invention can detect novel coronavirus N protein, influenza A virus and influenza B virus, and meets the detection requirement of sensitivity.
2. Specificity of
The standard solutions of adenovirus, syncytial virus, mycoplasma pneumoniae, and chlamydia pneumoniae were used as interfering substances and were respectively dropped 500 μ L into the rapid combination test card prepared in example 5 to perform the test, and the test results were observed with the naked eye after 15 minutes, and are shown in table 8.
TABLE 8
Figure RE-GDA0002506458290000114
Figure RE-GDA0002506458290000121
As shown in the test results in Table 8, the rapid combined test card of the present invention has strong specificity and no cross reaction with respiratory viruses such as other adenoviruses, syncytial viruses, Mycoplasma pneumoniae, Chlamydia pneumoniae, etc.
Attached: preparation of the required solution
(1) Specimen preservation solution
Figure RE-GDA0002506458290000122
The purified water is fixed to the volume of 1000m L;
(2) P.B buffer solution
Disodium hydrogen phosphate 51.68g
Sodium dihydrogen phosphate 9.92g
The purified water was made to 1000m L.
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 (11)

1. A rapid combined detection card is characterized by comprising a new corona/influenza A/influenza B detection test strip and a card shell for detecting a sample; the card shell comprises a bottom groove and an upper cover which are mutually clamped, and the new crown/A flow/B flow detection test strip is arranged on the bottom groove of the card shell.
2. The rapid assay card of claim 1, wherein the neo-corona/microfluidic/yi-flow assay strip comprises a base plate, and a sample pad, a label pad, a coating pad and a water absorbent pad are fixed on the base plate, wherein the sample pad, the label pad, the coating pad and the water absorbent pad are sequentially connected; one end of the coating pad, which is close to the detection line, is connected with a marker pad, and one end of the coating pad, which is close to the quality control line, is connected with a water absorption pad; the coating pad is sequentially provided with a first detection line, a second detection line, a third detection line and a quality control line, and the sample pad and the marker pad are connected into a whole.
3. The rapid assay kit of claim 2, wherein the top cover has wells at positions corresponding to the sample pads of the new corona/flow a/flow b assay strip.
4. The rapid joint detection card according to claim 3, wherein the upper cover is provided with observation windows for data collection of the detection line and the quality control line respectively corresponding to the first detection line, the second detection line, the third detection line and the quality control line of the new crown/first flow/second flow detection strip.
5. The rapid combined detection card according to claim 4, wherein the new corona/influenza A/B detection test strip is characterized in that the first detection line, the second detection line or the third detection line are respectively coated with one of a monoclonal antibody against a novel coronavirus N protein, a monoclonal antibody against influenza A virus and a monoclonal antibody against influenza B virus, and the substances coated on the three detection lines are different; the quality control line is coated with a goat anti-rabbit polyclonal antibody.
6. The rapid combination test card of claim 5, wherein the marker pad is coated with another monoclonal antibody against the N protein of the novel coronavirus labeled with a marker, another monoclonal antibody against the influenza A virus labeled with a marker, another monoclonal antibody against the influenza B virus labeled with a marker, and rabbit IgG labeled with a marker.
7. The rapid combination test card of claim 6, wherein the molar ratio of the marker-labeled another anti-novel coronavirus N protein monoclonal antibody, the marker-labeled another anti-influenza A virus monoclonal antibody, the marker-labeled another anti-influenza B virus monoclonal antibody, and the marker-labeled rabbit IgG is 1:1:1: (0.1-4).
8. The rapid assay kit of claim 7, wherein the label is selected from the group consisting of fluorescent microspheres, colloidal gold, colloidal selenium, colored latex, and magnetic microspheres.
9. The rapid combination test card of claim 1, wherein the sample is selected from the group consisting of a nasal swab, a pharyngeal swab, a cavity swab, sputum, saliva, alveolar lavage, and blood.
10. A method for preparing a rapid combination test card according to any one of claims 1 to 9, comprising the steps of:
1) uniformly mixing another anti-novel coronavirus N protein monoclonal antibody marked by a marker, another anti-influenza A virus monoclonal antibody marked by the marker, another anti-influenza B virus monoclonal antibody marked by the marker and rabbit IgG marked by the marker, and then coating the obtained mixture on a glass cellulose membrane to prepare a marker pad;
2) respectively and optionally coating a monoclonal antibody against novel coronavirus N protein, a monoclonal antibody against influenza A virus and a monoclonal antibody against influenza B virus on a first detection line, a second detection line and a third detection line, wherein the substances coated on the three detection lines are different, and coating a polyclonal antibody against goat rabbit on a quality control line to prepare a coating pad;
3) the coated pad is adhered to the bottom plate, the water absorbing pad is lapped at one end close to the quality control line of the coated pad, the marker pad and the sample pad connected with the marker pad are lapped at one end close to the detection line of the coated pad, the test strip is cut into test strips by a slitter, and the test strips are put into a card shell to prepare the new crown/A flow/B flow rapid combined detection card.
11. A method for using a rapid joint detection card according to any one of claims 1 to 9, comprising the steps of:
treating the collected sample with a sample preservation solution to obtain a sample to be detected, adding 30-200 mu L sample to be detected to a sample adding hole of the new crown/first flow/second flow rapid combined detection card, standing for 5-30 min,
inserting the new corona/A flow/B flow rapid combined detection card into a fluorescence immunochromatographic analyzer for detection, automatically calculating T/C values corresponding to detection lines and quality control lines by the analyzer, and comparing the respective T/C values with the corresponding normal value ranges of the novel coronavirus N protein, the influenza A virus and the influenza B virus to judge whether the corona/A flow/B flow rapid combined detection card is negative or positive;
or directly observing with naked eyes or observing with naked eyes under ultraviolet light, and under the premise that a colored strip or a fluorescent strip appears on the quality control line, when the colored strip or the fluorescent strip appears on any detection line, the detection substance corresponding to the detection line is positive.
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