WO2022105896A1 - A test strip for rapid detection of contamination by a variety of viruses or pathogens - Google Patents

A test strip for rapid detection of contamination by a variety of viruses or pathogens Download PDF

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Publication number
WO2022105896A1
WO2022105896A1 PCT/CN2021/131924 CN2021131924W WO2022105896A1 WO 2022105896 A1 WO2022105896 A1 WO 2022105896A1 CN 2021131924 W CN2021131924 W CN 2021131924W WO 2022105896 A1 WO2022105896 A1 WO 2022105896A1
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Prior art keywords
antibody
pad
viruses
test strip
contamination
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PCT/CN2021/131924
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French (fr)
Inventor
Hong Li
Zhenjuan WU
Shijie Xie
Benlong SU
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Hangke Zhongtou Biotechnology (Beijing) Co., Ltd.
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Publication of WO2022105896A1 publication Critical patent/WO2022105896A1/en

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    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G01N33/54388—Immunochromatographic test strips based on lateral flow
    • 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
    • G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54346—Nanoparticles
    • 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
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, 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
    • G01N33/569—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
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/10—Detection of antigens from microorganism in sample from host
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the disclosure relates to a test strip for rapid detection of contamination by a variety of viruses or pathogens, and belongs to the field of rapid virus detection.
  • Virus is a kind of submicroscopic particle with cellular infectivity, and is substantially a DNA or RNA, which is wrapped with a protective outer shell. Through the mechanism of infection, such simple organism is able to utilize the cell system of a host to replicate itself, without the ability to grow and replicate independently.
  • the detection and diagnosis methods for some viruses include enzyme-linked immunosorbent assay (ELISA) , chemiluminescent immunoassay, gold immune-chromatography assay, biochip and so on, but some disadvantages exist in these detection and diagnosis methods such as longer operation time, high cost and complicated operation. Meanwhile, one kind of virus can only be detected for most of the existing virus detection test strip and the detection efficiency is relatively low. Therefore, how to design a test strip that can detect multiple viruses at the same time has become an important problem to be solved.
  • ELISA enzyme-linked immunosorbent assay
  • chemiluminescent immunoassay chemiluminescent immunoassay
  • gold immune-chromatography assay gold immune-chromatography assay
  • the disclosure provides the following technical solutions.
  • a test strip for rapid detection of contamination by a variety of viruses or pathogens is characterized by including a sampling pad, a conjugate pad, a reaction and coloration membrane, an liquid-absorbing pad and a supporting plate, where the sampling pad, the conjugate pad, the reaction and coloration membrane, and the liquid-absorbing pad are arranged on the supporting plate in sequence from left to right; the sampling pad and the liquid-absorbing pad are respectively arranged on the opposite sides of the supporting plate; the conjugate pad is located on the right side of the sampling pad, the adjacent sides of the conjugate pad and the sampling pad having a connecting overlap area; the reaction and coloration membrane is closely adjacent to the right side of the conjugate pad, the conjugate pad covering the adjacent side of the reaction and coloration membrane; the liquid-absorbing pad is located on the right side of the reaction and coloration membrane, and covers the adjacent side of the reaction and coloration membrane; the sampling pad is made of an liquid-absorbing material and configured to carry an object to be detected; and the liquid-absorbing pad is made of an liquid-absorbing material.
  • the conjugate pad includes a biotinylated anti-rabbit IgG antibody having a concentration of 10-30 ⁇ g/ml.
  • the conjugate pad also includes a biotinylated anti-RNA antibody, a biotinylated anti-DNA antibody, or other biotinylated antiviral antibody, the biotinylated anti-RNA antibody having a concentration of 30-50 ⁇ g/ml, the biotinylated anti-DNA antibody having a concentration of 30-50 ⁇ g/ml and the other biotinylated antiviral antibody having a concentration of 50-100 ⁇ g/ml.
  • a biotinylated anti-RNA antibody having a concentration of 30-50 ⁇ g/ml
  • the biotinylated anti-DNA antibody having a concentration of 30-50 ⁇ g/ml
  • the other biotinylated antiviral antibody having a concentration of 50-100 ⁇ g/ml.
  • reaction and coloration membrane is made from nitrocellulose membrane, on the surface of which there is a positive control marker.
  • a positive control marker There is also a specific anti-RNA antibody marker, a specific anti-DNA antibody marker or other specific antiviral antibody marker on the surface of the nitrocellulose membrane.
  • Each of the markers includes avidinylated nanometer colloidal gold particles at a concentration of 50-100 ⁇ g/ml.
  • the positive control marker, the specific anti-RNA antibody marker, the specific anti-DNA antibody marker, and the other specific antiviral antibody marker are separated from each other.
  • the positive control marker includes a non-specific rabbit IgG at a concentration of 10-30 ⁇ g/ml.
  • the specific anti-RNA antibody marker includes a specific anti-RNA antibody at a concentration of 30-50 ⁇ g/ml.
  • the specific anti-DNA antibody marker includes a specific anti-DNA antibody at a concentration of 30-50 ⁇ g/ml.
  • the other specific antiviral antibody marker respectively includes a highly specific antiviral antibody, such as influenza virus antibody, SARs virus antibody or coronavirus antibody, at a concentration of 50-100 ⁇ g/ml.
  • the supporting plate is made of a non-liquid-absorbing material, such as polyvinyl chloride (PVC) , transparent plastic film or other hard material.
  • PVC polyvinyl chloride
  • the sample to be detected When the dilute of a sample to be detected is loaded on the sampling pad, if the contamination, caused by a virus or pathogen such as influenza virus, coronavirus, pathogenic DNA or pathogenic RNA, is present in the sample to be detected, the sample to be detected firstly binds with the biotinylated antiviral antibody, the biotinylated anti-RNA antibody or the biotinylated anti-DNA antibody on the conjugate pad to form a complex, the complex including (1) influenza virus-anti-influenza virus antibody-biotin, or (2) coronavirus-anti-coronavirus antibody-biotin, or (3) pathogenic DNA-anti-DNA antibody-biotin, or (4) pathogenic RNA-anti-RNA antibody-biotin.
  • influenza virus-anti-influenza virus antibody-biotin or (2) coronavirus-anti-coronavirus antibody-biotin, or (3) pathogenic DNA-anti-DNA antibody-biotin, or (4) pathogenic RNA-anti-RNA
  • the conjugate pad also includes a biotinylated anti-rabbit IgG antibody.
  • the complex and the biotinylated anti-rabbit IgG antibody move forward along the direction of chromatography under capillary action and continue to flow in the direction of the liquid-absorbing pad up to the reaction and coloration membrane containing the avidinylated colloidal gold.
  • the aforementioned complexes will be respectively conjugated with specific anti-influenza virus antibody, specific anti-coronavirus antibody, specific anti-pathogenic DNA antibody or specific anti-pathogenic RNA antibody, which is sprayed on different markers of the reaction and coloration membrane, to form different types of conjugates of “specific antiviral antibody-virus-antiviral antibody-biotin-avidin-colloidal gold” .
  • the specific antiviral antibodies solidified on different markers will enrich corresponding viruses, and a large amount of colloidal gold particles will be accumulated when the detection result is positive, thereby exhibiting a red color visible to the naked eye on the corresponding markers.
  • Different types of viral contamination and its contamination degree may be identified by the coloration of colloidal gold on different markers and their color shades.
  • the biotinylated anti-rabbit IgG antibody continues to move forward for chromatography and reacts with the rabbit IgG solidified on a positive control marker to form a “rabbit IgG-anti-rabbit IgG antibody-biotin-avidin-colloidal gold” conjugate, thereby forming a red color visible to the naked eye on the positive control marker.
  • the positive control marker Under the capillary action, the positive control marker will adsorb the chromatographed anti-rabbit IgG and certainly develop color; if the color is absent, it shows that the test result is invalid. If there is no contamination due to viruses or pathogens such as influenza virus, coronavirus, pathogenic DNA or pathogenic RNA in the object to be detected, no conjugate will be formed, and only visible red will be present on the positive control marker and there is no color change on other markers.
  • viruses or pathogens such as influenza virus, coronavirus, pathogenic DNA or pathogenic RNA
  • the application may be implemented without instruments and equipment and professional testing personals.
  • the detection operation is very simple, virus infection may be identified intuitively, and the results may be shown with the simplest test strip color.
  • the disclosure introduces biotin and avidin so that the sensitivity of detection is greatly enhanced. If there is a more accurate requirement on the test result, the chemiluminescence at the amplification level of biotin and avidin may also be used to make a more accurate determination by a microplate reader or spectrophotometer, or the color on the reaction and coloration membrane may be compared with the comparison table or card for standard result at the end of the reaction.
  • the testing method is simple, has a rapid testing speed and an obvious testing effect, and can be used as an important means for preliminary screening.
  • the disclosure makes the detection range and results more comprehensive and accurate by detecting virus and viral DNA or RNA.
  • the detection of virus encompasses not only the detection of viral protein, but also the detection of viral nucleic acid.
  • the detection of viral protein improves the accuracy of the detection results, and the detection of viral nucleic acid improves the sensitivity of the detection results.
  • This kind of preliminary screening test strip material may be made into a separate test strip and applied to the mask or other protective equipment, and may also aid in judging the lifetime of the mask or protective equipment and whether the virus infection occurs via the color change.
  • Fig. 1 is a schematic structural diagram of a test strip for rapid detection of contamination by a variety of viruses or pathogens of the disclosure, the bold arrow indicating the direction of liquid flow.
  • 110 a sampling pad
  • 120 a conjugate pad
  • 130 a reaction and coloration membrane
  • 100 an liquid-absorbing pad.
  • Fig. 2 is a schematic diagram of a test strip for rapid detection of contamination by a variety of viruses or pathogens according to a specific embodiment of the disclosure.
  • Fig. 3 is a brief theoretical guidance diagram of a test strip for rapid detection of contamination by a variety of viruses or pathogens of the disclosure.
  • Fig. 4 shows the detection results of influenza virus (H1N1 strain) and novel coronavirus (COVID-19) in chicken embryo culture by a test strip for rapid detection of contamination by a variety of viruses or pathogens according to one embodiment of the disclosure.
  • a test strip for rapid detection of contamination by a variety of viruses or pathogens is characterized by including a sampling pad, a conjugate pad, a reaction and coloration membrane, an liquid-absorbing pad and a supporting plate, where the sampling pad, the conjugate pad, the reaction and coloration membrane, and the liquid-absorbing pad are arranged on the supporting plate in sequence from left to right; the sampling pad and the liquid-absorbing pad are respectively arranged on the opposite sides of the supporting plate; the conjugate pad is located on the right side of the sampling pad, the adjacent sides of the conjugate pad and the sampling pad having a connecting overlap area; the reaction and coloration membrane is closely adjacent to the right side of the conjugate pad, the conjugate pad covering the adjacent side of the reaction and coloration membrane; the liquid-absorbing pad is located on the right side of the reaction and coloration membrane, and covers the adjacent side of the reaction and coloration membrane; the sampling pad is made of an liquid-absorbing material and configured to carry an object to be detected; and the liquid-absorbing pad is made of an liquid-absorbing material.
  • the conjugate pad includes a biotinylated anti-rabbit IgG antibody having a concentration of 10-30 ⁇ g/ml.
  • the conjugate pad also includes a biotinylated anti-RNA antibody, a biotinylated anti-DNA antibody, or other biotinylated antiviral antibody, the biotinylated anti-RNA antibody having a concentration of 30-50 ⁇ g/ml, the biotinylated anti-DNA antibody having a concentration of 30-50 ⁇ g/ml and the other biotinylated antiviral antibody having a concentration of 50-100 ⁇ g/ml.
  • the reaction and coloration membrane is made from nitrocellulose membrane, on the surface of which there is a positive control marker.
  • a positive control marker There is also a specific anti-RNA antibody marker, a specific anti-DNA antibody marker or other specific antiviral antibody marker on the surface of the nitrocellulose membrane.
  • Each of the markers includes avidinylated nanometer colloidal gold particles at a concentration of 50-100 ⁇ g/ml.
  • the positive control marker, the specific anti-RNA antibody marker, the specific anti-DNA antibody marker, and the other specific antiviral antibody marker are separated from each other.
  • the positive control marker includes a non-specific rabbit IgG at a concentration of 10-30 ⁇ g/ml.
  • the specific anti-RNA antibody marker includes a specific anti-RNA antibody at a concentration of 30-50 ⁇ g/ml.
  • the specific anti-DNA antibody marker includes a specific anti-DNA antibody at a concentration of 30-50 ⁇ g/ml.
  • the other specific antiviral antibody marker includes a highly specific antiviral antibody, such as influenza virus antibody, SARs virus antibody or coronavirus antibody, at a concentration of 50-100 ⁇ g/ml.
  • the supporting plate is made of a non-liquid-absorbing material, such as polyvinyl chloride (PVC) , transparent plastic film or other hard material.
  • PVC polyvinyl chloride
  • the conjugate pad of the test strip was sprayed with biotin-marked mouse anti-novel coronavirus antibody and biotin-marked mouse anti-influenza virus antibody, each antibody having a concentration of 50 ⁇ g/ml.
  • the nitrocellulose membrane was provided with two markers, namely, an antibody marker that reacted specifically with goat anti-novel coronavirus and an antibody marker that reacted specifically with goat anti-influenza virus, each marker having a concentration of 50 ⁇ g/ml.
  • the two markers were each also sprayed with avidinylated colloidal gold particles with a concentration of 50 ⁇ g/ml.
  • the conjugate pad was also sprayed with biotinylated anti-rabbit IgG antibody with a concentration of 10 ⁇ g/ml; the positive control marker on the nitrocellulose membrane was sprayed with non-specific rabbit IgG with a concentration of 10 ⁇ g/ml.
  • the inventors used this test strip to detect whether the virus in the culture supernatants of chicken embryo inoculated with novel coronavirus and of chicken embryo inoculated with influenza virus was present.
  • the culture supernatant of chicken embryo without virus infection was used as a control.
  • Fig. 4 shows the detection results of influenza virus (H1N1 strain) and novel coronavirus (COVID-19) in chicken embryo cultures by a test strip for rapid detection of contamination by a variety of viruses or pathogens.
  • PCR amplification technique was applied to detect the transcription of novel coronavirus (COVID-19) mRNA in the supernatant with different extents of dilution of chicken embryo culture (at 37°C for 2 days) inoculated with novel coronavirus. It was proved that there was a high concentration of novel coronavirus in the supernatant of chicken embryo culture.
  • PCR amplification technique was applied to detect the transcription of influenza virus mRNA in the supernatant with different extents of dilution of chicken embryo culture inoculated with influenza virus (H1N1) (at 37°C for 2 days) . It was proved that there was a high concentration of influenza virus (H1N1) in the supernatant of chicken embryo culture.
  • test strip The results showed that the detection results of the test strip were completely consistent with those of PCR amplification technique for novel coronavirus and influenza virus.
  • novel coronavirus and influenza virus in the samples to be tested firstly reacted with the biotinylated mouse anti-novel coronavirus antibody and the biotinylated mouse anti-influenza virus antibody respectively, moved towards the liquid-absorbing pad by capillarity, and then were conjugated with the antibodies on the nitrocellulose membrane that respectively react specifically with goat anti-novel coronavirus and with goat anti-influenza virus.
  • Two kinds of conjugates were each formed on two different markers, namely, (1) antibody specifically reacted with goat anti-novel coronavirus-novel coronavirus-mouse anti-novel coronavirus antibody-biotin-avidin-colloidal gold conjugate, and (2) antibody specifically reacted with goat anti-influenza virus-influenza virus-mouse anti-influenza virus antibody-biotin-avidin-colloidal gold conjugate.
  • a great amount of colloidal gold particles were enriched on two different antibody markers to develop a color.
  • the biotinylated anti-rabbit IgG antibody was sprayed on a conjugate pad, and the rabbit IgG was sprayed on the positive control marker on the nitrocellulose membrane.
  • the biotinylated anti-rabbit IgG antibody continued to move forward along the direction of chromatography and reacted with the rabbit IgG on the positive control marker to form a “rabbit IgG-anti-IgG antibody-biotin-avidin-colloidal gold” conjugate, thereby forming a red color visible to the naked eye on the positive control marker.
  • the positive control marker must show color, if not, then the test results were invalid. If the object to be detected does not include virus such as influenza virus, novel coronavirus, etc. or pollutants, no conjugate will be formed, only the positive control marker will form red visible to the naked eye and other markers will not show color change.
  • the negative result should exclude the interference of the following factor, namely, the test method is wrong.
  • the positive result should exclude the interference of the following factor: cross-contamination of other virus.
  • the judgment of the result based on the above color also includes the following four kinds of situations:
  • test result for negative detection should be negative, otherwise it means that the environment is unqualified.
  • test result for positive control should be positive, otherwise it means that the test strip is invalid.
  • test results for three times were all negative the test results were determined to be negative.
  • test result was determined to be positive.
  • a conjugate pad of the test strip was sprayed with biotinylated mouse anti-novel coronavirus antibody and biotinylated mouse anti-influenza virus antibody.
  • such antibodies may also be any combination of biotinylated anti-RNA antibody, biotinylated anti-DNA antibody or other biotinylated antiviral antibody.
  • there are two antibody markers on the nitrocellulose membrane i.e. the antibody markers that react specifically with goat anti-novel coronavirus and goat anti-influenza virus.
  • these markers may also be any combination of specific anti-RNA antibody, specific anti-DNA antibody or other specific antiviral antibody.
  • the detection of virus by the test strip may encompass not only the detection of viral protein, but also the detection of viral nucleic acid.
  • the detection of viral protein improves the accuracy of the detection results, and the detection of viral nucleic acid improves the sensitivity of the detection results.
  • the chemiluminescence at the amplification level of biotin and avidin may also be used to make a more accurate determination by a microplate reader or spectrophotometer, or the color on the reaction and coloration membrane may be compared with the comparison table or card for standard result at the end of the reaction.
  • the testing method is simple, has a rapid testing speed and an obvious testing effect and can be used as an important means for preliminary screening.
  • This kind of preliminary screening test strip material may be made into a separate test strip and may be applied to the mask or other protective equipment, and can also aid in judging the lifetime of the mask or protective equipment and whether the virus infection occurs through the color change.
  • test strip involved in this application is convenient for patient self-examination and can get results through a simple detection method, it may only be used as a means of screening for virus infection, and the false positive and false negative results caused by many factors cannot be completely excluded. Therefore, it is necessary to use the results of the test strip and clinical symptoms, in combination with the experience of doctor to make a final determination. If the test result is positive, it is essential to seek medical treatment in time.

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Abstract

A test strip for rapid detection of contamination by a variety of viruses or pathogens, which is composed of a sampling pad (110), a conjugate pad (120), a reaction and coloration membrane (130), an adsorbent pad (100) and a supporting plate, the sampling pad (110) and the liquid-absorbing pad (100) are respectively arranged on opposite sides of the supporting plate. The conjugate pad (120) includes biotinylated antiviral antibody and anti-rabbit IgG antibody. The reaction and coloration membrane (130) is made of a nitrocellulose membrane, on the surface of which a positive control marker including non-specific rabbit IgG as well as a variety of complex specific antiviral antibody markers are provided. By combining multiple complex antibodies and multiple specific virus antibody markers, it is possible to detect the pollution of multiple viruses or pathogens simultaneously, thereby improving efficiency and reducing cost.

Description

A TEST STRIP FOR RAPID DETECTION OF CONTAMINATION BY A VARIETY OF VIRUSES OR PATHOGENS TECHNICAL FIELD
The disclosure relates to a test strip for rapid detection of contamination by a variety of viruses or pathogens, and belongs to the field of rapid virus detection.
BACKGROUND
Virus is a kind of submicroscopic particle with cellular infectivity, and is substantially a DNA or RNA, which is wrapped with a protective outer shell. Through the mechanism of infection, such simple organism is able to utilize the cell system of a host to replicate itself, without the ability to grow and replicate independently. At present, the detection and diagnosis methods for some viruses include enzyme-linked immunosorbent assay (ELISA) , chemiluminescent immunoassay, gold immune-chromatography assay, biochip and so on, but some disadvantages exist in these detection and diagnosis methods such as longer operation time, high cost and complicated operation. Meanwhile, one kind of virus can only be detected for most of the existing virus detection test strip and the detection efficiency is relatively low. Therefore, how to design a test strip that can detect multiple viruses at the same time has become an important problem to be solved.
SUMMARY
In order to solve the above problems, the disclosure provides the following technical solutions.
A test strip for rapid detection of contamination by a variety of viruses or pathogens is characterized by including a sampling pad, a conjugate pad, a reaction and  coloration membrane, an liquid-absorbing pad and a supporting plate, where the sampling pad, the conjugate pad, the reaction and coloration membrane, and the liquid-absorbing pad are arranged on the supporting plate in sequence from left to right; the sampling pad and the liquid-absorbing pad are respectively arranged on the opposite sides of the supporting plate; the conjugate pad is located on the right side of the sampling pad, the adjacent sides of the conjugate pad and the sampling pad having a connecting overlap area; the reaction and coloration membrane is closely adjacent to the right side of the conjugate pad, the conjugate pad covering the adjacent side of the reaction and coloration membrane; the liquid-absorbing pad is located on the right side of the reaction and coloration membrane, and covers the adjacent side of the reaction and coloration membrane; the sampling pad is made of an liquid-absorbing material and configured to carry an object to be detected; and the liquid-absorbing pad is made of an liquid-absorbing material.
Further, the conjugate pad includes a biotinylated anti-rabbit IgG antibody having a concentration of 10-30 μg/ml.
Further, the conjugate pad also includes a biotinylated anti-RNA antibody, a biotinylated anti-DNA antibody, or other biotinylated antiviral antibody, the biotinylated anti-RNA antibody having a concentration of 30-50 μg/ml, the biotinylated anti-DNA antibody having a concentration of 30-50 μg/ml and the other biotinylated antiviral antibody having a concentration of 50-100 μg/ml.
Further, the reaction and coloration membrane is made from nitrocellulose membrane, on the surface of which there is a positive control marker. There is also a specific anti-RNA antibody marker, a specific anti-DNA antibody marker or other specific antiviral antibody marker on the surface of the nitrocellulose membrane. Each of the markers includes avidinylated nanometer colloidal gold particles at a concentration of 50-100 μg/ml.
Further, the positive control marker, the specific anti-RNA antibody marker, the specific anti-DNA antibody marker, and the other specific antiviral antibody marker are separated from each other.
Further, the positive control marker includes a non-specific rabbit IgG at a concentration of 10-30 μg/ml.
Further, the specific anti-RNA antibody marker includes a specific anti-RNA  antibody at a concentration of 30-50 μg/ml.
Further, the specific anti-DNA antibody marker includes a specific anti-DNA antibody at a concentration of 30-50 μg/ml.
Further, the other specific antiviral antibody marker respectively includes a highly specific antiviral antibody, such as influenza virus antibody, SARs virus antibody or coronavirus antibody, at a concentration of 50-100 μg/ml.
Further, the supporting plate is made of a non-liquid-absorbing material, such as polyvinyl chloride (PVC) , transparent plastic film or other hard material.
When the dilute of a sample to be detected is loaded on the sampling pad, if the contamination, caused by a virus or pathogen such as influenza virus, coronavirus, pathogenic DNA or pathogenic RNA, is present in the sample to be detected, the sample to be detected firstly binds with the biotinylated antiviral antibody, the biotinylated anti-RNA antibody or the biotinylated anti-DNA antibody on the conjugate pad to form a complex, the complex including (1) influenza virus-anti-influenza virus antibody-biotin, or (2) coronavirus-anti-coronavirus antibody-biotin, or (3) pathogenic DNA-anti-DNA antibody-biotin, or (4) pathogenic RNA-anti-RNA antibody-biotin. The conjugate pad also includes a biotinylated anti-rabbit IgG antibody. The complex and the biotinylated anti-rabbit IgG antibody move forward along the direction of chromatography under capillary action and continue to flow in the direction of the liquid-absorbing pad up to the reaction and coloration membrane containing the avidinylated colloidal gold. The aforementioned complexes will be respectively conjugated with specific anti-influenza virus antibody, specific anti-coronavirus antibody, specific anti-pathogenic DNA antibody or specific anti-pathogenic RNA antibody, which is sprayed on different markers of the reaction and coloration membrane, to form different types of conjugates of “specific antiviral antibody-virus-antiviral antibody-biotin-avidin-colloidal gold” . The specific antiviral antibodies solidified on different markers will enrich corresponding viruses, and a large amount of colloidal gold particles will be accumulated when the detection result is positive, thereby exhibiting a red color visible to the naked eye on the corresponding markers. Different types of viral contamination and its contamination degree may be identified by the coloration of colloidal gold on different markers and their color shades. Meanwhile, the biotinylated anti-rabbit IgG antibody  continues to move forward for chromatography and reacts with the rabbit IgG solidified on a positive control marker to form a “rabbit IgG-anti-rabbit IgG antibody-biotin-avidin-colloidal gold” conjugate, thereby forming a red color visible to the naked eye on the positive control marker. Under the capillary action, the positive control marker will adsorb the chromatographed anti-rabbit IgG and certainly develop color; if the color is absent, it shows that the test result is invalid. If there is no contamination due to viruses or pathogens such as influenza virus, coronavirus, pathogenic DNA or pathogenic RNA in the object to be detected, no conjugate will be formed, and only visible red will be present on the positive control marker and there is no color change on other markers.
Beneficial effects
Compared with the prior art, the disclosure achieves the following beneficial effects:
1. The application may be implemented without instruments and equipment and professional testing personals. The detection operation is very simple, virus infection may be identified intuitively, and the results may be shown with the simplest test strip color.
2. By combining a variety of complex antibodies and a variety of virus-specific antibody display markers, contamination from multiple viruses or pathogens may be detected simultaneously, thereby improving the detection efficiency and reducing the detection cost effectively.
3. Considering the disadvantage that the test strip detection device on the market is fast but not sensitive enough, the disclosure introduces biotin and avidin so that the sensitivity of detection is greatly enhanced. If there is a more accurate requirement on the test result, the chemiluminescence at the amplification level of biotin and avidin may also be used to make a more accurate determination by a microplate reader or spectrophotometer, or the color on the reaction and coloration membrane may be compared with the comparison table or card for standard result at the end of the reaction. The testing method is simple, has a rapid testing speed and an obvious testing effect, and can be used as an important means for preliminary screening.
4. The disclosure makes the detection range and results more comprehensive and accurate by detecting virus and viral DNA or RNA. The detection of virus encompasses not  only the detection of viral protein, but also the detection of viral nucleic acid. The detection of viral protein improves the accuracy of the detection results, and the detection of viral nucleic acid improves the sensitivity of the detection results.
5. In the disclosure, it can be preliminarily judged whether there is virus infection or not by detecting the color change of the test strip. This kind of preliminary screening test strip material may be made into a separate test strip and applied to the mask or other protective equipment, and may also aid in judging the lifetime of the mask or protective equipment and whether the virus infection occurs via the color change.
BRIEF DESCRIPTION OF DRAWINGS
Fig. 1 is a schematic structural diagram of a test strip for rapid detection of contamination by a variety of viruses or pathogens of the disclosure, the bold arrow indicating the direction of liquid flow.
Description of reference numbers: 110, a sampling pad; 120, a conjugate pad; 130, a reaction and coloration membrane; 100, an liquid-absorbing pad.
Fig. 2 is a schematic diagram of a test strip for rapid detection of contamination by a variety of viruses or pathogens according to a specific embodiment of the disclosure.
Fig. 3 is a brief theoretical guidance diagram of a test strip for rapid detection of contamination by a variety of viruses or pathogens of the disclosure.
Fig. 4 shows the detection results of influenza virus (H1N1 strain) and novel coronavirus (COVID-19) in chicken embryo culture by a test strip for rapid detection of contamination by a variety of viruses or pathogens according to one embodiment of the disclosure.
DESCRIPTION OF EMBODIMENTS
With reference to the accompanying drawings in embodiments of the present disclosure, the following clearly and comprehensively describes the technical solutions in embodiments of the present disclosure. The described embodiments are merely a part rather than all embodiments of the present disclosure.
Embodiment
A test strip for rapid detection of contamination by a variety of viruses or pathogens is characterized by including a sampling pad, a conjugate pad, a reaction and coloration membrane, an liquid-absorbing pad and a supporting plate, where the sampling pad, the conjugate pad, the reaction and coloration membrane, and the liquid-absorbing pad are arranged on the supporting plate in sequence from left to right; the sampling pad and the liquid-absorbing pad are respectively arranged on the opposite sides of the supporting plate; the conjugate pad is located on the right side of the sampling pad, the adjacent sides of the conjugate pad and the sampling pad having a connecting overlap area; the reaction and coloration membrane is closely adjacent to the right side of the conjugate pad, the conjugate pad covering the adjacent side of the reaction and coloration membrane; the liquid-absorbing pad is located on the right side of the reaction and coloration membrane, and covers the adjacent side of the reaction and coloration membrane; the sampling pad is made of an liquid-absorbing material and configured to carry an object to be detected; and the liquid-absorbing pad is made of an liquid-absorbing material.
The conjugate pad includes a biotinylated anti-rabbit IgG antibody having a concentration of 10-30 μg/ml.
The conjugate pad also includes a biotinylated anti-RNA antibody, a biotinylated anti-DNA antibody, or other biotinylated antiviral antibody, the biotinylated anti-RNA antibody having a concentration of 30-50 μg/ml, the biotinylated anti-DNA antibody having a concentration of 30-50 μg/ml and the other biotinylated antiviral antibody having a concentration of 50-100 μg/ml.
The reaction and coloration membrane is made from nitrocellulose membrane, on the surface of which there is a positive control marker. There is also a specific anti-RNA antibody marker, a specific anti-DNA antibody marker or other specific antiviral antibody marker on the surface of the nitrocellulose membrane. Each of the markers includes avidinylated nanometer colloidal gold particles at a concentration of 50-100 μg/ml.
The positive control marker, the specific anti-RNA antibody marker, the specific anti-DNA antibody marker, and the other specific antiviral antibody marker are separated from each other.
The positive control marker includes a non-specific rabbit IgG at a concentration of 10-30 μg/ml.
The specific anti-RNA antibody marker includes a specific anti-RNA antibody at a concentration of 30-50 μg/ml.
The specific anti-DNA antibody marker includes a specific anti-DNA antibody at a concentration of 30-50 μg/ml.
The other specific antiviral antibody marker includes a highly specific antiviral antibody, such as influenza virus antibody, SARs virus antibody or coronavirus antibody, at a concentration of 50-100 μg/ml.
The supporting plate is made of a non-liquid-absorbing material, such as polyvinyl chloride (PVC) , transparent plastic film or other hard material.
In a specific embodiment of the disclosure, the conjugate pad of the test strip was sprayed with biotin-marked mouse anti-novel coronavirus antibody and biotin-marked mouse anti-influenza virus antibody, each antibody having a concentration of 50 μg/ml. The nitrocellulose membrane was provided with two markers, namely, an antibody marker that reacted specifically with goat anti-novel coronavirus and an antibody marker that reacted specifically with goat anti-influenza virus, each marker having a concentration of 50 μg/ml. The two markers were each also sprayed with avidinylated colloidal gold particles with a concentration of 50 μg/ml. At the same time, the conjugate pad was also sprayed with biotinylated anti-rabbit IgG antibody with a concentration of 10 μg/ml; the positive control marker on the nitrocellulose membrane was sprayed with non-specific rabbit IgG with a concentration of 10 μg/ml. The inventors used this test strip to detect whether the virus in the culture supernatants of chicken embryo inoculated with novel coronavirus and of chicken embryo inoculated with influenza virus was present. The culture supernatant of chicken embryo without virus infection was used as a control. Fig. 4 shows the detection results of influenza virus (H1N1 strain) and novel coronavirus (COVID-19) in chicken embryo cultures by a test strip for rapid detection of contamination by a variety of viruses or pathogens.
A. One drop (about 20 microliter) of supernatant, which is diluted as 1x10 -3, of normal chicken embryo culture (at 37℃ for 2 days) without inoculation of any virus was applied to the sampling pad, leaving for five minutes at room temperature. A result was as  shown.
B. One drop (about 20 microliter) of supernatant, which is diluted as 1x10 -3, of chicken embryo culture (at 37℃ for 2 days) inoculated with novel coronavirus was applied to the sampling pad, leaving for five minutes at room temperature. A result was as shown.
C. One drop (about 20 microliter) of supernatant, which is diluted as 1x10 -5, of chicken embryo culture (at 37℃ for 2 days) inoculated with influenza virus was applied to the sampling pad, leaving for five minutes at room temperature. A result was as shown.
D. PCR amplification technique was applied to detect the transcription of novel coronavirus (COVID-19) mRNA in the supernatant with different extents of dilution of chicken embryo culture (at 37℃ for 2 days) inoculated with novel coronavirus. It was proved that there was a high concentration of novel coronavirus in the supernatant of chicken embryo culture.
E. PCR amplification technique was applied to detect the transcription of influenza virus mRNA in the supernatant with different extents of dilution of chicken embryo culture inoculated with influenza virus (H1N1) (at 37℃ for 2 days) . It was proved that there was a high concentration of influenza virus (H1N1) in the supernatant of chicken embryo culture.
The results showed that the detection results of the test strip were completely consistent with those of PCR amplification technique for novel coronavirus and influenza virus.
In this embodiment, novel coronavirus and influenza virus in the samples to be tested firstly reacted with the biotinylated mouse anti-novel coronavirus antibody and the biotinylated mouse anti-influenza virus antibody respectively, moved towards the liquid-absorbing pad by capillarity, and then were conjugated with the antibodies on the nitrocellulose membrane that respectively react specifically with goat anti-novel coronavirus and with goat anti-influenza virus. Two kinds of conjugates were each formed on two different markers, namely, (1) antibody specifically reacted with goat anti-novel coronavirus-novel coronavirus-mouse anti-novel coronavirus antibody-biotin-avidin-colloidal gold conjugate, and (2) antibody specifically reacted with goat anti-influenza virus-influenza virus-mouse anti-influenza virus antibody-biotin-avidin-colloidal gold conjugate. A great amount of colloidal gold particles were enriched on two different antibody markers to develop  a color. Meanwhile, the biotinylated anti-rabbit IgG antibody was sprayed on a conjugate pad, and the rabbit IgG was sprayed on the positive control marker on the nitrocellulose membrane. The biotinylated anti-rabbit IgG antibody continued to move forward along the direction of chromatography and reacted with the rabbit IgG on the positive control marker to form a “rabbit IgG-anti-IgG antibody-biotin-avidin-colloidal gold” conjugate, thereby forming a red color visible to the naked eye on the positive control marker. The positive control marker must show color, if not, then the test results were invalid. If the object to be detected does not include virus such as influenza virus, novel coronavirus, etc. or pollutants, no conjugate will be formed, only the positive control marker will form red visible to the naked eye and other markers will not show color change.
The negative result should exclude the interference of the following factor, namely, the test method is wrong.
The positive result should exclude the interference of the following factor: cross-contamination of other virus.
The judgment of the result based on the above color also includes the following four kinds of situations:
1. The test result for negative detection should be negative, otherwise it means that the environment is unqualified.
2. The test result for positive control should be positive, otherwise it means that the test strip is invalid.
3. If the test results for three times were all negative, the test results were determined to be negative.
4. If at least one of test results was positive, the test result was determined to be positive.
In this example, a conjugate pad of the test strip was sprayed with biotinylated mouse anti-novel coronavirus antibody and biotinylated mouse anti-influenza virus antibody. In practical application, such antibodies may also be any combination of biotinylated anti-RNA antibody, biotinylated anti-DNA antibody or other biotinylated antiviral antibody. In this embodiment, there are two antibody markers on the nitrocellulose membrane, i.e. the antibody markers that react specifically with goat anti-novel coronavirus and goat  anti-influenza virus. In practical application, these markers may also be any combination of specific anti-RNA antibody, specific anti-DNA antibody or other specific antiviral antibody. The detection of virus by the test strip may encompass not only the detection of viral protein, but also the detection of viral nucleic acid. The detection of viral protein improves the accuracy of the detection results, and the detection of viral nucleic acid improves the sensitivity of the detection results. If there is a more accurate requirement on the test results, the chemiluminescence at the amplification level of biotin and avidin may also be used to make a more accurate determination by a microplate reader or spectrophotometer, or the color on the reaction and coloration membrane may be compared with the comparison table or card for standard result at the end of the reaction. The testing method is simple, has a rapid testing speed and an obvious testing effect and can be used as an important means for preliminary screening.
In the disclosure, it can be preliminarily judged whether there is virus infection or not by detecting the color change of the test strip. This kind of preliminary screening test strip material may be made into a separate test strip and may be applied to the mask or other protective equipment, and can also aid in judging the lifetime of the mask or protective equipment and whether the virus infection occurs through the color change.
Even though the test strip involved in this application is convenient for patient self-examination and can get results through a simple detection method, it may only be used as a means of screening for virus infection, and the false positive and false negative results caused by many factors cannot be completely excluded. Therefore, it is necessary to use the results of the test strip and clinical symptoms, in combination with the experience of doctor to make a final determination. If the test result is positive, it is essential to seek medical treatment in time.
Finally, it should be noted that the above description is merely a preferred embodiment of the present application and is not intended to limit the present application. Although the present application is described in detail with reference to the foregoing embodiments, persons of ordinary skill in the art should understand that they may still make modifications to the technical solutions described in the foregoing embodiments or make equivalent substitutions to some technical features thereof. Within the spirit and principles of  the present application, any modification, equivalent replacement and improvement should be included within the protection scope of the present application.

Claims (12)

  1. A test strip for rapid detection of contamination by a variety of viruses or pathogens, comprising a sampling pad, a conjugate pad, a reaction and coloration membrane, an liquid-absorbing pad and a supporting plate, wherein the sampling pad, the conjugate pad, the reaction and coloration membrane, and the liquid-absorbing pad are arranged on the supporting plate in sequence from left to right; the sampling pad and the liquid-absorbing pad are respectively arranged on the opposite sides of the supporting plate; the conjugate pad is located on the right side of the sampling pad, the adjacent sides of the conjugate pad and the sampling pad having a connecting overlap area; the reaction and coloration membrane is closely adjacent to the right side of the conjugate pad, the conjugate pad covering the adjacent side of the reaction and coloration membrane; the liquid-absorbing pad is located on the right side of the reaction and coloration membrane, and covers the adjacent side of the reaction and coloration membrane; the sampling pad is made of an liquid-absorbing material and configured to carry an object to be detected; and the liquid-absorbing pad is made of an liquid -absorbing material.
  2. The test strip for rapid detection of contamination by a variety of viruses or pathogens according to claim 1, wherein the conjugate pad comprises a biotinylated anti-rabbit IgG antibody at a concentration of 10-30 μg/ml.
  3. The test strip for rapid detection of contamination by a variety of viruses or pathogens according to claim 1, wherein the conjugate pad further comprises a biotinylated anti-RNA antibody, a biotinylated anti-DNA antibody, or other biotinylated antiviral antibody, the biotinylated anti-RNA antibody having a concentration of 30-50 μg/ml, the biotinylated anti-DNA antibody having a concentration of 30-50 μg/ml and the other biotinylated antiviral antibody having a concentration of 50-100 μg/ml.
  4. The test strip for rapid detection of contamination by a variety of viruses or pathogens according to claim 1, wherein the reaction and coloration membrane is made from nitrocellulose membrane, on the surface of which there is a positive control marker, and there is also a specific anti-RNA antibody marker, a specific anti-DNA antibody marker or other  specific antiviral antibody marker on the surface of the nitrocellulose membrane, each of the markers comprising avidinylated nanometer colloidal gold particles at a concentration of 50-100 μg/ml.
  5. The test strip for rapid detection of contamination by a variety of viruses or pathogens according to claim 4, wherein the positive control marker, the specific anti-RNA antibody marker, the specific anti-DNA antibody marker, and the other specific antiviral antibody marker are separated from each other.
  6. The test strip for rapid detection of contamination by a variety of viruses or pathogens according to claim 4, wherein the positive control marker comprises a non-specific rabbit IgG at a concentration of 10-30 μg/ml.
  7. The test strip for rapid detection of contamination by a variety of viruses or pathogens according to claim 4, wherein the specific anti-RNA antibody marker comprises a specific anti-RNA antibody at a concentration of 30-50 μg/ml.
  8. The test strip for rapid detection of contamination by a variety of viruses or pathogens according to claim 4, wherein the specific anti-DNA antibody marker comprises a specific anti-DNA antibody at a concentration of 30-50 μg/ml.
  9. The test strip for rapid detection of contamination by a variety of viruses or pathogens according to claim 4, wherein the other specific antiviral antibody marker comprises a highly specific antiviral antibody at a concentration of 50-100 μg/ml.
  10. The test strip for rapid detection of contamination by a variety of viruses or pathogens according to claim 9, wherein the highly specific antiviral antibody is influenza virus antibody, SARs virus antibody or coronavirus antibody.
  11. The test strip for rapid detection of contamination by a variety of viruses or pathogens according to claim 1, wherein the supporting plate is made of a non-liquid-absorbing material.
  12. The test strip for rapid detection of contamination by a variety of viruses or pathogens according to claim 11, wherein the non-liquid-absorbing material is polyvinyl chloride (PVC) , transparent plastic film or other hard material.
PCT/CN2021/131924 2020-11-20 2021-11-19 A test strip for rapid detection of contamination by a variety of viruses or pathogens WO2022105896A1 (en)

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