Severe acute respiratory syndrome (SARS)—associated coronavirus diagnostics
The invention relates to the diagnosis of a SARS-associated coronavirus, such as a SARS CoV-2 infection and SARS-CoV infection, using the SARS-CoV and SARS-CoV-2 nucleocapsid proteins and antibodies binding to these proteins. The invention encompasses reagents, methods and kits for the detection of a SARS-associated coronavirus.
1. A method for the detection of a severe acute respiratory syndrome (SARS)
associated coronavirus infection in a biological sample comprising:
providing a severe acute respiratory syndrome-associated coronavirus 2 (SARS-CoV-2) nucleocapsid (N) protein expressed and purified from E. coli strain BL21 (DE3) pDIA17 transformed with recombinant plasmid pETM11/N-nCov E. coli 3-(His)6-Nter or pETM11/N-nCov E. coli 4-(His)6-Nter deposited at the Collection Nationale de Cultures de Microorganismes (CNCM) at the Institut Pasteur, 25, Rue du Docteur Roux, 75724 Paris, FR, on May 11, 2020, under the deposit numbers CNCM 1-5510 and CNCM 1-5511, respectively;
providing a biological sample from an individual or a patient suspected to be infected with a severe acute respiratory syndrome-associated coronavirus (SARS-CoV) coronavirus;
contacting said SARS-CoV-2 N protein with said biological sample; and
visualizing the antigen-antibody complexes formed.
2. The method of claim 1 , wherein it comprises detection of IgG or IgM or IgA.
3. The method of claim 1 , wherein it comprises detection of IgG, IgM and IgA.
4. The method of claim 1 , comprising an ELISA, lateral flow immunoassays, bead based immunoassays, or multiplex bead based immunoassays.
5. The method of claim 1 , wherein the SARS-CoV-2 N protein comprises the sequence of SEQ ID NO:1.
6. The method of claim 1 , wherein the SARS-CoV-2 N protein consists of the sequence of SEQ ID NO:1.
7. A method for the detection of a SARS-associated coronavirus infection in a biological sample comprising:
providing a SARS-CoV-1 N protein and a SARS-CoV-2 N protein,
wherein said SARS-CoV-2 N protein is expressed and purified from E. coli strain BL21 (DE3) PDIA17 transformed with recombinant plasmid PETM11/N-nCov E. coli 3-(His)6-Nter or pETM11/N-nCov E. coli 4-(His)6-Nter deposited at the Collection Nationale de Cultures de Microorganismes (CNCM) at the Institut Pasteur, 25, Rue du Docteur Roux, 75724 Pars, FR, on May 11, 2020, under the deposit numbers CNCM I-5510 and CNCM 1-5511, respectively:
providing a biological sample from an individual or a patient suspected to be infected with a coronavirus:
contacting said SARS-CoV-1 N and SARS-CoV-2 N proteins with said biological sample; and
visualizing the antigen-antibody complexes formed.
8. The method of claim 7 , comprising an ELISA, lateral flow immunoassays, bead based immunoassays, or multiplex bead based immunoassays.
9. The method of claim 7 , wherein it comprises detection of IgG or IgM or IgA.
10. The method of claim 7 , wherein it comprises detection of IgG, IgM and IgA.
11. A kit for the detection of a SARS-CoV coronavirus infection in a biological sample comprising a SARS-CoV-2 N protein expressed and purified from E. coli BL21 (DE3) pDIA17 transformed with recombinant plasmid pETM11/N-nCov E. coli 3-(His)6-Nter or pETM11/N-nCov E. coli 4-(His)6-Nter deposited at the Collection Nationale de Cultures de Microorganismes (CNCM) at the Institut Pasteur, 25, Rue du Docteur Roux, 75724 Paris, FR, on May 11, 2020, under the deposit numbers CNCM 1-5510 and CNCM 1-5511, respectively.
12. The kit of claim 11 , comprising serum from an animal immunized with a SARS-CoV-2 N protein.
13. The kit of claim 11 , comprising a SARS-CoV-1 N protein.
14. The kit of claim 13 , comprising serum from an animal immunized with a SARS-CoV-2 N protein.
15. A method for producing a SARS-CoV-2 N protein comprising the following steps:
Culturing a bacterium transformed with a recombinant plasmid encoding for a SARS-CoV-2 N protein deposited at the Collection Nationale de Cultures de Microorganismes (CNCM) at the Institut Pasteur, 25, Rue du Docteur Roux, 75724 Paris, FR, on May 11, 2020, under the deposit number CNCM 1-5510 or the deposit number CNCM 1-5511 in a suitable bacterial growth medium;
inducing the production of SARS-CoV-2 N protein;
recovering and purifying SARS-CoV-2 N protein.
16. The method of claim 15 , wherein recovering of SARS-CoV-2 N protein is performed by pelleting and breakage of bacteria.
17. The method of claim 16 , wherein purifying of SARS-CoV-2 N protein is performed by metal chelate affinity chromatography, and/or gel filtration.
18. The method of claim 17 , wherein purifying of SARS-CoV-2 N protein is performed by metal chelate affinity chromatography, and/or gel filtration.