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Methods for identification of coronaviruses

  • US 8,057,993 B2
  • Filed: 04/22/2004
  • Issued: 11/15/2011
  • Est. Priority Date: 04/26/2003
  • Status: Expired due to Fees
First Claim
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1. A multiplex polymerase chain reaction method for identifying a coronavirus, comprising:

  • a) providing a sample suspected of comprising one or more coronavirus family members, wherein the identity of said coronavirus family members is unknown;

    b) providing coronavirus RNA from said sample, wherein the identity of the coronavirus source of said coronavirus RNA is unknown;

    c) providing corresponding DNA from said RNA wherein said DNA comprises sequences encoding at least a portion of a nsp11 gene and at least a portion of a RdRp gene;

    d) amplifying said DNA to produce first and second amplification products using two or more oligonucleotide primer pairs, wherein said first amplification product comprises sequences encoding at least a portion of said nsp11 gene and said second amplification product comprises sequences encoding at least a portion of said RdRp gene;

    e) determining the base compositions of said first and second amplification products by mass spectrometry without sequencing said first and second amplification products, wherein said base compositions identifies the number of A residues, C residues, T residues, G residues, U residues, analogs thereof and mass tag residues thereof in said first and second amplification products;

    f) comparing said base compositions of said first and second amplification products to calculated or measured base compositions of corresponding amplification products of one or more known coronaviruses present in a database with the proviso that sequencing of said first and second amplification products is not used to identify said coronavirus, wherein a match between said determined base compositions and said calculated or measured base composition in said database identifies the identity of said one or more coronavirus family members in said sample; and

    g) determining the identity of said one or more coronavirus family members by identifying a match between said determined based compositions and said calculated or measured base composition in said database.

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