Enzymatic diagnostic test for sars and other viral diseases

Abstract

The present invention is directed towards methods, compositions and kits for testing for a virus in a sample. The methods determine the presence of a viral enzyme by contacting the sample with a peptidal compound capable of being cleaved by the viral enzyme to form peptidal compound fragments. Detection of a peptidal compound fragment confirms the presence of the virus.

1 - 22 . (canceled) 
     
     
         23 . A method for determining the presence of a virus in a sample, the method comprising:
 contacting the sample with a peptide comprising at least four amino acid residues and capable of being cleaved at a cleavage point by a protease to form a cleaved first peptide fragment and a second peptide fragment; wherein a signaling moiety is linked to a portion of the peptide present in the first peptide fragment; and   detecting the virus by observing a signal from the signaling moiety linked to the cleaved first peptide fragment,   wherein the protease is encoded by nucleic acid of the virus and is present in the sample, and wherein said peptide comprises an amino acid sequence identical to a sequence of a polyprotein cleavage region of said protease and including said cleavage point.   
     
     
         24 . The method of  claim 23 , wherein a quenching moiety is linked to a portion of the peptide present in the second peptide fragment and wherein the signaling moiety and the quenching moiety are linked to the peptide at relative positions such that the quenching moiety quenches the a signal of the signaling moiety unless the peptide is cleaved at the cleavage point. 
     
     
         25 . The method of  claim 23 , further comprising contacting the sample with a second peptide comprising at least four amino acid residues and capable of being cleaved at a cleavage point by a second protease, wherein the second protease is encoded by the viral nucleic acid, and wherein said second peptide comprises an amino acid sequence identical to a sequence of a polyprotein cleavage region of said second protease and including said cleavage point. 
     
     
         26 . The method of  claim 23 , wherein said protease is a protease required for replication of the virus. 
     
     
         27 . The method of  claim 23 , wherein the signal of the signaling moiety is selected from a group consisting of a moiety emitting a fluorescent signal, a colorimetric signal, and a chemiluminescent signal. 
     
     
         28 . The method of  claim 27 , wherein the signal of the signaling moiety emits a fluorescent signal. 
     
     
         29 . The method of  claim 23 , wherein the virus is a virus selected from the group consisting of Nidovirus, Herpesvirus, Adenovirus, Retrovirus, Picornavirus and Potyvirus. 
     
     
         30 . The method of  claim 29 , wherein the sample is selected from a group consisting of mucus, saliva, blood, serum, plasma, urine, spinal fluid, sputum, tissue biopsy, broncheoalveolar fluid, and tears. 
     
     
         31 . The method of  claim 23 , wherein the virus is the Severe Acute Respiratory Syndrome (SARS) virus. 
     
     
         32 . The method of  claim 31 , wherein said peptide comprises the amino acid sequence VQAG and wherein said cleavage point is Q/AG. 
     
     
         33 . The method of  claim 23 , wherein said virus is an Enterovirus. 
     
     
         34 . The method of  claim 23 , wherein the virus is a Rhinovirus. 
     
     
         35 . The method of  claim 23 , wherein said virus is a Picornavirus, wherein said peptide comprises the amino acid sequence EALFQGP and wherein said cleavage point is Q/GP. 
     
     
         36 . The method of  claim 35 , wherein said peptide comprises at least nine amino acid residues and wherein said cleaved first peptide fragment comprises at least five amino acid residues. 
     
     
         37 . The method of  claim 23 , wherein the method is a homogenous assay method. 
     
     
         38 . The method of  claim 23 , wherein the method is a heterogenous assay method. 
     
     
         39 . The method of  claim 23 , wherein said peptide comprises at least seven amino acid residues. 
     
     
         40 . The method of  claim 23 , wherein said peptide comprises at least ten amino acid residues. 
     
     
         41 . A method for determining the presence of a Picornavirus or Nidovirus virus in a sample, the method comprising:
 contacting the sample with a peptide comprising at least four amino acid residues and capable of being cleaved at a cleavage point by a protease to form a cleaved first peptide fragment and a second peptide fragment; wherein a signaling moiety is linked to a portion of the peptide present in the first peptide fragment; and   detecting the virus by observing the signal from the signaling moiety linked to the cleaved first peptide fragment,   wherein the protease is encoded by nucleic acid of the virus and is present in the sample, and wherein said peptide comprises an amino acid sequence identical to a sequence of a polyprotein cleavage region of said protease and including said cleavage point, and wherein said method is a homogenous assay method.   
     
     
         42 . A kit for detecting the presence of a virus in a sample, the kit comprising a reagent comprising a peptide comprising at least four amino acid residues, and capable of being cleaved at a cleavage point by a proteolytic enzyme of said virus to form a cleaved first peptide fragment and a second peptide fragment; wherein a signaling moiety is linked to a portion of the peptide present in the first peptide fragment, wherein the enzyme is encoded by nucleic acid of the virus and is present in the sample, wherein said at least four amino acid residues comprise a polyprotein cleavage region of said protease including said cleavage point. 
     
     
         43 . The kit of  claim 42 , wherein the virus is a virus selected from the group consisting of Nidovirus, Herpesvirus, Adenovirus, Retrovirus, Picornavirus and Potyvirus families. 
     
     
         44 . The kit of  claim 42 , wherein a quenching moiety is linked to a portion of the peptide present in the second peptide fragment, wherein the signaling moiety and the quenching moiety are linked to the peptide at relative positions such that the quenching moiety quenches the a signal of the signaling moiety unless the peptide is cleaved at the cleavage point. 
     
     
         45 . The kit of  claim 42 , further comprising a second peptide capable of being cleaved at a cleavage point by a second enzyme, wherein the second enzyme is encoded by the viral nucleic acid. 
     
     
         46 . The kit of  claim 42 , wherein the signal of the signaling moiety is selected from a group consisting of a fluorescent signal, a colorimetric signal, and a chemiluminescent signal. 
     
     
         47 . The kit of  claim 42 , further comprising a protease inhibitor for inhibiting non-viral proteases in said sample. 
     
     
         48 . The kit of  claim 42 , wherein said peptide comprises at least seven amino acid residues. 
     
     
         49 . A composition for detecting a virus in a sample, the composition comprising:
 a reagent comprising a peptide comprising at least four amino acid residues, and capable of being cleaved at a cleavage point by a proteolytic enzyme of said virus to form a cleaved first peptide fragment and a second peptide fragment; wherein a signaling moiety is linked to a portion of the peptide present in the first peptide fragment, wherein the enzyme is encoded by nucleic acid of the virus and is present in the sample, wherein said at least four amino acid residues comprise a polyprotein cleavage region of said protease including said cleavage point and wherein the virus is selected from a group consisting of the Nidovirus, Herpesvirus, Adenovirus, Retrovirus, Picornavirus and Potyvirus families, and   a buffer.