Novel methods and compositions to evaluate and determine inactivation of hazardous biological material

Abstract

Novel time and temperature integrator (TTI) assays, kits containing the components of the assays, and the novel components for those assays are provided herein. These novel TTI assays evaluate and/or determine the inactivation of biological material in/on a sample by quantifying the degradation of DNA using qPCR. The sample can be a food product (e.g., fruits, vegetables, meat from animals, or eggs) while the item can be any object (e.g., medical equipment, especially reusable medical equipment) for which one needs to determine that the amount of inactivation of specific hazardous biological material on the object or in a sample is at or below a pre-determined amount.

We, the inventors, claim: 
     
         1 . A kit comprising:
 a pair of primers comprising a first polynucleotide and a second polynucleotide;   a label;   optionally DNase; and   optionally instructions on using said first polynucleotide and said second polynucleotide in a method to determine the amount of inactivation of a biological material,   wherein said first polynucleotide has the sequence in one of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO: 7, and   wherein when said first polynucleotide has the sequence in SEQ ID NO: 1, said second polynucleotide has the sequence in SEQ ID NO: 2; when said first polynucleotide has the sequence in SEQ ID NO: 3, said second polynucleotide has the sequence in SEQ ID NO: 4; when said first polynucleotide has the sequence in SEQ ID NO: 5, then said second polynucleotide has the sequence in SEQ ID NO: 6; and when said first polynucleotide has the sequence in SEQ ID NO: 7, then said second polynucleotide has the sequence in SEQ ID NO: 8.   
     
     
         2 . The kit of  claim 1  wherein said label is selected from the group consisting of a fluorescent composition, an intercalating dye, a spectroscopic label, a photochemical label, a biochemical label, an immunochemical label, and a chemical label. 
     
     
         3 . The kit of  claim 1  wherein said label comprises:
 a probe; 
 a quencher dye; and 
 a fluorescent dye, 
 wherein said quencher dye and said fluorescent dye are linked to said probe, 
 wherein when the sequence of said first polynucleotide is SEQ ID NO: 1, said probe has a sequence of between approximately 15 contiguous bases and approximately 45 contiguous bases of SEQ ID NO: 14 or the reverse complement thereof, 
 wherein when the sequence of said first polynucleotide is SEQ ID NO: 3, said probe has a sequence of between approximately 15 contiguous bases and approximately 45 contiguous bases of SEQ ID NO: 15 or the reverse complement thereof, 
 wherein when the sequence of said first polynucleotide is SEQ ID NO: 5, said probe has a sequence of between approximately 15 contiguous bases and approximately 45 contiguous bases of SEQ ID NO: 16 or the reverse complement thereof, and 
 wherein when the sequence of said first polynucleotide is SEQ ID NO: 7, said probe has a sequence of between approximately 15 contiguous bases and approximately 45 contiguous bases of SEQ ID NO: 17 or the reverse complement thereof. 
 
     
     
         4 . A kit comprising:
 a pair of primers comprising a first polynucleotide and a second polynucleotide;   a label;   optionally DNase; and   instructions on using said first polynucleotide and said second polynucleotide in a method to determine the amount of inactivation of a biological material,   wherein said first polynucleotide has the sequence in one of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO: 7, and   wherein when said first polynucleotide has the sequence in SEQ ID NO: 1, said second polynucleotide has the sequence in SEQ ID NO: 2; when said first polynucleotide has the sequence in SEQ ID NO: 3, said second polynucleotide has the sequence in SEQ ID NO: 4; when said first polynucleotide has the sequence in SEQ ID NO: 5, then said second polynucleotide has the sequence in SEQ ID NO: 6; and when said first polynucleotide has the sequence in SEQ ID NO: 7, then said second polynucleotide has the sequence in SEQ ID NO: 8.