Oligonucleotide sequences that identify species of animal

Abstract

The present invention provides a method for identifying animal species, said method comprises a step of amplifying a DNA fragment by PCR using a DNA in a sample as a template and animal-specific DNA sequences as a primer pair, wherein the animal-specific DNA sequences are derived from a ATP synthase subunit 8 gene or a region proximal thereto of a mitochondrial genome; and a step of detecting the amplified DNA fragment.

1 . A method of detecting animal-derived components in a sample, comprising:
 providing a sample comprising DNA;   adding a pair of DNA primers and a DNA polymerase to the sample to form a PCR mixture, the pair of DNA primers having a structure to prime a PCR reaction if the sample comprises components derived from animals or a specific grouping or species of animals;   subjecting the PCR mixture to conditions suitable for promoting PCR including bringing the PCR mixture to a first temperature sufficient to denature the DNA in the sample followed by a second temperature, less than the first temperature, sufficient to anneal the pair of DNA primers to a complementary DNA sequence; and   determining the source of components in the sample using one of the following criteria:   1) observing a single PCR product when the sample comprises components derived from animals or a specific grouping or species of animals; and   2) observing a lack of a PCR product when the sample does not comprise components derived from animals or a specific grouping or species of animals,   wherein the animals or the specific groups or species of animals are selected from the group consisting of mammal, ruminant, cattle, sheep, goat, deer, pig, horse, rabbit, whale, chicken, and fish.   
     
     
         2 . The method of  claim 1 , wherein the animals or the specific grouping or species of animals are mammals, and wherein the pair of DNA primers comprises a first primer having a DNA sequence of SEQ ID NO: 1 and a second primer having a DNA sequence of SEQ ID NO: 2. 
     
     
         3 . The method of  claim 1 , wherein the animals or the specific grouping or species of animals are ruminants, and wherein the pair of DNA primers comprises a first primer having a DNA sequence of SEQ ID NO: 3 and a second primer having a DNA sequence of SEQ ID NO: 4. 
     
     
         4 . The method of  claim 1 , wherein the animals or the specific grouping or species of animals are ruminants, and wherein the pair of DNA primers comprises a first primer having a DNA sequence of SEQ ID NO: 5 and a second primer having a DNA sequence of SEQ ID NO: 6. 
     
     
         5 . The method of  claim 1 , wherein the animals or the specific grouping or species of animals are cattle, wherein the pair of DNA primers comprises a combination of a first primer having a DNA sequence and a second primer having a DNA sequence, and wherein the combination of the first and the second primers is selected from the group consisting of the following DNA sequence combinations: SEQ ID NO: 9 and SEQ ID NO: 13; SEQ ID NO: 9 and SEQ ID NO: 12; SEQ ID NO: 11 and SEQ ID NO: 13; SEQ ID NO: 10 and SEQ ID NO: 12; SEQ ID NO: 11 and SEQ ID NO: 12; SEQ ID NO: 8 and SEQ ID NO: 12; and SEQ ID NO: 14 and SEQ ID NO: 15. 
     
     
         6 . The method of  claim 1 , wherein the animals or the specific grouping or species of animals are pigs, wherein the pair of DNA primers comprises a combination of a first primer having a DNA sequence and a second primer having a DNA sequence, and wherein the combination of the first and the second primers is a combination of a DNA sequence of SEQ ID NO: 17 and a DNA sequence of SEQ ID NO: 19; or a combination of a DNA sequence of SEQ ID NO: 18 and a DNA sequence of SEQ ID NO: 22. 
     
     
         7 . The method of  claim 1 , wherein the animals or the specific grouping or species of animals are sheep, and wherein the pair of DNA primers comprises a first primer having a DNA sequence comprising SEQ ID NO: 23 and a second primer having a DNA sequence comprising SEQ ID NO: 24. 
     
     
         8 . The method of  claim 1 , wherein the animals or the specific grouping or species of animals are goats, and wherein the pair of DNA primers comprises a first primer having a DNA sequence comprising SEQ ID NO: 25 and a second primer having a DNA sequence comprising SEQ ID NO: 26. 
     
     
         9 . The method of  claim 1 , wherein the animals or the specific grouping or species of animals are chickens, and wherein the pair of DNA primers comprises a first primer having a DNA sequence of SEQ ID NO: 28 and a second primer having a DNA sequence of SEQ ID NO: 30. 
     
     
         10 . The method of  claim 1 , wherein the animals or the specific grouping or species of animals are fish, and wherein the pair of DNA primers comprises a first primer having a DNA sequence comprising one selected from the group consisting of SEQ ID NOS: 32, 34, 38, and 39; and a second primer having a DNA sequence comprising one selected from the group consisting of SEQ ID NOS: 33, 35, 36, 37, 40, and 41. 
     
     
         11 . A method of detecting ruminant-derived components in a sample, comprising:
 providing a sample comprising DNA;   adding a pair of DNA primers and a DNA polymerase to the sample to form a PCR mixture, the pair of DNA primers having a structure to prime a PCR reaction if the sample comprises components derived from ruminants or a specific grouping or species of ruminants;   subjecting the PCR mixture to conditions suitable for promoting PCR including bringing the PCR mixture to a first temperature sufficient to denature the DNA in the sample followed by a second temperature, less than the first temperature, sufficient to anneal the pair of DNA primers to a complementary DNA sequence; and   determining the source of components in the sample using one of the following criteria:   1) observing a single PCR product when the sample comprises components derived from ruminants or a specific grouping or species of ruminants; and   2) observing a lack of a PCR product when the sample does not comprise components derived from ruminants or a specific grouping or species of ruminants.   
     
     
         12 . The method of  claim 11 , wherein the pair of DNA primers comprises a first primer having a DNA sequence of SEQ ID NO: 3 and a second primer having a DNA sequence of SEQ ID NO: 4. 
     
     
         13 . The method of  claim 11 , wherein the pair of DNA primers comprises a first primer having a DNA sequence of SEQ ID NO: 5 and a second primer having a DNA sequence of SEQ ID NO: 6. 
     
     
         14 . The method of  claim 11 , wherein the ruminants or the specific grouping or species of ruminants are cattle. 
     
     
         15 . The method of  claim 14 , wherein pair of DNA primers comprises a combination of a first primer having a DNA sequence and a second primer having a DNA sequence, and wherein the combination of the first and the second primers is selected from the group consisting of the following DNA sequence combinations: SEQ ID NO: 9 and SEQ ID NO: 13; SEQ ID NO: 9 and SEQ ID NO: 12; SEQ ID NO: 11 and SEQ ID NO: 13; SEQ ID NO: 10 and SEQ ID NO: 12; SEQ ID NO: 11 and SEQ ID NO: 12; SEQ ID NO: 8 and SEQ ID NO: 12; and SEQ ID NO: 14 and SEQ ID NO: 15. 
     
     
         16 . The method of  claim 11 , wherein the ruminants or the specific grouping or species of ruminants are sheep. 
     
     
         17 . The method of  claim 16 , and wherein the pair of DNA primers comprises a first primer having a DNA sequence comprising SEQ ID NO: 23 and a second primer having a DNA sequence comprising SEQ ID NO: 24. 
     
     
         18 . The method of  claim 11 , wherein the ruminants or the specific grouping or species of ruminants are goats. 
     
     
         19 . The method of  claim 18 , wherein the pair of DNA primers comprises a first primer having a DNA sequence comprising SEQ ID NO: 25 and a second primer having a DNA sequence comprising SEQ ID NO: 26. 
     
     
         20 . The method of  claim 11 , wherein the pair of DNA primers comprise a DNA sequence having a structure to anneal to a mitochondrial ATP synthase subunit 8 gene or a region proximal thereto of a mitochondrial genome. 
     
     
         21 . The method of  claim 11 , wherein ruminants include one or more selected from the group consisting of cattle, sheep, goats, and deer. 
     
     
         22 . The method of  claim 11 , further comprising preparing the sample comprising DNA by extracting mitochondrial DNA from a sample comprising one or more of animal and plant material. 
     
     
         23 . The method of  claim 22 , wherein the sample is substantially free from non-mitochondrial DNA. 
     
     
         24 . A method of detecting ruminant-derived components in a sample, comprising:
 providing a sample comprising DNA;   adding a DNA primer pair having a structure to anneal to a ruminant-derived DNA target and prime a PCR reaction to a mitochondrial ATP synthase subunit 8 gene or a region proximal thereto of a mitochondrial genome of ruminants and having a structure to not prime a PCR reaction of a non-ruminant DNA target, to form a PCR mixture;   subjecting the PCR mixture to conditions and reagents suitable for promoting PCR including bringing the PCR mixture to a first temperature sufficient to denature the DNA in the sample followed by a second temperature, less than the first temperature, sufficient to anneal the pair of DNA primers to a complementary DNA sequence; and   determining the source of components in the sample using one of the following criteria:   1) observing a single PCR product and assigning the sample as containing ruminant-derived components; and   2) observing a lack of a PCR product and assigning the sample as not containing ruminant-derived components.   
     
     
         25 . The method of  claim 24 , wherein the pair of DNA primers comprises a first primer having a DNA sequence of SEQ ID NO: 3 and a second primer having a DNA sequence of SEQ ID NO: 4. 
     
     
         26 . The method of  claim 24 , wherein the pair of DNA primers comprises a first primer having a DNA sequence of SEQ ID NO: 5 and a second primer having a DNA sequence of SEQ ID NO: 6. 
     
     
         27 . The method of  claim 24 , wherein the ruminant is cattle. 
     
     
         28 . The method of  claim 27 , wherein pair of DNA primers comprises a combination of a first primer having a DNA sequence and a second primer having a DNA sequence, and wherein the combination of the first and the second primers is selected from the group consisting of the following DNA sequence combinations: SEQ ID NO: 9 and SEQ ID NO: 13; SEQ ID NO: 9 and SEQ ID NO: 12; SEQ ID NO: 11 and SEQ ID NO: 13; SEQ ID NO: 10 and SEQ ID NO: 12; SEQ ID NO: 11 and SEQ ID NO: 12; SEQ ID NO: 8 and SEQ ID NO: 12; and SEQ ID NO: 14 and SEQ ID NO: 15. 
     
     
         29 . The method of  claim 24 , wherein the ruminants are sheep. 
     
     
         30 . The method of  claim 29 , and wherein the pair of DNA primers comprises a first primer having a DNA sequence comprising SEQ ID NO: 23 and a second primer having a DNA sequence comprising SEQ ID NO: 24. 
     
     
         31 . The method of  claim 24 , wherein the ruminants are goats. 
     
     
         32 . The method of  claim 31 , wherein the pair of DNA primers comprises a first primer having a DNA sequence comprising SEQ ID NO: 25 and a second primer having a DNA sequence comprising SEQ ID NO: 26. 
     
     
         33 . The method of  claim 24 , wherein ruminants include one or more selected from the group consisting of cattle, sheep, goats, and deer. 
     
     
         34 . A method of detecting ruminant-derived components in a sample, comprising:
 providing a sample comprising DNA;   adding a pair of ruminant-specific DNA primers consisting of a combination of a first primer having a DNA sequence and a second primer having a DNA sequence, and wherein the combination of the first and second primers is a combination of a DNA sequence of SEQ ID NO. 3 and a DNA sequence of SEQ ID NO. 4; and a combination of a DNA sequence of SEQ ID NO. 5 and a DNA sequence of SEQ ID NO. 6;   amplifying a DNA sequence from the DNA in the sample by PCR, wherein the amplified sequence comprises one or more sequences selected from the group consisting of the sequence of a mitochondrial ATP synthase subunit 8 gene and a region proximal thereto in a mitochondrial genome of a ruminant; and   observing a single PCR product and assigning the sample as containing ruminant-derived components.   
     
     
         35 . The method of  claim 34 , wherein the sample comprises DNA derived from one or more selected from the group consisting of cattle, sheep, goats, and deer.