Methods and kits useful in the differentiation of
The invention pertains to sequences, methods, and kits useful in the identification of and differentiation between Burkholderia pseudomallei and Burkholderia mallei . The methods generally involve the addition of oligonucleotides to mixtures containing nucleic acid isolated from a sample and performing nucleic acid amplification on the mixture.
1. A method of determining whether a sample contains B. mallei or B. pseudomallei comprising the steps of: receiving said sample, wherein said sample comprises nucleic acid; forming a mixture by adding oligonucleotides represented by SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, and SEQ ID NO. 4 to said nucleic acid; subjecting said mixture to conditions that allow amplification of said nucleic acid; and identifying said sample as containing or not containing B. mallei or B. pseudomallei based upon a result of the nucleic acid amplification.
2. The method of claim 1 , further comprising the step of performing DNA sequencing upon one or more products of the nucleic acid amplification.
3. The method of claim 1 , wherein said oligonucleotide represented by SEQ ID NO. 3 comprises a first label and wherein said oligonucleotide represented by SEQ ID NO. 4 comprises a second label different from said first label.
4. The method of claim 1 , wherein said first oligonucleotide is affixed to a solid substrate.
5. The method of claim 1 , wherein said sample comprises an environmental sample.
6. The method of claim 1 , wherein said sample is derived from a subject selected from the group consisting of a human, a companion animal, and a livestock animal.
7. The method of claim 1 , wherein said result comprises one or more DNA sequences.
8. The method of claim 1 , wherein said result comprises one or more ΔCt values.
9. A kit that facilitates determining whether a nucleic acid-containing sample contains B. mallei or B. pseudomallei comprising:
a first oligonucleotide represented by SEQ ID NO. 1;
a second oligonucleotide represented by SEQ ID NO. 2;
a third oligonucleotide represented by SEQ ID NO. 3;
a fourth oligonucleotide represented by SEQ ID NO. 4; and
an indication of a result that signifies classification of the sample as containing a bacteria selected from the group consisting of B. mallei and B. pseudomallei when said nucleic acid from said sample is mixed with said first and said second oligonucleotides;
wherein at least the first oligonucleotide is affixed to a substrate, and further wherein the third oligonucleotide comprises a first label and the fourth oligonucleotide comprises a second label different from said first label.
10. The kit of claim 9 , wherein said result is in a form selected from the group consisting of a ΔCt value and a nucleic acid sequence.
11. The kit of claim 9 , wherein said indication is selected from the group consisting of a positive control, a writing, and software configured to detect the result as input and identification of the sample as containing B. mallei or B. pseudomallei as output.