Sequence of the tropheryma whippelii bacteria rpoB gene and oligonucleotide for molecular diagnosis of whipple's disease
View Patent ↗The invention concerns the total sequence of the Tropheryma whippelii bacteria rpoB gene, characterized in that it consists of the nucleotide sequence SEQ ID no. °9, and the nucleotide sequences derived from said sequence specific of the Tropheryma whippelii bacteria and their use as probe and primer.
1. An isolated nucleic acid sequence of the rpoB gene of Tropheryma whippelii bacteria, comprising at least one of: the nucleotide sequence set forth in SEQ ID NO: 9 and a full length complement thereof.
2. A process for determining the presence of bacteria of the species Tropheryma Whippelii in a biological sample, comprising:
placing said biological sample in contact with at least one probe consisting of an oligonucleotide consisting of 10–100 contiguous nucleotides of the portions of SEQ ID NO: 9 outside SEQ ID NO: 3, and
detecting formation or absence of formation of a hybridization complex between said probe and a nucleic acid of said sample, wherein the formation or absence of formation of said hybridization complex determines, respectively, the presence or absence of said bacteria in said sample.
3. The process according to claim 2 , wherein said probe consists a nucleotide sequence selected from the group consisting of SEQ ID NOs: 5–8, 10, 11, 12, and 17, and full length complementary sequences of SEQ ID NOs: 10, 11, 12.
4. The process according to claim 2 , wherein said probe consists of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 11, 12, and full length complementary sequences of SEQ ID NOs: 11, 12.
5. An amplification primer set comprising at least one forward primer and at least one reverse primer, wherein each primer is 18–35 bases in length, wherein the primers are non-complementary to one another, wherein the primers have corresponding melting temperatures, and wherein the primers together frame an about 500 base region of nucleotides 1–2014 and 2625–3767 of SEQ ID NO: 9, the sequence of said region hybridizing specifically to Thropheryma whippelii.
6. The amplification primer set of claim 5 , wherein the forward primer and the reverse primer together frame SEQ ID NO: 10.
7. The amplification primer set of claim 5 , wherein the forward primer consists of SEQ ID NO: 11, and wherein the reverse primer consists of SEQ ID NO:12.
8. An amplification primer set, comprising at least one forward primer and at least one reverse primer wherein each primer is 18–35 bases in length, consisting of contiguous nucleotides of the portion of SEQ ID NO: 9 outside SEQ ID NO: 3, said contiguous nucleotides forming a sequence hybridizing specifically to Thropheryma whippelii.
9. A process for determining the presence or absence of a bacteria of the species Tropheryma Whippelii in a biological sample, comprising
carrying out amplification of DNA of said sample by the means of the primer set of claim 5 , and
detecting formation or absence of formation of an amplified DNA, wherein the formation or absence of formation of said amplified DNA determines, respectively, the presence or absence of said bacteria.
10. The process according to claim 9 , wherein wherein the forward primer and the reverse primer together frame SEQ ID NO: 10.
11. The process according to claim 9 , wherein wherein the forward primer consists of SEQ ID NO: 11, and wherein the reverse primer consists of SEQ ID NO:12.
12. A process according to claim 9 , wherein the sequence of said amplified DNA is compared to the sequence set forth in SEQ ID NO: 9 and the presence or absence of the sequence of the amplified DNA in the sequence set forth in SEQ ID NO: 9 determines, respectively, the presence or absence of said bacteria.
13. A process according to claim 12 , wherein the sequence of said amplified DNA is determined in carrying out sequencing of said amplified DNA.
14. A process according to claim 9 , wherein said two primers include a primer consisting of the sequence set forth in SEQ ID NO: 11 and a primer consisting of the sequence set forth in SEQ ID NO: 12, and the sequence of the amplified DNA is compared to the sequence set forth in SEQ ID NO: 10 to determine the presence or absence of said bacteria.
15. An oligonucleotide consisting of a sequence selected from the group consisting of SEQ ID NOS: 5–8, 10–12, 17, and full-length complements of SEQ ID NOS: 5–8, 10–12, 17.
16. The oligonucleotide of claim 15 , wherein the oligonucleotide is selected from the group consisting of SEQ ID NOS: 5–8, and full-length complements of SEQ ID NOS: 5–8.
17. The oligonucleotide of claim 15 , wherein the oligonucleotide is selected from the group consisting of SEQ II) NOS: 10–12 and 17, and full-length complements of SEQ ID NOS: 10–12 and 17.
18. A process for determining the presence or absence of a bacteria of the species Tropheryma Whippelii in a biological sample, comprising:
carrying out amplification of DNA of said sample by the means of the primer set of claim 8 , and
detecting formation or absence of formation of an amplified DNA, wherein the formation or absence of formation of said amplified DNA determines, respectively, the presence or absence of said bacteria.