Assay and compositions for detection of
View Patent ↗The invention includes compositions and methods of detection of Bacillus anthracis that use oligonucleotide probes specific for genetic material contained in the pXO1 and pXO2 plasmids in nucleic acid hybridization reactions. Embodiments of the method may include additional probes specific for other gene sequences to distinguish B. anthracis from other bacterial species present in a sample or to provide an indication that the assay was performed properly even when no Bacillus sequence is detected.
1. A composition comprising a synthetic oligonucleotide consisting of 25 to 40 nucleotides that hybridizes specifically to a B. anthracis pagA target sequence selected from the group consisting of SEQ ID Nos. 1, 2, 3, 4, 5, 6, 7, 8, 21, 22, 23, and 24, sequences fully complementary to SEQ ID Nos. 1, 2, 3, 4, 5, 6, 7, 8, 21, 22, 23, and 24, RNA equivalents of SEQ ID Nos. 1, 2, 3, 4, 5, 6, 7, 8, 21, 22, 23, and 24, and RNA sequences fully complementary to SEQ ID Nos. 1, 2, 3, 4, 5, 6, 7, 8, 21, 22, 23, and 24.
2. The composition of claim 1 , wherein the synthetic oligonucleotide that hybridizes specifically to the pagA target sequence consists of SEQ ID NO:1 or SEQ ID NO:2.
3. The composition of claim 1 , wherein the synthetic oligonucleotide that hybridizes specifically to the pagA target sequence consists of SEQ ID NO:3 or SEQ ID NO:4.
4. The composition of claim 1 , wherein the synthetic oligonucleotide that hybridizes specifically to the pagA target sequence consists of SEQ ID NO:5 or SEQ ID NO:6.
5. The composition of claim 1 , wherein the synthetic oligonucleotide that hybridizes specifically to the pagA target sequence consists of SEQ ID NO:7 or SEQ ID NO:8.
6. The composition of claim 1 further comprising a capB-specific synthetic oligonucleotide that hybridizes specifically to a capB target sequence, wherein the capB-specific oligonucleotide consists of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13 or SEQ ID NO:14.
7. The composition of claim 1 , wherein the synthetic oligonucleotide has a DNA or RNA backbone, or mixed DNA and RNA backbone, or contains at least one 2′-methoxy RNA group linking the bases.
8. The composition of claim 1 , wherein the synthetic oligonucleotide has a signal-producing label linked directly or indirectly to the oligonucleotide.
9. A kit comprising a synthetic oligonucleotide consisting of 25 to 40 nucleotides that hybridizes specifically to a sequence contained in a B. anthracis pagA target sequence selected from the group consisting of SEQ ID Nos. 1, 2, 3, 4, 5, 6, 7, 8, 21, 22, 23, and 24, sequences fully complementary to SEQ ID Nos. 1, 2, 3, 4, 5, 6, 7, 8, 21, 22, 23, and 24, RNA equivalents of SEQ ID Nos. 1, 2, 3, 4, 5, 6, 7, 8, 21, 22, 23, and 24, and RNA sequences fully complementary to SEQ ID Nos. 1, 2, 3, 4, 5, 6, 7, 8, 21, 22, 23, and 24.
10. The kit of claim 9 , further comprising a capB-specific probe oligonucleotide consisting of SEQ ID NO: 9, 10, 11, 12, 13 or 14.
11. A method of detecting B. anthracis nucleic acid in a sample comprising the steps of:
providing a sample containing B. anthracis nucleic acids;
providing a pagA-specific probe that hybridizes specifically to a pagA target sequence, wherein the pagA-specific probe is a synthetic oligonucleotide of 25 to 40 nucleotides selected from the group consisting of SEQ ID Nos. 1, 2, 3, 4, 5, 6, 7, 8, 21, 22, 23, and 24, sequences fully complementary to SEQ ID Nos. 1, 2, 3, 4, 5, 6, 7, 8, 21, 22, 23, and 24, RNA equivalents of SEQ ID Nos. 1, 2, 3, 4, 5, 6, 7, 8, 21, 22, 23, and 24, and RNA sequences fully complementary to SEQ ID Nos. 1, 2, 3, 4, 5, 6, 7, 8, 21, 22, 23, and 24;
hybridizing specifically at least one pagA-specific probe to the pagA target sequence; and
detecting the presence of at least one pagA-specific probe hybridized to the pagA target sequence to indicate the presence of B. anthracis in the sample.
12. The method of claim 11 further comprising a capB-specific probe oligonucleotide consisting of SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:14.