Methods and probes for detecting a vancomycin resistance gene
Compositions and methods for the detection of vancomycin-resistant pathogens using primers and/or probes to the vanA and vanB genes.
1. A method for testing for the presence of a vancomycin resistance gene in a sample, comprising:
providing said sample;
contacting said sample with an amplification primer pair that hybridizes to and is capable of amplifying sequences from a vanB gene under conditions that enable nucleic acid amplification to generate a vanB amplicon if said sample comprises a pathogen comprising said vanB gene, wherein said amplification primer pair comprises an oligonucleotide consisting of SEQ ID NO: 1096 or the complement thereof, or variants of SEQ ID NO: 1096, wherein said variants optionally have up to three nucleotide changes compared to SEQ ID NO: 1096, wherein said variants are capable of hybridizing to and amplifying nucleic acids from a vanB gene in a nucleic acid amplification assay; and
determining whether or not said vanB amplicon is present wherein the presence of a vanB amplicon is indicative of the presence of a vancomycin resistance gene in the sample, thereby testing for the presence of a vancomycin resistance gene in a sample.
2. The method of claim 1 , further comprising:
contacting said sample with an amplification primer pair that hybridizes to and is capable of amplifying sequences from a vanA gene under said conditions that enable nucleic acid amplification to generate a vanA amplicon if said sample comprises a pathogen comprising said vanA gene; and
determining whether or not said vanA amplicon is present.
3. The method of claim 1 , further comprising:
contacting said sample with an amplification primer pair that hybridizes to and is capable of amplifying sequences from a vanC gene under said conditions that enable nucleic acid amplification, to generate a vanC amplicon if said sample comprises a pathogen comprising said vanC gene; and
determining whether or not said vanC amplicon is present.
4. The method of claim 2 , further comprising:
contacting said sample with an amplification primer pair that hybridizes to and is capable of amplifying sequences from a vanC gene under said conditions that enable nucleic acid amplification, to generate a vanC amplicon if said sample comprises a pathogen comprising said vanC gene; and
determining whether or not said vanC amplicon is present.
5. The method of claim 1 , wherein said method further comprises contacting said sample with a probe comprising an oligonucleotide that hybridizes to a portion of the vanB amplicon under said conditions that enable nucleic acid amplification.
6. The method of claim 5 , wherein said probe comprises a fluorescent moiety.
7. The method of claim 6 , wherein said probe is a molecular beacon.
8. The method of claim 1 wherein said method comprises performing a nucleic acid amplification assay selected from the group consisting of:
(a) polymerase chain reaction (PCR),
(b) ligase chain reaction,
(c) nucleic acid sequence-based amplification,
(d) self-sustained sequence replication,
(e) strand displacement amplification,
(f) branched DNA signal amplification,
(g) nested PCR, and
(h) multiplex PCR.
9. The method of claim 8 , wherein said nucleic acid amplification assay comprises PCR.
10. The method of claim 8 , wherein said nucleic acid amplification assay comprises multiplex PCR.
11. A composition for the detection of a vancomycin resistant pathogen in a sample using a nucleic acid amplification assay, comprising:
an amplification primer pair, said amplification primer pair comprising an oligonucleotide consisting of SEQ ID NO: 1096 or the complement thereof, or variants of SEQ ID NO: 1096, wherein said variants optionally have up to three nucleotide changes compared to SEQ ID NO: 1096, wherein said variants are capable of hybridizing to and amplifying nucleic acids from a vanB gene in said nucleic acid amplification assay.
12. The composition of claim 11 , wherein said amplification primer pair further comprises an oligonucleotide consisting of SEQ ID NO: 1095 or the complement thereof, or variants of SEQ ID NO: 1095, wherein said variants optionally have up to three nucleotide changes compared to SEQ ID NO: 1095, wherein said variants are capable of hybridizing to and amplifying nucleic acids from a vanB gene in said nucleic acid amplification assay.
13. The composition of claim 12 , wherein each oligonucleotide optionally includes a detectable moiety.
14. The composition of claim 11 , further comprising a probe comprising an oligonucleotide that hybridizes to a portion of the vanB amplicon.
15. The composition of claim 14 , wherein said probe comprises a fluorescent moiety.
16. The composition of claim 15 , wherein said probe is a molecular beacon.
17. The composition of claim 11 , further comprising dideoxynucleotide triphosphates.
18. The composition of claim 11 , further comprising a polymerase.
19. The composition of claim 11 , further comprising a buffer.
20. The composition of claim 11 , further comprising an internal control nucleic acid.