Compositions and methods for detecting
Four highly conserved genes, encoding translation elongation factor Tu, translation elongation factor G, the catalytic subunit of proton-translocating ATPase and the RecA recombinase, are used to generate species-specific, genus-specific, family-specific, group-specific and universal nucleic acid probes and amplification primers to rapidly detect and identify algal, archaeal, bacterial, fungal and parasitical pathogens from clinical specimens for diagnosis. The detection of associated antimicrobial agents resistance and toxin genes are also under the scope of the present invention.
1. A composition for the detection of Klebsiella pneumoniae in a sample using a nucleic acid amplification assay, comprising an amplification primer pair, said amplification primer pair consisting of an oligonucleotide consisting of SEQ ID NO: 1331 or the complement thereof, and an oligonucleotide consisting of SEQ ID NO: 1332 or the complement thereof, or variants of either SEQ ID NO: 1331 or 1332, or both,
wherein said variants differ from SEQ ID NO: 1331 or 1332 in that they have up to three nucleotide changes compared to SEQ ID NO:1331 or 1332,
wherein said variants are capable of hybridizing to and amplifying K. pneumoniae nucleic acids in said nucleic acid amplification assay
and wherein each oligonucleotide optionally includes a detectable moiety.
2. The composition of claim 1 , further comprising a probe that hybridizes to a portion of the atpD gene amplified by said amplification primer pair.
3. The composition of claim 2 , wherein said probe comprises a fluorescent moiety.
4. The composition of claim 2 , wherein said probe is a molecular beacon.
5. A method of detecting Klebsiella pneumoniae in a sample comprising:
a) contacting the sample with the composition according to claim 1 ;
b) amplifying target nucleic acid in the sample of a) to generate amplification product(s); and
c) detecting the presence or amount of amplification product(s) as an indication of the presence of the Klebsiella pneumoniae in said sample.
6. The method of claim 5 , wherein amplification step comprises a method 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.
7. The method of claim 6 , wherein said amplification step comprises PCR.
8. The method of claim 5 , further comprising contacting the sample with a probe that hybridizes to a portion of the atpD gene amplified by said amplification primer pair.
9. The method of claim 8 , wherein said probe comprises a fluorescent moiety.