Highly conserved genes and their use to generate probes and primers for detection of microorganisms
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 capable of detecting and identifying E. coli and Shigella bacteria in a sample using a nucleic acid amplification assay, said composition comprising:
an amplification primer pair comprising a first and a second oligonucleotide, wherein said primer pair is adapted to amplify a region of the E. coli and Shigella DNA between and including said first and second oligonucleotides to produce a detectable amplification product that consists of sequences from the tuf gene, wherein said first oligonucleotide is, or is fully complementary to, SEQ ID NO: 1661 and optionally includes one degenerate nucleotide, or said first oligonucleotide consists of at least 15 contiguous nucleotides of SEQ ID NO: 1661 or the complement thereof, and wherein said second oligonucleotide is, or is fully complementary to SEQ ID NO: 1665 and optionally includes one degenerate nucleotide, or said second oligonucleotide consists of at least 15 contiguous nucleotides of SEQ ID NO: 1665 or the complement thereof.
2. The composition of claim 1 , wherein said first and second oligonucleotides are SEQ ID NOs: 1661 and 1665.
3. The composition of claim 2 , further comprising a probe, wherein said probe hybridizes to the amplification product under a set of conditions that is the same as the condition used in the nucleic acid amplification assay.
4. The composition of claim 3 , wherein said probe comprises a detectable label.
5. The composition of claim 4 , wherein said detectable label is fluorescent.
6. The composition of claim 1 , further comprising a DNA polymerase.
7. A method of detecting and identifying E. coli and Shigella bacteria in a sample, comprising:
providing said sample;
contacting said sample with the composition of claim 1 under conditions that allow amplification of the region of E. coli and Shigella DNA between and including the first and second oligonucleotides in the presence of E. coli and Shigella DNA in the sample, to generate an amplification product; and
testing for the presence of the amplification product, wherein the presence of the amplification product is indicative of the presence of E. coli or Shigella in the sample.
8. The method of claim 7 , wherein said testing comprises:
contacting said sample with a probe comprising a detectable label, wherein contacting the probe to the amplification product is performed under a set of conditions that is the same as the condition used in the nucleic acid amplification step; and measuring the amount of probe hybridized to the amplification product.
9. The method of claim 8 , further comprising:
recording the amount of probe hybridized to the amplification product.
10. The method of claim 7 , wherein said first and second oligonucleotides are SEQ ID NOs: 1661 and 1665.
11. The method of claim 8 , wherein said detectable label is fluorescent.