Methods for detecting, enumerating, quantifying, classifying and/or identifying total organismal DNA in a sample
First Claim
1. A pair of oligonucleotide primers useful for amplifying a target nucleotide sequence that is substantially conserved among organismal nucleic acids, wherein the forward primer is SEQ ID NO. 1 or a sequence having at least 80% identity to SEQ ID NO. 1 or a sequence capable of hybridizing to SEQ ID NO. 1 under low stringency conditions and the reverse primer is SEQ ID NO. 2 or a sequence having at least 80% identity to SEQ ID NO. 2 or a sequence capable of hybridizing to SEQ ID NO. 2 under low stringency conditions.
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Abstract
A universal primer pair (SEQ. ID 1 and 2) is used to amplify total organismal DNA in a sample. Probes from any or all three taxonomic domains may be used to further classify the amplified DNA by organelle (e.g., chloroplast) or by taxonomic DNA levels (i.e., kingdom, phylum, class, order, or family) in relation to total DNA. The universal primer pair amplifies regions of the 16s (eukaryotic 18s) ribosomal DNA gene by hybridizing to regions highly conserved among all organismal DNA. Less conserved regions within the amplicon produced by the universal primers are targeted by probes, so that a general taxonomic breakdown of the DNA present can be determined. The primers and probes of the present invention are conveniently used with real-time PCR or similar or related technology to detect and enumerate any organismal DNA from the three major taxonomic groups (bacteria, archaea and eucarya) and enable improved methods of environmental surveillance and quick identification of unknown biological material.
10 Citations
33 Claims
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1. A pair of oligonucleotide primers useful for amplifying a target nucleotide sequence that is substantially conserved among organismal nucleic acids, wherein the forward primer is SEQ ID NO. 1 or a sequence having at least 80% identity to SEQ ID NO. 1 or a sequence capable of hybridizing to SEQ ID NO. 1 under low stringency conditions
and the reverse primer is SEQ ID NO. 2 or a sequence having at least 80% identity to SEQ ID NO. 2 or a sequence capable of hybridizing to SEQ ID NO. 2 under low stringency conditions.
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2. An oligonucleotide probe useful for detecting 16s(18s) ribosomal DNA nucleic acid from an organism, of sequence (SEQ ID NO. 3), (SEQ ID NO. 4), (SEQ ID NO. 5), (SEQ ID NO. 6), (SEQ ID NO. 7), (SEQ ID NO. 8), or a sequence fully complementary to one of said sequences.
- 3. A kit useful for detecting and enumerating total DNA content in a biological sample, comprising a first nucleic acid sequence of SEQ ID NO. 1, and a second nucleic acid sequence of SEQ ID NO. 2.
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8. An oligonucleotide probe for detecting 16s(18s) ribosomal DNA from an organism which has the sequence (SEQ ID NO. 3), (SEQ ID NO. 4), (SEQ ID NO. 5), (SEQ ID NO. 6), (SEQ ID NO. 7), (SEQ ID NO. 8), (SEQ ID NO. 9), (SEQ ID NO. 10), (SEQ ID NO. 11), (SEQ ID NO. 12), (SEQ ID NO. 13), (SEQ ID NO. 14), (SEQ ID NO. 15), (SEQ ID NO. 16), (SEQ ID NO. 17), (SEQ ID NO. 18), (SEQ ID NO. 19), (SEQ ID NO. 20), (SEQ ID NO. 21), (SEQ ID NO. 22), (SEQ ID NO. 23), (SEQ ID NO. 24), or a sequence fully complementary to one of said sequences.
- 9. A method for detecting total organismal nucleic acid contained in a sample, comprising amplifying a region of nucleic acid(s) from a 16s(18s) ribosomal DNA gene, wherein the amplification is achieved by a polymerase chain reaction using a pair of primers having the sequence (SEQ ID NO. 1) and (SEQ ID NO. 2) or a sequence having at least 80% identity to one of said sequences or capable of hybridizing to said sequences under low stringency conditions.
- 14. A method for determining total DNA content in a sample, comprising amplifying a target nucleotide sequence using at least two primers complementary to sequences which are present in substantially all organismal species.
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22. A method according to 14 wherein the organismal DNA is amplified with a primer pair comprising a forward primer having the sequence set forth in SEQ ID NO:
- I or a sequence having at least about 80% identity thereto or a sequence capable of hybridizing to SEQ ID NO;
1 or its complementary form under low stringency conditions. - View Dependent Claims (23, 25, 27, 28, 29, 30, 31)
- I or a sequence having at least about 80% identity thereto or a sequence capable of hybridizing to SEQ ID NO;
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32. A combination comprising an oligonucleotide of (SEQ. ID NO 1) or a sequence having at least 80% identity to (SEQ. ID NO 1) or a sequence capable of hybridizing to (SEQ ID NO. 1) under low stringency conditions and
an oligonucleotide of (SEQ. ID NO 2), or a sequence having at least 80% identity to (SEQ. ID NO 2) or a sequence capable of hybridizing to (SEQ ID NO. 2) under low stringency conditions.
Specification