Internal control nucleic acids for amplification
Nucleic acids sequences that can be used for nucleic acid amplification, for example quantitative nucleic acid amplification, are provided herein.
1. A method for quantifying the amount of a polynucleotide in a sample, the method comprising:
combining a quantity of the polynucleotide with the sample, the polynucleotide comprising a sequence at least about 97% identical to the sequence of SEQ ID NO: 1;
contacting the polynucleotide with a forward primer and a reverse primer, wherein the forward primer comprises an oligonucleotide at least about 85% identical to SEQ ID NO: 3 or 5, and wherein the reverse primer comprises an oligonucleotide at least about 85% identical to SEQ ID NO: 4 or 6;
extending the forward primer and reverse primer, thereby producing at least one target amplicon; and
detecting a signal proportional to the quantity of the at least one target amplicon.
2. The method of claim 1 , wherein the forward primer is at least about 95% identical to SEQ ID NO: 3.
3. The method of claim 1 , wherein the reverse primer is at least about 95% identical to SEQ ID NO: 4.
4. The method of claim 1 , wherein the forward primer comprises SEQ ID NO: 3, and the reverse primer comprises SEQ ID NO: 4.
5. The method of claim 1 , wherein the forward primer comprises SEQ ID NO: 5, and the reverse primer comprises SEQ ID NO: 6.
6. The method of claim 1 , wherein detecting a signal proportional to the quantity of the at least one target amplicon comprises contacting the amplicon with a single-stranded oligonucleotide probe that anneals to the amplicon at a temperature of at least 50° C., wherein the probe is configured to emit a detectable signal following hybridization to a substantially complementary nucleic acid, but to not emit the detectable signal when it is single-stranded.
7. The method of claim 6 , wherein the single-stranded oligonucleotide probe comprises a sequence at least about 95% identical to SEQ ID NO: 2, or the reverse complement thereof.
8. The method of claim 6 , wherein the single-stranded oligonucleotide probe further comprises a least one fluorophore, and at least one quencher.
9. The method of claim 1 , further comprising extracting nucleic acid from the sample, wherein the polynucleotide is combined with the sample prior to extracting nucleic acid from the sample.
10. The method of claim 1 , wherein extending the forward primer and reverse primer comprises at least one of polymerase chain reaction (PCR), strand displacement amplification (SDA), loop-mediated isothermal amplification (LAMP), ligase chain reaction (LCR), immuno-amplification, transcription-mediated amplification (TMA), nucleic acid sequence based amplification (NASBA), self-sustained sequence replication (3SR), and rolling circle amplification.
11. The method of claim 1 , wherein the forward primer is at least about 95% identical to SEQ ID NO: 5.
12. The method of claim 1 , wherein the reverse primer is at least about 95% identical to SEQ ID NO: 6.