Polynucleotides for the amplification and detection of chlamydia trachomatis
The invention provides methods and compositions for the detection of Chlamydia trachomatis in a test sample. Its presence or absence in the sample is determined by nucleic acid based testing methods using primers and/or probes and or molecular beacons that bind to the 23S ribosomal genes or gene transcripts.
1. A molecular beacon comprising a fluorophore, a quencher, and a polynucleotide, wherein the polynucleotide comprises a sequence selected from the group consisting of SEQ ID NOs: 97-101 and 103-130.
2. The composition of claim 1 , wherein the polynucleotide sequence consists of SEQ ID NO: 115.
3. The molecular beacon of claim 2 , wherein the fluorophore is FAM and the quencher is BHQ1.
4. The molecular beacon of claim 1 , wherein the fluorophore is FAM and the quencher is BHQ1.
5. The molecular beacon of claim 1 , wherein the polynucleotide consists of a sequence selected from the group consisting of SEQ ID NOs: 97-101 and 103-130.
6. The molecular beacon of claim 1 , wherein the fluorophore is ATTO 565 or Alexa 594 and the quencher is BHQ1 or BHQ2.
7. A method of detecting Chlamydia trachomatis in a test sample, the method comprising:
(a) extracting nucleic acid from the test sample;
(b) amplifying a target sequence by reacting the nucleic acid extracted in step (a) with a reaction mixture comprising a strand displacement DNA polymerase and a sequence-specific primer set, wherein said sequence-specific primer set comprises a forward inner primer comprising the nucleotide sequence of SEQ ID NO: 83 and a backward inner primer comprising the nucleotide sequence of SEQ ID NO: 84; and
(c) detecting the presence or absence of an amplified product of step (b); wherein detecting the presence or absence of the amplified product comprises hybridizing the amplified product with a molecular beacon comprising a fluorophore, a quencher, and a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 97-101 and 103-130; and further wherein the presence of said amplification product is indicative of the presence of Chlamydia trachomatis in the test sample.
8. The method of claim 7 , wherein the amplification in step (b) of the target sequence is performed at between about 60° C. and about 67° C. for less than fifteen minutes.
9. The method of claim 8 , wherein the amplification step is performed for less than ten minutes.
10. The method of claim 7 , wherein the reaction mixture further comprises a reverse transcriptase.
11. The method of claim 7 , wherein the polynucleotide sequence of the molecular beacon is SEQ ID NO: 115.
12. The method of claim 7 , wherein Chlamydia trachomatis is present in the test sample at a concentration of ≤100 IFU/mL.
13. The method of claim 12 , wherein Chlamydia trachomatis is present in the test sample at a concentration of ≤5 IFU/ml and the amplification step is performed for less than 15 minutes.
14. The method of claim 12 , wherein Chlamydia trachomatis is present in the test sample at a concentration of ≤10 IFU/ml and the amplification step is performed for less than six minutes.
15. The method of claim 7 , wherein the fluorophore is FAM and the quencher is BHQ1.
16. The method of claim 7 , wherein the sequence-specific primer set further comprises a forward outer primer comprising the nucleotide sequence of SEQ ID NO: 81 and a backward outer primer comprising the nucleotide sequence of SEQ ID NO: 82.
17. The method of claim 16 , wherein the sequence-specific primer set further comprises a forward loop primer comprising the nucleotide sequence of SEQ ID NO: 85 and a backward loop primer comprising the nucleotide sequence of SEQ ID NO: 86.