Oligonucleotides for detecting human papilloma virus in a test sample
View Patent ↗Oligonucleotides targeted to HPV Type 16 and/or Type 18 nucleic acid sequences which are particularly useful to aid in detecting HPV type 16 and or 18 are described. The oligonucleotides can aid in detecting HPV Type 16 and/or Type 18 in different ways such as by acting as hybridization assay probes, helper probes, and/or amplification primers.
1. A composition for amplification and detection of mRNA of HPV type 16 comprising:
amplification oligonucleotides selected from the group consisting of
a promoter-primer made up of a 5′ promoter sequence covalently linked to a 3′ target hybridizing region consisting of SEQ ID NO:89 and a primer consisting of SEQ ID NO:21 , and
a promoter-primer made up of a 5′ promoter sequence covalently linked to a 3′ target hybridizing region consisting of SEQ ID NO:21 and a primer consisting of SEQ ID NO:13, and
a detection probe oligonucleotide consisting of the sequence of SEQ ID NO:25 or the completely complementary sequence of SEQ ID NO:25.
2. A kit comprising the composition of claim 1 .
3. The kit of claim 2 further comprising a detection probe consisting of SEQ ID NO:29.
4. The kit of claim 2 further comprising a detection probe oligonucleotide consisting of SEQ ID NO:17.
5. The composition of claim 1 wherein the promoter-primer is made up of a 5′ promoter consisting of SEQ ID NO:97 covalently linked to the 3′ target hybridizing region consisting of SEQ ID NO:89, the primer consists of SEQ ID NO:21, and the detection probe is labeled with an acridinium ester label.
6. The composition of claim 1 , wherein the promoter-primer is made up of a 5′ promoter sequence consisting of SEQ ID NO:97 covalently linked to the 3′ target hybridizing region consisting of SEQ ID NO:21, the primer consists of SEQ ID NO:13, and the detection probe consists of SEQ ID NO:25 labeled with an acridinium ester.
7. The composition of claim 1 , wherein the detection probe oligonucleotide is labeled with a non-isotopic label selected from the group consisting of fluorescent molecules, chemiluminescent molecules, enzymes, co-factors, enzyme substrates, haptens or other ligands.
8. The composition of claim 1 , wherein the detection probe oligonucleotide is labeled with a chemiluminescent molecule.
9. A method of detecting a HPV Type 16 nucleic acid in a sample comprising the steps of:
a) contacting a sample containing HPV type 16 nucleic acid with a mixture of amplification oligonucleotides consisting of a promoter-primer made up of a 5′ promoter sequence covalently linked to a 3′ target hybridizing region consisting of SEQ ID NO:21and a primer consisting of SEQ ID NO:13;
b) amplifying the HPV type 16 nucleic acid in an in vitro amplification reaction that includes at lease one polymerase that acts with an amplification oligonucleotide in the mixture to produce one or more HPV type 16 amplification products by using the HPV type 16 nucleic acid as a template;
c) contacting the HPV type 16 amplification products with at least one hybridization assay probe consisting of SEQ ID NO:25 that forms a detectable SEQ ID NO:25 probe: HPV type 16 target hybrid under stringent hybridization conditions; and
d) detecting a signal from the SEQ ID NO:25 probe: MN type 16 target hybrid, thereby indicating the presence of HPV type 16 in the sample.
10. The method of claim 9 , further comprising additional steps after the amplifying step that include a contacting step wherein the HPV type 16 amplification products are contacted with a hybridization assay probe consisting of SEQ ID NO:29 to form a detectable SEQ ID NO:29 probe: HPV type 16 target hybrid under stringent hybridization conditions, and a detecting step that detects a signal from the SEQ ID NO:29 probe: HPV type 16 target hybrid, thereby indicating the presence of an amplification product made from an HPV type 16 E6 mRNA.
11. The method of claim 10 , wherein the detecting step detects a signal from a chemiluminescent label attached to the probe in the SEQ ID NO:29probe:
HPV type 16 target hybrid.
12. The method of claim 9 , further comprising additional steps after the amplifying step that include a contacting step wherein the HPV type 16 amplification products are contacted with a hybridization assay probe consisting of SEQ ID NO:17 to form a SEQ ID NO:17 detectable probe: HPV type 16 target hybrid under stringent hybridization conditions, and a detecting step that detects a signal from the SEQ ID NO:17 probe: HPV type 16 target hybrid, thereby indicating the presence of an amplification product made from an HPV type 16 E6** mRNA.
13. The method of claim 12 , wherein the detecting step detects a signal from a chemiluminescent label attached to the probe in the SEQ ID NO:17probe:
HPV type 16 target hybrid.
14. The method of claim 9 , wherein the promoter-primer is made up of a 5′ promoter sequence consisting of SEQ ID NO:97 covalently linked to the 3′ target hybridizing region.
15. The method of claim 9 , wherein the detecting step detects a signal from a chemiluminescent label attached to the probe in the SEQ ID NO:25 probe: HPV type 16 target hybrid.
16. A method of detecting a HPV Type 16 nucleic acid in a sample comprising the steps of:
a) contacting a sample containing HPV type 16 nucleic acid with a mixture of amplification oligonucleotides consisting of a promoter-primer made up of a 5′ promoter sequence covalently linked to a 3′ target hybridizing region consisting of SEQ ID NO:89and a primer consisting of SEQ ID NO:21;
b) amplifying the HPV type 16 nucleic acid in an in vitro amplification reaction that includes at lease one polymerase that acts with an oligonucleotide in the mixture to produce one or more HPV type 16 amplification products by using the HPV type 16 nucleic acid as a template;
c) contacting the HPV type 16 amplification products with at least one hybridization assay probe consisting of a completely complementary sequence of SEQ ID NO:25 that binds to an amplification product and forms a detectable probe: HPV type 16 target hybrid under stringent hybridization conditions; and
d) detecting a signal from the probe: HPV type 16 target hybrid, thereby indicating the presence of HPV type 16 in the sample.
17. The method of claim 16 , wherein the detecting step detects a signal from a non-isotopic label attached to the probe in the probe: HPV type 16 target hybrid.
18. The method of claim 17 , wherein the detecting step detects a signal from a chemiluminescent label attached to the probe in the probe: HPV type 16 target hybrid.