Detection and typing of human papillomavirus using PNA probes
View Patent ↗The invention provides methods for detection and typing of HPV infection using PNA probes. More specifically, methods are provided for detecting high-risk types of HPV infection with minimal numbers of PNA probes or using PNA probes to selectively amplify only high-risk types of HPV.
1. A method for detecting the presence of a target nucleic acid of a human papilloma virus (HPV) in a sample cell, comprising the steps of:
suspending a sample cell that may comprise a target nucleic acid of a HPV in a cell preservative solution
separating said a target nucleic acid from said sample cell wherein said separated target nucleic acid remains in said cell preservative solution wherein said cell preservative solution comprises an alcohol in an amount sufficient to preserve said sample cells without coagulation;
contacting said target nucleic acid in said cell preservative solution with at least one probe comprising at least one peptide nucleic acid (PNA), said at least one probe being substantially complementary to portions of nucleic acids of multiple HPV types;
allowing said at least one probe to hybridize with said target nucleic acid, if present, in said cell preservative solution; and
detecting hybridization between said at least one probe and said target nucleic acid in said solution;
wherein the detection of said hybridization is indicative of the presence of a target nucleic acid of a HPV in a sample cell.
2. The method of claim 1 , wherein said sample cells come from a subject and wherein the presence of said target nucleic acid sequence indicates a risk of tumor growth in said subject.
3. The method of claim 2 , wherein said tumor growth is associated with either cervical or endocervical carcinoma.
4. The method of claim 1 , wherein the presence of said target nucleic acid sequence is indicative of the presence of a particular type of HPV.
5. The method of claim 4 , wherein said particular type of HPV is selected from the group consisting of types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68 and 70.
6. The method of claim 1 , wherein absence of said target nucleic acid sequence is diagnostic of absence of infection by HPV.
7. The method of claim 1 , wherein absence of said target nucleic acid sequence is diagnostic of absence of infection by HPV types selected from the group consisting of types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68 and 70.
8. The method of claim 1 , wherein absence of said target nucleic acid sequence is diagnostic of absence of infection by high-risk types of HPV.
9. The method of claim 1 , further comprising amplification of said target nucleic acid.
10. The method of claim 9 , wherein said amplification step comprises conducting a polymerase chain reaction.
11. The method of claim 1 , further comprising capturing said target nucleic acid onto a solid support through PNA-DNA interaction.
12. The method of claim 1 , wherein each of said at least one probe comprises at least 8 bases.
13. The method of claim 1 , wherein said at least one probe comprises a nucleotide different from PNA.
14. The method of claim 1 , wherein said at least one probe is selected from the group consisting of SEQ. ID. NOS. 1-5.
15. The method of claim 1 , wherein said at least one probe is labeled with a detectable marker.
16. The method of claim 1 wherein said at least one probe comprises a molecular beacon probe.
17. The method of claim 1 , further comprising using an antibody to recognize said hybridization.
18. The method of claim 1 , wherein said cell preservative further comprises, an anti-clumping agent, and a buffer agent that maintains said solution at a pH within a range of about 4 to about 7.