Composition and method for determination of CK19 expression
원문보기
IPC분류정보
국가/구분
United States(US) Patent
등록
국제특허분류(IPC7판)
C12Q-001/68
C07H-021/04
출원번호
US-0031051
(2013-09-19)
등록번호
US-9593374
(2017-03-14)
우선권정보
GR-20050100430 (2005-08-17)
발명자
/ 주소
Evriklia, Lianidou
Stathopoulou, Aliki
Mavroudis, Dimitrios
Georgoulias, Vasileios
출원인 / 주소
Evriklia, Lianidou
대리인 / 주소
Fedrick, Michael F.
인용정보
피인용 횟수 :
0인용 특허 :
104
초록▼
Disclosed is a method for quantitative determination of CK-19 mRNA positive cells in a biological sample. The method can be used, for instance, with peripheral blood to detect cancer in a patient. In one embodiment, the method can be used to detect the cancer before initiation of adjuvant treatment,
Disclosed is a method for quantitative determination of CK-19 mRNA positive cells in a biological sample. The method can be used, for instance, with peripheral blood to detect cancer in a patient. In one embodiment, the method can be used to detect the cancer before initiation of adjuvant treatment, thereby providing information about therapeutic efficacy. Practice of the invention method is sensitive, reliable, and easy to perform.
대표청구항▼
1. A primer pair consisting of one primer having the sequence of SEQ ID NO: 1 and one primer having the sequence of SEQ ID NO: 2. 2. The primer pair according to claim 1, wherein the primers comprise nucleotides, analogues of nucleotides or labels. 3. A method of quantitatively determining the prese
1. A primer pair consisting of one primer having the sequence of SEQ ID NO: 1 and one primer having the sequence of SEQ ID NO: 2. 2. The primer pair according to claim 1, wherein the primers comprise nucleotides, analogues of nucleotides or labels. 3. A method of quantitatively determining the presence in a test sample of a mRNA derived from a gene comprising at least one intron using a pair of primers, wherein at least one of said primers comprises at least one intron-spanning site comprising the steps of (i) forming a reaction mixture comprising nucleic acid amplification reagents, the primer pair of claim 1 and a test sample;(ii) subjecting the mixture to amplification conditions to generate at least one copy of a nucleic acid sequence complementary to the target sequence; and(iii) determining the amount of the mRNA in the sample using real-time monitoring during PCR. 4. The method according to claim 3, wherein the test sample is subjected to reverse transcription prior to forming the reaction mixture of step (i). 5. The method according to claim 3, wherein the test sample is selected from a blood sample, a sample from the bone marrow and a sample derived from the lymph nodes. 6. The method according to claim 5, wherein the sample is a blood sample. 7. The method according to claim 6, wherein the blood sample is centrifuged before forming the reaction mixture. 8. The method according to claim 3, wherein the reaction mixture of step (i) further comprises a housekeeping primer pair. 9. The method according to claim 8, wherein the housekeeping primer pair has the sequence according to SEQ ID NO:5 and SEQ ID NO: 6. 10. A method of determining the prospects of adjuvant therapy in a patient suffering from cancer comprising the steps of (i) providing a biological sample from the patient; (ii) isolating nucleic acids from the biological sample; (iii) optionally reverse transcribing the isolated nucleic acids, when the origin of the nucleic acid is RNA; (iv) forming a reaction mixture comprising nucleic acid amplification reagents, the primer pair of claim 1 and an aliquot of the nucleic acids isolated in step (ii) or the reverse transcribed nucleic acid of step (iii); (v) subjecting the mixture to amplification conditions to generate at least one copy of a nucleic acid sequence complementary to the target sequence; (vi) quantitating the CK-19 mRNA positive cells in the sample using real-time monitoring during PCR; and (vii) based on the amount of CK-19 mRNA positive cells in the sample determining the prospects of adjuvant therapy. 11. The method according to claim 10, wherein the cancer is breast cancer. 12. The Diagnostic method according to claim 10, wherein the biological sample is a blood sample. 13. The method according to claim 10, wherein the reaction mixture of step (iv) further comprises a housekeeping primer pair. 14. The method according to claim 13, wherein the housekeeping primer pair has the sequence according to SEQ ID NO:5 and SEQ ID NO: 6. 15. A diagnostic kit for amplifying a CK19 target sequence comprising: (i) the primer pair of claim 1;(ii) optionally sequences hybridizing to additional markers on cancer cells;(iii) amplification reagents. 16. The diagnostic kit according to claim 15, wherein the amplification reagents comprise the hybridization probes of SEQ ID NO: 3 and SEQ ID NO: 4. 17. The diagnostic kit according to claim 15, wherein the kit further comprises a housekeeping primer pair having the sequences according to SEQ ID NO: 5 and SEQ ID NO: 6. 18. The diagnostic kit according to claim 15, wherein the amplification reagents and the primer pair(s) are lyophilized. 19. A method of determining presence of CK-19 mRNA in a biological sample, the method comprising the following steps: a) separating any mononuclear cells from the biological fluid,B) contacting the separated mononuclear cells with an antibody that specifically binds antigen expressed by the mononuclear cells (or antigen binding fragment thereof), wherein the antibody (or fragment) is bound to a solid support, the contacting being sufficient to form a binding complex between the cells, the antibody (or fragment) and solid support,c) separating the complex from any unbound material,d) isolating nucleic acid from endothelial mononuclear cells bound to the complex,e) forming a reaction mixture comprising nucleic acid amplification reagents, the primer pair of claim 1 and the nucleic acid,f) subjecting the mixture to amplification conditions to generate at least one copy of a nucleic acid sequence complementary to the CK-19 target sequence; andg) detecting CK-19 mRNA in the biological sample using PCR. 20. The method of claim 19, wherein the antibody is a monoclonal that specifically binds a glycoprotein expressed by the endothelial mononuclear cells. 21. The method of claim 20, wherein the monoclonal antibody is Ber-EP4. 22. The method of claim 19, wherein the biological fluid is peripheral blood.
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