Isothermal amplification based assay for the detection and quantitation of alpha-fetoprotein mRNA

• US 20040076962A1
• Filed: 10/22/2002
• Published: 04/22/2004
• Est. Priority Date: 10/22/2002
• Status: Active Grant

• Current Assignee: Biomerieux Incorporated (Compagnie Merieux Alliance SAS)
• Original Assignee: Biomerieux Incorporated (Compagnie Merieux Alliance SAS)
• Inventors: Lee, Eun Mi, Shurtliff, Roxanne N.
• Granted Patent: US 7,074,561 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2531/143 Promoter based amplification, e.g. NASBA, 3SR, TAS
• CPC Class Codes: C12Q 2600/158 Expression markers
• CPC Class Codes: Y10S 435/81 Packaged device or kit

Abstract

The present invention is directed to isothermal transcription based assays for the detection and quantification of alpha-fetoprotein (AFP) mRNA. The present invention is also directed to oligonucleotides for amplifying AFP mRNA and probes for use in the detection and quantification of the amplification product. The present invention is also directed to detecting hepatocellular carcinoma, metastasis thereof and tumor recurrence by analyzing peripheral blood for the presence of AFP mRNA.

Claims

1. A method for the detection or quantitation of alpha-fetoprotein (AFP) mRNA in a sample, comprising:

(a) obtaining a sample which may contain AFP mRNA;

(b) performing isothermal transcription based amplification on the sample with two oligonucleotide primers, a first primer which comprises at least 10 consecutive nucleotides of a sequence selected from the group consisting of SEQ ID NO;

2 and SEQ ID NO;

3, and a second primer which comprises at least 10 consecutive nucleotides of a sequence selected from the group consisting of SEQ ID NO;

6 and SEQ ID NO;

7; and

(c) detecting or quantitating the amplification product of step (b) whereby detection or quantitation of the amplification product indicates the presence or quantity of AFP mRNA in the sample.

2. The method of claim 1, wherein detection of the amplification product uses a labeled wild-type probe comprising a sequence according to SEQ ID NO:

8, whereby hybridization of the wild-type probe to the amplification product indicates the presence of AFP mRNA in the sample.

3. The method of claim 2, further comprising adding a known amount of control RNA Q prior to step (b), and detecting amplification product of Q by using a labeled probe comprising the sequence of SEQ ID NO:

10, whereby the quantity of AFP mRNA in the sample is calculated by comparing the signals of the probes for Q and the wild-type probe.

4. The method of claim 2, wherein detection of the amplification product further uses a capture probe according to SEQ ID NO:

9.

5. The method of claim 1, wherein the sample comprises cells and RNA is extracted from the cells in the sample prior to step (b).

6. The method of claim 1, wherein the first primer further comprises a RNA polymerase promoter sequence operably attached to the 5′

end thereof.

7. The method of claim 6, wherein the RNA polymerase promoter sequence is a T7 RNA polymerase promoter as set forth in SEQ ID NO:

1.

8. The method of claim 1, wherein the isothermal transcription based amplification is nucleic acid sequence based amplification (NASBA).

9. An oligonucleotide selected from the group consisting of SEQ ID NO:

2, SEQ ID NO;

3, SEQ ID NO;

6, SEQ ID NO;

7, SEQ ID NO;

8, SEQ ID NO;

9 and SEQ ID NO;

10.

10. The oligonucleotide of claim 9, wherein the oligonucleotide is selected from the group consisting of SEQ ID NO:

2, SEQ ID NO;

3, SEQ ID NO;

6 and SEQ ID NO;

7.

11. An oligonucleotide of about 15-26 nucleotides, comprising at least 10 consecutive nucleotides of a sequence selected from the group consisting of SEQ ID NO:

2, SEQ ID NO;

3, SEQ ID NO;

6, SEQ ID NO;

7, SEQ ID NO;

8, SEQ ID NO;

9 and SEQ ID NO;

10.

12. The oligonucleotide of claim 11, wherein the oligonucleotide is selected from the group consisting of SEQ ID NO:

2, SEQ ID NO;

3, SEQ ID NO;

6 and SEQ ID NO;

7.

13. The oligonucleotide of claim 11, wherein the oligonucleotide is selected from the group consisting of SEQ ID NO:

2 and SEQ ID NO;

3, and the oligonucleotide further comprises a RNA polymerase promoter sequence operatively attached to the 5′

end thereof.

14. The oligonucleotide of claim 13, wherein the RNA polymerase promoter sequence is a T7 RNA polymerase promoter as set forth in SEQ ID NO:

1.

15. A pair of oligonucleotides for the detection or quantitation of AFP mRNA, a first oligonucleotide of said pair being about 15-26 nucleotides in length and comprising at least 10 consecutive nucleotides of a sequence selected from the group consisting of SEQ ID NO:

2 and SEQ ID NO;

3, and a second oligonucleotide of said pair being about 15-26 nucleotides in length and comprising at least 10 consecutive nucleotides of a sequence selected from the group consisting of SEQ ID NO;

6 and SEQ ID NO;

7.

16. The pair of oligonucleotides of claim 15, wherein the first oligonucleotide further comprises a RNA polymerase promoter sequence operably attached to the 5′

end thereof.

17. The pair of oligonucleotides of claim 16, wherein the RNA polymerase promoter sequence is a T7 RNA polymerase promoter as set forth in SEQ ID NO:

1.

22. A kit for the detection or quantitation of AFP mRNA in a sample, comprising a pair of oligonucleotides of claim 15.

18. A primer pair for the detection or quantitation of AFP mRNA in a sample, comprising a first primer selected from the group consisting of SEQ ID NO:

2 and SEQ ID NO;

3, and a second primer selected from the group consisting of SEQ ID NO;

6 and SEQ ID NO;

7.

19. The primer pair of claim 18, wherein the first primer further comprises a RNA polymerase promoter sequence operably attached to the 5′

end thereof.

20. A kit for the detection or quantitation of AFP mRNA in a sample, comprising the primer pair of claim 18 and at least one probe selected from the group consisting of SEQ ID NO:

8 and SEQ ID NO;

10.

21. The kit of claim 20, further comprising a capture probe according to SEQ ID NO:

9.