Isothermal amplification based assay for the detection and quantitation of alpha-fetoprotein mRNA
First Claim
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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.
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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.
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Citations
19 Claims
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1. A method for the detection or quantitation of alpha-fetoprotein (AFP) mRNA in a sample, comprising:
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(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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8)
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9. An oligonucleotide selected from the group consisting of SEQ ID NO:
- 2, SEQ ID NO;
3 and SEQ ID NO;
7.
- 2, SEQ ID NO;
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10. 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 and SEQ ID NO;
3, wherein the oligonucleotide further comprises a RNA polymerase promoter sequence operatively attached to the 5′
end thereof. - View Dependent Claims (11)
- 26 nucleotides, comprising at least 10 consecutive nucleotides of a sequence selected from the group consisting of SEQ ID NO;
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12. 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. - View Dependent Claims (13, 14, 19)
- 26 nucleotides in length and comprising at least 10 consecutive nucleotides of a sequence selected from the group consisting of SEQ ID NO;
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15. 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. - View Dependent Claims (16, 17, 18)
- 2 and SEQ ID NO;
Specification