Method for assaying nucleic acids by fluorescence

1. A method for determining the amount of nucleic acid present in a biological sample, comprising the steps of:

adding to the biological sample a single fluorophore capable of interacting with the nucleic acid wherein the fluorophore is ([N-bis-(3-dimethylaminopropyl)-amino]-4-[2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methyllidene]-1-phenyl-quinolinium]+);

measuring fluorescence intensities, I 1 , I 2 and, optionally I 3 , emitted by the single fluorophore at at least two emission wavelengths, λ 1 , λ 2 and, optionally a third emission wavelength λ 3 , in response to light stimulations at at least two excitation wavelengths, λ′ 1 , λ′ 2 and, optionally a third excitation wavelength λ′ 3 , respectively, wherein the difference (delta-lambda) between each excitation wavelength and corresponding emission wavelength is 30 nm;

deducing the amount of nucleic acid present in the sample from the at least two measured fluorescence intensities, wherein the deducing step comprises calculating a value F according to at least one of the formulas:

F

=



I

2

-

I

1



⁢

⁢

and

⁢

/

⁢

or

⁢

⁢

F

=

I

2

-

[

I

1

-

I

3

λ

1

-

λ

3

⁢

λ

2

-

λ

1

⁢

I

3

-

λ

3

⁢

I

1

λ

1

-

λ

3

]

,

and determining the amount of nucleic acid in the sample from the value of F by using a calibration curve.

2. The method according to claim 1 , wherein the nucleic acid is DNA or RNA.

3. The method according to claim 1 , wherein the sample is is diluted from 1/20 to 1/400 in a buffer.

4. The method of claim 3 , wherein the buffer is a TRIS-Borate-EDTA (TBE) buffer.

5. The method according to claim 1 , wherein the sample originates from a patient and the method determines whether the patient has suffered cellular disruption of a physio-pathological origin.

6. The method according to claim 1 , wherein the fluorophore is a nucleic acid intercalating agent.

7. The method according to claim 1 , wherein the fluorescence intensities (I 1 , I 2 and I 3 ), emitted by the fluorophore are measured at three emission wavelengths λ 1 , λ 2 and λ 3 in response to light stimulations at three excitation wavelengths, λ′ 1 , λ′ 2 and λ′ 3 , respectively, with λ 1 <λ 2 <λ 3 and λ 1 , λ 2 and λ 3 being predetermined.

8. The method according to claim 7 , wherein the amount of nucleic acid is deduced from the following value of F:

F

=

I

2

-

[

I

1

-

I

3

λ

1

-

λ

3

⁢

λ

2

-

λ

1

⁢

I

3

-

λ

3

⁢

I

1

λ

1

-

λ

3

]

.

9. The method according to claim 8 , wherein the amount of nucleic acid in the sample is deduced from the value of F using a calibration curve.

10. The method according to claim 7 , wherein the fluorophore is ([N-bis-(3-dimethylaminopropyl)-amino]-4-[2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methyllidene]-1-phenyl-quinolinium]+) and the wavelengths are as follows:

λ′ 1 =472±10 nm λ 1 =502±10 nm

λ′ 2 =496±10 nm λ 2 =526±10 nm

λ′ 3 =538±10 nm λ 3 =568±10 nm.

11. The method according to claim 1 , wherein the fluorescence intensities, I 1 and I 2 , emitted by the fluorophore at two emission wavelengths λ 1 and λ 2 in response to light stimulations at two predetermined excitation wavelengths, λ′ 1 and λ′ 2 , respectively are measured.

12. The method according to claim 11 , wherein the amount of nucleic acid is deduced from the absolute value of the difference between I 1 and I 2 , i.e. the following value of F:

F=|I 2 −I 1 |.

13. The method according to claim 11 , wherein the fluorophore is ([N-bis-(3-dimethylaminopropyl)-amino]-4-[2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methyllidene]-1-phenyl-quinolinium]+) and the wavelengths are as follows:

λ′ 1 =472±10 nm λ 1 =502±10 nm, and

λ′ 2 =496±10 nm λ 2 =526±10 nm, or

λ′ 1 =496±10 nm λ 1 =526±10 nm, and

λ′ 2 =538±10 nm λ 2 =568±10 nm.

14. The method of claim 1 , wherein the biological sample is a serum or plasma sample from a patient.

15. The method of claim 14 , wherein the patient is selected from the group consisting of a patient suffering or suspected of suffering from a cancer, a patient undergoing chemotherapy, a patient having undergone a surgical operation, a traumatised patient and a patient having undergone a myocardial infarction.

16. The method of claim 15 , wherein the patient is a cancer patient.

17. The method of claim 14 , further comprising obtaining a serum or plasma sample from the patient, wherein the method is performed without amplifying, purifying or isolating said nucleic acid in said sample.

18. The method of claim 1 , wherein the method is performed without amplifying, purifying or isolating said nucleic acid in said sample.