Method of quantifying polynucleotide analytes from dried samples
Presented are methods, systems, and software products useful for determining the concentration of an analyte in a fluid specimen used to produce a dried sample, where the dried sample serves as a source of the analyte in a detection and quantification procedure. Particularly illustrated is the use of dried blood spots for quantifying a polynucleotide analyte.
1. A method of quantifying a polynucleotide analyte present in a fluid blood sample that dried to produce a dried blood spot (DBS), the method comprising the steps of:
(a) performing a nucleic acid amplification reaction using the DBS as a source of templates to produce amplification products and obtain a measured result, the measured result A indicating a concentration or an amount of the polynucleotide analyte;
(b) multiplying the measured result by a correction factor to obtain a corrected result,
wherein the correction factor is the solution to an equation that specifies the correction factor as a function of the measured result, and
wherein the equation comprises a non-linear equation,
and (c) quantifying the polynucleotide analyte present in the fluid blood sample and recording the corrected result.
2. A method of quantifying a polynucleotide analyte present in a fluid blood sample that created a dried blood spot (DBS), the method comprising the steps of:
(a) performing a nucleic acid amplification reaction using the DBS as a source of templates to produce amplification products and obtain a measured result, the measured result indicating a concentration or an amount of the polynucleotide analyte;
(b) solving an equation to determine a correction factor,
wherein the equation specifies the correction factor as a function of the measured result, and
wherein the equation comprises a non-linear equation;
(c) multiplying the measured result by the correction factor to obtain a corrected result,
and (d) quantifying the polynucleotide analyte present in the fluid blood sample and recording the corrected result.
3. The method of claim 2 , wherein the non-linear equation comprises coefficients optimized in a mathematical curve fitting procedure to define a fitted curve.
4. The method of claim 3 , wherein the non-linear equation comprises four coefficients.
5. The method of claim 2 , wherein step (a) comprises performing with an automated nucleic acid analyzer that amplifies the polynucleotide analyte and detects amplification products as the nucleic acid amplification reaction is occurring.
6. The method of claim 5 , wherein the non-linear equation in step (b) is a non-linear equation prepared using results obtained from an automated nucleic acid analyzer different from the automated nucleic acid analyzer used for performing the nucleic acid amplification reaction in step (a).
7. The method of claim 2 , wherein step (a) comprises performing with an automated nucleic acid analyzer that isolates the polynucleotide analyte, and then amplifies the isolated polynucleotide analyte.
8. The method of claim 7 , wherein the automated nucleic acid analyzer further detects synthesis of amplification products as the nucleic acid amplification reaction is occurring.
9. The method of claim 2 , wherein the measured result indicates a concentration of the polynucleotide analyte in a plasma sample.
10. The method of claim 2 , wherein the nucleic acid amplification reaction is an isothermal nucleic acid amplification reaction.
11. The method of claim 10 , wherein the isothermal nucleic acid amplification reaction is a transcription-associated nucleic acid amplification reaction.
12. The method of claim 11 , wherein the transcription-associated nucleic acid amplification reaction comprises a transcription mediated amplification (TMA) reaction.
13. The method of claim 6 , wherein the polynucleotide analyte comprises a segment of a viral genome.
14. The method of claim 13 , wherein the viral genome comprises RNA.
15. The method of claim 14 , wherein the polynucleotide analyte comprises a segment of an HIV-1 genome.
16. The method of claim 2 , wherein the fluid blood sample comprises whole blood.
17. A method of quantifying an analyte present in a bodily fluid sample that dried to produce a dried sample, the method comprising the steps of:
(a) performing a reaction using the dried sample as a source of analyte to obtain a A measured result, the measured result indicating a concentration or an amount of the analyte;
(b) multiplying the measured result by a correction factor to obtain a corrected result,
wherein the correction factor is the solution to an equation that specifies the correction factor as a function of the measured result, and
wherein the equation comprises a non-linear equation,
and (c) quantifying the analyte present in the bodily fluid sample and recording the corrected result.
18. The method of claim 17 , wherein the non-linear equation comprises coefficients optimized in a mathematical curve fitting procedure to define a fitted curve.
19. The method of claim 18 , wherein the non-linear equation comprises four coefficients.
20. The method of claim 17 , wherein the analyte is a polynucleotide analyte, and wherein step (a) comprises performing with an automated nucleic acid analyzer that amplifies the polynucleotide analyte and detects amplification products as the nucleic acid amplification reaction is occurring.
21. The method of claim 20 , wherein the non-linear equation in step (b) comprises coefficients optimized in a mathematical curve fitting procedure to define a fitted curve, and wherein the non-linear equation is prepared using results obtained from an automated nucleic acid analyzer different from the automated nucleic acid analyzer used for performing the nucleic acid amplification reaction in step (a).
22. The method of claim 20 , wherein step (a) comprises performing with an automated nucleic acid analyzer that isolates the polynucleotide analyte, and then amplifies the isolated polynucleotide analyte.
23. The method of claim 22 , wherein the measured result indicates a concentration of the polynucleotide analyte in a plasma sample.
24. The method of claim 22 , wherein the nucleic acid amplification reaction is an isothermal nucleic acid amplification reaction.
25. The method of claim 24 , wherein the isothermal nucleic acid amplification reaction is a transcription-associated nucleic acid amplification reaction.
26. The method of claim 25 , wherein the transcription-associated nucleic acid amplification reaction comprises a transcription mediated amplification (TMA) reaction.
27. The method of claim 20 , wherein the polynucleotide analyte comprises a segment of a viral genome.
28. The method of claim 27 , wherein the viral genome comprises RNA.
29. The method of claim 28 , wherein the polynucleotide analyte comprises a segment of an HIV-1 genome.
30. The method of claim 17 , wherein the bodily fluid sample is selected from the group consisting of a whole blood sample, a plasma sample, a urine sample, and a saliva sample.