Signal encoding and decoding in multiplexed biochemical assays
This disclosure provides methods, systems, compositions, and kits for the multiplexed detection of a plurality of analytes in a sample. In some examples, this disclosure provides methods, systems, compositions, and kits wherein multiple analytes may be detected in a single sample volume by acquiring a cumulative measurement or measurements of at least one quantifiable component of a signal. In some cases, additional components of a signal, or additional signals (or components thereof) are also quantified. Each signal or component of a signal may be used to construct a coding scheme which can then be used to determine the presence or absence of any analyte.
1. A method of unambiguously detecting any unique combination of presence or absence of a plurality of analytes, the method comprising:
(a) providing a sample from a subject comprising, or potentially comprising, said plurality of analytes,
(b) forming a mixture of said sample and hybridization probes, wherein at least one of said hybridization probes is configured to bind to at least one of said plurality of analytes corresponding with a respiratory viral pathogen, and wherein said hybridization probes further comprise at least one fluorophore and at most four fluorophores;
(c) exciting said at least one fluorophore to generate one or more signals if one or more of said plurality of analytes is present, wherein said one or more signals comprise at least one signal generated by excitement of said one or more fluorophores;
(d) measuring said one or more signals to generate a cumulative intensity measurement, wherein said cumulative intensity measurement corresponds to the presence of a unique combination of presence or absence of said plurality of analytes in said sample; and
(e) determining whether said at least one of said plurality of analytes corresponding with said respiratory viral pathogen is present, in any unique combination of presence or absence, based on said cumulative intensity measurement, wherein the method does not require any step of immobilization of said plurality of polynucleotide analytes or mass spectrometry, and
wherein each analyte is derived from a respiratory viral pathogen.
2. The method of claim 1 , wherein said respiratory viral pathogen is selected from influenza virus and respiratory syncytial virus (RSV).
3. The method of claim 1 , wherein said plurality of analytes are derived from at least three respiratory viral pathogens.
4. The method of claim 1 , wherein said plurality of analytes comprise at least five analytes.
5. The method of claim 1 , wherein said plurality of analytes comprise at least seven analytes.
6. The method of claim 1 , wherein the sample is derived from saliva, blood, or urine from the subject.
7. The method of claim 1 , wherein the detecting the presence or absence of at least one of said plurality of analytes indicates the presence of an infection with said at least one pathogen in said subject.
8. The method of claim 1 , further comprising providing information concerning the presence or absence of said plurality of analytes to a health care professional, wherein the health care professional makes a clinical decision based on said information.
9. The method of claim 8 , wherein said clinical decision is a treatment decision.
10. The method of claim 9 , wherein said treatment decision comprises treatment with an anti-viral.
11. The method of claim 1 , further comprising amplifying the analytes.