We present a novel method for the encoding and decoding of multiplexed biochemical assays. The method enables a theoretically unlimited number of independent targets to be detected and uniquely identified in any combination in the same sample. For example, the method offers easy access to 12-plex and larger PCR assays, as contrasted to the current 4-plex assays. This advancement would allow for large panels of tests to be run simultaneously in the same sample, saving reagents, time, consumables, and manual labor, while also avoiding the traditional loss of sensitivity due to sample aliquoting. Thus the presented method is a major technological breakthrough with far-reaching impact on biotechnology, biomedical science, and clinical diagnostics. Herein, we present the mathematical theory behind the method as well as its experimental proof of principle using Taqman PCR on sequences specific to infectious diseases.

While many coding schemes are possible under the same method, let's focus on the simplest one, which we call the binary scheme. The idea is that the same color can be used to code for multiple sequences if the signal outputs of each sequence are digitized to be 1x, 2x, 4x, 8x, etc. of some chosen signal intensity. This can be ensured by the respective choice of starting Taqman probe molecules for each sequence. Then a result of 7x uniquely means the sequences for 1x, 2x, and 4x are present, but none other; while a result of 3x means only 1x and 2x are present. This scheme is infinite and non-degenerate by construction, i.e. all results uniquely decode to a unique combination of present targets.

To test the binary scheme experimentally, three sequences were chosen, Dengue Virus, HIV TPP, and HIV P17, in just one color (blue). The fluorescence signals from their probes in positive-control end-point PCR reactions were measured and used to calculate the respective probe concentrations that would produce a 1x, 2x, and 4x signal intensity, respectively. Then all 7 non-null combinations of target occurrence were processed in a batch experiment using the same Masterplex mixture of primers and probes. The fluorescence signal of each case was plotted in a chromatogram (Figure 1). The multiplicity levels were assembled as the expected integer multiples of the 1x signal intensity, while their widths were calculated as the propagated uncertainty of the 1x measurement. That uncertainty was equated to the standard deviation of the fluorescence signals of the last five PCR cycles in saturation.

The results in Figure 2 show a virtually perfect agreement between expectations and experiment. In every case, the result correctly decodes to the exact list of present sequences. Furthermore, the experimental set is exhaustive, as all theoretical combinations are accounted for and measured for the case of 1 color and 3 sequences. These results show proof of principle for the binary coding scheme and the general method.

While proven for Taqman PCR assays, the general coding method is applicable to any other biochemical assay where the output signal can be reliably measured and quantized for coding.