Methods and devices for single-cell based digital high resolution melt
Provided are devices, systems, and methods for the identification, quantification, and profiling of microscopic organisms. The methods for the identification, quantification, and profiling of microscopic organisms include, for example, the selective enrichment of microscopic organisms from a heterogeneous sample; subsequent loading of the microscopic organisms into microfluidic channels or reaction chambers; direct amplification of nucleic acids from single, isolated microscopic organisms; and examination of amplification products using digital High Resolution Melting (HRM) analysis.
1. A method for detecting one or more species of microbial organisms, comprising:
partitioning a heterogeneous sample into a plurality of fixed reaction chambers comprising at least 5000 fixed reaction chambers, wherein the heterogeneous sample comprises the one or more species of microbial organisms and one or more of non-microbial cells, mammalian cells, microbial nucleic acid and mammalian nucleic acid, and wherein each of the at least 5000 fixed reaction chambers receives a partition of the heterogeneous sample;
performing a lysis step simultaneously in each of the at least 5000 fixed reaction chambers to create a lysed partition in each of the at least 5000 fixed reaction chambers, wherein the lysis step lyses any microbial organisms that are present in each of the partitions;
performing a nucleic acid amplification step in the presence of the lysed partition simultaneously in each fixed reaction chamber of the at least 5000 fixed reaction chambers; and
performing a nucleic acid analysis simultaneously in each of the at least 5000 fixed reaction chambers, wherein the nucleic acid analysis detects a nucleic acid amplification product resulting from the one or more species of microbial organisms in the one or more of the at least 5000 fixed reaction chambers.
2. The method of claim 1 , wherein performing the nucleic acid analysis comprises one or more of digital polymerase chain reaction (dPCR), isothermal amplification, loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), or melt curve analysis.
3. The method of claim 1 , wherein each fixed reaction chamber of the at least 5000 fixed reaction chambers comprises two or fewer microbial organisms.
4. The method of claim 1 , wherein the heterogeneous sample is distributed to provide a Poisson distribution of the one or more species of microbial organisms into the at least 5000 fixed reaction chambers.
5. The method of claim 4 , wherein 99.5% of the at least 5000 fixed reaction chambers having a positive signal generated in the step of nucleic acid analysis contain a single microbial organism.
6. The method of claim 4 , wherein the partitioning comprises distributing the one or more species of microbial organisms at a density of one organism per 20 fixed reaction chambers of the at least 5000 fixed reaction chambers.
7. The method of claim 1 , wherein performing the nucleic acid analysis comprises performing nucleic acid amplification to amplify an amplicon having a size greater than 500 bp.
8. The method of claim 1 , wherein performing the nucleic acid analysis comprises performing nucleic acid amplification to amplify an amplicon having a size greater than 1000 bp.
9. The method of claim 1 , further comprising enriching the heterogeneous sample for microbial organisms prior to the partitioning step.
10. The method of claim 9 , wherein the enriching comprises removing or lysing non-microbial organisms from the heterogeneous sample prior to the partitioning step.
11. The method of claim 1 , further comprising phenotypic profiling of the one or more species of microbial organisms in each fixed reaction chamber of the at least 5000 fixed reaction chambers prior to the lysis step.
12. The method of claim 11 , wherein the phenotypic profiling comprises antibiotic resistance, cell imaging, or a combination thereof.
13. The method of claim 1 , wherein the at least 5000 fixed reaction chambers comprises at least 10,000 fixed reaction chambers.
14. The method of claim 1 , wherein performing the nucleic acid analysis comprises quantification of the one or more species of microbial organisms in the heterogeneous sample.
15. The method of claim 1 , wherein performing nucleic acid analysis comprises determining cell populations within the heterogeneous sample.
16. The method of claim 1 , wherein performing nucleic acid analysis comprises obtaining a melt curve signature for each fixed reaction chamber of the at least 5000 fixed reaction chambers.
17. The method of claim 1 , wherein performing nucleic acid analysis comprises simultaneously heating and imaging each fixed reaction chamber of the plurality of at least 5000 fixed reaction chambers.