System and method for determining copies-per-unit-volume using pcr and flow control of droplets

Abstract

Methods and systems for quantification of a target nucleic acid in a sample are provided. The method includes forming a plurality of discrete sample portions. Each of the plurality of discrete sample portions comprising a portion of the sample, and a reaction mixture. The method further includes amplifying the plurality of discrete sample portions to form a plurality of discrete processed sample portions. At least one discrete processed sample portion containing nucleic acid amplification reaction products. Fluorescence signals are detected from the at least one of the plurality of discrete processed sample portions to determine a presence of the at least one target nucleic acid. The method also includes determining the respective volumes of the plurality of the plurality of discrete processed sample portions, and estimating the number of copies-per-unit-volume of the at least one target nucleic acid in the sample. Estimating the number of copies-per-unit-volume is based on the number of discrete processed sample portions determined to contain the at least one target nucleic acid therein.

1 - 22 . (canceled) 
     
     
         23 . A method, comprising:
 forming a plurality of discrete sample portions of a sample comprising a target nucleic acid, each of the plurality of discrete sample portions comprising a portion of the sample;   amplifying the plurality of discrete sample portions to form at least one discrete processed sample portion containing a nucleic acid amplification reaction product of the target nucleic acid;   detecting a fluorescence signal from the at least one of the plurality of discrete processed sample portions;   using an optical imager, determining the respective volumes of the plurality of the plurality of discrete processed sample portions by analyzing a reference dye signal to determine a dimension of each discrete processed sample portion; and   determining an amount of the target nucleic acid based on the number of discrete processed sample portions containing the target nucleic acid therein.   
     
     
         24 . The method of  claim 23 , using the optical imager to determine the respective volumes of the plurality of discrete processed sample portions by analyzing a time-of-flight of the processed sample portions. 
     
     
         25 . The method of  claim 23 , further comprising estimating a portion size of the discrete sample portions to provide a predetermined average number of copies a target nucleic acid within the discrete sample portions. 
     
     
         26 . The method of  claim 23 , wherein the plurality of discrete sample portions comprises discrete sample portions of substantially two different sizes and/or of substantially a plurality of predetermined sizes. 
     
     
         27 . The method of  claim 23 , wherein each of the plurality of sample portions is at least partially surrounded by a medium that is at least substantially immiscible with the plurality of discrete sample portions and wherein the medium that is substantially immiscible with the plurality of discrete sample portions optionally comprises at least one of a mineral oil, a silicone oil, a paraffin oil, a fluorinated fluid, or a perfluorinated polyether. 
     
     
         28 . The method of  claim 23 , wherein the plurality of discrete sample portions comprises at least one of porous beads or magnetic beads. 
     
     
         29 . The method of  claim 23 , wherein determining the respective volumes comprises imaging the plurality of discrete processed sample portions. 
     
     
         30 . The method of  claim 23 , further comprising estimating the number of copies-per-unit-volume of the target nucleic acid in the sample based on the number of discrete processed sample portions determined to contain the at least one target nucleic acid therein. 
     
     
         31 . A system, comprising:
 a droplet apparatus, in a first configuration, capable of forming a plurality of discrete sample portions of a sample comprising a target nucleic acid, each of the plurality of discrete sample portions comprising a portion of the sample;   an amplification apparatus configured to amplify the plurality of discrete sample portions to form at least one discrete processed sample portion containing nucleic acid amplification reaction product of the target nucleic acid;   a detection apparatus configured to detect a fluorescence signal from the at least one of the plurality of discrete processed sample portions in response to a presence of the at least one target nucleic acid;   an optical imager configured to determine the respective volumes of the plurality of discrete processed sample portions by analyzing a reference dye signal to determine a dimension of each discrete processed sample portion; and   a processor configured to determine an amount of the target nucleic acid based on the number of discrete processed sample portions containing the target nucleic acid therein.   
     
     
         32 . The system of  claim 31 , wherein the optical imager is configured to determine the respective volumes of the plurality of discrete processed sample portions by analyzing a time-of-flight of the processed sample portions. 
     
     
         33 . The system of  claim 31 , further comprising a processor configured to estimate a portion size of the discrete sample portions to provide a predetermined average number of copies a target nucleic acid within the discrete sample portions. 
     
     
         34 . The system of  claim 31 , further comprising an optical imager configured to determine the respective volumes of the plurality of the plurality of discrete processed sample portions. 
     
     
         35 . The system of  claim 31 , wherein the droplet apparatus is configured to form the plurality of discrete sample portions of at least one of a plurality of sizes, substantially two different sizes, or substantially a plurality of predetermined sizes. 
     
     
         36 . The system of  claim 31 , wherein each of the plurality of discrete sample portions is at least partially surrounded by a medium that is at least substantially immiscible with the plurality of discrete sample portions. 
     
     
         37 . The system of  claim 36 , wherein the medium that is substantially immiscible with the plurality of sample portions comprises at least one of a mineral oil, a silicone oil, a paraffin oil, a fluorinated fluid, or a perfluorinated polyether. 
     
     
         38 . The system of  claim 31 , wherein the plurality of discrete sample portions comprises at least one or porous beads or magnetic beads. 
     
     
         39 . The system of  claim 31 , wherein determining the respective volumes comprises imaging the plurality of discrete processed sample portions. 
     
     
         40 . The system of  claim 31 , further comprising a processor configured to estimate the number of copies-per-unit-volume of the target nucleic acid in the sample based on the number of discrete processed sample portions determined to contain the at least one target nucleic acid therein. 
     
     
         41 . A method, comprising:
 forming a plurality of discrete sample portions of a sample comprising a target nucleic acid, each of the plurality of discrete sample portions comprising a portion of the sample;   amplifying the plurality of discrete sample portions to form at least one discrete processed sample portion containing a nucleic acid amplification reaction product of the target nucleic acid;   detecting a fluorescence signal from the at least one of the plurality of discrete processed sample portions;   using an optical imager, determining the respective volumes of the plurality of the plurality of discrete processed sample portions by imaging the plurality of discrete processed sample portions; and   determining an amount of the target nucleic acid based on the number of discrete processed sample portions containing the target nucleic acid therein.   
     
     
         42 . A system, comprising:
 a droplet apparatus, in a first configuration, capable of forming a plurality of discrete sample portions of a sample comprising a target nucleic acid, each of the plurality of discrete sample portions comprising a portion of the sample;   an amplification apparatus configured to amplify the plurality of discrete sample portions to form at least one discrete processed sample portion containing nucleic acid amplification reaction product of the target nucleic acid;   a detection apparatus configured to detect a fluorescence signal from the at least one of the plurality of discrete processed sample portions in response to a presence of the at least one target nucleic acid;   an optical imager configured to determine the respective volumes of the plurality of discrete processed sample portions by imaging the plurality of discrete processed sample portions; and   a processor configured to determine an amount of the target nucleic acid based on the number of discrete processed sample portions containing the target nucleic acid therein.