DIGITAL BRIDGE PCR

摘要

The present disclosure, among other things, provides methodologies for quantifying targets of interest in samples by 1) capturing single target entities on individual solid phase particles in a manner that permits that those particles that contain captured targets to be optically distinguished from those that do not, and 2) optically analyzing the particles so that those with captured target entities are “counted”. In some embodiments, provided methods and compositions in the present application comprise a population of particles including one or more sub-populations distinguishable from one another.

主权项

1. A method comprising steps of:
a) providing a population of particles wherein the population of the particles each carries one or more copies of a single pair of primers, which pair comprises a capture/first primer and a second primer for amplification of a target nucleic acid; b) contacting the population with a sample comprising a quantity of the target nucleic acid, under conditions that permit the target nucleic acid in the sample to hybridize with the capture/first primer, thereby being captured to the particles, the step of contacting further being performed under conditions so that, on average, not more than one copy of the target nucleic acid from the sample hybridizes to any individual particle; c) performing at least bridge PCR on the particles so that one or more optical aspects of those particles that hybridize and amplify the target nucleic acid are altered, and the particles that contain at least one copy of an amplified nucleic acid of the target nucleic acid are optically distinguishable from those that do not; d) optically characterizing the population, so as to determine the number of particles that contain the at least one amplified nucleic acid of the target nucleic acid, which number reflects the quantity of the target nucleic acid in the sample.