Microfluidic measurements of the response of an organism to a drug
Inventors
Ismagilov, Rustem F. • KHOROSHEVA, Eugenia • Schlappi, Travis S. • CURTIS, Matthew S. • Schoepp, Nathan G. • Maamar, Hedia • Shen, Feng • JUE, Erik B.
Assignees
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Publication Number
US-11542545-B2
Publication Date
2023-01-03
Expiration Date
Abstract
Disclosed herein are methods and devices for rapid assessment of whether a microorganism present in a sample is susceptible or resistant to a treatment.
Core Innovation
The disclosed technology addresses determining a resistance or susceptibility to an antibiotic in a population of bacteria by comparing a first portion treated with an antibiotic for a period of no more than 2 hours and a second portion not treated. Target molecules include a segment of a bacterial chromosome or plasmid, and the presence or absence of the target is evaluated after distributing each sample among analysis regions.
Each of the first and second analysis regions is contacted with a reagent for performing a reaction to detect the presence or absence of a threshold number of target molecules in each analysis region. Detection is carried out by determining whether the threshold number of target molecules is present or absent in each region, and the distribution is effected such that the threshold number is detected in some regions and not detected in some regions after the reaction.
The regional presence/absence results are used to infer resistance or susceptibility of the population of bacteria to the antibiotic. In a related formulation, a method is provided for detecting the presence or absence of a threshold number of target analytes in distributed analysis regions using a first sample from a first portion treated with a drug for no more than 2 hours and a second sample from a second portion not treated.
Claims Coverage
The provided independent claims are directed to three related methods that share a common structure: split bacteria or cells into treated and untreated portions, distribute each portion into analysis regions, react with a reagent to detect presence or absence of a threshold number of chromosomal/plasmid segments, and interpret the region-by-region presence/absence outcome. Across the independent claims, the inventive features center on antibiotic or drug treatment of portions, distribution among analysis regions, detection of a threshold number, and downstream determination of resistance or susceptibility from the results.
Two-portion antibiotic-treated versus untreated population assay with threshold detection
Providing a first sample comprising a target molecule from a first portion of a population of bacteria treated with an antibiotic for a period of no more than 2 hours, and a second sample comprising a target molecule from a second portion not treated; distributing each sample among a plurality of analysis regions; contacting each analysis region with a reagent for performing a reaction to detect the presence or absence of a threshold number of target molecules where the distribution is effected so the threshold is detected in some regions and not detected in some regions after the reaction.
Resistance or susceptibility determination from treated/untreated threshold region results
Determining a resistance or susceptibility to an antibiotic in a population of bacteria by providing antibiotic-treated and untreated samples containing target molecules comprising a segment of a bacterial chromosome or plasmid; distributing each sample among analysis regions; detecting, after contacting with a reagent and performing a reaction, the presence or absence of a threshold number of target molecules region-by-region such that the threshold is detected in some regions and not detected in some regions; determining resistance or susceptibility from the results of said detection.
Drug-treated versus untreated cell portion analysis for threshold analyte detection
Providing a first sample comprising a target analyte from a first portion of a population of cells treated with a drug for a period of no more than 2 hours and a second sample not treated; distributing each sample among analysis regions; contacting each analysis region with a reagent for performing a reaction to detect presence or absence of a threshold number of target analytes where the target analytes comprise a segment of a bacterial chromosome or plasmid contacted with the reagent in at least some regions; detecting so that the threshold number of target analytes is detected in some regions and not detected in some regions after the reaction.
Across the independent claims, the core coverage is directed to region-distributed assays that compare a treated portion with an untreated portion, detect presence or absence of a threshold number of chromosomal or plasmid segments in distributed analysis regions after contacting with a reagent, and determine resistance or susceptibility from the region-by-region detection results.
Stated Advantages
Rapid antimicrobial susceptibility testing with a total AST turnaround workflow.
High specificity, stated as greater than 99%.
Digital quantification suitable for low-abundance detection.
Provides quantified dynamic ranges for bacterial load and gene-expression markers.
Improved resolution of digital PCR over qPCR, as stated in the examples.
Enables comparisons between antibiotic-treated versus untreated samples.
Documented Applications
Antimicrobial susceptibility testing (AST) on bacterial populations, including comparisons of antibiotic-treated versus untreated portions.
Detecting changes in rDNA copy in ciprofloxacin-treated susceptible versus resistant E. coli, as demonstrated in example experiments.
Assessing gene-expression profiling using marker targets (e.g., FeoB/RecA) and related markers, as described in the example context.
Determining resistance or susceptibility to an antibiotic in a population of bacteria using distributed analysis regions and threshold detection of chromosomal or plasmid segments.
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