Systems and Methods for Detecting Antibiotic Resistance

A method for determining a disease condition of a subject, the method comprising the steps of:

• (a) obtaining a sample from a patient;
• (b) isolating nucleic acid material from said sample;
• (c) amplifying a target locus from said nucleic acid material;
• (d) contacting said target locus with a set of probes (resequencing probes), wherein said set comprising 8 probes (4 probes based on the sense strand and 4 probes based on the anti-sense strand) per nucleotide base interrogated in each target locus;
• (e) determining hybridization signal strengths across the set of probes;
• (f) determining hybridization signal strengths for a plurality of different interrogation probes, each of which is complementary to a section within said target locus;
• (g) determining the sequence of the target locus by analysis of the hybridization signal strengths of the resequencing probes;
• (h) comparing the target locus sequence with a set of known sequences to determine the presence and/or antibiotic resistance repertoire of one or more target organisms;
• (j) defining therapeutic strategy for said patient based on the results of step (h);
• (l) classifying, diagnosing, prognosing, and/or predicting an outcome of said condition based on the results of step (h).

A method for parallel detection and strain level-identification of a panel of more than 44 organisms, in parallel with antibiotic resistance profiling of said organisms comprising the steps of:

• a) extraction of nucleic acids from a patient sample using a rapid optimized protocol;
• b) amplifying target loci from that sample using multiplex polymerase chain reaction;
• c) pooling target locus amplified products;
• d) labeling pooled amplified products;
• e) contacting the labeled amplified pool of products with a plurality of resequencing probes which target both the sense and anti-sense strands of the target loci represented in SEQ ID NOS:1-1323;
• f) determining hybridization signal strength for each of said probes, wherein said determination identifies the specific sequence of the target locus, providing either strain level organism identification in parallel with single nucleotide polymorphism resolution antibiotic resistance determinant sequence information.

A method of designing and fabricating an array system comprising:

• identifying organism-specific and antibiotic resistance element sequences corresponding to a plurality of organisms and resistance elements of interest;
• selecting target loci unique to each organism and resistance element;
• selecting resequencing probes;
• and fabricating said array system.