Sequence-specific detection and phenotype determination
Disclosed herein are materials and methods for achieving sequence-specific organism detection and/or phenotype(s) using sequence-specific oligonucleotides. Also disclosed are related kits, cultures, and cells for detecting and/or phenotyping microorganisms in a sequence-specific manner.
1. A method for measuring a mechanism that imparts a non-susceptible phenotype, comprising:
providing a sample comprising an Enterobacteriaceae that is not susceptible to carbapenem;
contacting the sample with carbapenem;
contacting the Enterobacteriaceae with an oligonucleotide molecule that targets a gene associated with carbapenem resistance;
contacting the sample with a non-replicative transduction particle (NRTP) comprising a reporter nucleic acid molecule encoding a reporter molecule under conditions such that the NRTP inserts into the microorganism the reporter nucleic acid molecule and such that the reporter molecule provides a detectable indication of viability; wherein the NRTP is produced by a method comprising, (a) inducing a lytic phase of a bacterial cell packaging system, wherein said bacterial cell packaging system comprises: a host bacteria cell; a first nucleic acid construct inside the host bacteria cell, comprising of a bacteriophage genome having a non-functional packaging initiation site sequence, wherein the non-functional packaging initiation site sequence prevents packaging of the bacteriophage genome into the NRTP; and a second nucleic acid construct inside the host bacteria cell and separate from the first nucleic acid construct, comprising of a reporter nucleic acid molecule having a reporter gene and a bacteriophage gene that contains a functional packaging initiation site sequence for facilitating packaging of a replicon of the reporter nucleic add molecule into the NRTP, wherein the functional packaging initiation site sequence within the bacteriophage gene on the second nucleic acid construct complements the non-functional packaging initiation site sequence in the bacteriophage genome on the first nucleic acid construct; (b) allowing the replicon of the reporter nucleic acid molecule to be packaged to produce the NRTP; and (c) collecting a lysate comprising a plurality of NRTPs; and
measuring the mechanism of carbapenem non-susceptibility for the Enterobacteriaceae based on the presence or absence of the detectable indication of viability of the Enterobacteriaceae when the Enterobacteriaceae is in contact with the carbapenem and the oligonucleotide molecule, wherein the presence of the detectable indication of viability indicates that the gene targeted by the oligonucleotide molecule is not the mechanism of carbapenem nonsusceptibility, and wherein the absence of the detectable indication of viability indicates that the gene targeted by the oligonucleotide molecule is the mechanism of carbapenem non-susceptibility.
2. The method of claim 1 wherein the reporter nucleic acid molecule is a gene encoding a light-emitting molecule and the detectable indication of viability is light emission.
3. The method of claim 1 , wherein the oligonucleotide molecule is selected from the group consisting of a peptide nucleic acid (PNA), a peptide-PNA, a CRISPR RNA (crRNA), an antisense oligonucleotide, and a double-stranded RNA (dsRNA).
4. The method of claim 3 wherein the oligonucleotide molecule is selected from the group consisting of SEQ. ID NO 2, SEQ ID NO 3, a CRISPR/Cas9 System comprising SEQ ID NO 4, and, a CRISPR/Cas9 System comprising SEQ ID NO 5.