DETECTION OF GENOMIC SEQUENCES AND PROBE MOLECULES THEREFOR
Methods of identifying homologous genomic sequences that may be present in a sample utilizing virtual probes, arrays for distinguishing homologous genomic sequences, systems for distinguishing homologous genomic sequences, and probe molecules useful in the methods, arrays, and systems of the disclosure.
1 . A method of detecting if a first organism having a first genome or a second organism having a second genome is present in a test sample or an initial sample from which the test sample was prepared, wherein the first and second organisms are Staphylococci , the method comprising:
(a) probing the test sample with a virtual probe comprising a plurality of probe molecules, wherein
(i) the virtual probe comprises at least two probe molecules is capable of specifically hybridizing to one or more target nucleic acids corresponding to a 16S rRNA gene of the first genome and/or the second genome,
(ii) the virtual probe optionally further comprises one or more probe molecules capable of specifically hybridizing to one or more target nucleic acids corresponding to a 16S-23S intergenic spacer region of the first genome and/or the second genome;
(iii) the probe molecules cannot individually distinguish the target nucleic acids corresponding to the first and second genomes, and
(iv) the probe molecules hybridize non-identically to the target nucleic acids corresponding to the first and second genomes, such that the hybridizing of the probe molecules to the target nucleic acids corresponding to the first and second genomes can distinguish between the target nucleic acids corresponding to the first and second genomes; and
(b) detecting and/or quantifying signals from hybridization of the probe molecules in the virtual probe to nucleic acids, if any, in the test sample.
2 . The method of claim 1 , wherein the one or more target nucleic acids corresponding to the first genome are a first amplicon set and the one or more target nucleic acids corresponding to the second genome are a second amplicon set, and wherein each probe molecule in the virtual probe is capable of specifically hybridizing to one or more amplicons in the first amplicon set and/or the second amplicon set, and wherein the probe molecules hybridize non-identically to the amplicons in the first amplicon set and the amplicons in the second amplicon set, such that the hybridizing of the probe molecules to the amplicons in the first amplicon set and the second amplicon set can distinguish between the first amplicon set and the second amplicon set.
3 . The method of claim 2 , which further comprises preparing the test sample by performing a PCR amplification reaction on the initial sample using PCR primers capable of hybridizing to, and initiating a PCR amplification from, both the first genome and the second genome, resulting in the first amplicon set and a second amplicon set, respectively, when the first genome and second genome are present in the initial sample.
4 . The method of claim 3 , wherein the PCR primers comprise more than one primer pair and wherein the first amplicon set comprises a plurality of first amplicons and/or the second amplicon set comprises a plurality of second amplicons.
5 . The method of claim 4 , wherein the plurality of first amplicons comprises nucleotide sequences corresponding to a portion of the 16S rRNA gene and the 16S-23S intergenic spacer region of to the first genome and/or the plurality of second amplicons comprises nucleotide sequences corresponding to a portion of the 16S rRNA gene and the 16S-23S intergenic spacer region of the second genome.
6 . The method of claim 3 , wherein the PCR primers comprise a single primer pair and the first amplicon set consists of a single first amplicon corresponding to a portion of the 16S rRNA gene of the first genome and the second amplicon set consists of a single second amplicon corresponding to a portion of the 16S rRNA gene of the second genome.
7 . The method of claim 1 , wherein the probe molecules of the virtual probe are positionally addressable probe molecules present on an array, each at a discrete location on the array.
8 - 25 . (canceled)
26 . The method of claim 1 , wherein the first microorganism is a coagulase negative Staphylococcus sp. and the second microorganism is a coagulase positive Staphylococcus sp.
27 . The method of claim 26 , wherein at least one probe molecule in the virtual probe has a nucleotide sequence comprising CCAGTCTTATAGGTAGGTTAYCCACG (SEQ ID NO:1) and another probe molecule in the virtual probe has a nucleotide sequence comprising GCTTCTCGTCCGTTCGCTCG (SEQ ID NO:2).
28 . The method of claim 5 , wherein the first microorganism is a coagulase negative Staphylococcus sp. and the second microorganism is a coagulase positive Staphylococcus sp.
29 . The method of claim 28 , wherein at least one probe molecule in the virtual probe has a nucleotide sequence comprising CCAGTCTTATAGGTAGGTTAYCCACG (SEQ ID NO:1) and another probe molecule in the virtual probe has a nucleotide sequence comprising GCTTCTCGTCCGTTCGCTCG (SEQ ID NO:2).
30 . The method of claim 6 , wherein the first microorganism is a coagulase negative Staphylococcus sp. and the second microorganism is a coagulase positive Staphylococcus sp.
31 . The method of claim 30 , wherein at least one probe molecule in the virtual probe has a nucleotide sequence comprising CCAGTCTTATAGGTAGGTTAYCCACG (SEQ ID NO:1) and another probe molecule in the virtual probe has a nucleotide sequence comprising GCTTCTCGTCCGTTCGCTCG (SEQ ID NO:2).
32 . The method of claim 1 , wherein the initial sample or test sample is a biological sample, an environmental sample, or a food product.
33 . The method of claim 32 , wherein the initial sample or test sample is a biological sample selected from blood, serum, saliva, urine, gastric fluid, digestive fluid, tears, stool, semen, vaginal fluid, interstitial fluid, fluid derived from tumorous tissue, ocular fluid, sweat, mucus, earwax, oil, glandular secretions, breath, spinal fluid, hair, fingernails, skin cells, plasma, fluid obtained from a nasal swab, fluid obtained from a nasopharyngeal wash, cerebrospinal fluid, a tissue sample, fluid or tissue obtained from a throat swab, fluid or tissue obtained from a wound swab, biopsy tissue, placental fluid, amniotic fluid, peritoneal dialysis fluid, cord blood, lymphatic fluids, cavity fluids, sputum, pus, microbiota, meconium, breast milk, or a sample processed, extracted or fractionated from any of the foregoing.
34 . The method of claim 1 , wherein the initial sample or test sample is:
(a) urine, sputum or a sample processed, extracted or fractionated from urine;
(b) sputum or a sample processed, extracted or fractionated from sputum;
(c) a wound swab or a sample processed, extracted or fractionated from a wound swab;
(d) blood or a sample processed, extracted or fractionated from blood; or
(e) peritoneal dialysis fluid or a sample processed, extracted or fractionated from peritoneal dialysis fluid.