Assays and methods for determining microbial resistance
Assays and methods for detecting resistance to beta-lactam antibiotics including detection of multiple β-lactamase family specific gene targets by polymerase chain reaction or microarray. One or more kits including primers and/or probes for identification of β-lactamase genes selected from the group consisting of one or more of the following: MOX-like, FOX-like, ACC-like, ACT/MIR-like, CMY-2-like, DHA-like, CTX-M-14-like, CTX-M-15-like, VIM-like, NDM-like, IMP-like, KPC-like, and OXA-48-like, OXA-51-like, OXA-143-like, OXA-58-like, OXA-23-like, OXA-24/40-like, TEM-like, and SHV-like. A kit may also include one or more primers and/or probes for the identification a non-beta lactamase gene family which confers antibiotic resistance, such as the MCR-1 gene.
1. A kit for identification of one or more β-lactamase genes, wherein the one or more β-lactamase genes are selected from the group consisting of: CMY-2, DHA, MOX, ACC, FOX, and ACT/MIR genes, the kit comprising probes comprising the following sequences: AGGATGGCAAGGCCCACTATTTCA (SEQ ID NO: 194), AGCCATTACGTTCCAGAGTTGCGT (SEQ ID NO: 197), TCTGCTGAAGTTTRYCGAGGCMAA (SEQ ID NO: 200), TGAGACACGGTCCAGACTCCTACG (SEQ ID NO: 203), AAACCGGGCGATATGCGTCTGTAT (SEQ ID NO: 206), GATGCCATTGCYCGSGGTGAAAT (SEQ ID NO: 209), and ATGTTGGCCTGAACCCAGCG (SEQ ID NO: 212), wherein each probe comprises a fluorophore and/or a fluorescent quencher.
2. The kit of claim 1 , including an endogenous internal control.
3. The kit of claim 2 , wherein the endogenous internal control targets a conserved region in Gram-negative bacteria.
4. The kit of claim 2 , wherein the internal control detects a 16S rRNA gene.
5. The kit of claim 2 , wherein the internal control detects a 16S rRNA gene for E. coli, Pseudomonas, Acinetobacter, Klebsiella , or Salmonella.
6. The kit of claim 1 , including at least one control DNA mix.
7. The kit of claim 1 , including exactly two control DNA mixes.
8. The kit of claim 1 , including exactly three control DNA mixes.
9. The kit of claim 1 , including a composition containing a tracking dye.
10. A method for identifying one or more β-lactamase genes selected from the group consisting of CMY-2, DHA, MOX, ACC, FOX, and ACT/MIR, the method comprising assaying a biological sample using the kit of claim 1 to identify the one or more β-lactamase genes, wherein the assaying comprises real-time polymerase chain reaction.
11. The method of claim 10 , wherein 0.1 copy to 10,000,000,000 copies of target DNA sequence is detected.
12. The method of claim 10 , wherein the biological sample is a clinical sample of blood, plasma, urine or feces, or of rectal, nose, throat, skin, wound or cerebrospinal fluid specimens.
13. The kit of claim 1 , further comprising the following primers: GCTGCTCAAGGAGCACAGGAT (SEQ ID NO: 192), CACATTGACATAGGTGTGGTGC (SEQ ID NO: 193), AACAGCCTCAGCAGCCGGTTA (SEQ ID NO: 195), TTCGCCGCAATCATCCCTAGC (SEQ ID NO: 196), GCCGAGGCTTACGGGATCAAG (SEQ ID NO: 198), CAAAGCGCGTAACCGGATTGG (SEQ ID NO: 199), GAGAGGATGAYCAGCCACAC (SEQ ID NO: 201), and CGCCCATTGTSCAATATTCC (SEQ ID NO: 202).
14. The kit of claim 13 , further comprising the following primers: AACTTTCACAGGTGTGCTGGGT (SEQ ID NO: 204), CCGTACGCATACTGGCTTTGC (SEQ ID NO: 205), CTTCCACTGCGGCTGCCAGTT (SEQ ID NO: 208), CCGAAGCCTATGGCGTGAAATCC (SEQ ID NO: 210), GCAATGCCCTGCTGGAGCG (SEQ ID NO: 211), GAGAGGATGAYCAGCCACAC (SEQ ID NO: 213), and CGCCCATTGTSCAATATTCC (SEQ ID NO: 214).
15. The kit of claim 14 , wherein the contents of the kit are enclosed in one or more containers.
16. The kit of claim 1 , further comprising the following primers: AACTTTCACAGGTGTGCTGGGT (SEQ ID NO: 204), CCGTACGCATACTGGCTTTGC (SEQ ID NO: 205), CTTCCACTGCGGCTGCCAGTT (SEQ ID NO: 208), CCGAAGCCTATGGCGTGAAATCC (SEQ ID NO: 210), GCAATGCCCTGCTGGAGCG (SEQ ID NO: 211), GAGAGGATGAYCAGCCACAC (SEQ ID NO: 213), and CGCCCATTGTSCAATATTCC (SEQ ID NO: 214).
17. The kit of claim 1 , wherein each probe is labeled with the fluorophore at one end and the fluorescent quencher at the other end.
18. The kit of claim 1 , wherein the fluorophore is selected from the group consisting of fluorescein, hexachlorofluorescein, FAM, HEX, TEX615 and TYE665.
19. The kit of claim 1 , wherein the fluorophore excites between 450 nm and 763 nm and emits between 500 nm and 800 nm.
20. The kit of claim 1 , wherein peak absorbance of the fluorescent quencher is at 531 nm, 534 nm, 578 nm, or 656 nm.
21. The kit of claim 1 , wherein the contents of the kit are enclosed in one or more containers.