Compositions and methods for detecting microorganisms
A technique for detecting samples, binding constants, applied in microorganism-based methods, biochemical equipment and methods, microorganisms, etc., and can solve problems such as difficulty, death, disease, etc.
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[0120] Polynucleotides may also contain analogous forms of ribose or deoxyribose sugars commonly known in the art, including 2'-O-methyl-, 2'-O-allyl, 2'-fluoro- or 2'-stack Nitro-ribose, carbocyclic sugar analogs, alpha-anomeric sugars, epimeric sugars such as arabinose, xylose or lyxose, pyranose, furanose, sedum heptulose, acyclic analogs and Abasic nucleoside analogs such as methyl nucleosides. As mentioned above, one or more of the phosphodiester linkages can be replaced by other linking groups. These alternative linking groups include embodiments in which the phosphate is replaced with the following groups: P(O)S ("thioester"), P(S)S ("dithioester"), (O)NR 2 ("amidated"), P(O)R, P(O)OR', CO or CH 2 ("formacetal"), wherein each R or R' is independently H or substituted or unsubstituted alkyl (1-20C), aryl optionally containing ether (-O-) linkages , alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not all linkages in a polynucleotide have to be the same. Substitution of...
Embodiment 1
[0302] Example 1: Selection and identification of aptamers with binding specificity to S. aureus proteins
[0303] This example provides representative methods to select and generate aptamers with binding specificities for the following 10 surface-associated S. aureus proteins: SpA, CIfA, CIfB, FnbA, FnbB, SasD, IsdA, IsdB, IsdC, and IsdH .
[0304] Purification of Staphylococcus aureus targets
[0305] The relevant portion of the gene encoding the desired target or target domain was PCR amplified with primers from S. aureus NRS384 (USA300) genomic DNA and cloned into pCR-Script SK+ (Stratgene). The clfA, clfB, fnbA, sasD, isdA, isdB, isdC, and isdH genes were transferred as BamHI-SacI cassettes into His with an amino-terminal streptavidin tag and a carboxy-terminal His 10 The marker expression vector pET-51b (EMD-Novagen). One of the targets, fnbB, was cloned into pET-14b (EMD-Novagen) as an NdeI-BamHI fragment with an amino-terminal His 10 Mark. The plasmids were sequen...
Embodiment 2
[0330] Example 2: Bacterial cell binding and selective capture by aptamer
[0331] This example shows that selected and identified aptamers that bind S. aureus cell surface proteins also bind to whole cells and are capable of selectively capturing S. aureus cells in mixed bacterial cultures.
[0332] Aptamer Equilibrium and Whole Cell Radiolabel Binding Assay
[0333] The aptamers were properly folded prior to binding assays by heating at 95°C for 5 min and then cooling to room temperature over a 10-15 min period.
[0334] Serial dilutions of protein (0.001-100 nmol l) were used as described (Gold et al., 2010). -1 ) and Zorbax PSM-300A (Agilent Technologies) resin for separation onto filter plates in radiolabeled aptamers (10-20 pmol 1 -1 ) in the equilibrium solution binding assay to determine affinity (K d ).
[0335] Before cloning, aptamer pools were also tested for specific binding to S. aureus in a 2-h equilibrium binding assay using S. epidermidis, S. hemolyticus, ...
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