Multiplexed Signal Ion Emission Reactive Release Amplification (SIERRA) Assay for the Culture-Free Detection of Gram-Negative and Gram-Positive Bacteria and Antimicrobial Resistance Genes
| Autor: | Tiyash Bose, Gerald A. Denys, Michael Pugia, M. Regina Barron, Frédérique Deiss, Marco Tjioe, Monica Barron, Ian McLuckey, Chandan K. Sen, Karamjeet Kaur, Anna Vorsilak, Zane Baird, Amitava Das, Cao Zehui, Jessica Carpenter, Sashwati Roy, Dylan Frabutt |
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| Rok vydání: | 2021 |
| Předmět: |
Staphylococcus aureus
Klebsiella pneumoniae Gram-positive bacteria Microbial Sensitivity Tests Gram-Positive Bacteria 010402 general chemistry medicine.disease_cause 01 natural sciences Article Analytical Chemistry Microbiology Anti-Infective Agents Drug Resistance Bacterial Gram-Negative Bacteria Escherichia coli medicine Humans Pathogen biology Pseudomonas aeruginosa Chemistry 010401 analytical chemistry biology.organism_classification 16S ribosomal RNA Anti-Bacterial Agents 0104 chemical sciences Bacteria |
| Zdroj: | Anal Chem |
| ISSN: | 1520-6882 0003-2700 |
| DOI: | 10.1021/acs.analchem.0c00453 |
| Popis: | The global prevalence of antibiotic-resistant bacteria has increased the risk of dangerous infections, requiring rapid diagnosis and treatment. The standard method for diagnosis of bacterial infections remains dependent on slow culture-based methods, carried out in central laboratories, not easily extensible to rapid identification of organisms, and thus not optimal for timely treatments at the point-of-care (POC). Here, we demonstrate rapid detection of bacteria by combining electrochemical immunoassays (EC-IA) for pathogen identification with confirmatory quantitative mass spectral immunoassays (MS-IA) based on signal ion emission reactive release amplification (SIERRA) nanoparticles with unique mass labels. This diagnostic method uses compatible reagents for all involved assays and standard fluidics for automatic sample preparation at POC. EC-IA, based on alkaline phosphatase-conjugated pathogen-specific antibodies, quantified down to 10(4) bacteria per sample when testing Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa lysates. EC-IA quantitation was also obtained for wound samples. The MS-IA using nanoparticles against S. aureus, E. coli, Klebsiella pneumoniae, and P. aeruginosa allowed selective quantitation of ~10(5) bacteria per sample. This method preserves bacterial cells allowing extraction and amplification of 16S ribosomal RNA genes and antibiotic resistance genes, as was demonstrated through identification and quantitation of two strains of E. coli, resistant and nonresistant due to β-lactamase cefotaximase genes. Finally, the combined immunoassays were compared against culture using remnant deidentified patient urine samples. The sensitivities for these immunoassays were 83, 95, and 92% for the prediction of S. aureus, P. aeruginosa, and E. coli or K. pneumoniae positive culture, respectively, while specificities were 85, 92, and 97%. The diagnostic platform presented here with fluidics and combined immunoassays allows for pathogen isolation within 5 min and identification in as little as 15 min to 1 h, to help guide the decision for additional testing, optimally only on positive samples, such as multiplexed or resistance gene assays (6 h). |
| Databáze: | OpenAIRE |
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