Rapid Minimum Inhibitory Concentration (MIC) Analysis Using Lyophilized Reagent Beads in a Novel Multiphase, Single-Vessel Assay.
Rapid Minimum Inhibitory Concentration (MIC) Analysis Using Lyophilized Reagent Beads in a Novel Multiphase, Single-Vessel Assay.
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DOI：
10.3390/antibiotics12111641
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发表时间：
2023-11-19
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--
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抗生素耐药性（AMR）是一种全球性威胁，由抗生素药物的不正确（和过度使用）引发，导致多重和极端耐药菌株的进化。与金标准细菌培养方法相关的较长的抗生素给药时间（TTA）是抗生素经验性使用的原因，也是AMR上升的关键因素。虽然聚合酶链反应（PCR）和其他核酸扩增方法正在迅速取代传统的培养方法，但它们的范围主要限于检测耐药性的基因型决定因素，几乎没有提供关于抗生素表型敏感性的信息。本文介绍的工作旨在通过将短生长期（~3-4 h）与下游PCR检测配对，提供表型抗菌药物敏感性检测（AST）信息，以最终预测抗生素治疗的最低抑菌浓度（MIC）值。为了进一步简化AST和PCR测定的双重工作流程，这些反应在使用新型冻干试剂珠（LRB）的单容器格式（PCR管）中进行，所述冻干试剂珠沿着储存干燥的PCR试剂以及引物和酶，并且分别储存抗生素药物。这两个反应使用熔化石蜡密封在空间和时间上分开，从而消除了在不同耗材之间转移试剂的需要，并最大限度地减少了用户交互。最后，这些两步单容器反应通过使用微流体歧管进行多重化，该歧管允许同时测试未知细菌样品对不同浓度的不同抗生素的抵抗。微流控系统中使用的LRB对细菌生长和PCR检测没有干扰，并为快速床旁诊断（POC-Dx）提供了一个创新平台。
Antimicrobial resistance (AMR) is a global threat fueled by incorrect (and overuse) of antibiotic drugs, giving rise to the evolution of multi- and extreme drug-resistant bacterial strains. The longer time to antibiotic administration (TTA) associated with the gold standard bacterial culture method has been responsible for the empirical usage of antibiotics and is a key factor in the rise of AMR. While polymerase chain reaction (PCR) and other nucleic acid amplification methods are rapidly replacing traditional culture methods, their scope has been restricted mainly to detect genotypic determinants of resistance and provide little to no information on phenotypic susceptibility to antibiotics. The work presented here aims to provide phenotypic antimicrobial susceptibility testing (AST) information by pairing short growth periods (~3–4 h) with downstream PCR assays to ultimately predict minimum inhibitory concentration (MIC) values of antibiotic treatment. To further simplify the dual workflows of the AST and PCR assays, these reactions are carried out in a single-vessel format (PCR tube) using novel lyophilized reagent beads (LRBs), which store dried PCR reagents along with primers and enzymes, and antibiotic drugs separately. The two reactions are separated in space and time using a melting paraffin wax seal, thus eliminating the need to transfer reagents across different consumables and minimizing user interactions. Finally, these two-step single-vessel reactions are multiplexed by using a microfluidic manifold that allows simultaneous testing of an unknown bacterial sample against different antibiotics at varying concentrations. The LRBs used in the microfluidic system showed no interference with the bacterial growth and PCR assays and provided an innovative platform for rapid point-of-care diagnostics (POC-Dx).
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DOI：
10.1016/s1473-3099(20)30477-1
发表时间：
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期刊：
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影响因子：
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作者：
Kadri SS;Lai YL;Warner S;Strich JR;Babiker A;Ricotta EE;Demirkale CY;Dekker JP;Palmore TN;Rhee C;Klompas M;Hooper DC;Powers JH 3rd;Srinivasan A;Danner RL;Adjemian J;forming the National Insititutes of Health Antimicrobial Resistance Outcomes Research Initiative (NIH-ARORI)
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