Multiplex detection of the big five carbapenemase genes using solid-phase recombinase polymerase amplification
Multiplex detection of the big five carbapenemase genes using solid-phase recombinase polymerase amplification
复制标题
使用固相重组酶聚合酶扩增多重检测五大碳青霉烯酶基因
DOI：
10.1039/d3an01747h
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发表时间：
2024
期刊：
影响因子：
--
通讯作者：
Johnson C
中科院分区：
文献类型：
--
作者：
Johnson C
五种碳青霉烯酶，被称为“五大酶”，基于其广泛的底物亲和力和全球流行性，已被确定为全球抗生素耐药性的最大威胁。在这里，我们展示了利用重组酶聚合酶扩增（RPA）的等温扩增方法对5种碳青霉烯酶的基因序列进行分子检测的方法。我们证明了使用空间分离的多重扩增策略，以飞摩尔检测限成功检测到五大碳青霉烯酶基因中的每一个。该方法使用加尾寡核苷酸进行杂交，与经典RPA检测策略相比，降低了测定的复杂性和成本。报告探针辣根过氧化物酶在台式酶标仪上产生可测量的输出，但更值得注意的是，我们的研究利用了便携式拉曼光谱仪的功能，使检测限提高了19倍。值得注意的是，开发方法采用固相RPA形式，其中靶向五种碳青霉烯酶基因中每一种的正向引物固定在链霉亲和素包被的微孔板上。采用这种固相方法是实现成功的发展途径时，采用这种精简的方法是至关重要的。该测定需要2小时直到结果，包括在37 °C下40分钟的RPA扩增步骤。这是第一个使用固相RPA检测大五的例子，代表了使用RPA开发大五自动化护理点诊断的里程碑。
Five carbapenemase enzymes, coined the ‘big five’, have been identified as the biggest threat to worldwide antibiotic resistance based on their broad substrate affinity and global prevalence. Here we show the development of a molecular detection method for the gene sequences from the five carbapenemases utilising the isothermal amplification method of recombinase polymerase amplification (RPA). We demonstrate the successful detection of each of the big five carbapenemase genes with femtomolar detection limits using a spatially separated multiplex amplification strategy. The approach uses tailed oligonucleotides for hybridisation, reducing the complexity and cost of the assay compared to classical RPA detection strategies. The reporter probe, horseradish peroxidase, generates the measureable output on a benchtop microplate reader, but more notably, our study leverages the power of a portable Raman spectrometer, enabling up to a 19-fold enhancement in the limit of detection. Significantly, the development approach employed a solid-phase RPA format, wherein the forward primers targeting each of the five carbapenemase genes are immobilised to a streptavidin-coated microplate. The adoption of this solid-phase methodology is pivotal for achieving a successful developmental pathway when employing this streamlined approach. The assay takes 2 hours until result, including a 40 minutes RPA amplification step at 37 °C. This is the first example of using solid-phase RPA for the detection of the big five and represents a milestone towards the developments of an automated point-of-care diagnostic for the big five using RPA.