| Detection of Pathogens and Ampicillin-resistance Genes Using Multiplex Padlock Probes
|
|
Author:
Date:
2017-08-20
[Abstract] Diagnostic assays for pathogen identification and characterization are limited either by the number of simultaneously detectable targets, which rely on multiplexing methods, or by time constraints due to cultivation-based techniques. We recently presented a 100-plex method for human pathogen characterization to identify 75 bacterial and fungal species as well as 33 clinically relevant β-lactamases (Barišić et al., 2016). By using 16S rRNA gene sequences as barcode elements in the padlock probes, and two different fluorescence channels for species and antibiotic resistance identification, we managed to cut the number of microarray probes needed by half. Consequently, we present here the protocol of an assay with a runtime of approx. 8 h and a detection limit of 105 cfu ...
[摘要] 用于病原体鉴定和表征的诊断测定法由依赖于多重方法的同时可检测目标的数量或由于基于培养的技术的时间限制来限制。 我们最近提出了一种用于人类病原体鉴定的100plex方法，以鉴定75种细菌和真菌物种以及33种临床相关β-内酰胺酶（Barišić等，2016）。 通过使用16S rRNA基因序列作为挂锁探针中的条形码元件，以及用于物种和抗生素抗性鉴定的两种不同的荧光通道，我们设法将需要的微阵列探针的数量减少一半。 因此，我们在这里介绍一个运行时间约为的测定方案。 8 h，检测限为105 cfu ml-1。 正确鉴定了89％的β-内酰胺酶和93.7％的物种。
【背景】β-内酰胺酶是一类提供抗β-内酰胺抗生素的抗生素抗性基因，其结构模拟D-丙氨酰-D-丙氨酸，细菌细胞壁的一个组分，从而抑制细菌细胞壁合成。 β-内酰胺酶能够水解β内酰胺抗生素β-内酰胺环的中心成分，并使其无效（Kong et al。，2010）。今天，描述了超过1000种β-内酰胺酶，并且存在巨大的潜在环境储层（Bush，2010; Brandt等，2017）。 ...
|
|
|
| Preparation of Pneumococcal Proteins for Western Blot Analysis
|
|
Author:
Date:
2013-07-05
[Abstract] This protocol was developed in a study aimed to determine the cellular localization of the lysin of pneumococcal phage SV1 (Frias et al., 2013). We obtained proteins from the surface of Streptococcus pneumoniae by elution with choline or those secreted to the medium. The analysis by Western blot of these fractions allowed us to demonstrate that the phage lysin localizes to the cell wall, associating with choline residues in the teichoic acids. Hence, protein extracts can be used to determine the localization of uncharacterized proteins and can also be useful for other biochemical analyses such as protein identification. This protocol can be easily adapted to different pneumococcal strains and growth conditions and it is well suited to isolate other proteins of interest.
[摘要] 该方案是在旨在确定肺炎球菌噬菌体SV1的溶素的细胞定位的研究中开发的（Frias等人，2013）。 我们通过用胆碱或分泌到培养基的那些洗脱从肺炎链球菌的表面获得蛋白质。 通过这些级分的Western印迹分析允许我们证明噬菌体溶素定位于细胞壁，与磷壁酸中的胆碱残基相关。 因此，蛋白质提取物可用于确定未表征蛋白质的定位，并且还可用于其他生物化学分析，例如蛋白质鉴定。 该方案可以容易地适应于不同的肺炎球菌菌株和生长条件，并且其非常适合于分离其他感兴趣的蛋白质。
|
|
|