Department of Cell and Molecular Biology & Microbiology, Faculty of Biological Science and Technology, University of Isfahan, Isfahan, Iran.
NanoBioPhotonics Lab, Department of Biological Engineering, Utah State University, Utah, United States.
J Appl Microbiol. 2021 Feb;130(2):493-503. doi: 10.1111/jam.14799. Epub 2020 Aug 10.
Diagnosis of Staphylococcus aureus is important in various diseases from hospital-acquired infections to foodborne diseases. This work reports two new luminescent affiprobes for specific detection of S. aureus.
To develop advanced luminescent affiprobes, enhanced green fluorescent protein (EGFP) was flanked by single and double repeats of ZpA963 affibody using molecular biology studies. The recombinant proteins including fluorescent monomeric affibody (fA ) and fluorescent dimeric affibody (fA ) were expressed in the bacterial expression system, purified and used to identify the S. aureus. Fluorescence microscope and flow cytometry results demonstrated that the treated samples with fA and fA had relatively high fluorescent mean intensities in comparison to the untreated S. aureus cells. Moreover, it was revealed that 'fA ' affiprobe had lower dissociation constant value (about 25-fold) and was more effective for detection of S. aureus than the 'fA ' affiprobe. In addition, the binding of the affiprobes for some other pathogenic bacteria i.e. Escherichia coli, Bacillus cereus, Enterococcus faecalis and Staphylococcus saprophyticus was examined. Expectedly, no cross-reaction was observed for binding the constructed affiprobes to these bacteria, eliminating possibilities for false positive results.
The results show that 'fA ' affiprobe and 'fA ' affiprobe are two new efficient luminescent affiprobes for detecting S. aureus.
We developed a new approach for detection of Staphylococcus aureus in a simple one-step process and in low concentrations of probes. In the best of our knowledge, this is the first study to direct detection of bacterial cells by affiprobes and may be used to develop new diagnostic kits.
金黄色葡萄球菌的诊断在各种疾病中都很重要，从医院获得性感染到食源性疾病。本工作报道了两种用于特异性检测金黄色葡萄球菌的新型发光亲和探针。
为了开发先进的发光亲和探针，使用分子生物学研究方法在增强型绿色荧光蛋白（EGFP）的两侧分别串联和并联 ZpA963 亲和体的单重复和双重复。包括荧光单体亲和体（fA）和荧光二聚体亲和体（fA）在内的重组蛋白在细菌表达系统中表达、纯化，并用于鉴定金黄色葡萄球菌。荧光显微镜和流式细胞术结果表明，与未经处理的金黄色葡萄球菌细胞相比，用 fA 和 fA 处理的样品具有相对较高的荧光平均强度。此外，结果表明“fA”亲和探针具有更低的解离常数值（约 25 倍），并且比“fA”亲和探针更有效地检测金黄色葡萄球菌。此外，还研究了这些亲和探针与其他一些病原菌，即大肠杆菌、蜡样芽孢杆菌、粪肠球菌和腐生葡萄球菌的结合情况。不出所料，这些构建的亲和探针与这些细菌的结合没有交叉反应，消除了出现假阳性结果的可能性。
结果表明，fA 亲和探针和 fA 亲和探针是两种用于检测金黄色葡萄球菌的新型高效发光亲和探针。
我们开发了一种新的方法，用于在简单的一步过程中和在探针的低浓度下检测金黄色葡萄球菌。据我们所知，这是首次使用亲和探针直接检测细菌细胞的研究，可用于开发新的诊断试剂盒。
