Papadopoulou E, Gale N, Goodchild S A, Cleary D W, Weller S A, Brown T, Bartlett P N
Chemistry , University of Southampton , Highfield , Southampton SO17 1BJ , UK . Email:
ATDBio Ltd , Chemistry , University of Southampton , Highfield , Southampton SO17 1BJ , UK.
Chem Sci. 2015 Mar 1;6(3):1846-1852. doi: 10.1039/c4sc03084b. Epub 2015 Jan 7.
Strain discrimination within genetically highly similar bacteria is critical for epidemiological studies and forensic applications. An electrochemically driven melting curve analysis monitored by SERS has been utilised to reliably discriminate strains of the bacterial pathogen , the causative agent of plague. DNA amplicons containing Variable Number Tandem Repeats (VNTRs) were generated from three strains of : CO92, Harbin 35 and Kim. These amplicons contained a 10 base pair VNTR repeated 6, 5, and 4 times in CO92, Harbin 35 and Kim respectively. The assay also included a blocker oligonucleotide comprising 3 repeats of the 10-mer VNTR sequence. The use of the blocker reduced the effective length of the target sequence available to bind to the surface bound probe and significantly improved the sensitivity of the discrimination. The results were consistent during three replicates that were carried out on different days, using different batches of PCR product and different SERS sphere segment void (SSV) substrate. This methodology which combines low cost, speed and sensitivity is a promising alternative to the time consuming current electrophoretic methods.
在基因高度相似的细菌中进行菌株鉴别对于流行病学研究和法医应用至关重要。一种通过表面增强拉曼光谱（SERS）监测的电化学驱动熔解曲线分析方法已被用于可靠地区分鼠疫病原体（鼠疫的致病因子）的菌株。从三种菌株：CO92、哈尔滨35和Kim中生成了包含可变数目串联重复序列（VNTRs）的DNA扩增子。这些扩增子分别在CO92、哈尔滨35和Kim中包含一个10个碱基对的VNTR，重复次数分别为6次、5次和4次。该检测还包括一个包含10聚体VNTR序列3次重复的阻断寡核苷酸。阻断剂的使用减少了可与表面结合探针结合的靶序列的有效长度，并显著提高了鉴别的灵敏度。在不同日期使用不同批次的PCR产物和不同的SERS球段空隙（SSV）底物进行的三次重复实验中，结果是一致的。这种结合了低成本、速度和灵敏度的方法是目前耗时的电泳方法的一种有前途的替代方法。
