AIT Austrian Institute of Technology, Molecular Diagnostics, 1190 Vienna, Austria.
Diagn Microbiol Infect Dis. 2013 Oct;77(2):118-25. doi: 10.1016/j.diagmicrobio.2013.06.013. Epub 2013 Aug 12.
The elucidation of resistance mechanisms is of central importance to providing and maintaining efficient medical treatment. However, molecular detection methods covering the complete set of resistance genes with a single test are still missing. Here, we present a novel 100-plex assay based on padlock probes in combination with a microarray that allows the simultaneous large-scale identification of highly diverse β-lactamases. The specificity of the assay was performed using 70 clinical bacterial isolates, recovering 98% of the β-lactamase nucleotide sequences present. Additionally, the sensitivity was evaluated with PCR products and genomic bacterial DNA, revealing a detection limit of 10(4) DNA copies per reaction when using PCR products as the template. Pre-amplification of genomic DNA in a 25-multiplex PCR further facilitated the detection of β-lactamase genes in dilutions of 10(7) cells/mL. In summary, we present an efficient, highly specific, and highly sensitive multiplex detection method for any gene.
阐明耐药机制对于提供和维持有效的医疗至关重要。然而，目前仍缺乏一种能够通过单次检测涵盖整套耐药基因的分子检测方法。在这里，我们提出了一种新的基于发夹探针与微阵列相结合的 100 重检测方法，可同时大规模鉴定高度多样化的β-内酰胺酶。该检测方法的特异性通过 70 株临床分离菌进行评估，可回收 98%存在的β-内酰胺酶核苷酸序列。此外，还使用 PCR 产物和基因组细菌 DNA 评估了该方法的灵敏度，结果表明当使用 PCR 产物作为模板时，检测限为每个反应 10(4)个 DNA 拷贝。使用 25 重 PCR 对基因组 DNA 进行预扩增，进一步促进了对 10(7)细胞/mL 稀释度的β-内酰胺酶基因的检测。总之，我们提出了一种高效、高特异性和高灵敏度的多重检测方法，可用于任何基因。
