用序列比对设计的茎环结构探针检测金黄色葡萄球菌

Stem-loop Structured Probes Designed by Sequence Comparisons for Detection of Staphylococcus aureus

基于金黄色葡萄球菌16S rRNA基因序列,采用序列比对设计了一种茎环结构的寡聚核苷酸探针。探针的环序列即为金黄色葡萄球菌16S rRNA基因序列的其中一个片段,同其他菌种的16S rRNA基因序列误配2个以上的核苷酸,因此能高度专一、灵敏的检测金黄色葡萄球菌16S rRNA。根据分子信标技术和酶联免疫分析的原理,评估一个实验方法,即利用能构象转换的、固定化的茎环结构探针酶联检测靶核酸。由于探针的特异性加强,这个检测系统能有效的排除假阳性即不会出现误配一个核苷酸的情况。采用微量浓度测定分析,最低下限可检测出大约4ng的金葡球菌16SrRNA。这种方法的灵敏度比其他常规检测方法高出了至少一个数量级。

Based on Staphylococcus aureus 16S rRNA gene sequences, sequence comparisons have been applied to design a kind of stem-loop structured oligonucleotide probes whose loop sequence mismatched other bacterial strains 16S rRNA gene sequences in more than two positions with high specificity and sensitivity. According to the principle of molecular beacon technology and enzyme linked immunosorbent assay, a method has been evaluated using immobilized stem-loop structured oligonucleotides probe as the conformation switch which is applied to enzymatic detection of nucleic acid targets. As its specificity has been strengthened, the system is able to successfully eliminate false-positive result that is mismatch in an oligonucleotide. Employing a microtiter assay format, 4ng of S. aureus 16S rRNA could be detected at least. This approach is more sensitive than other conventional method at least one order of magnitude.