Wu Shaw-Jye, Chan Alex, Kado Clarence I
Department of Life Science, National Central University, Jhong-li City, Taoyuan, Taiwan.
J Microbiol Methods. 2004 Mar;56(3):395-400. doi: 10.1016/j.mimet.2003.11.005.
For rapid and inexpensive detection of polymerase chain reaction (PCR) amplicons, a novel microsphere agglutination assay has been developed. PCR is carried out using biotinylated forward and reverse primers, and the amplified DNA fragments are able to agglutinate streptavidin-coated microspheres (5.7 microm in diameter). Purification of PCR amplicons is unnecessary when initial primer concentrations are 250 nM. Agglutination can be identified visually within 2 min without any additional equipment or reagents. Using listeriolysin (lisA)-specific biotinylated primers, we have successfully detected and identified Listeria monocytogenes lisA+ cells among Salmonella typhimurium, Staphylococcus aureus, Campylobacter jejuni and Escherichia coli O157:H7 cells. The simplicity of this protocol considerably reduces the time and cost of diagnostic PCR experiments. This procedure is potentially useful for various studies and field applications.
为了快速且低成本地检测聚合酶链反应（PCR）扩增子，已开发出一种新型微球凝集试验。使用生物素化的正向和反向引物进行PCR，扩增的DNA片段能够凝集链霉亲和素包被的微球（直径5.7微米）。当初始引物浓度为250 nM时，无需纯化PCR扩增子。无需任何额外设备或试剂，2分钟内即可通过肉眼识别凝集现象。使用李斯特菌溶血素（lisA）特异性生物素化引物，我们已成功在鼠伤寒沙门氏菌、金黄色葡萄球菌、空肠弯曲菌和大肠杆菌O157:H7细胞中检测并鉴定出单核细胞增生李斯特菌lisA+细胞。该方案的简便性大大减少了诊断性PCR实验的时间和成本。此方法对各种研究和现场应用可能有用。
