Houhoula Dimitra, Papaparaskevas Joseph, Zatsou Katerina, Nikolaras Nikolaos, Malkawi Hanan I, Mingenot-Leclercq Marie-Paule, Konteles Spyros, Koussisis Stamatis, Tsakris Athanassios, Charvalos Ekatherina
Department of Food Technology, Technological Educational Institution of Athens, 12210, Athens, Greece.
Department of Microbiology, Medical School, National and Kapodistrian University of Athens, 11527, Athens, Greece.
New Microbiol. 2017 Jul;40(3):165-169. Epub 2017 May 17.
This paper evaluated magnetic nanoparticle-enhanced PCR for the detection and identification of Staphylococcus aureus and Salmonella enteritidis. Two different types of magnetic nanoparticles designated MPIO (iron concentration 2.5 mg/ml, size 1 µm) and NP (iron concentration 8.7 mg/ml, size 60 nm), both conjugated with S. aureus or S. enteritidis antibodies were evaluated as an enrichment procedure for PCR-detection of the pathogens in Trypticase Soy Broth, milk, blood and meat broth. Bacterial suspensions (1.5x108 cfu/ml) were prepared and serial diluted 10-1. The MPIO and NP nanoparticles were added, followed by incubation for 1 hour at room temperature, magnetic separation of the pellet, DNA extraction and PCR, targeting the femA and invA sequences. The nanoparticle-free and the NP-supplemented dilutions were positive down to the 1.5x102 cfu/ml concentration for both bacteria. The MPIO-supplemented dilutions were positive down to approx. 2x100 cfu/ml concentration, respectively. Bacteria-free TSB was negative by PCR. MPIO nanoparticles (size 1 µm) enhanced the detection of S. aureus and S. enteritidis by PCR, whilst NP nanoparticles (size 60 nm) did not, thus indicating that the size of the magnetic nanoparticles play a significant role in the enrichment procedure.
本文评估了磁性纳米颗粒增强型PCR技术用于检测和鉴定金黄色葡萄球菌和肠炎沙门氏菌的效果。两种不同类型的磁性纳米颗粒，分别为MPIO（铁浓度2.5mg/ml，粒径1µm）和NP（铁浓度8.7mg/ml，粒径60nm），均与金黄色葡萄球菌或肠炎沙门氏菌抗体偶联，被评估作为在胰蛋白胨大豆肉汤、牛奶、血液和肉汁中对这些病原体进行PCR检测的富集方法。制备细菌悬液（1.5x108 cfu/ml）并进行10倍系列稀释至10-1。加入MPIO和NP纳米颗粒，随后在室温下孵育1小时，对沉淀进行磁性分离、DNA提取和PCR，以femA和invA序列为靶标。对于两种细菌，未添加纳米颗粒和添加NP的稀释液在浓度低至1.5x102 cfu/ml时均呈阳性。添加MPIO的稀释液在浓度低至约2x100 cfu/ml时分别呈阳性。不含细菌的胰蛋白胨大豆肉汤经PCR检测为阴性。MPIO纳米颗粒（粒径1µm）通过PCR增强了对金黄色葡萄球菌和肠炎沙门氏菌的检测，而NP纳米颗粒（粒径60nm）则没有，这表明磁性纳米颗粒的大小在富集过程中起着重要作用。
