Highly sensitive and rapid detection of Pseudomonas aeruginosa based on magnetic enrichment and magnetic separation.
Highly sensitive and rapid detection of Pseudomonas aeruginosa based on magnetic enrichment and magnetic separation.
复制标题
基于磁富集和磁分离的铜绿假单胞菌高灵敏快速检测
作者：
Tang Y;Zou J;Ma C;Ali Z;Li Z;Li X;Ma N;Mou X;Deng Y;Zhang L;Li K;Lu G;Yang H;He N
本文介绍了一种基于磁富集和磁分离技术的高灵敏、快速检测铜绿假单胞菌的方法。样品裂解后，磁性纳米颗粒（MNP）被用于吸附基因组DNA。直接对DNA结合MNP进行聚合酶链反应（PCR）以扩增铜绿假单胞菌gyrB特异性序列。生物素标记的PCR产物与探针修饰的MNP和碱性磷酸酶（ALP）标记的链霉亲和素（SA）共同孵育后，用化学发光法检测。琼脂糖凝胶电泳分析证实了原位PCR方法的可靠性。详细研究了影响荧光强度的因素。结果表明，粒径为400 nm的纳米粒子有利于检测。探针的序列长度和与靶序列的结合位点对检测有明显的影响。最佳探针浓度为10 μM，杂交温度为60 ℃，杂交时间为60 min。基于MNP的原位PCR方法可以大大提高DNA模板的利用率，最终提高检测灵敏度。实验结果表明，该引物和探针具有较高的特异性，可成功检测出铜绿假单胞菌，检测限低至10 cfu/mL，且可实现对单一铜绿假单胞菌的检测。
A method for highly sensitive and rapid detection of Pseudomonas aeruginosa, based on magnetic enrichment and magnetic separation, is described in this paper. The magnetic nanoparticles (MNPs) were applied to adsorb genome DNA after the sample was lysed. The DNA binding MNPs were directly subjected to polymerase chain reaction (PCR) to amplify gyrB specific sequence of Pseudomonas aeruginosa. The biotin labeled PCR products were detected by chemiluminescence when they were successively incubated with the probes-modified MNPs and alkaline phosphatase (ALP) labeled streptavidin (SA). Agarose gel electrophoresis analyses approved the method of in situ PCR to be highly reliable. The factors which could affect the chemiluminiscence were studied in detail. The results showed that the MNPs of 400 nm in diameter are beneficial to the detection. The sequence length and the binding site of the probe with a target sequence have obvious effects on the detection. The optimal concentration of the probes, hybridization temperature and hybridization time were 10 μM, 60 ºC and 60 mins, respectively. The method of in situ PCR based on MNPs can greatly improve the utilization rate of the DNA template ultimately enhancing the detection sensitivity. Experiment results proved that the primer and probe had high specificity, and Pseudomonas aeruginosa was successfully detected with detection limits as low as 10 cfu/mL by this method, while the detection of a single Pseudomonas aeruginosa can also be achieved.
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影响因子：
10.8
作者：
Fornara, Andrea;Johansson, Petter;Johansson, Christer
通讯作者：
Johansson, Christer
影响因子：
4
作者：
Lee CS;Wetzel K;Buckley T;Wozniak D;Lee J
通讯作者：
Lee J
DOI：
10.1016/j.diagmicrobio.2010.07.012
发表时间：
2010-12-01
影响因子：
2.9
作者：
Logan, Catriona;Habington, Adele;O'Sullivan, Niamh
通讯作者：
O'Sullivan, Niamh
影响因子：
5.7
作者：
Cattoir V;Gilibert A;Le Glaunec JM;Launay N;Bait-Mérabet L;Legrand P
通讯作者：
Legrand P
影响因子：
12.4
作者：
Chen XS
通讯作者：
Chen XS