Rapid detection of Listeria monocytogenes by nanoparticle-based immunomagnetic separation and real-time PCR

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• 作者：Hua Yang ; Liangwei Qu ; Adrienne N. Wimbrow ; Xiuping Jiang ; Yaping Sun
• 关键词：Listeria monocytogenes ; Magnetic nanoparticles ; Immunomagnetic separation ; Real-time PCR
• 刊名：International Journal of Food Microbiology
• 出版年：2007
• 期刊代码：63_01681605
• 类别：abs
• 出版时间：15 September 2007
• 卷：118
• 期：2
• 页码：132-138
• 文件大小：352 K

摘要

The objective of this study was to develop a method combining nanoparticle-based immunomagnetic separation (IMS) with real-time PCR for a rapid and quantitative detection of Listeria monocytogenes. Carboxyl modified magnetic nanoparticles were covalently bound with rabbit anti-L. monocytogenes via the amine groups. Several factors, such as the amount of immunomagnetic nanoparticles (IMNPs), reaction and collection times, and washing step, were optimized, and the nanoparticle-based IMS in combination with real-time PCR was further evaluated for detecting L. monocytogenes from artificially contaminated milk. The cell numbers calculated from the means of threshold cycles (C_T) of PCR amplification curves were compared to those from plate counts in order to determine the correspondence degree of quantitative data. The capture efficiency (CE) by plating from IMNP-based IMS was 1.4 to 26 times higher than those of Dynabeads^®-based IMS depending on the initial cell concentrations inoculated into milk samples. When combined with real-time PCR, L. monocytogenes DNA was detected in milk samples with L. monocytogenes ≥ 10² CFU/0.5 ml. In the range of 10³ to 10⁷ L. monocytogenes CFU/0.5 ml, cell numbers calculated from C_T values were 1.5 to 7 times higher than those derived from plate counts. Our results demonstrated that both the use of nanoparticles and the choice of anti-L. monocytogenes in our IMNP-based IMS in combination with real-time PCR has improved the sensitivity of L. monocytogenes detection from both nutrient broth and milk samples.