The Novel Multiple Inner Primers-Loop-Mediated Isothermal Amplification (MIP-LAMP) for Rapid Detection and Differentiation of Listeria monocytogenes

Molecules

Yi Wang²  Yan Wang²  Aijing Ma²  Dongxun Li³  Lijuan Luo²  Dongxin Liu¹  Shoukui Hu²  Dong Jin²  Kai Liu²  Changyun Ye² 

[1] Pathogenic Biology Institute, University of South China, Hengyang 421000, Hunan, China;State Key Laboratory of Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Chinese Center for Disease Control and Prevention, Changping, Changbai Road 155, Beijing 102206, China;Changping District Center for Disease Control and Prevention, Changping, Beijing 102200, China;

关键词: MIP-LAMP;    LAMP;    LoD;    L. monocytogenes detection;    HlyA gene amplification;   

DOI  :  10.3390/molecules201219787

来源: mdpi

【 摘 要 】

Here, a novel model of loop-mediated isothermal amplification (LAMP), termed multiple inner primers-LAMP (MIP-LAMP), was devised and successfully applied to detect Listeria monocytogenes. A set of 10 specific MIP-LAMP primers, which recognized 14 different regions of target gene, was designed to target a sequence in the hlyA gene. The MIP-LAMP assay efficiently amplified the target element within 35 min at 63 °C and was evaluated for sensitivity and specificity. The templates were specially amplified in the presence of the genomic DNA from L. monocytogenes. The limit of detection (LoD) of MIP-LAMP assay was 62.5 fg/reaction using purified L. monocytogenes DNA. The LoD for DNA isolated from serial dilutions of L. monocytogenes cells in buffer and in milk corresponded to 2.4 CFU and 24 CFU, respectively. The amplified products were analyzed by real-time monitoring of changes in turbidity, and visualized by adding Loop Fluorescent Detection Reagent (FD), or as a ladder-like banding pattern on gel electrophoresis. A total of 48 pork samples were investigated for L. monocytogenes by the novel MIP-LAMP method, and the diagnostic accuracy was shown to be 100% when compared to the culture-biotechnical method. In conclusion, the MIP-LAMP methodology was demonstrated to be a reliable, sensitive and specific tool for rapid detection of L. monocytogenes strains.

【 授权许可】

CC BY   
© 2015 by the authors; licensee MDPI, Basel, Switzerland.