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Methods ARTICLE Provisionally accepted The full-text will be published soon. Notify me

Front. Cell. Infect. Microbiol. | doi: 10.3389/fcimb.2019.00078

Rapid detection of Brucella spp. and elimination of carryover using multiple cross displacement amplification coupled with nanoparticles-based lateral flow biosensor

 Shijun Li1, Ying Liu1, Yue Wang1, Ming Wang1, Chunting Liu1 and  Yi Wang2*
  • 1Guizhou Centre for Disease Control and Prevention, China
  • 2National Clinical Research Center for Respiratory Diseases, China

Brucella spp. is capable of causing disease in a range of animal hosts, and human brucellosis is regarded as a life-threating disease. A novel isothermal amplification technique, termed multiple cross displacement amplification (MCDA), was employed for detecting all Brucella species strains. Brucella-MCDA targets the Bscp31 gene (Brucella species-specific gene) to specifically design a set of ten primers. The Brucella-MCDA can be coupled with nanoparticles-based lateral flow biosensor (LFB) for highly specific, simple, rapid and visual detection of Brucella-specific amplicons. Using the protocol, a MCDA amplification followed by 2-min LFB resulted in visualization of DNA products trapped at the LFB test line. Various species of Gram-positive and Gram-negative strains are applied for optimizing and evaluating the target assay. Optimal MCDA condition is found to be 63°C for 40 min, with detection limits at 10 fg of templates in the pure cultures. The specificity of MCDA-LFB technique is of 100%, and no cross-reactions to non-Brucella strains are observed according to the specificity examination. Furthermore, dUTP and AUDG enzyme are added into the MCDA reaction mixtures, which are used for removing false-positive amplification generating from carryover contamination. Thus, 20 min for rapid template extraction followed by AUDG digestion (5 min), MCDA (40 min) combined with LFB detection (2 min) resulted in a total assay time of approximately 70 min. In sum, Brucella-MCDA-LFB technique is a rapid, simple, reliable and sensitive method to detect all Brucella species strains, and can be used as potential screening tool for Brucella strains in various laboratories.

Keywords: Brucellosis; , Isothermal amplification technique; , Nano-biosensor; , Rapid diagnosis; , Limit of Detection

Received: 11 Jan 2019; Accepted: 06 Mar 2019.

Edited by:

Bing Gu, Affiliated Hospital of Xuzhou Medical College, China

Reviewed by:

Erin Graf, Perelman School of Medicine, University of Pennsylvania, United States
Hui Li, Guizhou Medical University (GMU), China  

Copyright: © 2019 Li, Liu, Wang, Wang, Liu and Wang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

* Correspondence: Dr. Yi Wang, National Clinical Research Center for Respiratory Diseases, Guangzhou, China, wildwolf0101@163.com