Rapid Detection of Brucella spp. and Elimination of Carryover Using Multiple Cross Displacement Amplification Coupled With Nanoparticles-Based Lateral Flow Biosensor.

Shijun Li,Ying Liu,Yi Wang,Yue Wang,Chunting Liu,Ming Wang

doi:10.3389/fcimb.2019.00078

Abstract

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 10 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 ~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.

Highlights

Brucella spp. are facultative intracellular bacteria that consists of six classical species: Brucella melitensis (B. melitensis), B. abortus, B. canis, B. ovis, B. suis, B. neotomae, one human origin species B. innoponita, and two marine species: B. ceti and B. pennipedilis (De et al, 2008)
We examined the analytical specificity of multiple cross displacement amplification (MCDA)-lateral flow biosensor (LFB) using DNA templates from 79-Brucella strains to 18 non-Brucella strains (Table 2)
Biotin-labeled products form complex with SA-PNPs via biotin-streptavidinbiotin interactions at the conjugated pad, and biotin/MCDA complexes were captured at the test line (TL) by interaction between anti-FITC and FITC

Summary

Introduction

Brucella spp. are facultative intracellular bacteria that consists of six classical species: Brucella melitensis (B. melitensis), B. abortus, B. canis, B. ovis, B. suis, B. neotomae, one human origin species B. innoponita, and two marine species: B. ceti and B. pennipedilis (De et al, 2008). Brucella species are responsible for brucellosis in a range of animal hosts, including marine mammals, wildlife, domesticated livestock, and humans (Tiller et al, 2010). These bacteria are typically transmitted to humans through exposure to tissues or fluids from infected animals, or consumption of unpasteurized dairy products (Traxler et al, 2013). Brucella spp. can cause economic losses to the livestock industry, and serious health problems to animals and humans. To effectively control and prevent these pathogens, the suitable diagnostic techniques using a sensitive, reliable, and rapid assay are required in field, clinical, and veterinary laboratories

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Conclusion