Establishment and Application of Visual Loop-mediated Isothermal Amplification of Brucella spp.
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Abstract:
Brucellosis is a zoonotic disease caused by Brucella, which affects the healthy development of animal husbandry, even threatens the health and safety of human beings, and causes serious economic and social losses. Therefore, the prevention and control of Brucellosis is very necessary. In this study, based on Omp2a, a conserved gene of Brucella, three pairs of primers were designed by online website, and the reaction conditions and reaction system were optimized. At the same time, the established LAMP method was compared with PCR detection method, and was used for the detection of actual samples by Center for Disease Control and Prevention of Shaanxi. The results showed that the amplification results of LAMP could be observed by naked eyes based on HNB indicator. The LAMP detection method can accurately distinguish Brucella from non-Brucella, and the detection limit is 2.56×10-4 ng/μL. In the actual sample detection, the sensitivity and specificity of LAMP were 100% compared with conventional separation and identification methods. Therefore, the LAMP detection method established in this study has the advantages of strong specificity and high sensitivity. The detection results can be observed by naked eye after 45 min amplification, which is suitable for field and grass-roots detection.
