AUTHOR=Gumaa M. M. , Li Zhaocai , Cao Xiaoan , Zhang Nianzhang , Lou Zhongzi , Zhou Jizhang , Fu Baoquan 

TITLE=Specific Detection and Differentiation Between Brucella melitensis and Brucella abortus by a Duplex Recombinase Polymerase Amplification Assay

JOURNAL=Frontiers in Veterinary Science

VOLUME=Volume 7 - 2020

YEAR=2020

URL=https://www.frontiersin.org/journals/veterinary-science/articles/10.3389/fvets.2020.539679

DOI=10.3389/fvets.2020.539679

ISSN=2297-1769

ABSTRACT=Brucellosis is a highly contagious zoonosis. The disease caused by the species members under the genus Brucella. Duplex A duplex recombinase polymerase amplification (Duplex RPA) assay for the specific detection of Brucella melitensis and Brucella abortus has beenwas developed in this study. Primers were designed targeting hypothetical protein gene and membrane transporter genes of B. melitensis and B. abortus respectively. The newly developed assay was validated for its analytical sensitivity and specificity. Different samples were collected from the Qinghai, Inner Mongolia, and Xinjiang provinces. After DNA extraction, the samples were analysed by Duplex RPA, real-time PCR, and multiplex AMOS PCR to estimate the The test was applied for the detection of two species of Brucella in disease outbreaks occurred prevalence of brucellosis in sheep and yak in the West of China. The new developed assay was validated to its analytical sensitivity (detection limit) and specificity. Different field samples from Qinghai, Inner Mongolia, and Xinjiang provinces were analyzed by Duplex RPA, Real-time PCR, and multiplex AMOS (abortus-melitensis-ovis-suis) PCR. The analytical sensitivitysensitivities of Duplex-RPA was were 9x102 plasmid copies of Brucella melitensis and 9x101 plasmid copies of B. abortus, but by mixing the reaction tubes after 4 minutes of incubationstarting , the sensitivity sensitivities were 4×100 and 5×100 copies of B. melitensis and B. abortus respectively. There was no cross-reactivity with Brucella suis, Chlamydia abortus, Salmonella typhimurium, E. coli, and Toxoplasma gondii. Screening of field samples by Duplex-RPA reveals revealed that a highthe prevalence of B. melitensis in sheep and yak was 75.8% and low the prevalence of B. abortus was 4.8%. Whilst multiplex Multiplex AMOS PCR showed that the prevalence of B. melitensis was 19.3%, and that of B. abortus was 4.8%. It was concluded that the developed Duplex RPA is a rapidsensitive and specific technique for specific the detection of and differentiation between of Brucella melitensis and Brucella abortus and Brucella melitensis. It iswhich will  usefulbe useful in epidemiological surveillance and in the clinical field.