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=7

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=<p>Brucellosis is a highly contagious zoonosis caused by a species under the genus <italic>Brucella</italic>. A duplex recombinase polymerase amplification (Duplex RPA) assay for the specific detection of <italic>Brucella melitensis</italic> and <italic>Brucella abortus</italic> was developed in this study. Primers were designed targeting hypothetical protein genes and membrane transporter genes of <italic>B. melitensis</italic> and <italic>B. abortus</italic>, 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 analyzed by Duplex RPA, real-time PCR, and multiplex AMOS PCR to estimate the prevalence of brucellosis in sheep and yak in West China. The analytical sensitivities of Duplex RPA were 9 × 10<sup>2</sup> plasmid copies of <italic>B. melitensis</italic> and 9 × 10<sup>1</sup> plasmid copies of <italic>B. abortus</italic>, but by mixing the reaction tubes after 4 min of incubation, the sensitivities were 4 × 10<sup>0</sup> and 5 × 10<sup>0</sup> copies of <italic>B. melitensis</italic> and <italic>B. abortus</italic>, respectively. There was no cross-reactivity with <italic>Brucella suis, Chlamydia abortus, Salmonella typhimurium, Escherichia coli</italic>, and <italic>Toxoplasma gondii</italic>. The screening of field samples by Duplex RPA revealed that the prevalence of <italic>B. melitensis</italic> in sheep and yak was 75.8% and the prevalence of <italic>B. abortus</italic> was 4.8%. Multiplex AMOS PCR showed that the prevalence of <italic>B. melitensis</italic> was 19.3%, and that of <italic>B. abortus</italic> was 4.8%. It was concluded that the developed Duplex RPA is sensitive and specific to the detection of and differentiation between <italic>B. melitensis</italic> and <italic>B. abortus</italic> which will be useful in epidemiological surveillance and in the clinical settings.</p>