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

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