A Novel, Rapid, and Simple PMA-qPCR Method for Detection and Counting of Viable Brucella Organisms.

INTRODUCTION: The plate counting method widely used at present to discern viable from non-viable Brucella in the host or cell is time-consuming and laborious. Therefore, it is necessary to establish a rapid, simple method for detecting and counting viable Brucella organisms. MATERIAL AND METHODS: Using propidium monoazide (PMA) to inhibit amplification of DNA from dead Brucella, a novel, rapid PMA-quantitative PCR (PMA-qPCR) detection method for counting viable Brucella was established. The standard recombinant plasmid with the target BCSP31 gene fragment inserted was constructed for drawing a standard curve. The reaction conditions were optimised, and the sensitivity, specificity, and repeatability were analysed. RESULTS: The optimal exposure time and working concentration of PMA were 10 min and 15 µg/mL, respectively. The correlation coefficient (R2) of the standard curve was 0.999. The sensitivity of the method was 103 CFU/mL, moreover, its specificity and repeatability also met the requirements. The concentration of B. suis measured by the PMA-qPCR did not differ significantly from that measured by the plate counting method, and the concentrations of viable bacteria in infected cells determined by the two methods were of the same order of magnitude. CONCLUSION: In this study, a rapid and simple PMA-qPCR counting method for viable Brucella was established, which will facilitate related research.

Characterization of a Novel Interleukin-1 Receptor Antagonist from Sheep (Ovis aries).

Interleukin-1 receptor antagonist (IL-1Ra) is an antagonist of IL-1ß binding IL-1ß receptors but does not induce intracellular responses or signal transduction. In this study, the full-length complementary DNA (cDNA) of the IL-1Ra gene (OaIL-1Ra) was identified from sheep (Ovis aries) using rapid amplification of cDNA ends PCR and submitted to GenBank with the accession number KC425613. The OaIL-1Ra cDNA comprised an open reading frame of 525 bp encoding a protein of 19765.8 Da, a 5'-untranslated region (UTR) of 27 bp, and a 3'-UTR of 676 bp with a poly(A) tail. Recombinant OaIL-1Ra with bioactivity was expressed in a prokaryotic expression system, and a monoclonal antibody against native OaIL-1Ra was prepared. Through Western blot analyses, the OaIL-1Ra protein was widely expressed in lung, heart, spleen, liver, kidney, muscle, intestine, lymphonodi, rumen, and white blood cells, with the highest levels in liver and spleen. The expression of OaIL-1Ra in primary cultured white blood cells of sheep were highly induced in a time-dependent manner when challenged with different bacteria. These results implied that OaIL-1Ra is associated with immune responses during bacterial infections.