| Abstrakt: |
Streptococcus parauberis, a gram-positive bacterium, causes significant mortality in cultured fish. In Korea, S. parauberis exhibits diverse serotypes (Ia, Ib/c, and II) from diseased fish including olive flounder (Paralichthys olivaceus), each with different pathogenicity. Therefore, the development of a reliable detection and serotyping assay is essential for effective disease surveillance. From the biochemical traits, although S. parauberis can be categorized into two subgroups associated with serotypes I and II, based on pyrrolidonyl arylamidase (PYR) activity, biochemical characterization is limited in differentiating between subserotypes. Furthermore, even though PCR-based serotyping assays (multiplex PCR; mPCR) have been reported, we aimed to develop an isothermal amplification-based assay for rapid and simultaneous detection and serotyping without the need for thermal cycling equipment. Based on the consensus sequence of the capsular polysaccharide (cps) locus, multiplex recombinase polymerase amplification (mRPA) primers were designed for the simultaneous detection and serotyping of S. parauberis. The optimized primer concentrations for mRPA were 280 nM for the detection of S. parauberis, 240 nM for serotypes Ia and II, and 200 nM for serotype Ib, with optimal amplification conditions determined to be 30 min at 42 °C. In the specificity test, no cross-reactivity with other fish pathogens was observed. In addition, the sensitivity of mRPA was 0.02 ng for detecting S. parauberis and serotypes Ia and Ib/c, while it was 0.2 ng for serotyping type II. Using DNA from cultured bacteria, the novel mRPA showed a detection rate consistent with that of mPCR. Moreover, using DNA from artificially infected tissues, the detection rate by mRPA was 96.7% (29/30), which was different from the results of mPCR (80%, 24/30), which exhibited an 80% detection rate. Therefore, the novel mRPA is an effective and rapid diagnostic tool for the simultaneous detection and serotyping of S. parauberis without requiring thermal cycling equipment as well as a specific bacterial culture step. [ABSTRACT FROM AUTHOR] |
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