Development and comparison of SYBR green– and TaqMan probe–based quantitative polymerase chain reaction (qPCR) assays for detection of microsporidian parasite Microsporidium seriolae (beko disease) in cultured yellowtail, Seriola quinqueradiata

Abstract

Microsporidium seriolae, an intracellular fungal-related parasite, causes microsporidiosis, also referred to as beko disease, in farmed yellowtail (Seriola quinqueradiata) and amberjack (S. dumerili) in Japan. This disease causes the formation of cysts in the muscles of fry to adult fish. In this study, we described novel SYBR green– and TaqMan probe–based real-time quantitative polymerase chain reaction (qPCR) assays for the detection of M. seriolae based on the newly identified short conserved small subunit ribosomal RNA (SSU rRNA) sequence at the 5′ region. The partial sequences of SSU rRNA of the eight M. seriolae strains were 1814–1820 bp. The developed qPCR primers specifically targeted to the SSU rRNA of M. seriolae but not that of Spraguea sp. in plasmid DNA or clinical sample. The efficiency of SYBR green qPCR assay and TaqMan qPCR assay ranged from 97.1 to 98.0% and 97.2 to 98.4%, respectively, and the sensitivity of both assays using diluted plasmid was 0.5 × 10⁴ copies/20 μl reaction. Both assays could detect M. seriolae at 0.4 ng of total genomic DNA from a M. seriolae-infected yellowtail. No difference in qPCR amplification efficiency was observed between the two methods when clinical samples were used. These results suggest that the developed qPCR assay is a useful tool for the rapid detection of M. seriolae and can facilitate epidemiological investigations in a yellowtail aquaculture.