Multiplex PCR development for the simultaneous and rapid detection of two pathogenic flukes, Dactylogyrus spp. and Centrocestus formosanus, in ornamental fishes

Both monogenean Dactylogyrus spp. and digenean Centrocestus formosanus are major pathogens for aquaculture and the fish industry, especially in goldfish (Carassius auratus). The resulting infections in the fish cause extensive morbidity and mortality. However, the accurate detection of both parasitic diseases requires highly experienced personnel and takes a long time. Reliable and sensitive diagnostic methods are needed for detecting the infections in various fish species. To overcome this problem, this study developed the multiplex polymerase chain reaction (m-PCR) assay to rapidly identify and diagnose Dactylogyrus spp. and C. formosanus simultaneously on the target genes (18S and cytb gene) in field-collected samples under one reaction. Two pairs of species-specific primers were manually designed. The optimal reaction conditions, specificity, and sensitivity of the m-PCR assay were investigated. The results demonstrated that the multiplex PCR assay can be used to detect Dactylogyrus spp. and C. formosanus specifically, without cross-amplification with other parasite-related species and their host. The sensitivity showed that the m-PCR system could amplify the two target genes with the lowest DNA concentration at 0.078 ng/mu L for Dactylogyrus spp. and 0.625 ng/mu L for C. formosanus. The detection limit in the case of genomic combination between samples with goldfish was detected at the ratios of 1:13 and 1:2 ng/mu L for Dactylogyrus spp. and C. formosanus, respectively. Moreover, this assay can also be developed and applied to detect the adult, metacercariae, cercariae, and egg stages of C. formosanus as well. Additionally, a total of 50 goldfish samples collected from several farms were found to be positive for Dactylogyrus spp., C. formosanus, and co-infection (fifteen, fourteen, and eighteen, respectively). The developed multiplex PCR shows advantages in specificity and sensitivity which prove to be an invaluable tool for diagnoses and monitoring the epidemic situation.