DEVELOPMENT OF A SYBR GREEN REAL-TIME PCR ASSAY FOR DETECTION OF MYXOBOLUS HONGHUENSIS AND ITS APPLICATION

Pharyngeal myxosporidiosis caused by Myxobolus honghuensis is one of the most important limiting factors for the culture industry of the gibel carp, Carassius auratus gibelio (Bloch), in China. Pathogen abundance in the culture system directly determines the consequence of disease outbreaks in cultured aquatic animals. Therefore, a quantitative detection method to monitor the pathogen during the whole culture cycle will not only be applied in early diagnosis but also provide a technical basis for assessing disease risk and evaluating the effects of applied preventative and control measures. Here, a SYBR Green I real-time fluorescent quantitative PCR assay (QPCR) was developed to detect and monitor the abundance of M. honghuensis with a newly designed primer pair, HHF/R, which was based on the ITS loci. The specificity, sensitivity, repeatability and applicability of the assay were deeply evaluated. The results indicated that the developed method could specifically detect M. honghuensis without cross-reactivity with Henneguya doneci, Myxobolus nielii, Myxobolus pronini and Myxobolus wulii, a genetically similar pathogen causing hepatic myxosporidiosis of gibel carp. The lowest detection limit was 3.02×10¹ copies, which was 1000 times more sensitive than conventional PCR. The intraassay and interassay coefficients of variation were below 2%. Importantly, this method could detect all life cycle stages of M. honghuensis, which included it’s trophozoite and presporogonic stages, distributed not only in the tissue of infected fish but also in the water column and sediments. Therefore, the developed QPCR assay has high specificity, sensitivity and repeatability, which can be applied for quantitative monitoring of all life cycle stages of M. honghuensis distributed in a culture system during the whole culture cycle of gibel carp and will be the basis for the development of targeted and precise control strategies for pharyngeal myxosporidiosis of gibel carp.