DAO 124:31-39 (2017)  -  DOI: https://doi.org/10.3354/dao03100

Development of a multiplex PCR method for the simultaneous detection of four myxosporeans infecting gibel carp Carassius auratus gibelio

Dan Li1,2,3, Yanhua Zhai1,2,3, Zemao Gu1,2,3, Yang Liu1,2,3,*

1Department of Aquatic Animal Medicine, College of Fisheries, Huazhong Agricultural University, Wuhan 430070, PR China
2Freshwater Aquaculture Collaborative Innovation Center of Hubei Province, Wuhan 430070, PR China
3Key Lab of Freshwater Animal Breeding, Ministry of Agriculture, Wuhan 430070, PR China
*Corresponding author:

ABSTRACT: Gibel carp Carassius auratus gibelio (Bloch), a commercially important freshwater-cultured fish in China, is threatened by myxosporeans, particularly Thelohanellus wuhanensis, Myxobolus honghuensis, M. wulii and M. turpisrotundus. Here, we developed a multiplex PCR assay for simultaneous detection of these 4 myxosporeans. The specific primers for each species were designed based on the 28S rDNA gene of T. wuhanensis, the ITS–5.8S rDNA of M. honghuensis and M. wulii, and the 18S rDNA gene of M. turpisrotundus. Specificity testing confirmed that the 4 primer sets have no cross-reactivity with other related myxosporean species tested. Detection limits of the multiplex PCR assay were 0.2, 0.3, 3.1 and 3.8 spores for T. wuhanensis, M. honghuensis, M. wulii and M. turpisrotundus, respectively. Following screening of 104 field samples, the analytical sensitivity of the present multiplex PCR assay was found to be similar to the sensitivity obtained by the singleplex PCR assays and was higher than that of microscopic examination. Moreover, Kappa analysis showed a strong agreement between the results of the singleplex and multiplex PCR assays, indicating that the developed multiplex PCR assay was an efficient approach for the diagnosis of the 4 myxosporeans infecting gibel carp.


KEY WORDS: Multiplex PCR · Molecular detection · Myxosporeans · Gibel carp · rDNA


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Cite this article as: Li D, Zhai Y, Gu Z, Liu Y (2017) Development of a multiplex PCR method for the simultaneous detection of four myxosporeans infecting gibel carp Carassius auratus gibelio. Dis Aquat Org 124:31-39. https://doi.org/10.3354/dao03100

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