title: Development of a multiplex PCR method for the simultaneous detection of four myxosporeans infecting gibel carp Carassius auratus gibelio.
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.
authors.affiliation: Department of Aquatic Animal Medicine, College of Fisheries, Huazhong Agricultural University, Wuhan 430070, PR China.
authors.forename: Dan
authors.initials: D
authors.lastname: Li
authors.forename: Yanhua
authors.initials: Y
authors.lastname: Zhai
authors.forename: Zemao
authors.initials: Z
authors.lastname: Gu
authors.forename: Yang
authors.initials: Y
authors.lastname: Liu
mesh_terms: D000818:Animals; D004247:DNA / Q000235:genetics; D005393:Fish Diseases / Q000469:parasitology*; D006054:Goldfish; D055764:Myxozoa / Q000235:genetics*; D010273:Parasitic Diseases, Animal / Q000175:diagnosis / Q000469:parasitology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D004247:DNA
keywords: Gibel carp; Molecular detection; Multiplex PCR; Myxosporeans; rDNA