Li Dan, Zhai Yanhua, Gu Zemao, Liu Yang
Department of Aquatic Animal Medicine, College of Fisheries, Huazhong Agricultural University, Wuhan 430070, PR China.
Dis Aquat Organ. 2017 Mar 30;124(1):31-39. doi: 10.3354/dao03100.
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.
银鲫（Carassius auratus gibelio，Bloch）是中国一种具有重要商业价值的淡水养殖鱼类，受到粘孢子虫的威胁，尤其是武汉碘泡虫（Thelohanellus wuhanensis）、洪湖粘体虫（Myxobolus honghuensis）、伍氏粘体虫（M. wulii）和圆形丑陋粘体虫（M. turpisrotundus）。在此，我们开发了一种多重PCR检测方法，用于同时检测这4种粘孢子虫。基于武汉碘泡虫的28S rDNA基因、洪湖粘体虫和伍氏粘体虫的ITS-5.8S rDNA以及圆形丑陋粘体虫的18S rDNA基因，为每个物种设计了特异性引物。特异性测试证实，这4组引物与测试的其他相关粘孢子虫物种无交叉反应。多重PCR检测方法对武汉碘泡虫、洪湖粘体虫、伍氏粘体虫和圆形丑陋粘体虫的检测限分别为0.2、0.3、3.1和3.8个孢子。在对104份野外样本进行筛查后，发现本多重PCR检测方法的分析灵敏度与单重PCR检测方法获得的灵敏度相似，且高于显微镜检查的灵敏度。此外，Kappa分析表明单重和多重PCR检测结果之间具有高度一致性，表明所开发的多重PCR检测方法是诊断感染银鲫的这4种粘孢子虫的有效方法。
