Development of a multiplex PCR method for the simultaneous detection of four myxosporeans infecting gibel carp Carassius auratus gibelio.
Development of a multiplex PCR method for the simultaneous detection of four myxosporeans infecting gibel carp Carassius auratus gibelio.
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DOI：
10.3354/dao03100
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发表时间：
2017-03
影响因子：
1.4
通讯作者：
Dan Li;Yanhua Zhai;Z. Gu;Yang Liu
中科院分区：
文献类型：
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作者：
Dan Li;Yanhua Zhai;Z. Gu;Yang Liu
异育银鱼是中国重要的经济养殖淡水鱼，受到粘孢子虫的威胁，尤其是武汉粘孢子虫、洪湖粘孢子虫、乌拉锦鲤和团头锦鲤。在这里，我们建立了一种同时检测这4种粘孢子菌的多重PCR方法。根据武汉山毛虫的28S rDNA基因、洪湖和乌拉木的ITS-5.8S rDNA，以及大菱形吸虫的18S rDNA基因，设计了针对各物种的特异引物。特异性试验证实，这4组引物与所测试的其他相关粘孢子菌没有交叉反应。多重聚合酶链式反应的检测限分别为0.2、0.3、3.1和3.8个孢子，对乌汉锥虫、华支睾吸虫、乌拉木和松材线虫的检测限分别为0.2、0.3、3.1和3.8个孢子。通过对104个现场样品的筛选，发现本方法的分析灵敏度与单重聚合酶链式反应的灵敏度相似，高于镜检法。Kappa分析表明，单链和多重聚合酶链式反应的结果具有很好的一致性，表明所建立的复合聚合酶链式反应方法是诊断银鱼4株粘孢子菌的有效方法。
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.