Establishment And Preliminary Application Of PCR Detection Methods For Myxosporeans In Carassius Auratus Gibelio

Carassius auratus gibelio Bloch is an economically important freshwater cultured fish in China. With the successful cultivation of new variety and promotion of aquaculture techniques, this species has been widely extended for culture. Meanwhile, more and more pathogenic organisms have been reported to restrict development of its aquaculture industry, of which myxosporeans are emerging as the major parasite group.Among them,the best known species are Thelohanellus wuhanensis,Myxobolus honghuensis, M. wulii,M. turpisrotunds, which are responsible for the morbidity, mortality and sever economic losses. In order to effectively control the disease, it is urgent to establish a rapid, accurate and practical method for detection of them. Therefore, in the present study, a single-round PCR and multiplex PCR method were established to detect the four important myxosporeans and applied to actual detection.1. Establishment of the single-round PCR method: Based on the sequence alignment and analysis of four myxosporeans ribosomal genes, four specific primers were screened targeting on 28 S r DNA gene of T. wuhanensis, ITS1-5.8S-ITS2 sequence of M.honghuensis and M. wulii, and 18 S rDNA gene ofM. turpisrotundus, which generates four specific amplifications of 245, 447, 590 and 894 bp,respectively. The assays did not cross react with other closely related myxosporean species. The diagnostic sensitivities of all the four myxosporean species reached pg level of parasite DNA.2. Establishment of the multiplex PCR method: Based on the four single-round PCR methods, the multiplex PCR reaction system and condition were optimized and four specific products could be successfully amplified in one reaction. Specificity test confirmed four primer sets have no cross-reactive with other related myxosporean species.The diagnostic sensitivities of all the four myxosporean species reached ng level of parasite DNA.3. Preliminary application of two PCR methods: 104 DNA samples extracted from mixed tissue of gibel carp were used to confirm the unity of two PCR methods compared with microscopic exanmination. Results showed that positive detection rate by two PCR methods were higher than that of microscopic exanmination, and that of single PCR was higher than multiplex PCR method.