Discrimination and simultaneous detection of two myxozoan parasites belonging to genus Thelohanellus by multiplex polymerase chain reaction.

Thelohanellus kitauei and Thelohanellus hovorkai are myxozoan parasites pathogenic in cyprinid fish especially adult Israel carp and common carp. In the present study, the complete 18S rRNA-ITS1-5.8S rRNA-ITS2 sequences of these two Thelohanellus species were cloned with primers designed from information from Genbank and previous studies. The results revealed that ITS1 and ITS2 sequences of T. kitauei and T. hovorkai were clearly distinguished although the 18S rRNA regions of the two parasites were highly conserved. Based on these sequences, new primer sets were designed for specific identification of these two parasites by multiplex PCR. Both single and multiplex PCR methods using these primers could identify these two myxozoan parasites from mixed DNA samples successfully. Our findings provide a powerful tool for the differentiation of the highly similar pathogenic Thelohanellus species for specific detection for the early diagnosis of diseases.