Abstract: Objective To establish a loop-mediated isothermal amplification (LAMP) method for detecting the metacercariae in freshwater fish infected with Clonorchis sinensis. Methods ITS2 gene sequence of Clonorchis sinensis was used as the target sequence,and three sets of LAMP primers,including Cs-1,Cs-2 and Cs-3 were synthesized. The specificity of the three sets of primers was compared based on the samples of Clonorchis sinensis metacercaria as a template,and reference samples using DNA extracted from adult Schistosoma japonicum,adult tapeworms,roundworm eggs and metacercariae of other trematode. The sensitivity of the three sets of primers was compared in compliance with different concentration of genomic DNA from adult Clonorchis sinensis as a template. Finally,the sensitivity and specificity of LAMP were concurrently evaluated by comparing PCR findings in detecting the metacercariae in 16 samples of Clonorchis sinensis and 23 samples from other flukes. Results Of the three sets of primers,Cs-3 was the best in sensitivity and specificity. LAMP reaction system established on Cs-3 basis can reveal the DNA in adult Clonorchis sinensis by up to 3. 33×10^-4 ng / μL at the minimal detection concentration,and the sensitivity and specificity were identical to PCR measurement,both of which were 100%. Conclusion A LAMP method for detecting metacercaria of Clonorchis sinensis in freshwater fish was successfully established,which can provide a quick and efficient detection approach to monitoring of parasitic diseases in freshwater fish.

Key words: LAMP, Clonorchis sinensis, Metacercaria, Freshwater fish