Abstract:

Objective  To develop a detection method of the third-stage larvae of Angiostrongylus cantonensis by real-time PCR and high-resolution melt curve analysis. 　Methods　 A pair of specific primers was designed based on the internal transcribed spacer 1 （ITS1） region of the nuclear ribosomal DNA of A. cantonensis. The third-stage larvae of A. cantonensis were detected by real-time PCR and high-resolution melt curve analysis. The specificity of the method was analyzed by testing DNAs of A. cantonensis, Clonorchis sinensis, and Gnathostoma spinigerum. The genomic DNA were extracted from 1 to 10 third-stage larvae of A. cantonensis, respectively, and used to identify the sensitivity of the method. 　Results　 This method could specifically detect A. cantonensis and the detection limit reached to one larva. No amplification curve and melt curve were found in C. sinensis and G. spinigerum.　　Conclusion　 Real-time PCR and high-resolution melt curve analysis show good specificity and sensitivity for detecting the third-stage larvae of A. cantonensis.

Key words:  , Angiostrongylus cantonensis；Real-time PCR；High-resolution melt curve