Evaluation of the classical nested PCR method for Cyclospora cayetanensis detection

doi:10.12140/j.issn.1000-7423.2026.04.011

Abstract

Abstract:

Objective To systematically evaluate a classical nested PCR assay targeting the 18S rRNA gene of Cyclospora cayetanensis for the detection of human infections. Methods A nested PCR assay targeting the 18S rRNA gene of C. cayetanensis was performed with optimized reaction conditions. The specificity was evaluated using DNA templates from 8 non-Eimeriidae parasites, including Clonorchis sinensis, Schistosoma japonicum, Echinococcus granulosus, and Cryptosporidium parvum, as well as 4 Eimeriidae coccidia, including Eimeria caprovina, Cystoisospora canis, E. tenella, and E. bovis. The sensitivity was assessed using 10-fold serial dilutions of recombinant plasmids containing the target fragment (10⁴-10⁰ copies/μL). Results The specificity test showed that the positive DNA of C. cayetanensis from human origin yielded a specific amplicon of approximately 500 bp, while no amplification was observed for the 8 non-Eimeriidae parasites or the negative control. However, the 4 Eimeriidae coccidia (E. caprovina, I. canis, E. tenella, and E. bovis) all produced amplicons of approximately 500 bp. Sequencing confirmed that the sequences shared > 99% similarity with reference sequences of both Cyclospora and Eimeria. The sensitivity test showed that the detection limit of the nested PCR for recombinant plasmids was 10¹ copies/μL. Conclusion The assay exhibits high sensitivity for detecting C. cayetanensis in human fecal samples but shows cross-reaction with Eimeriidae coccidia. Given the strict host specificity of these coccidia, which typically do not infect humans, this method is applicable for screening C. cayetanensis in human-derived samples but is not suitable for detection in animal feces, environmental, or food samples, to avoid overestimation of Cyclospora infection levels. Results should be interpreted with caution.

Key words: Cyclospora cayetanensis, 18S rRNA gene, Nested PCR, Specificity, Sensitivity

CLC Number:

R382.3

ZHOU Hao, LIU Hua, JIANG Yanyan, SHEN Yujuan, LI Zhen, YIN Jianhai, ZHENG Bin, LI Shizhu. Evaluation of the classical nested PCR method for Cyclospora cayetanensis detection[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2026, 44(4): 532-537.

Figures/Tables 3

References 20

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