Establishment and application of a dual-probe fluorescent recombinase polymerase amplification assay for detection of Echinococcus granulosus

doi:10.12140/j.issn.1000-7423.2025.01.010

Abstract

Abstract:

Objective To establish a rapid and convenient assay for detection of Echinococcus and identification of E. granulosus based on recombinant polymerase amplification (RPA). Methods The Echinococcus cytochrome C oxidase subunit 1 (co1) and NADH dehydrogenase subunit 5 (nd5) of genes were selected as target sequences, and specific primers and probes were designed with the software Primer Premier 6 and screened to establish a dual-probe fluorescent RPA assay for detection of Echinococcus nucleic acid. The sensitivity of the dual-probe fluorescent RPA assay was evaluated with co1 and nd5 recombinant plasmid DNA at concentrations of 10⁴, 10³, 10², 10, and 1 copies/μl, and the specificity of the assay was evaluated with genomic DNA from E. multilocularis, Taenia solium, Schistosoma japonicum, Necator americanus, Nippostrongylus brasiliensis, Clonorchis sinensis, Giardia lamblia, Cryptosporidium parvum, Toxoplasma gondii and Babesia microti. The feasibility of the method was evaluated using 23 simulated samples (prepared by mixing E. granulosus protoscolex DNA with DNA extracted from negative canine feces) and 5 field-collected dog fecal samples positive for E. granulosus (confirmed by PCR). Results The optimal reaction procedure of the FAM/HEX dual-probe fluorescence RPA assay for detection of Echinococcus genomic DNA was oscillation in a metal bath at 39 ℃, 300 r/min for 4 minutes, followed by detection of the fluorescence signal for 12.5 minutes using real-time quantitative PCR (qPCR) assay. The total reaction time of performance was 16.5 minutes. The minimum detection limits of the dual-probe fluorescent RPA assay were 10 copies/μl for the co1 recombinant plasmid and 100 copies/μl for the nd5 recombinant plasmid, respectively, and the assay yielded the FAM/HEX dual fluorescence signals for E. granulosus, FAM single fluorescence signal for E. multilocularis, and no specific reaction to the genomic DNA from T. solium, S. japonicum, N. americanus, N. brasiliensis, C. sinensis, G. lamblia, C. parvum, T. gondii and B. microti. In simulated DNA samples, FAM/HEX double positive fluorescence signals were generated in 16 samples containing E. granulosus genomic DNA, including 8 simulated samples containing E. granulosus genomic DNA alone and 8 samples containing both E. granulosus and E. multilocularis genomic DNA, and the FAM single positive fluorescence signal was produced in 7 samples containing E. multilocularis genomic DNA alone. In addition, FAM/HEX double positive fluorescence signals were found in 5 field captured dog fecal samples positive for E. granulosus genomic DNA. No positive signals were observed in other DNA samples. The detection results of the dual-probe fluorescent RPA assay were consistent with those by PCR assay. Conclusion A dual-probe fluorescent RPA assay has been successfully developed for detection of Echinococcus and distinguishing E. granulosus, which is low in the detection limit, high in specificity, and simple and rapid in procedures.

Key words: Echinococcus, E. granulosus, Recombinase polymerase amplification, Rapid identification

CLC Number:

R383.33

DU Jianbo, SU Yaxin, HUO Lele, WANG Ying, WANG Xu, JIANG Bin, CHEN Yuqing, SHEN Yujuan. Establishment and application of a dual-probe fluorescent recombinase polymerase amplification assay for detection of Echinococcus granulosus[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2025, 43(1): 61-68.

Figures/Tables 6

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引物及探针名称 Name of primers and probes	序列（5′→3′） Sequence (5′→3′)
co1-F	TGTTTGGGTTCTATGGGTTGTTGTTTGCTATGT
co1-R	AACACACAAGCAGACAAAACTATACCCGTAACTC
nd5-F	GGCGGTGATTATGTTGGGTTTATGTTGTTGAAGT
nd5-R	AACCAAACACCAGCAGCAACCAAAGTAGAAG
HEX-P	AGAGTGCTGGTTATCCTTTTATTAGGTGG(iHEXdT)T(idsp)(iBHQ1dT)TGGAGGCGATGCGGGC-C3 Spacer
FAM-P	GGTAGCAGGGTTTGGGGTCATCATATGT(i6FAMdT)T(idsp)C(iBHQ1dT)GTTGGGTTGGATGTGAA-C3 Spacer

样品 Sample	双探针荧光RPA方法 Dual-probe fluorescent RPA assay			PCR
样品 Sample	FAM/HEX	FAM	阴性 Negative	阳性 Positive
细粒棘球绦虫模拟样品 E. granulosus simulation sample	8	0	0	8
多房棘球绦虫模拟样品 E. multilocularis simulation sample	0	7	0	7
细粒棘球绦虫和多房棘球绦虫混合模拟样品 E. granulosus + E. multilocularis simulation sample	8	0	0	8
犬粪样 Dog fecal sample	5	0	0	5
合计 Total	21	7	0	28