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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References 23

[1]	Wen H, Vuitton L, Tuxun T, et al. Echinococcosis: Advances in the 21st century[J]. Clin Microbiol Rev, 2019, 32(2): e00075-18.
[2]	蒉嫣, 薛垂召, 王旭, 等. 2022年全国棘球蚴病防治工作进展[J]. 中国寄生虫学与寄生虫病杂志, 2024, 42(1): 8-16. doi: 10.12140/j.issn.1000-7423.2024.01.002
	Kui Y, Xue CZ, Wang X, et al. Progress of echinococcosis control in China, 2022[J]. Chin J Parasitol Parasit Dis, 2024, 42(1): 8-16. (in Chinese)
[3]	Wang ZH, Wang XM, Liu XQ. Echinococcosis in China, a review of the epidemiology of Echinococcus spp.[J]. Ecohealth, 2008, 5(2): 115-126.
[4]	Vola A, Manciulli T, De Silvestri A, et al. Diagnostic performances of commercial ELISA, indirect hemagglutination, and western blot in differentiation of hepatic echinococcal and non-echinococcal lesions: A retrospective analysis of data from a single referral centre[J]. Am J Trop Med Hyg, 2019, 101(6): 1345-1349.
[5]	Lobato IM, O’Sullivan CK. Recombinase polymerase amplification: Basics, applications and recent advances[J]. Trends Analyt Chem, 2018, 98: 19-35.
[6]	Liu XQ, Yan QY, Huang JF, et al. Influence of design probe and sequence mismatches on the efficiency of fluorescent RPA[J]. World J Microbiol Biotechnol, 2019, 35(6): 95.
[7]	Nakao M, McManus DP, Schantz PM, et al. A molecular phylogeny of the genus Echinococcus inferred from complete mitochondrial genomes[J]. Parasitology, 2007, 134(Pt 5): 713-722. doi: 10.1017/S0031182006001934 pmid: 17156584
[8]	Alvarez Rojas CA, Romig T, Lightowlers MW. Echinococcus granulosus sensulato genotypes infecting humans: Review of current knowledge[J]. Int J Parasitol, 2014, 44(1): 9-18.
[9]	Khan SN, Ali R, Khan S, et al. Cystic echinococcosis: An emerging zoonosis in southern regions of Khyber Pakhtunkhwa, Pakistan[J]. BMC Vet Res, 2021, 17(1): 139. doi: 10.1186/s12917-021-02830-z pmid: 33794898
[10]	Moro P, Schantz PM. Echinococcosis: A review[J]. Int J Infect Dis, 2009, 13(2): 125-133. doi: 10.1016/j.ijid.2008.03.037 pmid: 18938096
[11]	Shang JY, Zhang GJ, Liao S, et al. A multiplex PCR for differential detection of Echinococcus granulosus sensustricto, Echinococcus multilocularis and Echinococcus canadensis in China[J]. Infect Dis Poverty, 2019, 8: 68.
[12]	Notomi T, Okayama H, Masubuchi H, et al. Loop-mediated isothermal amplification of DNA[J]. Nucleic Acids Res, 2000, 28(12): E63.
[13]	张艳艳, 叶倩, 王正荣, 等. 基于cox2基因的细粒棘球绦虫环介导等温扩增检测方法的初步建立[J]. 中国寄生虫学与寄生虫病杂志, 2017, 35(2): 169-172.
	Zhang YY, Ye Q, Wang ZR, et al. Preliminary exploration of loop-mediated isothermal amplification based on cox2 gene of Echinococcus granulosus[J]. Chin J Parasitol Parasit Dis, 2017, 35(2): 169-172. (in Chinese)
[14]	吕蓓, 程海荣, 严庆丰, 等. 用重组酶介导扩增技术快速扩增核酸[J]. 中国科学: 生命科学, 2010, 40(10): 983-988.
	Lü B, Cheng HR, Yan QF, et al. Recombinase-aid amplification: A novel technology of in vitro rapid nucleic acid amplification[J]. Sci Sin Vitae, 2010, 40(10): 983-988. (in Chinese)
[15]	李婷, 杨坤. 等温扩增技术在寄生虫及其他病原体检测中的应用[J]. 中国血吸虫病防治杂志, 2018, 30(2): 232-236.
	Li T, Yang K. Application of isothermal amplification technology for pathogen detection in parasitic and other diseases[J]. Chin J Schisto Control, 2018, 30(2): 232-236. (in Chinese)
[16]	周鸿让, 茅光耀, 王晓玲, 等. 重组酶介导的多重核酸等温扩增法鉴别细粒棘球绦虫和多房棘球绦虫技术的建立与应用[J]. 中国寄生虫学与寄生虫病杂志, 2020, 38(3): 310-316. doi: 10.12140/j.issn.1000-7423.2020.03.09
	Zhou HR, Mao GY, Wang XL, et al. Establishment and application of a multiplex recombinase-aided isothermal amplification technique for identifying Echinococcus granulosus and Echinococcus multilocularis[J]. Chin J Parasitol Parasit Dis, 2020, 38(3): 310-316. (in Chinese)
[17]	Teoh BT, Sam SS, Tan KK, et al. Early detection of dengue virus by use of reverse transcription-recombinase polymerase amplification[J]. J Clin Microbiol, 2015, 53(3): 830-837.
[18]	梁家瑞, 徐斌, 胡薇, 等. 基于荧光重组酶聚合酶扩增技术快速检测美洲钩虫方法研究[J]. 中国热带医学, 2023, 23(7): 681-685. doi: 10.13604/j.cnki.46-1064/r.2023.07.01
	Liang JR, Xu B, Hu W, et al. Development and preliminary evaluation of a fluorescence RPA assay for the rapid detection of Necator americanus[J]. China Trop Med, 2023, 23(7): 681-685. (in Chinese)
[19]	Tian LB, Shi Y, Yang Y, et al. Rapid on-site detection of echinococcosis and schistosomiasis based on RPA[J]. Mem Inst Oswaldo Cruz, 2024, 119: e230244.
[20]	苏书晓, 赵帅阳, 刘军龙, 等. 环形泰勒虫SHERLOCK-LF检测方法的建立与初步应用[J]. 中国兽医科学, 2024, 54(9): 1182-1187.
	Su SX, Zhao SY, Liu JL, et al. Establishment and preliminary application of SHERLOCK-LF detection method for Theileria annulata[J]. Chin Vet Sci, 2024, 54(9): 1182-1187. (in Chinese)
[21]	高璟瑜, 李秋阳, 姚瑶, 等. 恶性疟原虫LF-RPA检测方法研究[J]. 寄生虫与医学昆虫学报, 2023, 30(4): 193-196, 209.
	Gao JY, Li QY, Yao Y, et al. Study on LF-RPA detection method of Plasmodium falciparum[J]. Acta Parasitol Med Entomol Sin, 2023, 30(4): 193-196, 209. (in Chinese)
[22]	王盛琳, 邓王平, 李银龙, 等. 重组酶聚合酶扩增技术快速检测日本血吸虫核酸方法的建立[J]. 中国寄生虫学与寄生虫病杂志, 2020, 38(3): 293-298. doi: 10.12140/j.issn.1000-7423.2020.03.006
	Wang SL, Deng WP, Li YL, et al. Establishment of recombinase polymerase amplification technique for rapid detection of Schistosoma japonicum nucleic acid[J]. Chin J Parasitol Parasit Dis, 2020, 38(3): 293-298. (in Chinese)
[23]	王丽萍, 吕超, 秦志强, 等. 基于重组酶聚合酶扩增的曼氏血吸虫核酸可视化检测技术的建立及初步评价[J]. 中国寄生虫学与寄生虫病杂志, 2022, 40(3): 337-343. doi: 10.12140/j.issn.1000-7423.2022.03.009
	Wang LP, Lv C, Qin ZQ, et al. Establishment and preliminary evaluation of a visualized detection technique for Schistosoma mansoni nucleic acid based on recombinase polymerase amplification[J]. Chin J Parasitol Parasit Dis, 2022, 40(3): 337-343. (in Chinese)

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