双探针荧光重组酶聚合酶扩增法检测细粒棘球绦虫技术的建立与应用

doi:10.12140/j.issn.1000-7423.2025.01.010

中国寄生虫学与寄生虫病杂志 ›› 2025, Vol. 43 ›› Issue (1): 61-68.doi: 10.12140/j.issn.1000-7423.2025.01.010

双探针荧光重组酶聚合酶扩增法检测细粒棘球绦虫技术的建立与应用

杜建伯()(), 苏雅馨, 霍乐乐, 王莹, 王旭, 姜斌, 陈雨晴, 沈玉娟^*()()

中国疾病预防控制中心寄生虫病预防控制所（国家热带病研究中心）；传染病溯源预警与智能决策全国重点实验室；国家卫生健康委员会寄生虫病原与媒介生物学重点实验室；世界卫生组织热带病合作中心；科技部国家级热带病国际联合研究中心，上海 200025

收稿日期:2024-12-05 修回日期:2025-01-07 出版日期:2025-02-28 发布日期:2025-03-26
通讯作者: *沈玉娟（ORCID：0000-0003-4386-6624），女，研究员，从事棘球蚴、隐孢子虫感染与免疫，核酸检测技术及药物研究。E-mail：shenyj@nipd.chinacdc.cn
作者简介:杜建伯（ORCID：0009-0002-5482-7989），男，硕士研究生，从事棘球蚴核酸检测技术研究。E-mail：Sebastian_Du@163.com
基金资助:
国家自然科学基金(82072307);国家自然科学基金(82372283)

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

DU Jianbo()(), SU Yaxin, HUO Lele, WANG Ying, WANG Xu, JIANG Bin, CHEN Yuqing, SHEN Yujuan^*()()

National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention; Chinese Center for Tropical Diseases Research; National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases; Key Laboratory on Parasite and Vector Biology, National Health Commission; WHO Collaborating Centre for Tropical Diseases; National Center for International Research on Tropical Diseases, Ministry of Science and Technology, Shanghai 200025, China

Received:2024-12-05 Revised:2025-01-07 Online:2025-02-28 Published:2025-03-26
Contact: *E-mail: shenyj@nipd.chinacdc.cn
Supported by:
National Natural Science Foundation of China(82072307);National Natural Science Foundation of China(82372283)

摘要/Abstract

摘要：

目的结合重组酶聚合酶扩增技术（RPA），建立一种快速、便捷的检测棘球绦虫及进一步鉴定细粒棘球绦虫的方法。方法以棘球绦虫细胞色素C氧化酶亚基1（co1）和NADH脱氢酶亚基5（nd5）的基因为靶序列，利用Primer Premier 6软件设计特异性引物和探针并进行筛选，建立棘球绦虫核酸双探针荧光RPA检测方法。采用不同DNA拷贝数（10⁴、10³、10²、10、1个拷贝/μl）的含co1和nd5的重组质粒，评价该方法的敏感度。以多房棘球绦虫、猪带绦虫、日本血吸虫、美洲钩虫、巴西日本圆线虫、华支睾吸虫、蓝氏贾第鞭毛虫、微小隐孢子虫、刚地弓形虫和田鼠巴贝虫基因组DNA评价该方法的特异性。利用23份模拟样品（棘球绦虫原头节DNA和阴性犬粪提取的DNA混合）和5份现场采集的犬粪细粒棘球绦虫阳性DNA样品（经PCR鉴定）评价该方法的可行性。结果针对棘球绦虫基因组DNA建立的FAM/HEX双探针荧光RPA检测法的最佳反应条件为39 ℃、金属浴300 rpm振荡反应4 min后，采用荧光定量PCR（qPCR）检测荧光信号12.5 min，总反应时长16.5 min。敏感度检测结果显示，该法对co1和nd5重组质粒的最低检出限分别为10拷贝/μl和100拷贝/μl。特异性检测结果显示，该法仅对细粒棘球绦虫有FAM/HEX双重荧光信号，对多房棘球绦虫有FAM单荧光信号，而对猪带绦虫、日本血吸虫、美洲钩虫和华支睾吸虫等9种寄生虫基因组DNA无特异性反应。可行性评价结果显示，含有细粒棘球绦虫基因组DNA的16份模拟样品产生FAM/HEX双阳性信号，其中8份为混合细粒棘球绦虫基因组DNA的模拟样品，8份为混合细粒和多房棘球绦虫基因组DNA的模拟样品；仅含有多房棘球绦虫基因组DNA的7份模拟样品产生FAM单阳性信号；5份现场采集的犬粪细粒棘球绦虫阳性DNA样品产生FAM/HEX双阳性信号；其他DNA样品均无任何阳性信号。该法的检测结果与PCR法的检测结果一致。结论本研究建立了基于双探针荧光RPA检测棘球绦虫的方法，且该方法可鉴定出细粒棘球绦虫，其检出限低、特异性强，操作简单快捷。

关键词: 棘球绦虫, 细粒棘球绦虫, 重组酶聚合酶扩增, 快速鉴别

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

中图分类号:

R383.33

杜建伯, 苏雅馨, 霍乐乐, 王莹, 王旭, 姜斌, 陈雨晴, 沈玉娟. 双探针荧光重组酶聚合酶扩增法检测细粒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, 2025, 43(1): 61-68.

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.

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参考文献 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

双探针荧光重组酶聚合酶扩增法检测细粒棘球绦虫技术的建立与应用

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

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[14]	仲顺虎, 孙玥, 郭小腊, 郑亚东, 陈轶霞. 多房棘球蚴感染小鼠脾淋巴细胞中差异表达miRNA的鉴定及其生物信息学分析[J]. 中国寄生虫学与寄生虫病杂志, 2022, 40(3): 288-294.
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