环境DNA技术检测日本血吸虫尾蚴的实验研究

doi:10.12140/j.issn.1000-7423.2025.04.023

中国寄生虫学与寄生虫病杂志 ›› 2025, Vol. 43 ›› Issue (4): 596-599.doi: 10.12140/j.issn.1000-7423.2025.04.023

环境DNA技术检测日本血吸虫尾蚴的实验研究

文雨松¹()(), 徐慧¹, 邱婷婷¹, 赵琴², 李婕², 徐建睿², 曾小军¹, 丁晟¹, 李召军¹^,^*()()

1 江西省寄生虫病防治研究所，江西南昌 330096
2 南昌大学公共卫生学院，江西南昌 330006

收稿日期:2025-03-31 修回日期:2025-05-21 出版日期:2025-08-30 发布日期:2025-10-09
通讯作者: *李召军（ORCID：0000-0003-1775-8945），男，硕士，研究员，从事血吸虫病预防控制研究。E-mail：13576086889@163.com
作者简介:文雨松（ORCID：0009-0006-1013-0492），男，硕士，助理研究员，从事血吸虫病预防控制研究。E-mail：343436758@qq.com
基金资助:
江西省科技计划(20203BBGL73168);江西省医学领先学科建设项目(2021030107);江西省卫生健康委科技计划(202211332);江西省卫生健康委科技计划(202410497);国家疾病预防控制局2024年度公共卫生人才培养支持项目

Detection of Schistosoma japonicum cercariae using environmental DNA assay

WEN Yusong¹()(), XU Hui¹, QIU Tingting¹, ZHAO Qin², LI Jie², XU Jianrui², ZENG Xiaojun¹, Ding Sheng¹, LI Zhaojun¹^,^*()()

1 Jiangxi Provincial Institute of Parasitic Diseases, Nanchang 330096, Jiangxi, China
2 School of Public Health, Nanchang University, Nanchang 330006, Jiangxi, China

Received:2025-03-31 Revised:2025-05-21 Online:2025-08-30 Published:2025-10-09
Contact: *E-mail: 13576086889@163.com
Supported by:
Jiangxi Provincial Science and Technology Project(20203BBGL73168);Jiangxi Provincial Leading Discipline Construction Program in Medical Sciences(2021030107);Science and Technology Project of Jiangxi Provincial Health Commission(202211332);Science and Technology Project of Jiangxi Provincial Health Commission(202410497);2024 Public Health Talent Training Support Project of the National Disease Control and Prevention Administration

摘要/Abstract

摘要：

为评估环境DNA（eDNA）技术检测日本血吸虫尾蚴的可行性及灵敏度，采用阳性钉螺逸蚴获得尾蚴，设不同密度尾蚴组，1、5、10、15、20、> 30条尾蚴为实验组，0条尾蚴为对照组，静置24 h后过滤尾蚴，收集水体，采用0.22 μm滤膜富集eDNA，使用水体基因组DNA试剂盒提取各组eDNA。以日本血吸虫线粒体细胞色素c氧化酶亚基1（cytochrome coxidase subunit 1，cox1）基因和16S rRNA基因为特异性引物，采用实时荧光定量PCR（qPCR）检测各组eDNA，记录循环阈值（Ct值），并对Ct值与尾蚴密度、eDNA浓度的相关性进行Spearman相关性分析。qPCR结果显示，引物cox1和16S rRNA均能检出不同密度尾蚴实验组的尾蚴DNA，DNA浓度分别为2.4、1.0、0.4、1.5、1.3、9.4 μg/ml。Spearman相关性分析结果显示，尾蚴密度与cox1引物Ct值呈负相关（r = -0.886，P < 0.05），与16S rRNA引物Ct值有负相关性趋势但无统计学意义（r = -0.771，P > 0.05）；eDNA浓度与cox1引物、16S rRNA引物Ct值均有负相关性趋势但无统计学意义（r = -0.314、-0.200，均P > 0.05）。结果提示环境DNA技术可以用于血吸虫尾蚴检测且时效性高，具有早期预警血吸虫感染高危环境的价值。

关键词: 日本血吸虫, 环境DNA, 血吸虫尾蚴, 钉螺

Abstract:

To investigate the feasibility and sensitivity of environmental DNA (eDNA) assay for detection of Schistosoma japonicum cercariae, cercariae shed from S. japonicum-infected Oncomelania hupensis snail were collected,with 1, 5, 10, 15, 20, and > 30 cercariae as experimental groups and 0 cercaria as the control group. After standing for 24 hours, cercariae were filtered and water samples were collected. Then, eDNA was enriched using membrane filter with 0.22 μm in pore size, and extracted using the Water DNA Isolation Kit. The Real-time fluorescence quantitative PCR (qPCR) assay was performed with S. japonicum mitochondrial cytochrome c coxidase subunit 1 (cox1) and 16S rRNA genes as specific primers to detect eDNA in each group. The cycle threshold (Ct value) was recorded, and the associations of Ct value with cercariae density and eDNA concentration were examined using Spearman correlation analysis. qPCR assay detected S. japonicum cercariae DNA in all experimental groups of different densities of cercariae with the primers cox1 and 16S rRNA. The DNA concentrations were 2.4, 1.0, 0.4, 1.5, 1.3, and 9.4 μg/ml, respectively. Spearman correlation analysis revealed that the cercariae density significantly negatively correlated with the Ct value with the primer cox1 gene (r = -0.886, P < 0.05), and negatively correlated with the Ct value with the primer 16S rRNA gene (r = -0.771, P > 0.05). The eDNA concentration negatively correlated with the Ct value with the primer cox1 gene (r = -0.314, P > 0.05) and the primer 16S rRNA gene (r = -0.200, P > 0.05). These findings suggest that eDNA assay is feasible for detection of S. japonicum cercariae with high timeliness, which has the value for early warning of environments at a high risk of S. japonicum infections.

Key words: Schistosoma japonicum, Environmental DNA, Schistosome cercaria, Oncomelania hupensis

中图分类号:

R383.24

文雨松, 徐慧, 邱婷婷, 赵琴, 李婕, 徐建睿, 曾小军, 丁晟, 李召军. 环境DNA技术检测日本血吸虫尾蚴的实验研究[J]. 中国寄生虫学与寄生虫病杂志, 2025, 43(4): 596-599.

WEN Yusong, XU Hui, QIU Tingting, ZHAO Qin, LI Jie, XU Jianrui, ZENG Xiaojun, Ding Sheng, LI Zhaojun. Detection of Schistosoma japonicum cercariae using environmental DNA assay[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2025, 43(4): 596-599.

图/表 2

参考文献 23

[1]	周晓农, 李石柱, 洪青标, 等. 不忘初心送瘟神科学防治谱新篇——纪念毛泽东主席《七律二首?送瘟神》发表60周年[J]. 中国血吸虫病防治杂志, 2018, 30(1): 1-4.
	Zhou XN, Li SZ, Hong QB, et al. Remain true to our original aspiration for farewell to the God of Plague, compose the new chapter for the national schistosomiasis control programme scientifically: Commemoration of 60th anniversary of publishing Chairman Mao Zedong’s two poems “Farewell to the God of Plague”[J]. Chin J Schisto Control, 2018, 30(1): 1-4. (in Chinese)
[2]	Zhou XN, Wang LY, Chen MG, et al. The public health significance and control of schistosomiasis in China: Then and now[J]. Acta Trop, 2005, 96(2/3): 97-105.
[3]	张利娟, 祝红庆, 王强, 等. 2020年长江流域洪涝灾害后血吸虫病传播风险分析[J]. 中国血吸虫病防治杂志, 2020, 32(5): 464-468, 475.
	Zhang LJ, Zhu HQ, Wang Q, et al. Assessment of schistosomiasis transmission risk along the Yangtze River basin after the flood disaster in 2020[J]. Chin J Schisto Control, 2020, 32(5): 464-468, 475. (in Chinese)
[4]	吴锋, 黄轶昕. 疫水测定在血吸虫病预测预警中的应用[J]. 中国血吸虫病防治杂志, 2010, 22(5): 500-503.
	Wu F, Huang YX. Application of determination of infested water in schistosomiasis forecast and early warning[J]. Chin J Schisto Control, 2010, 22(5): 500-503. (in Chinese)
[5]	陈琳, 曹淳力, 刘阳, 等. 迈向消除阶段我国血吸虫病突发疫情应急响应[J]. 中国血吸虫病防治杂志, 2021, 33(6): 570-574.
	Chen L, Cao CL, Liu Y, et al. Emergency responses to schistosomiasis outbreak during the stage moving towards elimination in China[J]. Chin J Schisto Control, 2021, 33(6): 570-574. (in Chinese)
[6]	曹淳力, 李石柱, 周晓农. 特大洪涝灾害对我国血吸虫病传播的影响及应急处置[J]. 中国血吸虫病防治杂志, 2016, 28(6): 618-623.
	Cao CL, Li SZ, Zhou XN. Impact of schistosomiasis transmission by catastrophic flood damage and emergency response in China[J]. Chin J Schisto Control, 2016, 28(6): 618-623. (in Chinese)
[7]	Grimes JET, Croll D, Harrison WE, et al. The roles of water, sanitation and hygiene in reducing schistosomiasis: A review[J]. Parasit Vectors, 2015, 8: 156.
[8]	利国, 楼理洋, 周晓蓉. 哨鼠监测中日本血吸虫成虫和虫卵玻片标本的制备方法[J]. 热带病与寄生虫学, 2017, 15(4): 207-208, 189.
	Li G, Lou LY, Zhou XR. Techniques for preparing the slide specimens of adult Schistosoma japonicum and its eggs obtained from sentinel mice[J]. J Trop Dis Parasitol, 2017, 15(4): 207-208, 189. (in Chinese)
[9]	余晴. 血吸虫尾蚴鉴定与检测技术研究现状[J]. 国际医学寄生虫病杂志, 2015, 42(3): 180-183, 188.
	Yu Q. Techniques for identification and detection of Schistosoma cercaria[J]. Int J Med Parasit Dis, 2015, 42(3): 180-183, 188. (in Chinese)
[10]	林祖昌, 徐冬梅, 陶海全, 等. 低密度尾蚴感染单只小鼠室内实验研究[J]. 中国血吸虫病防治杂志, 1991, 3(4): 224-225.
	Lin ZC, Xu DM, Tao HQ, et al. Laboratory experimental study on infection of single mouse with low density cercariae[J]. Chin J Schisto Control, 1991, 3(4): 224-225. (in Chinese)
[11]	Levy-Booth DJ, Campbell RG, Gulden RH, et al. Cycling of extracellular DNA in the soil environment[J]. Soil Biol Biochem, 2007, 39(12): 2977-2991.
[12]	Ficetola GF, Miaud C, Pompanon F, et al. Species detection using environmental DNA from water samples[J]. Biol Lett, 2008, 4(4): 423-425.
[13]	Giribet G, Wangensteen OS, Garcés-Pastor S, et al. Using eDNA to find Micrognathozoa[J]. Curr Biol, 2023, 33(14): R756-R757.
[14]	吴昀晟, 唐永凯, 李建林, 等. 环境DNA在长江江豚监测中的应用[J]. 中国水产科学, 2019, 26(1): 124-132.
	Wu YS, Tang YK, Li JL, et al. The application of environmental DNA in the monitoring of the Yangtze finless porpoise, Neophocaena phocaenoides asaeorientalis[J]. J Fish Sci China, 2019, 26(1): 124-132. (in Chinese)
[15]	Alzaylaee H, Collins RA, Shechonge A, et al. Environmental DNA-based xenomonitoring for determining Schistosoma presence in tropical freshwaters[J]. Parasit Vectors, 2020, 13(1): 63.
[16]	Sato MO, Rafalimanantsoa A, Ramarokoto C, et al. Usefulness of environmental DNA for detecting Schistosoma mansoni occurrence sites in Madagascar[J]. Int J Infect Dis, 2018, 76: 130-136.
[17]	Fornillos RJC, Sato MO, Tabios IKB, et al. Detection of Schistosoma japonicum and Oncomelania hupensis quadrasi environmental DNA and its potential utility to schistosomiasis Japonica surveillance in the Philippines[J]. PLoS One, 2019, 14(11): e0224617.
[18]	彭国华, 魏望远, 钱科, 等. 快速识别血吸虫易感性水体技术研究Ⅰ动物皮膜制作与检测效果初报[J]. 中国血吸虫病防治杂志, 2018, 30(2): 200-201, 210.
	Peng GH, Wei WY, Qian K, et al. Study on rapid recognition technique of schistosome susceptibility water body Ⅰ animal skin making and effect assessment[J]. Chin J Schisto Control, 2018, 30(2): 200-201, 210. (in Chinese)
[19]	兰炜明, 徐慧, 徐银, 等. 荧光定量PCR用于日本血吸虫感染高危环境早期预警的研究[J]. 中国寄生虫学与寄生虫病杂志, 2023, 41(4): 502-505.
	Lan WM, Xu H, Xu Y, et al. Study on early warning of high risk environment of Schistosoma japonicum infection by quantitative real-time PCR[J]. Chin J Parasitol Parasit Dis, 2023, 41(4): 502-505. (in Chinese)
[20]	阙斌. 基于数字全息及神经网络的血吸虫尾蚴检测技术[D]. 赣州: 赣南师范大学, 2022: 6, 42.
	Que B. Detection technology of schistosome cercariae based on digital holography and neural network[D]. Ganzhou: Gannan Normal University, 2022: 6, 42. (in Chinese)
[21]	易云, 汪廷华. 基于特征加权支持向量机的血吸虫尾蚴识别算法设计与应用[J]. 赣南师范学院学报, 2012, 33(3): 37-40.
	Yi Y, Wang TH. Design and application of schistosome cercariae recognition algorithm based on feature weighted SVM[J]. J Gannan Norm Univ, 2012, 33(3): 37-40. (in Chinese)
[22]	薛晓岩, 张振兴, 杨钦鸿, 等. 禽腺病毒Ⅰ群和禽腺病毒4型双重TaqMan探针荧光定量PCR检测方法的建立与应用[J]. 中国预防兽医学报, 2025, 47(2): 152-158.
	Xue XY, Zhang ZX, Yang QH, et al. Establishment and application of a duplex TaqMan real-time PCR for fowl adenovirus serotype Ⅰ and fowl adenovirus serotype 4 detection[J]. Chin J Prev Vet Med, 2025, 47(2): 152-158. (in Chinese)
[23]	李星颖, 谢梓民, 原耀贤, 等. 猫传染性腹膜炎病毒SYBR Green实时荧光定量PCR检测方法的建立与临床应用[J]. 中国兽医杂志, 2025, 61(3): 44-49.
	Li XY, Xie ZM, Yuan YX, et al. Establishment and clinical application of a SYBR green real-time fluorescence quantitative PCR detection method for feline infectious peritonitis virus[J]. Chin J Vet Med, 2025, 61(3): 44-49. (in Chinese)

组别	cox1		16S rRNA
组别	扩增成功率	Ct平均值	扩增成功率	Ct平均值
0条尾蚴	0/3		0/3
1条尾蚴	1/3	36.69	2/3	37.32
5条尾蚴	2/3	37.56	3/3	38.51
10条尾蚴	3/3	31.71	3/3	31.63
15条尾蚴	3/3	35.24	3/3	33.26
20条尾蚴	3/3	31.64	3/3	31.90
> 30条尾蚴	3/3	22.72	3/3	24.61

环境DNA技术检测日本血吸虫尾蚴的实验研究

Detection of Schistosoma japonicum cercariae using environmental DNA assay

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