卡耶塔环孢子虫经典巢式PCR检测方法的评价

doi:10.12140/j.issn.1000-7423.2026.04.011

中国寄生虫学与寄生虫病杂志 ›› 2026, Vol. 44 ›› Issue (4): 532-537.doi: 10.12140/j.issn.1000-7423.2026.04.011

卡耶塔环孢子虫经典巢式PCR检测方法的评价

周浩(), 刘华, 姜岩岩, 沈玉娟, 李真, 尹建海, 郑彬^*()(), 李石柱^*()()

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:2026-08-03 修回日期:2026-08-18 出版日期:2026-08-30 发布日期:2026-08-27
通讯作者: * 郑彬（ORCID：0000-0002-1768-7609），女，博士，研究员，从事寄生虫病防治研究。E-mail：zhengbin@nipd.chinacdc.cn；
李石柱（ORCID：0000-0003-3172-5218），男，博士，研究员，从事寄生虫病病原生物学和流行病学研究。E-mail：lisz@chinacdc.cn
作者简介:周浩，男，硕士，研究实习员，主要从事寄生虫感染与免疫研究。E-mail：zhouhao@nipd.chinacdc.cn
作者贡献
周浩负责论文撰写和实验操作，刘华和姜岩岩参与实验操作，沈玉娟参与论文修改，尹建海参与论文设计与修改，郑彬和李石柱负责论文设计、修改和审校。
基金资助:
上海市科学技术委员会专项(24DZ2203100);国家科技重大专项(2026ZD01909306);国家科技重大专项(2018ZX10201002-009)

Evaluation of the classical nested PCR method for Cyclospora cayetanensis detection

ZHOU Hao(), LIU Hua, JIANG Yanyan, SHEN Yujuan, LI Zhen, YIN Jianhai, ZHENG Bin^*()(), LI Shizhu^*()()

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; NHC Key Laboratory of Parasite and Vector Biology; WHO Collaborating Centre for Tropical Diseases; National Center for International Research on Tropical Diseases, Ministry of Science and Technology, Shanghai 200025, China

Received:2026-08-03 Revised:2026-08-18 Online:2026-08-30 Published:2026-08-27
Contact: * E-mail: zhengbin@nipd.chinacdc.cn; lisz@chinacdc.cn
Supported by:
Shanghai Municipal Science and Technology Commission Special Foundation(24DZ2203100);National Science and Technology Major Project(2026ZD01909306);National Science and Technology Major Project(2018ZX10201002-009)

摘要/Abstract

摘要：

目的对一种经典的卡耶塔环孢子虫18S rRNA基因巢式PCR检测方法进行系统评价。方法采用经典的卡耶塔环孢子虫18S rRNA基因巢式PCR引物，优化反应条件。以华支睾吸虫、日本血吸虫、细粒棘球绦虫、微小隐孢子虫等8种非艾美科寄生虫DNA，以及羊艾美球虫、犬等孢球虫、鸡柔嫩艾美球虫和牛艾美球虫等4种艾美科近缘球虫DNA为对照评价方法特异性；以10倍梯度稀释的含靶标片段的重组质粒（浓度范围为10⁴~10⁰拷贝/μL）为模板来评估该方法的敏感性。结果特异性试验结果显示，人来源卡耶塔环孢子虫阳性DNA扩增出约500 bp的目的条带，其余8种非艾美科寄生虫及阴性对照无扩增，但羊艾美球虫、犬等孢球虫、鸡柔嫩艾美球虫和牛艾美球虫等艾美科近缘球虫均产生约500 bp的扩增产物，测序证实该序列与卡耶塔环孢子虫及艾美球虫参考序列一致性达99%。敏感性试验结果显示，该巢式PCR对重组质粒的检测下限为10¹拷贝/μL。结论本方法对人粪便样本中卡耶塔环孢子虫的检测敏感性较高，但会与艾美科近缘球虫发生交叉反应。鉴于上述球虫具有严格的宿主特异性、通常不感染人类，因此本方法适用于人源样本的卡耶塔环孢子虫筛查检测，但不适用于动物粪便、环境及食品样本的检测，以免高估环孢子虫感染水平。解读检测结果时应保持审慎。

关键词: 卡耶塔环孢子虫, 18S rRNA基因, 巢式PCR, 特异性, 敏感性

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

中图分类号:

R382.3

周浩, 刘华, 姜岩岩, 沈玉娟, 李真, 尹建海, 郑彬, 李石柱. 卡耶塔环孢子虫经典巢式PCR检测方法的评价[J]. 中国寄生虫学与寄生虫病杂志, 2026, 44(4): 532-537.

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.

图/表 3

参考文献 20

[1]	Almeria S, Chacin-Bonilla L, Maloney JG, et al. Cyclospora cayetanensis: a perspective (2020-2023) with emphasis on epidemiology and detection methods[J]. Microorganisms, 2023, 11(9): 2171.
[2]	Herwaldt BL. Cyclospora cayetanensis: a review, focusing on the outbreaks of cyclosporiasis in the 1990s[J]. Clin Infect Dis, 2000, 31(4): 1040-1057.
[3]	Ortega YR, Sterling CR, Gilman RH, et al. Cyclospora species: a new protozoan pathogen of humans[J]. N Engl J Med, 1993, 328(18): 1308-1312.
[4]	Soave R, Dubey JP, Ramos LJ, et al. A new intestinal pathogen?[J]. Clin Res, 1986, 34: 533A.
[5]	Li JQ, Wang RJ, Chen YC, et al. Cyclospora cayetanensis infection in humans: biological characteristics, clinical features, epidemiology, detection method and treatment[J]. Parasitology, 2020, 147(2): 160-170.
[6]	苏庆平, 林秋君, 陈景龙. 我国圆孢子虫病首例报告[J]. 中国人兽共患病杂志, 1995, 11(5): 6.
	Su QP, Lin QJ, Chen JL. The first report of the cyclospore cayetanensis in China[J]. Chin J Zoonoses, 1995, 11(5): 6. (in Chinese)
[7]	Eberhard ML, Pieniazek NJ, Arrowood MJ. Laboratory diagnosis of Cyclospora infections[J]. Arch Pathol Lab Med, 1997, 121(8): 792-797.
[8]	Ortega YR, Sanchez R. Update on Cyclospora cayetanensis, a food-borne and waterborne parasite[J]. Clin Microbiol Rev, 2010, 23(1): 218-234.
[9]	Shields JM, Olson BH. Cyclospora cayetanensis: a review of an emerging parasitic coccidian[J]. Int J Parasitol, 2003, 33(4): 371-391.
[10]	肖淑敏, 李国清, 李韦华, 等. 牛源环孢子虫套式PCR检测方法的建立及初步应用[J]. 中国农业科学, 2007, 40(9): 2073-2078.
	Xiao SM, Li GQ, Li WH, et al. Development and application of nested PCR assay for detection of dairy cattle-derived Cyclospora sp[J]. Sci Agric Sin, 2007, 40(9): 2073-2078. (in Chinese)
[11]	Reiman DA, Schmidt TM, Gajadhar A, et al. Molecular phylogenetic analysis of Cyclospora, the human intestinal pathogen, suggests that it is closely related to Eimeria species[J]. J Infect Dis, 1996, 173(2): 440-445.
[12]	Li GQ, Xiao SM, Zhou RQ, et al. Molecular characterization of Cyclospora-like organism from dairy cattle[J]. Parasitol Res, 2007, 100(5): 955-961.
[13]	Hofstetter J, Arfken A, Kahler A, et al. Evaluation of Coccidia DNA in irrigation pond water and waste water sludge associated with Cyclospora cayetanensis 18S rRNA gene qPCR detections[J]. Microbiol Spectr, 2024, 12(8): e0090624.
[14]	Shields JM, Olson BH. PCR-restriction fragment length polymorphism method for detection of Cyclospora cayetanensis in environmental waters without microscopic confirmation[J]. Appl Environ Microbiol, 2003, 69(8): 4662-4669.
[15]	高振永, 李国清, 岳彩玲, 等. 猴源环孢子虫巢式PCR检测方法的建立[J]. 中国动物传染病学报, 2010, 18(5): 32-36.
	Gao ZY, Li GQ, Yue CL, et al. Development of nested PCR assay for detection of Cyclospora sp. from monkey[J]. Chin J Vet Parasitol, 2010, 18(5): 32-36. (in Chinese)
[16]	黄燕, 张龙现, 李晓虹, 等. 卡片PCR法检测果汁饮料中卡耶他环孢子虫技术的建立[J]. 中国动物传染病学报, 2010, 18(1): 59-63.
	Huang Y, Zhang LX, Li XH, et al. FTA-PCR method for detection of Cyclospora cayetanensis in juice[J]. Chin J Anim Infect Dis, 2010, 18(1): 59-63. (in Chinese)
[17]	王雅雪, 简金花, 刘华, 等. 云南省洱源县人群芽囊原虫和卡耶塔环孢子虫感染情况及其分子特征[J]. 中国寄生虫学与寄生虫病杂志, 2024, 42(2): 242-250.
	Wang YX, Jian JH, Liu H, et al. Infection of Blastocystis and Cyclospora cayetanensis in residents in Eryuan County, Yunnan Province and their molecular characterization[J]. Chin J Parasitol Parasit Dis, 2024, 42(2): 242-250. (in Chinese)
[18]	Liu H, Qin Y, Zuo QQ, et al. Molecular characterization of human pathogenic Enterocytozoon bieneusi, Giardia duodenalis and Cyclospora cayetanensis of diarrheal outpatients in Yangtze River Delta region, China[J]. BMC Microbiol, 2026, 26(1): 99.
[19]	Balan KV, Mammel M, Lipman D, et al. Development and single laboratory evaluation of a refined and specific real-time PCR detection method, using mitochondrial primers (Mit1C), for the detection of Cyclospora cayetanensis in produce[J]. J Food Prot, 2023, 86(2): 100037.
[20]	Zhu H, Kim BJ, Spizz G, et al. Development and evaluation/verification of a fully automated test platform for the rapid detection of Cyclospora cayetanensis in produce matrices[J]. Microorganisms, 2023, 11(11): 2805.

卡耶塔环孢子虫经典巢式PCR检测方法的评价

Evaluation of the classical nested PCR method for Cyclospora cayetanensis detection

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 3

参考文献 20

相关文章 15

编辑推荐

Metrics

本文评价

[1]	胡拉莱·哈德尔, 左清秋, 古丽巴努·艾麦尔, 李石柱, 郑彬, 沈玉娟. 卡耶塔环孢子虫流行特征、检测技术及防控研究进展[J]. 中国寄生虫学与寄生虫病杂志, 2026, 44(4): 468-476.
[2]	刘华, 倪红霞, 朱娜, 周浩, 左清秋, 曹建平, 郑彬, 尹建海. 浙江省宁波市儿童腹泻粪样卡耶塔环孢子虫的回顾性分子检测及遗传分析[J]. 中国寄生虫学与寄生虫病杂志, 2026, 44(4): 525-531.
[3]	张圣昀, 陈朱云, 肖丽贞, 王金章. 福建省常见库蠓种类巢式PCR分子鉴定及遗传分析方法的建立[J]. 中国寄生虫学与寄生虫病杂志, 2025, 43(3): 403-408.
[4]	宋绍征, 孟雅琴, 武颖超, 于康英, 周哲. 一种重组聚合酶扩增检测犬耳痒螨的方法建立及应用[J]. 中国寄生虫学与寄生虫病杂志, 2024, 42(6): 802-805.
[5]	任冀超, 甘露, 郑会珍, 冯秀娟, 崔泽云, 李佳欣, 金依璇, 张伟, 郭庆勇, 巴音查汗·盖力克, 李永畅. 驽巴贝虫metacaspase基因的克隆、表达、反应原性鉴定和生物信息分析[J]. 中国寄生虫学与寄生虫病杂志, 2024, 42(3): 325-331.
[6]	王雅雪, 简金花, 刘华, 秦源, 彭晓雪, 苏雅馨, 沈玉娟. 云南省洱源县人群芽囊原虫和卡耶塔环孢子虫感染情况及其分子特征[J]. 中国寄生虫学与寄生虫病杂志, 2024, 42(2): 242-250.
[7]	秦源, 刘华, 王雅雪, 张璟, 苏雅馨, 曹建平, 沈玉娟. 上海市宠物犬和猫毕氏肠微孢子虫和卡耶塔环孢子虫感染情况及分子特性研究[J]. 中国寄生虫学与寄生虫病杂志, 2024, 42(1): 63-68.
[8]	徐银, 刘婷, 徐慧, 曾小军, 兰炜明, 龚志红, 戴坤教, 邱婷婷, 郝先, 谢曙英. PCR-CRISPR/Cas12a华支睾吸虫囊蚴检测方法的建立和应用[J]. 中国寄生虫学与寄生虫病杂志, 2023, 41(4): 421-426.
[9]	黎玉琼, 于有利, 郜军荣, 刘芸芸, 李红兵, 牛晓昊. 宁夏银川地区奶牛毕氏肠微孢子虫的感染情况与基因型鉴定[J]. 中国寄生虫学与寄生虫病杂志, 2023, 41(4): 476-479.
[10]	曹天行, 刘军龙, 张志刚, 史康妍, 石苗, 关贵全, 李有全, 殷宏, 罗建勋. 环形泰勒虫重组TA04380蛋白的原核表达及ELISA的建立[J]. 中国寄生虫学与寄生虫病杂志, 2022, 40(3): 362-368.
[11]	吴冬妮, 孙凌聪, 张华勋, 万伦, 张娟, 曹慕民, 夏菁. 湖北省输入性卵形疟的实验室检测能力分析[J]. 中国寄生虫学与寄生虫病杂志, 2021, 39(3): 406-409.
[12]	张新峰, 刘运强, 张志君, 许艳, 李瑾, 张本光. 山东省泰安市2015—2019年输入性疟疾病例实验室诊断与复核结果分析[J]. 中国寄生虫学与寄生虫病杂志, 2021, 39(1): 129-132.
[13]	张苍林, 聂仁华, 徐丹, 吕高伟, 王剑, 杨亚明, 邓艳, 刘言, 周红宁. 中缅边境地区恶性疟原虫Pfcrt、Pfmdr和PfK13基因多态性与体外药物敏感性相关性的分析[J]. 中国寄生虫学与寄生虫病杂志, 2020, 38(5): 580-588.
[14]	周水茂, 涂祖武, 杨燕, 陈芳, 贾西帅. 环介导等温扩增检测恶性疟原虫与其他疟原虫的效果评价[J]. 中国寄生虫学与寄生虫病杂志, 2020, 38(4): 423-428.
[15]	赵亚男, 洪勇, 李朝品. Der p1 T细胞表位融合蛋白对哮喘小鼠的特异性免疫治疗效果[J]. 中国寄生虫学与寄生虫病杂志, 2020, 38(1): 74-79.