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

doi:10.12140/j.issn.1000-7423.2026.04.010

中国寄生虫学与寄生虫病杂志 ›› 2026, Vol. 44 ›› Issue (4): 525-531.doi: 10.12140/j.issn.1000-7423.2026.04.010

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

刘华¹(), 倪红霞², 朱娜³, 周浩¹, 左清秋¹, 曹建平¹, 郑彬¹, 尹建海¹^,^*()()

¹ 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
² 宁波市疾病预防控制中心，浙江宁波 315000
³ 宁波市医疗中心李惠利医院，浙江宁波 315040

收稿日期:2026-08-02 修回日期:2026-08-13 出版日期:2026-08-30 发布日期:2026-08-25
通讯作者: * 尹建海（ORCID：0000-0003-1333-8732），男，博士，研究员，从事寄生虫病原与媒介生物学研究。E-mail：yinjh@nipd.chinacdc.cn
作者简介:刘华，女，博士，副研究员，主要从事寄生虫感染与免疫研究。E-mail：liuhua@nipd.chinacdc.cn
作者贡献
刘华负责论文撰写和实验操作，倪红霞和朱娜负责样本采集，周浩参与实验操作，左清秋参与论文修改，曹建平和郑彬参与论文设计与修改，尹建海负责论文设计、修改和审校。
基金资助:
国家科技重大专项(2018ZX10201002-009);上海市加强公共卫生三年行动计划 (2020—2022)(GWV-10.1-XK13)

Retrospective molecular detection and genetic analysis of Cyclospora cayetanensis in stool samples from children with diarrhea in Ningbo City, Zhejiang Province

LIU Hua¹(), NI Hongxia², ZHU Na³, ZHOU Hao¹, ZUO Qingqiu¹, CAO Jianping¹, ZHENG Bin¹, YIN Jianhai¹^,^*()()

¹ 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
² Ningbo Center for Disease Control and Prevention, Ningbo 315000, Zhejiang, China
³ Ningbo Medical Center Lihuili Hospital, Ningbo 315040, Zhejiang, China

Received:2026-08-02 Revised:2026-08-13 Online:2026-08-30 Published:2026-08-25
Contact: * E-mail: yinjh@nipd.chinacdc.cn
Supported by:
National Science and Technology Major Project, China(2018ZX10201002-009);Three-Year Public Health Action Plan of Shanghai (2020-2022)(GWV-10.1-XK13)

摘要/Abstract

摘要：

目的回顾性检测浙江省宁波市儿童腹泻粪样中卡耶塔环孢子虫的感染情况，分析我国人源卡耶塔环孢子虫的遗传进化特征。方法取本实验室保存的2020—2021年浙江省宁波市儿童腹泻粪样，采用巢式PCR扩增卡耶塔环孢子虫的18S rRNA基因，对阳性PCR扩增产物进行双向测序，序列比对分析后采用MEGA 7软件以邻接法构建系统进化树。同时检索NCBI等公共数据库中我国人源卡耶塔环孢子虫18S rRNA基因序列，分析其遗传进化特征。结果 227份儿童腹泻粪样检测分析结果显示，7份（3.08%）为卡耶塔环孢子虫阳性样品，经序列比对和系统发育分析，其中6份阳性样品（GenBank登录号：PZ748322）具有完全一致的核苷酸序列，且与GenBank中人源卡耶塔环孢子虫18S rRNA序列（GenBank登录号：KJ569531）相似性达100%；另鉴定出1条新的基因序列（GenBank登录号：PZ737096），与KJ569531序列相似为99.6%。在基于18S rRNA基因构建的系统进化树上，上述7份阳性样品序列与KJ569531聚类于同一进化枝，具有高度亲缘关系。所有环孢子虫分离株均聚为独立进化枝，与艾美球虫（Eimeria）、田鼠巴贝虫（Babesia microti）及微小隐孢子虫（Cryptosporidium parvum）等外群物种显著分离，支持了环孢子虫属的单系性。整合筛选全国62条人源卡耶塔环孢子虫18S rRNA基因序列，最终得到25条差异化基因序列，共检出39个碱基差异位点。25条序列核苷酸多样性整体Pi值为0.015，表明随机两条序列在任意位点发生碱基差异的平均概率为1.5%。此外，与其他序列相比，本研究获得的新序列（GenBank登录号：PZ737096）在第404位和第406位分别发生了C→T和A→C的碱基置换。结论浙江省宁波市儿童腹泻粪样中环孢子虫感染率较高，且本地流行虫株与国内已报道人源虫株存在基因序列差异，补充了我国人群卡耶塔环孢子虫的遗传变异基础数据。

关键词: 卡耶塔环孢子虫, 儿童, 腹泻, 巢式PCR

Abstract:

Objective To retrospectively detect the presence of Cyclospora cayetanensis infections in stool samples from children with diarrhea in Ningbo City, Zhejiang Province, and to analyze the genetic and evolutionary characteristics of human-derived C. cayetanensis in China. Methods Stool samples from children with diarrhea in Ningbo City that were maintained in our laboratory were tested for C. cayetanensis 18S rRNA gene using nested PCR assay, and positive PCR amplification products were sequenced bidirectionally. Following sequence alignment and analysis, a phylogenetic tree was created using the neighbor joining method in the MEGA 7 software. In addition, the 18S rRNA gene sequences of human-derived C. cayetanensis isolates in China were retrieved in public databases, including NCBI, and the genetic and evolutionary characteristics of these gene sequences were analyzed. Results A total of 227 stool samples from children with diarrhea were included for retrospective testing, and there were 7 samples (3.08%) tested positive for C. cayetanensis. Sequence alignment and phylogenetic analysis demonstrated that six of these positive samples (GenBank accession no.: PZ748322) shared identical nucleotide sequences and exhibited 100% homology with a human-derived reference sequence (GenBank accession no.: KJ569531) from GenBank, and a novel gene sequence (GenBank accession no.: PZ737096) was identified, which shared 99.6% similarity with the sequence KJ569531. In the phylogenetic tree constructed based on the 18S rRNA gene, the gene sequences of these 7 positive samples were clustered with the sequence KJ569531 into the same clade, indicating a highly close genetic relationship. All C. cayetanensis isolates formed a distinct monophyletic clade, which were remarkably separated from outgroup species including Eimeria spp., Babesia microti, and Cryptosporidium parvum, supporting the monophyly of the genus Cyclospora. A total of 62 human-derived C. cayetanensis 18S rRNA gene sequences from China were integrated and screened, ultimately yielding 25 distinct gene sequences, in which 39 nucleotide polymorphic sites were identified. The overall nucleotide diversity (Pi) of the 25 gene sequences was 0.015, indicating that the average probability of a base difference occurring at any site between two random sequences was 1.5%. In addition, compared to other sequences, the new sequence (GenBank accession no.: PZ737096) obtained from this study showed base substitutions C→T at position 404 and A→C at position 406. Conclusion The prevalence of C. cayetanensis infection is high in stool samples from children with diarrhea in Ningbo City, Zhejiang Province, and the locally prevalent C. cayetanensis isolates exhibit genetic sequence differences from previously reported human-derived parasite isolates in China, which supplements the baseline data on genetic variation of C. cayetanensis in Chinese populations.

Key words: Cyclospora cayetanensis, Child, Diarrhea, Nested-PCR

中图分类号:

R382.3

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.

LIU Hua, NI Hongxia, ZHU Na, ZHOU Hao, ZUO Qingqiu, CAO Jianping, ZHENG Bin, YIN Jianhai. Retrospective molecular detection and genetic analysis of Cyclospora cayetanensis in stool samples from children with diarrhea in Ningbo City, Zhejiang Province[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2026, 44(4): 525-531.

图/表 7

表1

图1

表2

图2

表3

我国人群卡耶塔环孢子虫18S rRNA基因序列核苷酸差异

GenBank号 GenBank ID	核苷酸位点 Nucleotide positions
GenBank号 GenBank ID	27	28	29	37	45	52	53	58	59	62	66	67	81	89	90	91	98	100	103	109	114	116	119	125	152	187	193	194	195	201	208	223	329	342	382	404	406	415	426
AF111183	C	T	T	-	C	G	T	C	G	G	T	C	C	G	C	C	C	C	C	C	C	T	A	A	T	T	C	C	C	T	T	G	T	-	G	C	A	A	C
FJ009120	C	T	T	-	C	G	T	C	G	G	T	C	C	C	G	C	C	T	G	C	C	T	A	A	T	T	C	T	T	T	T	G	T	-	G	C	A	A	T
FJ009127	C	T	T	-	C	G	T	C	G	G	T	T	C	C	G	C	C	T	G	C	C	T	A	A	T	T	C	T	T	T	T	G	T	-	G	C	A	A	T
FJ009128	C	T	T	-	C	G	T	T	G	G	T	C	C	C	G	C	C	T	G	C	C	T	A	A	T	T	C	T	T	T	T	G	T	-	G	C	A	A	T
GQ292778	C	T	T	-	C	G	T	C	G	G	T	C	C	C	G	C	C	T	G	C	C	T	A	A	T	T	C	C	T	T	T	G	T	-	G	C	A	A	T
GQ292779	C	T	T	-	C	G	T	C	G	G	T	T	T	C	G	C	C	T	G	C	C	T	A	A	T	T	C	T	T	T	T	G	T	-	G	C	A	A	T
GQ292782	C	T	T	-	C	G	T	C	G	G	T	C	C	C	G	C	C	T	A	C	C	T	A	A	T	T	C	T	T	T	T	G	A	-	G	C	A	A	T
KJ569531	C	T	T	-	C	G	T	C	G	G	T	Y	C	C	G	C	C	T	G	C	C	T	A	A	T	T	C	T	T	T	T	G	T	-	G	C	A	A	C
PZ748322	C	T	T	-	C	G	T	C	G	G	T	Y	C	C	G	C	C	T	G	C	C	T	A	A	T	T	C	T	T	T	T	G	T	-	G	C	A	A	C
KJ569532	C	T	T	-	C	G	T	T	G	G	T	C	C	C	G	C	C	T	G	C	C	T	A	A	T	T	C	T	T	T	T	G	T	-	G	C	A	A	C
KJ569533	C	T	T	-	C	G	T	C	G	G	T	C	C	C	G	C	C	T	G	C	C	T	A	A	T	T	C	T	T	T	T	G	T	-	G	C	A	A	C
KJ569535	C	T	T	-	C	G	T	C	G	G	T	C	C	C	G	C	C	T	G	C	C	T	A	A	T	T	C	C	T	T	T	G	T	-	G	C	A	A	C
KY770758	C	T	T	-	C	G	T	C	G	G	T	T	C	C	G	C	C	T	G	C	C	T	A	A	T	T	C	T	T	T	T	G	T	-	G	C	A	A	C
KY770760	G	C	C	T	G	C	A	C	T	G	A	T	C	A	A	T	T	T	G	T	C	T	A	A	T	T	C	T	C	T	T	A	T	-	G	C	A	A	C
KY770761	G	C	C	T	G	C	A	C	T	G	A	T	C	A	A	T	T	T	G	T	C	T	A	A	T	G	C	T	C	T	T	A	T	-	G	C	A	A	C
KY770762	G	C	C	T	G	C	A	C	T	G	A	T	C	A	A	T	T	T	G	T	C	T	G	A	T	T	C	T	C	T	T	A	T	-	G	C	A	A	C
KY770763	G	T	C	T	G	C	A	C	T	G	A	T	C	A	A	T	T	T	G	T	C	T	A	A	T	T	C	T	C	T	T	A	T	-	G	C	A	A	C
MN893886	C	T	T	-	C	G	T	C	G	G	T	T	C	C	G	C	C	T	G	C	C	T	A	A	T	T	C	T	T	T	T	G	T	-	A	C	A	A	T
MN893888	C	T	T	-	C	G	T	C	G	G	T	T	C	C	G	C	C	T	G	C	G	T	A	A	T	T	C	T	T	G	T	G	T	-	G	C	A	A	T
OP143736	C	T	T	-	C	G	T	C	G	G	T	C	C	C	G	C	C	T	G	C	C	T	A	A	T	T	C	T	T	T	T	G	T	G	G	C	A	G	C
OQ363213	C	T	T	-	C	G	T	C	G	G	T	C	C	C	G	C	C	T	G	C	C	C	A	A	C	T	C	T	T	T	T	G	T	-	G	C	A	A	T
OQ363214	C	T	T	-	C	G	T	C	G	G	T	C	C	C	G	C	C	T	G	C	C	T	A	A	T	T	T	T	T	T	T	G	T	-	G	C	A	A	C
PP593967	C	T	T	-	C	G	T	C	G	G	T	C	C	C	G	C	C	T	G	C	C	T	A	G	T	T	C	T	T	T	C	G	T	-	G	C	A	A	C
PP593968	C	T	T	-	C	G	T	C	G	G	C	C	C	C	G	C	C	T	G	C	C	T	A	A	T	T	C	T	T	T	T	G	T	-	G	C	A	A	C
PP593970	C	T	T	-	C	G	T	C	G	T	T	C	C	C	G	C	C	T	G	C	C	T	A	A	T	T	C	T	T	T	T	G	T	-	G	C	A	A	T
PZ737096	C	T	T	-	C	G	T	C	G	G	T	C	C	G	C	C	C	C	C	C	C	T	A	A	T	T	C	C	C	T	T	G	T	-	G	T	C	A	C

表3

图3

表4

我国人群卡耶塔环孢子虫的感染情况

地区 Region	检测方法 Detection methods	靶基因 Target gene	人群 Population	样本数 No. examined	阳性样本数（阳性率/%） No. positive (Detection rate/%)	参考文献 Reference
云南 Yunnan	镜检 Microscopic examination		腹泻患者 Diarrhea patients	378	15（4.0）	[5]
安徽 Anhui	镜检 Microscopic examination		幼儿 Children	200	0（0）	[13]
			小学生 Primary School Students	200	1（0.5）
			腹泻患者 Diarrhea patients	178	10（5.62）
			免疫功能低下者 Immunosuppressive patients	32	3（9.38）
浙江 Zhejiang	镜检 Microscopic examination		腹泻患者 Diarrhea patients	860	42（4.88）	[16]
河南 Henan	镜检 Microscopic examination		医院患者 Hospital patients	11 554	81（0.7）	[6]
河南 Henan	镜检 Microscopic examination		医院患者 Hospital patients	6 579	35（0.53）	[20]
河南 Henan	巢式PCR Nested PCR	18S rRNA	医院病人 Hospital patients	6 579	76（1.2）	[17]
江苏 Jiangsu	巢式PCR Nested PCR	18S rRNA	腹泻患者 Diarrhea patients	3 696	7（0.19）	[18]
江苏 Jiangsu	巢式PCR Nested PCR	18S rRNA	腹泻患者 Diarrhea patients	1 388	6（0.43）	[18]
上海 Shanghai	巢式PCR Nested PCR	18S rRNA	医院患者 Hospital patients	291	5（1.7）	[20]
上海 Shanghai	巢式PCR Nested PCR	18S rRNA	腹泻患者 Diarrhea patients	1 645	6（0.36）	[18]
上海 Shanghai	巢式PCR Nested PCR	18S rRNA	腹泻患者 Diarrhea patients	682	5（0.73）	[19]
上海 Shanghai	巢式PCR Nested PCR	18S rRNA	腹泻患者 Diarrhea patients	252	0（0）	[20]
广西 Guangxi	巢式PCR Nested PCR	18S rRNA	实验动物从业人员/村民 Laboratory animal practitioners/Villagers	50	5（10）	[21]
黑龙江 Heilongjiang	巢式PCR Nested PCR	18S rRNA	HIV/AIDS患者 HIV/AIDS patients	601	7（1.2）	[22]
云南 Yunnan	巢式PCR Nested PCR	18S rRNA	村民 Villagers	100	2（2.0）	[23]
浙江 Zhejiang	巢式PCR Nested PCR	18S rRNA	腹泻患者 Diarrhea patients	489	3（0.6）	[24]
浙江 Zhejiang	巢式PCR Nested PCR	18S rRNA	腹泻患者 Diarrhea patients	650	8（1.23）	[19]
浙江 Zhejiang	巢式PCR Nested PCR	18S rRNA	腹泻儿童 Diarrhea children	227	7（3.08）	本研究 This study

表4

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引物名称 Primer name	引物序列（5'→3'） Primer sequence (5'→3')	产物长度/bp Amplication length/bp
CYCLF1	AATGTAAAACCCTTCCAGAGTAAC	~1 000
CYCLR1	GCAATAATCTATCCCCATCACG
CYCLF2	AATTCCAGCTCCAATAGTGTAT	~500
CYCLR2	CAGGAGAAGCCAAGGTAGGCRTTT

样本编号 Sample No.	性别 Sex	年龄/月 Age/moth	来源地区 Resource
161	男Male	4	海曙区 Haishu District
180	男 Male	13	余姚市 Yuyao City
204	女 Female	44	余姚市 Yuyao City
208	女 Female	36	余姚市 Yuyao City
215	男 Male	27	余姚市 Yuyao City
222	男 Male	10	余姚市 Yuyao City
225	男 Male	2	慈溪市 Cixi City

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

Retrospective molecular detection and genetic analysis of Cyclospora cayetanensis in stool samples from children with diarrhea in Ningbo City, Zhejiang Province

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