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

doi:10.12140/j.issn.1000-7423.2025.03.015

中国寄生虫学与寄生虫病杂志 ›› 2025, Vol. 43 ›› Issue (3): 403-408.doi: 10.12140/j.issn.1000-7423.2025.03.015

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

张圣昀¹()(), 陈朱云², 肖丽贞², 王金章¹^,²^,^*()()

1 福建医科大学公共卫生学院，福建福州 350122
2 福建省疾病预防控制中心，福建省人兽共患病研究重点实验室，福建福州 350012

收稿日期:2025-02-07 修回日期:2025-03-31 出版日期:2025-06-30 发布日期:2025-06-24
通讯作者: *王金章（ORCID：0009-0009-2926-533X），男，硕士，副主任医师，从事虫媒传染病防控研究工作。E-mail：wjz224@163.com
作者简介:张圣昀（ORCID：0009-0000-6651-4715），男，硕士研究生，从事虫媒传染病防控研究工作。Email：zsy@fjmu.edu.cn
基金资助:
福建省自然科学基金(2024J01167);福建省卫生健康科技计划(2023GGA042)

Development of a nested PCR assay-based molecular identification and genetic analysis of common Culicoides species in Fujian Province

ZHANG Shengyun¹()(), CHEN Zhuyun², XIAO Lizhen², WANG Jinzhang¹^,²^,^*()()

1 School of Public Health, Fujian Medical University, Fuzhou 350122, Fujian, China
2 Fujian Key Laboratory of Zoonosis, Fujian Provincial Center for Disease Control and Prevention, Fuzhou 350012, Fujian, China

Received:2025-02-07 Revised:2025-03-31 Online:2025-06-30 Published:2025-06-24
Contact: *E-mail: wjz224@163.com
Supported by:
Natural Science Foundation of Fujian Province(2024J01167);Health Science and Technology Project of Fujian Province(2023GGA042)

摘要/Abstract

摘要：

目的基于巢式PCR技术，建立一种福建省常见库蠓种类的分子鉴定方法，并开展库蠓的遗传进化分析。方法 2023年6月—2024年7月，在福建省内采用灯诱法采集蠓虫，制作成玻片标本后进行形态学鉴定。设计、合成库蠓线粒体细胞色素C氧化酶亚基1（cox1）序列的巢式PCR特异性引物。提取蠓虫基因组DNA，分别进行巢式PCR和普通PCR扩增，扩增产物经琼脂糖凝胶电泳后与形态学鉴定结果进行比较。巢式PCR扩增产物测序后使用SeqMan Pro软件拼接，BLAST比对核苷酸同源性。使用Clustal X软件对cox1序列进行比对分析。使用WinArl35、popart软件绘制单倍型网络图并计算单倍型多样性指数（Hd）。利用MEGA 11软件对样品序列进行特征分析，使用邻接法构建系统进化树。基于P距离模型，计算不同种类库蠓种群内、种群间的遗传距离。结果共采集35份蠓虫样品，经形态学鉴定为12种蠓虫，分别为荒川库蠓、原野库蠓、尖喙库蠓、霍飞库蠓、北京库蠓、苏岛库蠓、多斑库蠓、棒须库蠓、环基库蠓、东方库蠓、中华细蠓和武夷蠛蠓。巢式PCR结果显示，形态学鉴定为库蠓的29份DNA样品均能扩增出特异性条带，库蠓检出率为100%（29/29）；普通PCR结果显示，形态学鉴定为库蠓的29份DNA样品仅17份扩增出亮度较低的特异性条带，库蠓检出率为58.6%（17/29）。BLSAT结果显示，形态学鉴定为环基库蠓的4条cox1序列与GenBank中C. flumineus（ON002367）的序列一致性最高，为91.64%~91.78%，与环基库蠓参考序列（KY441782）的一致性仅为84.78%~84.87%；形态学鉴定为东方库蠓的3条cox1序列与C. flumineus（ON002367）的序列一致性最高，为91.64%~91.78%，与东方库蠓参考序列（KT352219）的一致性仅为81.93%~82.42%；其他22条cox1序列的BLSAT结果与形态学鉴定结果一致，序列一致性为86.78%~100%。cox1单倍型网络图结果显示，29条cox1序列共有22个单倍型，不同种类库蠓间的遗传变异水平差异较大（Hd = 0.968 0）。系统进化树结果显示，29条cox1序列均按照其所属亚属在进化树上聚集。遗传距离分析结果显示，各种类库蠓间遗传距离范围在0.000 4~0.222 9；种内遗传距离范围在0.000 0~0.006 2。结论建立了基于巢式PCR技术建立的分子鉴定方法，该法能够适用于福建省内多种常见库蠓的鉴定。

关键词: 库蠓, 巢式PCR, 线粒体细胞色素C氧化酶亚基1, 分子鉴定, 遗传进化

Abstract:

Objective To develop a molecular assay for identification of common Culicoides species in Fujian Province based on nested PCR assay, and to investigate the genetic evolution of Culicoides. Methods Culicoides biting midges were captured using light traps in Fujian Province from June 2023 to July 2024 and mounted on microscope slides for morphological identification. The specific primers for nested PCR assay targeting the mitochondrial cytochrome C oxidase subunit 1 (cox1) sequence of Culicoides were designed and synthesized. Genomic DNA was extracted from Culicoides for nested PCR assay and conventional PCR assay, and the amplification products were checked with agarose gel electrophoresis and then compared with morphological dentification results. Following nested PCR assay, the amplification products were sequenced and the resulting reads were assembled using the SeqMan Pro software. The nucleotide homology searches were conducted using the BLAST tool, and the cox1 sequences were aligned using the Clustal X software. The haplotype network was constructed and the haplotype diversity (Hd) index was calculated using the WinArl35 and popart software. The sample sequences were characterized using the MEGA 11 software, and a phylogenetic tree was built using the neighbor-joining method. The intra-population and inter-population genetic distances among different Culicoides species were calculated with P-distance model. Results A total of 35 biting midge samples were collected and morphologically identified as 12 species, including C. arakawai, C. homotomus, C. oxystoma, C. huffi, C. morisitai, C. sumatrae, C. maculatus, C. clavipalpis, C. circumbasalis, C. orientails, Leptoconops chinensis and Lasiohelea wuyiensis. Nested PCR assay successfully amplified specific bands for 29 DNA samples from morphologically identified Culicoides biting midges, with a 100% detection rate of Culicoides (29/29), and conventional PCR assay amplified 17 specific bands with low brightness for 29 DNA samples from morphologically identified Culicoides biting midges, with a 58.6% detection rate of Culicoides (17/29). The BLAST results demonstrated that 4 cox1 sequences of morphologically identified C. circumbasalis had the highest homology (91.64%-91.78%) with the sequence of C. flumineus (ON002367) in Genbank, and had 84.78%-84.87% homology with the reference sequence of C. circumbasalis (KY441782), while 3 cox1 sequences of morphologically identified C. orientails showed the highest homology (91.64%-91.78%) with the sequence of C. flumineus (ON002367) and had 81.93%-82.42% homology with the reference sequence of C. orientails (KT352219). The BLSAT results of other 22 cox1 sequences were consistent with morphological identification results, with sequence homology ranging from 86.78%-100%. The haplotype network diagram of the cox1 gene revealed that the 29 cox1 sequences contained 22 haplotypes, with considerable genetic variation among different Culicoides species (Hd = 0.968 0). Phylogenetic analysis revealed that the 29 cox1 sequences were clustered into the respective clades according to their corresponding subgenera. The inter- and intra-population genetic distances were 0.000 4-0.222 9 and 0.000 0-0.006 2 among different Culicoides species. Conclusion The nested PCR assay-based molecular identification was built, and it could be effectively applied to multiple common Culicoides species in Fujian Province.

Key words: Culicoides, Nested PCR, Mitochondria cytochrome C oxidase subunit 1, Molecular identification, Genetic evolution

中图分类号:

R384.52

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.

ZHANG Shengyun, CHEN Zhuyun, XIAO Lizhen, WANG Jinzhang. Development of a nested PCR assay-based molecular identification and genetic analysis of common Culicoides species in Fujian Province[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2025, 43(3): 403-408.

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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 Production length/bp
F1	TTCAGCCATTTAACCGCAACAATGA	1 335
R1	TAATCTGAGTATCGTCGAGG	1 335
F2	CCACTAATCATAAAGATATTGG	697
R2	GGGTGGCCAAAGAATCAAAATA	697

形态学种类 Morphological species	多斑库蠓 C. maculatus	苏岛库蠓 C. sumatrae	原野库蠓 C. homotomus	尖喙库蠓 C. oxystoma	棒须库蠓 C. clavipalpis	霍飞库蠓 C. huffi	东方库蠓 C. orientails	荒川库蠓 C. arakawai	环基库蠓 C. circumbasalis	北京库蠓 C. morisitai
多斑库蠓 C. maculatus	0.002 8^a
苏岛库蠓 C. sumatrae	0.189 0	-^b
原野库蠓 C. homotomus	0.179 2	0.214 1	0.001 0^a
尖喙库蠓 C. oxystoma	0.182 0	0.195 3	0.138 6	0.016 5^a
棒须库蠓 C. clavipalpis	0.191 8	0.218 3	0.161 5	0.137 2	0.003 1^a
霍飞库蠓 C. huffi	0.199 5	0.222 9	0.174 1	0.168 8	0.212 8	0.004 6^a
东方库蠓 C. orientails	0.182 0	0.194 2	0.165 3	0.143 0	0.189 6	0.165 5	0.000 0^a
荒川库蠓 C. arakawai	0.199 9	0.212 6	0.141 9	0.143 9	0.168 2	0.204 3	0.182 3	0.002 1^a
环基库蠓 C. circumbasalis	0.180 4	0.193 8	0.164 9	0.142 4	0.187 6	0.165 1	0.000 4	0.181 9	0.002 1^a
北京库蠓 C. morisitai	0.166 9	0.207 9	0.137 8	0.136 4	0.155 3	0.178 5	0.164 8	0.152 7	0.164 3	0.006 2^a

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

Development of a nested PCR assay-based molecular identification and genetic analysis of common Culicoides species in Fujian Province

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