白纹伊蚊和三带喙库蚊抗药性相关基因突变PCR-RFLP检测方法的建立

doi:10.12140/j.issn.1000-7423.2024.06.008

摘要/Abstract

摘要：

目的基于聚合酶链式反应-限制性片段长度多态性（PCR-RFLP）技术，建立白纹伊蚊和三带喙库蚊抗药性相关基因突变的快速检测方法。方法根据白纹伊蚊电压门控钠离子通道（VGSC）和三带喙库蚊VGSC、乙酰胆碱酯酶（AChE）基因序列设计PCR引物，优化酶切位点，建立检测白纹伊蚊和三带喙库蚊抗药性相关基因突变的PCR-RFLP方法。诱蚊灯法采集白纹伊蚊和三带喙库蚊，提取单只蚊虫基因组DNA，以单只蚊虫的基因组DNA为模板进行PCR，分别扩增含1016位点的白纹伊蚊VGSC基因、含1014位点的三带喙库蚊VGSC基因和含455位点的三带喙库蚊AChE基因片段。3种PCR扩增产物分别使用限制性内切酶BsaJ Ⅰ、Dde Ⅰ、Mbo Ⅰ进行酶切，根据酶切片段的长度多态性来区分个体的基因型。结果成功建立了检测白纹伊蚊VGSC-V1016G、三带喙库蚊VGSC-L1014F和三带喙库蚊AChE-F455W突变的方法。酶切结果显示，5个白纹伊蚊VGSC扩增产物中有3个野生型1016V纯合子、2个野生型1016V和突变型1016G杂合子，5个三带喙库蚊VGSC扩增产物中有2个野生型1014L纯合子、2个野生型1014L和突变型1014F杂合子、1个突变型1014F纯合子，5个三带喙库蚊AChE扩增产物中有2个野生型455F和突变型455W杂合子、3个突变型455W纯合子。结论建立的白纹伊蚊和三带喙库蚊抗药性相关基因突变PCR-RFLP检测方法操作方便快捷、成本更低且准确性高。

关键词: 白纹伊蚊, 三带喙库蚊, 电压门控钠离子通道, 乙酰胆碱酯酶, 抗性突变

Abstract:

Objective To establish a rapid detection method for detecting mutations in genes related to insecticide resistance in Aedes albopictus and Culex tritaeniorhynchus based on polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technology. Methods PCR primers were designed based on the gene sequences of voltage-gated sodium channel (VGSC) in Ae. albopictus, VGSC and acetylcholinesterase (AChE) in Cx. tritaeniorhynchus, and the restriction enzyme cutting sites were optimized to establish a PCR-RFLP method for detecting mutations in genes related to insecticide resistance in Ae. albopictus and Cx. tritaeniorhynchus. Mosquitoes were collected by light trap, and genomic DNA was extracted from individual mosquitoes. PCR was performed using genomic DNA from individual mosquitoes as templates to amplify fragments containing the 1016 site of the Ae. albopictus VGSC gene, the 1014 site of the Cx. tritaeniorhynchus VGSC gene and the 455 site of the Cx. tritaeniorhynchus AChE gene. The PCR products of 3 genes were digested with the restriction enzymes BsaJ Ⅰ, Dde Ⅰ and Mbo Ⅰ, respectively, and the genotypes of individuals were distinguished based on the length polymorphism of the digested fragments. Results The detection method for the VGSC-V1016G mutation in Ae. albopictus, the VGSC-L1014F mutation and the AChE-F455W mutation in Cx. tritaeniorhynchus was established successfully. The enzyme digestion results showed that among the five Ae. albopictus VGSC amplification products, three were homozygous for the wild-type 1016V, and two were heterozygous for the wild-type 1016V and mutant 1016G. Among the five Cx. tritaeniorhynchus VGSC amplification products, two were homozygous for the wild-type 1014L, two were heterozygous for the wild-type 1014L and mutant 1014F, and one was homozygous for the mutant 1014F. Among the five Cx. tritaeniorhynchus AChE amplification products, two were heterozygous for the wild-type 455F and mutant 455W, and three were homozygous for the mutant 455W. Conclusions The PCR-RFLP method established for detecting mutations in genes related to insecticide resistance in Ae. albopictus and Cx. tritaeniorhynchus is convenient, cost-effective and highly accurate.

Key words: Aedes albopictus, Culex tritaeniorhynchus, Voltage-gated sodium channel, Acetylcholinesterase, Insecticide resistance

中图分类号:

R384.11

刘鹃, 刘鹏, 王雅伟, 余小梅, 邱星辉. 白纹伊蚊和三带喙库蚊抗药性相关基因突变PCR-RFLP检测方法的建立[J]. 中国寄生虫学与寄生虫病杂志, 2024, 42(6): 744-747.

LIU Juan, LIU Peng, WANG Yawei, YU Xiaomei, QIU Xinghui. Establishment of a PCR-RFLP method for detecting mutations in genes related to insecticide resistance in Aedes albopictus and Culex tritaeniorhynchus[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2024, 42(6): 744-747.

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参考文献 19

[1]	Song X, Shi QQ, Cheng P, et al. Research progress in molecular mechanisms of vector insect’s resistance to insecticides[J]. Chin J Vector Biol Control, 2018, 29(6): 113-117, 121. (in Chinese)
	(宋晓, 史琦琪, 程鹏, 等. 病媒昆虫的抗药性分子机制研究进展[J]. 中国媒介生物学及控制杂志, 2018, 29(6): 113-117, 121.)
[2]	Liu NN. Insecticide resistance in mosquitoes: impact, mechanisms, and research directions[J]. Annu Rev Entomol, 2015, 60: 537-559. doi: 10.1146/annurev-ento-010814-020828 pmid: 25564745
[3]	Kasai S, Ng LC, Lam-Phua SG, et al. First detection of a putative knockdown resistance gene in major mosquito vector, Aedes albopictus[J]. Jpn J Infect Dis, 2011, 64(3): 217-221.
[4]	Kasai S, Caputo B, Tsunoda T, et al. First detection of a Vssc allele V1016G conferring a high level of insecticide resistance in Aedes albopictus collected from Europe (Italy) and Asia (Vietnam), 2016: a new emerging threat to controlling arboviral diseases[J]. Eur Commun Dis Bull, 2019, 24(5): 1700847.
[5]	Zhou XJ, Yang C, Liu N, et al. Knockdown resistance (kdr) mutations within seventeen field populations of Aedes albopictus from Beijing China: first report of a novel V1016G mutation and evolutionary origins of kdr haplotypes[J]. Parasit Vectors, 2019, 12(1): 180.
[6]	Tan A, Liu J, Wang YW, et al. Detection of mutations in the voltage-gated sodium channel genes of field Aedes albopictus populations in Neijiang, Sichuan Province, China[J]. Chin J Vector Biol Control, 2023, 34(3): 314-318. (in Chinese)
	(谭爱, 刘鹃, 王雅伟, 等. 四川省内江市白纹伊蚊野外群体电压门控钠离子通道基因突变检测分析[J]. 中国媒介生物学及控制杂志, 2023, 34(3): 314-318.) doi: 10.11853/j.issn.1003.8280.2023.03.006
[7]	Qiu XH. Current knowledge about the molecular mechanisms underlying insecticide resistance in Aedes albopictus[J]. Acta Parasitol Med Entomol Sin, 2019, 26(3): 194-198. (in Chinese)
	(邱星辉. 白纹伊蚊抗药性分子机制研究进展[J]. 寄生虫与医学昆虫学报, 2019, 26(3): 194-198.)
[8]	Liu J, Wang YW, Liu P, et al. Detection of target site mutations in the acetylcholinesterase and voltage-gated sodium channel in field populations of Culex quinquefasciatus and Cx. tritaeniorhynchus from southern Sichuan region of China[J]. J Am Mosq Control Assoc, 2023, 39(1): 57-60.
[9]	Jiang JY, Chen HY, Zhou HN, et al. Analysis of knowdown resistance gene mutation in Culex tritaeniorhynchus resistant to DDT and deltamethrin in Yunnan Province, China[J]. Chin J Parasitol Parasit Dis, 2017, 35(6): 536-540. (in Chinese)
	(姜进勇, 陈辉莹, 周红宁, 等. 云南省三带喙库蚊对DDT和溴氰菊酯抗性群体的击倒抗性基因突变分析[J]. 中国寄生虫学与寄生虫病杂志, 2017, 35(6): 536-540.)
[10]	Nabeshima T, Mori A, Kozaki T, et al. An amino acid substitution attributable to insecticide-insensitivity of acetylcholinesterase in a Japanese encephalitis vector mosquito, Culex tritaeniorhynchus[J]. Biochem Biophys Res Commun, 2004, 313(3): 794-801.
[11]	Wu ZM, Chu HL, Wang G, et al. Multiple-insecticide resistance and classic gene mutations to Japanese encephalitis vector Culex tritaeniorhynchus from China[J]. J Am Mosq Control Assoc, 2016, 32(2): 144-151.
[12]	Rinkevich FD, Zhang L, Hamm RL, et al. Frequencies of the pyrethroid resistance alleles of Vssc1 and CYP6D1 in house flies from the eastern United States[J]. Insect Mol Biol, 2006, 15(2): 157-167. pmid: 16640726
[13]	Xia XF, Sun BT, Gurr GM, et al. Gut microbiota mediate insecticide resistance in the diamondback moth, Plutella xylostella (L.)[J]. Front Microbiol, 2018, 9: 25.
[14]	Ministry of Health of the People’s Republic of China. Test methods of mosquito resistance to insecticides―Bioassay methods: GB/T 26347-2010[S]. Beijing: Standards Press of China, 2011: 3-5. (in Chinese)
	(中华人民共和国卫生部. 蚊虫抗药性检测方法生物测定法: GB/T 26347—2010[S]. 北京: 中国标准出版社, 2011: 3-5.)
[15]	Li X, Ling F, Wei SL, et al. Distribution of knockdown resistance genotypes in Aedes albopictus in Nanning, Guangxi Zhuang Autonomous Region, China, 2022[J]. Chin J Vector Biol Control, 2023, 34(4): 480-484. (in Chinese)
	(李雪, 凌峰, 韦舒琳, 等. 南宁市2022年白纹伊蚊击倒抗性基因型分布研究[J]. 中国媒介生物学及控制杂志, 2023, 34(4): 480-484.)
[16]	Zhu CY, Zhao CC, Wang YG, et al. Establishment of an innovative and sustainable PCR technique for 1534 locus mutation of the knockdown resistance (kdr) gene in the dengue vector Aedes albopictus[J]. Parasit Vectors, 2019, 12(1): 603.
[17]	Pichler V, Mancini E, Micocci M, et al. A novel allele specific polymerase chain reaction (AS-PCR) assay to detect the V1016G knockdown resistance mutation confirms its widespread presence in Aedes albopictus populations from Italy[J]. Insects, 2021, 12(1): 79.
[18]	Mu QZ, Zhao X, Li FF, et al. A novel strategy for screening mutations in the voltage-gated sodium channel gene of Aedes albopictus based on multiplex PCR-mass spectrometry minisequencing technology[J]. Infect Dis Poverty, 2023, 12: 74.
[19]	Zhang Y, Zhu CQ, Ai LL, et al. Rapid genotyping L1014F substitution in sodium channel of Culex tritaeniorhynchus with PCR-RFLP method[J]. Chin J Hyg Insect Equip, 2015, 21(4): 372-374. (in Chinese)
	(张燕, 朱长强, 艾乐乐, 等. 用PCR-RFLP法对三带喙库蚊钠通道基因L1014F突变快速检测与分型[J]. 中华卫生杀虫药械, 2015, 21(4): 372-374.)

抗性相关基因突变位点 Insecticide resistance-related mutation sites	引物序列（5'→3'） Primer sequences (5'→3')	扩增产物长度/bp Amplification product length/bp	限制性内切酶 Restriction enzyme	酶切位点 Digestion sites	酶切产物长度/bp Digestion product length/bp
白纹伊蚊电压门控钠离子通道1016 Ae. albopictus VGSC 1016	F：GAACTTCACCGACTTCATGC R：GCAATCTGGCTTGTTAACTTG	430	BsaJ Ⅰ	5'…C^▼CNNGG…3' 3'…GGNNC_▲C…5'	200，230
三带喙库蚊电压门控钠离子通道1014 Cx. tritaeniorhynchus VGSC 1014	F：CTTCACCGACTTCATGCACTC R：AATATTGTAACCACACTGAA	225	Dde Ⅰ	5'…C^▼TNAG…3' 3'…GANT_▲C…5'	86，139
三带喙库蚊乙酰胆碱酯酶455 Cx. tritaeniorhynchus AchE 455	F：AGCTGGTCGATAACGAGTGG R：CGGTGTACTCGAACACGATG	306	Mbo Ⅰ	5'…^▼GATC…3' 3'…CTAG_▲…5'	140，166