Establishment of a PCR-RFLP method for detecting mutations in genes related to insecticide resistance in Aedes albopictus and Culex tritaeniorhynchus

doi:10.12140/j.issn.1000-7423.2024.06.008

Abstract

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

CLC Number:

R384.11

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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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