目的 建立基于聚合酶链式反应-限制性片段长度多态性（PCR-RFLP）策略,可用于家蝇抗药性相关羧酸酯酶MdαE7 G137D和乙酰胆碱酯酶（AChE）V260L与F407Y突变的分子检测方法。方法 2022年6—7月,在四川省内江市资中、威远、隆昌、市中和东兴5个县（市、区）,采用诱蝇笼或捕虫网采集家蝇成虫,用无水乙醇保存,带回实验室操作。根据家蝇羧酸酯酶MdαE7和AChE基因序列设计引物,以单只家蝇的基因组DNA为模板,进行家蝇MdαE7基因和AChE编码基因片段的PCR扩增,用限制性内切酶酶切PCR产物,根据酶切产物的电泳检测结果来区分家蝇个体的基因型。结果 采用基因特异性引物,可扩增出长度分别为213、170和133 bp的PCR产物MdαE7-137,AChE-260和AChE-407。MdαE7-137经Bst XI酶切后,电泳检测显示26和187 bp 2条条带为137位点敏感纯合子（GG）,仅213 bp 1条条带为抗性纯合子（DD）,26、187和213 bp 3条条带为杂合子（G/D）。AChE-260经Sa1 I-HF®酶切后,电泳显示170 bp 1条条带的,为AChE 260位点敏感纯合子（VV）,显示26和144 bp 2条条带的,为抗性纯合子（LL）,显示26、144和170 bp 3 条条带的,为杂合子（V/L）。AChE-407经Eco RV-HF®酶切后,电泳条带存在133 bp 1条条带的,为AChE 407位点敏感纯合子（FF）,存在31和102 bp 2条条带的,为抗性纯合子（YY）,存在31、102和133 bp 3条条带的,为杂合子（F/Y）。结论 建立的PCR-RFLP基因分型方法简便、准确性高,可分别用于快速检测野外家蝇种群中抗药性相关的羧酸酯酶MdαE7 G137D、AChE V260L和AchE F407Y突变的频率。
Objective To establish a molecular method for detection of resistance-conferring mutations in carboxylesterase MdαE7 (G137D) and acetylcholinesterase (V260L and F407Y) of Musca domestica by adopting the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) strategy. Methods Fly traps or capture nets were used to collect adult houseflies from June to July 2022 in five counties, county-level cities, or districts, i.e., Zizhong, Weiyuan, Longchang, Shizhong, and Dongxing in Neijiang City, Sichuan Province, China. The houseflies were preserved in absolute ethanol and brought back to the laboratory for processing. DNA fragments of M.domestica acetylcholinesterase-encoding gene and MdαE7 gene were amplified by polymerase chain reaction (PCR) using gene-specific primers and individual genomic DNA as the template. Gene fragments were amplified by polymerase chain reaction (PCR) using individual genomic DNA as the template. The PCR products were digested using specific restriction endonucleases. The genotype of individual houseflies was determined according to the restriction fragment length polymorphisms of digestion products via agarose gel electrophoresis. Results Using gene-specific primers, the PCR products, MdαE7-137 (213 bp), AChE-260 (170 bp), and AChE-407 (133 bp), were obtained. After MdαE7-137 was incubated with Bst XI, the wild homozygote (GG), resistant homozygote (DD) and heterozygote (G/D) could produce two bands ( 26 and 187 bp), a band (213 bp), and three bands (26, 187, and 213 bp), respectively. After AChE-260 was treated with Sal I-HF®, a band (170 bp), two bands (26 and 144 bp), and three bands (26, 144, and 170 bp) could be seen on gel for wild homozygote (VV), resistant homozygote (LL) and heterozygote (V/L) respectively. The presence of one band (133 bp), two bands (31 and 102 bp) and three bands (31, 102, and 133 bp) on the gel loaded with Eco RV-HF® digestion products of AChE-407 was scored as wild homozygote (FF), resistant homozygote (YY), and heterozygote (F/Y) respectively. Conclusions The established PCR-RFLP genotyping method is simple and highly accurate. This method can be used for rapid determination of the frequencies of the resistance-related mutations G137D in carboxylesterase MdαE7 and V260L and F407Y in AChE in field M.domestica populations.
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