媒介蚊虫拟驱虫菊酯类杀虫剂抗性现场分子检测方法的研究

Insect vectors seriously harm human health. Although important advances continue to be made in the development of alternative control measures, insecticides will remain a vital part in the integrated control program of vector for the foreseeable future. Pyrethroid insecticides have become by far the most commonly used class of insecticides for indoor residual spray and bednet impregnation due to the low mammalian toxicity and rapid knockdown effect on insects. However, excessive and continuous application of insecticides has caused the development and spread of insecticide resistance, which has become a major obstacle to the control of mosquito-borne diseases. Accurately monitoring resistance status is essential to guide the rational use of insecticides and resistance management. Target site insensitivity due to point mutations in the para-sodium channel is an important mechanism of resistance to pyrethroid insecticides in mosquitoes and many other insects. It is the current focus to search the molecular markers for pyrethroid resistance detecting in natural mosquito populations from genes associated with knockdown resistance (kdr)..In this study, we will aim to determine the genetic diversity of the kdr gene in natural populations in Eastern China, and to examine the evolutionary response of kdr alleles to insecticide selection to illustrate the role of kdr alleles in pyrethroid resistance and we will compare the sensitivity and specificity of ASPCR method to direct sequencing, and develop a highly sensitive and reliable TaqMan probe method that can detect multiple forms of kdr alleles in Cx. pipiens pallens. To expect to determine the efficacy of the insecticides in the field, and research for alternative vector control strategies that minimize the use of insecticides or greatly reduce the spread of insecticide resistance.

虫媒病是严重危害人类健康的公共卫生问题, 媒介的化学防制是虫媒病防治的主要策略之一。杀虫剂的大量、连续使用导致了媒介抗药性的发生和发展，其已成为当前虫媒病防治中的突出问题。媒介抗药性治理的前提在于早期发现抗药性和监测抗药性的发展。然而由于方法学的限制，媒介对广泛使用的拟除虫菊酯类杀虫剂抗性的现场检测方法一直未能建立，严重制约了中国虫媒病的防治。.本研究选择我国重要家栖性病媒淡色库蚊为研究对象，采用PCR、基因序列分析等方法，以个体大样本、从种群水平研究抗药性的策略，鉴定蚊抗药性品系的kdr基因突变类型，分析kdr基因型与蚊抗药性表型的关系；检测多个自然种群中kdr基因的遗传多样性，进一步了解kdr等位基因频率与种群不同抗药性水平的关系，并初步探讨杀虫剂选择压力下kdr基因进化，以期为建立蚊拟除虫菊酯类杀虫剂抗性的现场检测和监测方法奠定基础，为深入阐明蚊抗药性的分子机理提供科学依据。

虫媒病是严重危害人类健康的公共卫生问题, 媒介的化学防制是虫媒病防治的主要策略之一。杀虫剂的大量、连续使用导致了媒介抗药性的发生和发展，其已成为当前虫媒病防治中的突出问题。媒介抗药性治理的前提在于早期发现抗药性和监测抗药性的发展。然而由于方法学的限制，媒介对广泛使用的拟除虫菊酯类杀虫剂抗性的现场检测方法一直未能建立，严重制约了中国虫媒病的防治。本研究以淡色库蚊为研究对象，采用PCR、基因序列分析等方法，鉴定蚊抗药性品系的kdr基因突变类型，分析kdr基因型与蚊抗药性表型的关系；检测多个自然种群中kdr基因的遗传多样性，探索建立蚊虫抗药性的现场检测方法。结果表明克隆出淡色库蚊成蚊的RNA、DNA和kdr等位基因，通过基因序列分析发现在淡色库蚊钠通道第II结构域S6节段1014位点上存在2种突变，即L1014S：TTA突变成为TCA，于是相应的亮氨酸(L)被丝氨酸(S)取代；L1014F：TTA突变成为TTT，同样相应的亮氨酸(L)被苯丙氨酸(F)取代。Kdr基因L1014F突变 和L1014S突变与淡色库蚊成蚊对溴氰菊酯的抗性表型皆表现为正性相关。PCR和AS-PCR技术都可以应用于蚊虫抗药性的大规模的现场检测，但TaqMan方法的检测结果与测序结果具有更高的一致性(K=0.963)。