The southern cattle fever tick, Rhipicephalus (Boophilus) microplus, is the most economically important ectoparasite of cattle worldwide. A limitation for sustainable control and eradication is the emergence of acaricide resistance among tick populations. Molecular diagnostic tools offer the opportunity to detect resistance rapidly, which can be complemented with confirmatory bioassays with larvae and adult ticks that are more resource and time consuming to generate. Synthetic pyrethroid resistance is one of the most prevalent and well-studied forms of resistance in arthropods, being linked with target site alterations in the sodium ion channel gene. Here, we report research on a novel molecular method to detect mutations in the para-sodium channel gene of R. microplus associated with acaricide resistance that is based on quantitative PCR high-resolution melt (HRM) analysis. Genomic DNA fragments of domains II and III of the para-sodium channel gene were amplified by real-time PCR in the presence of EVA®Green dye to test resistant and susceptible reference ticks from the U.S., Brazil, and Mexico. Larval packet tests with discriminating doses and a modified lethal time analysis were performed to confirm resistance to permethrin, cypermethrin, deltamethrin, and flumethrin in laboratory strains. Tick specimens collected from cattle that were inspected at the United States Port-of-Entry at the Texas-Mexico border were also genotyped. Previously described mutations associated with pyrethroid resistance (T170C, C190A, G184C, and T2134A) were successfully detected by qPCR-HRM in different genotypes and confirmed by sequencing. A novel non-synonymous SNP located at domain III (C2136A) and the G215T mutation in domain II, previously described only in Asian R. microplus and R. australis, were also detected with the HRM and confirmed by sequencing. This technique could be adapted for high-throughput screening, detection, and discovery of allele-specific mutations in cattle tick outbreak populations to inform eradication strategies in the USA. This knowledge could also be applied to integrated control programs in other parts of the world where R. microplus is endemic and where similar SNPs have been identified associated with pyrethroid resistance. This study highlights the existence of several mutations in the para-sodium channel gene in different combinations in field populations of R. microplus from Mexico.

中文翻译：

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高分辨率熔解（HRM）分析，用于检测与南部牛热tick虫Rhipicephalus（Boophilus）microplus（Acari：Ixodidae）中的拟除虫菊酯抗性相关的SNP。

南部的牛瘟tick（Rhipicephalus（Boophilus）microplus）是全世界牛中经济上最重要的体外寄生虫。可持续控制和根除的局限性是tick虫种群中出现了对杀螨剂的抗药性。分子诊断工具提供了快速检测抗药性的机会，可以与幼虫和成年tick虫的确证性生物检测相辅相成，这需要花费更多的资源和时间。合成拟除虫菊酯抗药性是节肢动物中最普遍和研究最充分的抗药性形式之一，与钠离子通道基因中的靶位点改变有关。在这里，我们报告了一种新型分子方法的研究，以检测R的对钠通道基因中的突变。基于定量PCR高分辨率熔体（HRM）分析的与杀螨剂抗性相关的microplus。在EVA®Green染料存在下，通过实时PCR扩增对钠通道基因结构域II和III的基因组DNA片段，以测试来自美国，巴西和墨西哥的抗性和易感参考reference。进行了区分剂量的幼虫数据包测试和经过修改的致死时间分析，以确认实验室菌株对氯菊酯，氯氰菊酯，溴氰菊酯和氟氰菊酯的抗性。还对在得克萨斯州-墨西哥边境的美国入境口岸检查过的从牛身上采集的ick虫标本进行了基因分型。先前描述的与拟除虫菊酯抗性相关的突变（T170C，C190A，G184C，qPCR-HRM成功检测了不同基因型的T2134A和T2134A），并通过测序进行了确认。还使用HRM检测到了位于结构域III（C2136A）和结构域II中的G215T突变的新型非同义SNP（以前仅在亚洲小micro和澳大利亚小。中进行了描述），并通过测序进行了确认。该技术可用于牛for暴发人群中的高通量筛选，检测和发现等位基因特异性突变，以为美国的根除策略提供信息。这些知识也可以应用于世界其他地方的综合控制计划，在这些地方，微小plus.rplus是地方性的，并且已经确定类似的SNP与拟除虫菊酯抗性相关。