PDF(Pubmed)
Abstract：
BACKGROUND: Culex pipiens L. is a principal vector of zoonotic arboviruses in Europe, acting in both an amplification role in enzootic transmission between avian hosts and as a bridge vector between avian hosts and mammals. The species consists of two forms which are indistinguishable using morphological methods but possess varying ecological and physiological traits that influence their vector capacity. In this study we validate methods that can be used to extract trace DNA from single pupal exuviae of Cx. pipiens for use in molecular speciation of samples. These DNA extraction methods are compared using measurement of the total yield and successful identification using a real-time polymerase chain reaction (PCR) assay.
RESULTS: Genomic DNA was initially extracted from colony-derived individuals using an ethanol precipitation method, two commercially available DNA extraction kits: DNeasy® Blood & Tissue Kit (Qiagen, UK) and Wizard® SV Genomic DNA Purification System (Promega, UK) and a direct real-time PCR method. Time elapsed between eclosion and processing of pupae significantly influenced Cx. pipiens form identification as nucleic acid concentration and PCR amplification success decreased with increased time elapsed. Real-time PCR amplification success, however, was not shown to vary significantly between the three extraction methods, with all methods successfully identifying all samples, but the direct real-time PCR method achieved a lesser amplification success rate of 70% (n = 20 for each treatment). More variable results were produced when field-derived exuviae were used, with no significant difference in real-time PCR amplification success found across the four methods and a lower overall rate of successful identification of 55-80%.
CONCLUSIONS: This study shows that both colony and field derived Cx. pipiens pupal exuviae can be a useful non-invasive source of trace DNA permitting accurate biotype differentiation for at least twenty-four hours post-eclosion. The significance and utility of this technique in ecological and behavioural studies of Cx. pipiens is discussed and recommendations made for use according to experimental scenario.
RESULTS: Genomic DNA was initially extracted from colony-derived individuals using an ethanol precipitation method, two commercially available DNA extraction kits: DNeasy® Blood & Tissue Kit (Qiagen, UK) and Wizard® SV Genomic DNA Purification System (Promega, UK) and a direct real-time PCR method. Time elapsed between eclosion and processing of pupae significantly influenced Cx. pipiens form identification as nucleic acid concentration and PCR amplification success decreased with increased time elapsed. Real-time PCR amplification success, however, was not shown to vary significantly between the three extraction methods, with all methods successfully identifying all samples, but the direct real-time PCR method achieved a lesser amplification success rate of 70% (n = 20 for each treatment). More variable results were produced when field-derived exuviae were used, with no significant difference in real-time PCR amplification success found across the four methods and a lower overall rate of successful identification of 55-80%.
CONCLUSIONS: This study shows that both colony and field derived Cx. pipiens pupal exuviae can be a useful non-invasive source of trace DNA permitting accurate biotype differentiation for at least twenty-four hours post-eclosion. The significance and utility of this technique in ecological and behavioural studies of Cx. pipiens is discussed and recommendations made for use according to experimental scenario.
摘要：
背景：淡色库蚊是欧洲人畜共患虫媒病毒的主要载体，在鸟类宿主之间的植物性传播中起放大作用，并作为鸟类宿主和哺乳动物之间的桥梁载体。该物种由两种形式组成，使用形态学方法无法区分，但具有影响其媒介容量的不同生态和生理特征。在这项研究中，我们验证了可用于从Cx的单个p中提取痕量DNA的方法。pipiens用于样品的分子形态。通过使用实时聚合酶链反应（PCR）测定的总产率测量和成功鉴定来比较这些DNA提取方法。
结果：最初使用乙醇沉淀法从菌落衍生的个体中提取基因组DNA，两种市售DNA提取试剂盒：DNeasy®血液和组织试剂盒（Qiagen，英国）和Wizard®SV基因组DNA纯化系统（Promega，英国）和直接实时PCR方法。羽化和p处理之间的时间显着影响Cx。pipiens形式鉴定为核酸浓度和PCR扩增成功率随时间的增加而降低。实时PCR扩增成功,然而,三种提取方法之间没有显着差异，所有方法都成功识别了所有样本，但是直接实时PCR方法的扩增成功率较低，为70％（每种处理n=20）。当使用现场衍生的漏洞时，产生了更多可变的结果，在四种方法中发现的实时PCR扩增成功率没有显着差异，并且总的成功率较低，为55-80％。
结论:本研究显示集落和田地来源的Cx。pipienspual可作为痕量DNA的有用非侵入性来源，可在羽化后至少24小时内进行准确的生物型分化。该技术在Cx的生态和行为研究中的意义和实用性。pipiens进行了讨论，并根据实验情况提出了使用建议。
结果：最初使用乙醇沉淀法从菌落衍生的个体中提取基因组DNA，两种市售DNA提取试剂盒：DNeasy®血液和组织试剂盒（Qiagen，英国）和Wizard®SV基因组DNA纯化系统（Promega，英国）和直接实时PCR方法。羽化和p处理之间的时间显着影响Cx。pipiens形式鉴定为核酸浓度和PCR扩增成功率随时间的增加而降低。实时PCR扩增成功,然而,三种提取方法之间没有显着差异，所有方法都成功识别了所有样本，但是直接实时PCR方法的扩增成功率较低，为70％（每种处理n=20）。当使用现场衍生的漏洞时，产生了更多可变的结果，在四种方法中发现的实时PCR扩增成功率没有显着差异，并且总的成功率较低，为55-80％。
结论:本研究显示集落和田地来源的Cx。pipienspual可作为痕量DNA的有用非侵入性来源，可在羽化后至少24小时内进行准确的生物型分化。该技术在Cx的生态和行为研究中的意义和实用性。pipiens进行了讨论，并根据实验情况提出了使用建议。
