Owens Carrie B, Szalanski Allen L
Department of Entomology, University of Arkansas, Fayetteville, AR 72701, USA.
J Med Entomol. 2005 Jul;42(4):709-11. doi: 10.1093/jmedent/42.4.709.
Proper DNA storage is critical for studies involving genetic analysis of insects and for molecular diagnostics of pathogens carried by them. Molecular surveillance of pathogens carried by insects can involve screening of thousands of insect DNA samples. Problems with storage and degradation of these samples can arise. In this study, a simple filter paper-based method for storage and preservation of insect DNA was evaluated using polymerase chain reaction (PCR). DNA was isolated from individual house fly, Musca domestica L., adults by using a cell lysis technique. From 50 house flies known to carry Campylobacter spp., a portion of the DNA sample was stored frozen and another portion was pipetted onto filter paper. At monthly intervals for 7 mo, PCR was conducted using 1 microl of the frozen DNA sample and a 2.0-mm disk from the filter paper samples as the PCR template. Two markers were used, a 450-bp region of the insect mitochondrial DNA (mtDNA) ND5 gene and a 857-bp region of the Campylobacter spp. mtDNA 16S rDNA gene. PCR amplification was successful for all of the samples regardless of the storage method. The filter paper method is a simple and economical way to store, preserve, and distribute DNA samples for PCR analysis.
合适的DNA储存对于涉及昆虫遗传分析的研究以及对昆虫携带病原体的分子诊断至关重要。对昆虫携带病原体的分子监测可能涉及对数千份昆虫DNA样本的筛选。这些样本可能会出现储存和降解问题。在本研究中,使用聚合酶链反应(PCR)评估了一种基于滤纸的简单昆虫DNA储存和保存方法。通过细胞裂解技术从家蝇(Musca domestica L.)成虫个体中分离DNA。从已知携带弯曲杆菌属的50只家蝇中,一部分DNA样本冷冻保存,另一部分移液到滤纸上。在7个月的时间里,每月进行一次PCR,使用1微升冷冻DNA样本和滤纸样本上2.0毫米的圆盘作为PCR模板。使用了两个标记,一个是昆虫线粒体DNA(mtDNA)ND5基因的450碱基对区域,另一个是弯曲杆菌属mtDNA 16S rDNA基因的857碱基对区域。无论储存方法如何,所有样本的PCR扩增均成功。滤纸法是一种简单且经济的储存、保存和分发用于PCR分析的DNA样本的方法。