Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI.
Vector Borne Diseases Unit, Papua New Guinea Institute of Medical Research, Madang, Papua New Guinea.
J Med Entomol. 2019 Jun 27;56(4):907-914. doi: 10.1093/jme/tjz013.
Genetic profiling has been used to link mosquito bloodmeals to the individual humans, but this analysis has not been done for other mammalian bloodmeals. In this study, we describe a microsatellite-based method for identifying individual pigs in mosquito bloodmeals based on their unique multilocus genotypes. Eleven tetranucleotide microsatellites and a sex-specific marker were selected based on Smith-Waterman DNA sequence alignment scores from the reference genome and primers were designed with features that reduce primer dimers, promote complete adenylation, and enable fluorescent labeling of amplicons. A multiplex polymerase chain reaction (PCR) assay was optimized and validated by analyzing DNA of individual pigs from several nuclear families and breeds before it was used to analyze genomic DNA of pig-derived mosquito bloodmeals from villages of Papua New Guinea. Population analysis of the nuclear families showed high expected and observed heterozygosity. The probability of observing two unrelated or sibling individuals sharing the same genotype at a single microsatellite locus or a combination of loci was vanishingly low. Samples had unique genotypes and gender was accurately predicted. Analysis of 129 pig bloodmeals identified 19 unique genotypes, which varied greatly in frequency in the mosquito bloodmeal samples. The high allelic diversity of the microsatellite loci and low probability of false attribution of identity show that this genotyping method reliably distinguishes distantly and closely related pigs and can be used to identify individual pigs from genotyped mosquito bloodmeals.
遗传分析已被用于将蚊子的血食与个体人类联系起来，但其他哺乳动物的血食分析尚未进行。在这项研究中，我们描述了一种基于微卫星的方法，可根据蚊子血食中的个体独特的多位点基因型来识别个体猪。根据参考基因组的 Smith-Waterman DNA 序列比对得分，选择了 11 个四核苷酸微卫星和一个性别特异性标记，并设计了具有减少引物二聚体、促进完全腺苷酸化和能够荧光标记扩增子的特征的引物。在分析了来自几个核家族和品种的单个猪的 DNA 后，优化并验证了多重聚合酶链反应 (PCR) 检测，然后用于分析巴布亚新几内亚村庄的猪源性蚊子血食的基因组 DNA。核家族的群体分析显示出高预期和观察到的杂合度。观察到两个不相关或兄弟姐妹个体在单个微卫星位点或多个位点上共享相同基因型的概率非常低。样本具有独特的基因型，性别也得到了准确预测。对 129 份猪血食样本的分析确定了 19 种独特的基因型，这些基因型在蚊子血食样本中的频率差异很大。微卫星位点的高等位基因多样性和身份错误归因的低概率表明，这种基因分型方法可靠地区分了远亲和近亲猪，并且可以用于从基因分型的蚊子血食中识别个体猪。
