背景:在媒介蚊子中PCR检测人类血餐中DNA所需的时间可能会有所不同,根据使用的分子标记,基于扩增子的大小和拷贝数。对自然界中蚊子种群的采血行为的详细了解是评估其媒介能力和评估个别脊椎动物作为媒介传播疾病的潜在宿主的作用的重要组成部分。
方法:进行了实验室实验,以比较PCR检测来自个体血液喂养的按蚊的人血餐中DNA的时间过程,使用具有不同特征的基因座,包括两个线粒体DNA(mtDNA)基因,血餐后不同时间点的cytB(228bp)和16S核糖体RNA(rRNA)(157bp)和核Alu重复元件(226bp)。
结果:在采血后84-120小时内可检测到人类DNA,取决于基因座的长度和拷贝数。我们的结果表明,16SrRNA和Alu-repeat标记可以在血餐后5天内从人类DNA中成功回收。16SrDNA和Alu重复序列基因座的下降速率明显(P=0.008)比cytB基因座慢。扩增子的中位检测周期(T50)为Alu重复序列的117、113和86.4小时,16SrDNA和cytB,分别,表明扩增子大小/拷贝数和消化时间之间的反线性关系。
结论:这项比较研究表明,Alu-repeat基因座是鉴定雌性蚊子血餐中人类DNA时程的最有效标记。它也是确定嗜血指数(AI)或人类血液指数(HBI)的有前途的工具,即人类血粉的比例,这通常被报道为不同蚊子媒介的食人症的相对衡量标准,因此衡量了在野外收集的蚊子的媒介能力。
BACKGROUND: The time required for PCR detection of DNA in human blood meals in vector mosquitoes may vary, depending on the molecular markers used, based on the size and copy number of the amplicons. Detailed knowledge of the blood-feeding behavior of mosquito populations in nature is an essential component for evaluating their vectorial capacity and for assessing the roles of individual vertebrates as potential hosts involved in the transmission of vector-borne diseases.
METHODS: Laboratory experiments were conducted to compare the time course of PCR detection of DNA in human blood meals from individual blood-fed Anopheles stephensi mosquitoes, using loci with different characteristics, including two mitochondrial DNA (mtDNA) genes, cytB (228 bp) and 16S ribosomal RNA (rRNA) (157 bp) and nuclear Alu-repeat elements (226 bp) at different time points after the blood meal.
RESULTS: Human DNA was detectable up to 84-120 h post-blood-feeding, depending on the length and copy number of the loci. Our results suggest that 16S rRNA and Alu-repeat markers can be successfully recovered from human DNA up to 5 days post-blood-meal. The 16S rDNA and Alu-repeat loci have a significantly (P = 0.008) slower decline rate than the cytB locus. Median detection periods (T50) for the amplicons were 117, 113 and 86.4 h for Alu-repeat, 16S rDNA and cytB, respectively, suggesting an inverse linear relationship between amplicon size/copy number and digestion time.
CONCLUSIONS: This comparative
study shows that the Alu-repeat locus is the most efficient marker for time-course identification of human DNA from blood meals in female mosquitoes. It is also a promising tool for determining the anthropophilic index (AI) or human blood index (HBI), i.e. the proportion of blood meals from humans, which is often reported as a relative measure of anthropophagy of different mosquito vectors, and hence a measure of the vector competence of mosquito species collected in the field.