Department of Vector Biology and Control of Diseases, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran.
Legal Medicine Research Center, Legal Medicine Organization, Tehran, Iran.
Parasit Vectors. 2023 Aug 14;16(1):284. doi: 10.1186/s13071-023-05884-0.
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.
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.
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.
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.
根据扩增子的大小和拷贝数，用于检测媒介蚊虫血餐中 DNA 的 PCR 所需时间可能因所使用的分子标记而异。详细了解自然状态下蚊虫种群的吸血行为是评估其媒介能力以及评估单个脊椎动物作为参与传播媒介传播疾病的潜在宿主的作用的重要组成部分。
实验室实验比较了使用具有不同特征的基因座（包括两个线粒体 DNA（mtDNA）基因 cytB（228 bp）和 16S 核糖体 RNA（rRNA）（157 bp）以及核 Alu 重复元件（226 bp）），检测个体吸血后不同时间点的人血餐中 DNA 的时间过程。
根据基因座的长度和拷贝数，人 DNA 可检测到 84-120 小时后。我们的结果表明，16S rRNA 和 Alu 重复标记物可以成功地从人 DNA 中回收长达 5 天的血餐。16S rDNA 和 Alu 重复序列的下降速度明显（P=0.008）比 cytB 序列慢。Alu 重复、16S rDNA 和 cytB 的扩增子中位检测期（T50）分别为 117、113 和 86.4 小时，这表明扩增子大小/拷贝数与消化时间之间存在逆线性关系。
这项比较研究表明，Alu 重复序列是鉴定雌性蚊虫血餐中人 DNA 的最有效标记物。它也是确定嗜人指数（AI）或人血指数（HBI）的有前途的工具，即来自人类的血餐比例，这通常被报道为不同蚊虫媒介嗜人程度的相对度量，也是评估野外采集的蚊子物种媒介能力的度量。
