A fast and inexpensive genotyping system for the simultaneous analysis of human and Aedes albopictus STRs

Abstract

Abstract Background Determination of the interactions between hematophagous mosquitoes and their human hosts is of great importance for better understanding the transmission dynamics of mosquito-borne arboviruses and developing effective strategies to mitigate risk. Genetic analysis of human and mosquito DNA can play a key role in this but commercial kits for human short tandem repeat (STR) genotyping are expensive and do not allow for the simultaneous STR analysis of host and vector. Here, we present an inexpensive and straightforward STR-loci multiplex system capable of simultaneously amplifying Ae. albopictus and human STRs from blood-fed mosquitoes. Additionally, we examined the effect of storage methods and post-feeding time on the integrity of host DNA. Methods 35 STRs (16 human and 19 Ae. albopictus STRs) subdivided in three multiplexes were tested for amplification and scoring reliability. Under laboratory conditions we compared the efficacy of two preservation methods (absolute ethanol vs. lysis buffer) on the integrity of host DNA in Ae. albopictus blood meals, we evaluated the effect of post-feeding time by sacrificing blood-fed mosquitoes at different times post-feed, and assessed our ability to detect multiple feedings. To determine if the methodology could be employed successfully under field conditions, we carried out a preliminary study using field-collected Ae. albopictus. Results All 35 STRs amplified consistently in the laboratory. Lysis buffer performed better than absolute ethanol in terms of allele peak height and clarity of electropherograms. Complete human DNA profiles could be obtained up to 48h following the blood meal. Analysis of multiple feedings confirmed that peak heights can be used as a proxy to determine post-feeding time and thus derive the number of different people bitten by a mosquito. In the field trial, amplification was successful for 32 STRs. We found human DNA signal in 38 of the 61 field-collected mosquitoes (62%), of which 34 (89%) had ingested a single blood meal, while 4 (11%) contained double meals. Conclusions Our new genotyping system will allow fast and reliable screening of both host and vector species, and can be further adapted to other mosquito species living in close contact with humans.