Robust comparative performance of genomic DNA extraction methods from non-engorged phlebotomine sandflies

The present study is a comparative analysis of DNeasy Blood & Tissue Qiagen (R) kit (Qiagen (R), Hilden, Alemanha) , salting out, HotShot and phenol-chloroform protocols to extract DNA from sandflies. In addition, a comparative test using sandflies with and without eyes evaluated the potential inhibitory effect in the cPCR. An inhibition test was performed using an exogenous DNA added to the qPCR. The genomic DNA quality of each sample was evaluated by cPCR based on the cytochrome c oxidase subunit I (cox1) gene. The DNA extraction protocols showed the following percentage of amplification: HotShot (91.6% [55/60]), salting out (71.6% [43/60]), phenol-chloroform (95% [57/60]) and kit DNeasy Blood & Tissue Qiagen (R) (73.3% [44/60]). The phenol-chloroform method achieved a significantly higher frequency of cox1 gene amplification. The pigment present in the phlebotomine's eyes seems to inhibit cPCR reactions since the frequency of amplification of the cox1 gene increased in the sandflies without eyes (p < 0.0001). The HotShot method showed the highest inhibitory potential. These manual extraction techniques can be an inexpensive and effective alternative to study vector-pathogen interactions.

Automated summary

This study compared four methods for DNA extraction from sandflies and found that the phenol-chloroform method had the highest amplification efficiency, while the HotShot method had the highest inhibition potential. Additionally, the pigment in the sandflies' eyes seemed to inhibit amplification reactions. These extraction techniques can be a cost-effective alternative for studying vector-pathogen interactions.