Comparative analysis of three methods from dried blood spots for expeditious DNA extraction from mosquitoes; suitable for PCR based techniques
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The objective of this work was to compare the quality, purity and quantity of DNA isolated from dried blood spots (DBS) by three methods (Chelex-100, QIAamp DNA mini kit, and TE (Tris EDTA)-Buffer). Sample collection was performed in six districts in Odisha, India and screened for cases of clinical malaria and dengue and vector density. Mosquito abdomens were spotted on Whatman 3MM (MERCK) Filter paper and dried for 10 min at room temperature. DNA was isolated from DBS using three methods (Chelex-100, QIAamp DNA mini kit, and TE-Buffer), and PCR was used to determine the feeding behaviours of vector mosquitoes. DNA was quantified using a UV-spectrophotometer, and q-PCR was used to determine the target gene copy number to compare the methods. The QIAamp DNA mini kit method was used as the reference method. The yield and purity of DNA extracted with Chelex-100 and TE were 14–72 ng/µl and 1.51–1.85 and 9–50 ng/µl and 1.68–2.1, respectively. DNA extracted using the Chelex-100 method was stored for over 1 month at − 20 °C and was suitable for later use. The Chelex-100 method had a sensitivity of 99.5% and specificity of 78%. A Bland–Altman plot suggested that the Chelex-100 method was similar to the QIAamp DNA mini kit method for determining the feeding behaviours of vector mosquitoes. The Chelex-100 method is simple, cost-effective, and safe and requires minimal time for DNA extraction from dried blood spots. In malaria and dengue research, detecting the feeding behaviours from mosquito DNA from dried blood spots on filter paper by PCR is an easy, minimally invasive and inexpensive molecular technique that can be performed in remote areas.
KeywordsChelex-100 TE-Buffer Dried blood spots Anopheles Aedes
We are grateful to Director RMRC for her support. We thank Entomology division staff, Santoshini Dash, Animesha Rath and Ipsita Mohanty of RMRC, BBSR for technical help.
BBP and RKH conceived the study and participated in its design and co-ordination; BBP and RKH carried out the field studies; BBP and NP drafted the manuscript, performed the statistical analysis; BBP, NP and RKH critically revised the manuscript for intellectual content; All authors read and approved the final manuscript; BBP and RKH are guarantors of the paper.
This work was supported by funds of DST and ICMR. Ms BBP supported the fellowship from DST-INSPIRE Fellowship, Govt. of India (Grant No. IF150310) and registered under Utkal University, Bhubaneswar, Odisha.
Compliance with ethical standards
Conflict of interest
The author declares that they have no conflict of interest.
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