<![CDATA[Background: Reliable PCR amplification from pooled mosquito DNA extracts is frequently compromised by high template concentrations and co-purified inhibitors, contributing to workflow inefficiencies in vector surveillance programs. Objectives: This study aimed to develop and validate a practical concentration stratified decision-making workflow for optimizing DNA template preparation prior to PCR by evaluating the effect of serial dilution on amplification success across samples with varying initial DNA concentrations. Methods: DNA was extracted from 10 pooled mosquito samples in each tube, there are 47 samples, quantified by Qubit fluorometric, and classified into four concentration categories (low <20 ng/µL; moderate 20-49 ng/µL; high 50-80 ng/µL; very high >80 ng/µL). Serial dilutions (undiluted, 1:10, 1:100, 1:200) were evaluated by agarose gel electrophoresis using a standardized visual scoring system (0-4). Results: A successful PCR reaction is defined as the presence of a single 680-bp amplicon, as expected, on agarose gel electrophoresis. Undiluted samples had a PCR eligibility rate of only 5%. Very high concentration samples required a minimum 1:100 dilution to achieve 85% eligibility, whereas high-concentration samples achieved 94.7% eligibility from 1:10 onward. Conclusion: Concentration stratified serial dilution combined with Qubit and electrophoresis-based scoring substantially improves PCR outcomes for pooled mosquito DNA. It can be concluded that dilution was associated with improved detection of the expected PCR product, particularly among high and very high concentration extracts. A concentration stratified dilution strategy may be useful, but the proposed thresholds require validation in independent sample sets and laboratories.]]>
