Stepwise Generation of a Verified DNA Standard: From in silico Primer Design to Quantitative PCR Calibration for <i>Neisseria flavescens</i>

Abstract

Background: Accurate molecular quantification of Neisseria flavescens is limited by a lack of commercially available reference standards, particularly in resource-limited settings. Objectives: This study aimed to develop and validate a reproducible workflow for absolute quantification of N. flavescens using an internally generated DNA standard. Methods: Five oropharyngeal swab samples were collected from healthy volunteers and screened using species-specific PCR. One positive sample was used to generate an internal standard. PCR products were confirmed by gel electrophoresis, excised, purified, and validated by Sanger sequencing. The confirmed DNA was quantified using NanoDrop spectrophotometry and used for qPCR amplification. A 7-point, 10-fold serial dilution series was prepared to construct a standard curve. Results: Sequencing analysis showed 100% identity with the target gene. The purified DNA concentration was 16 ng/µL, corresponding to approximately 7.49 × 1010 gene copies. The qPCR standard curve demonstrated excellent linearity and amplification efficiency. No amplification was observed in the no-template controls, and triplicate assays showed minimal variation, indicating high reproducibility. Conclusions: This stepwise approach, encompassing primer design and optimization, sequencing confirmation, and qPCR calibration, provides a validated and reproducible method for the absolute quantification of N. flavescens. The workflow can be readily adapted for other bacterial species in regions lacking access to commercial reference standards.