Rapid, Efficient Synthesis of Double-stranded DNA Standards for Quantitative PCR-Based Detection of HSV-2 using...

Synthetic DNA synthesis, also known as "gene synthesis", sequences ranging from 250 to 2000 base pairs, starting with single-stranded DNA oligonucleotides. Reliable, relevant positive controls are essential for accurate Real-Time PCR diagnostics, yet clinical or cultured positive materials are often scarce and costly, and they pose biosafety and logistical challenges.

This study aimed to provide a practical, affordable solution for laboratories seeking to establish transparent, comprehensive reporting of non-accessible pathogens, using HSV-2 as the model organism.

The US6 gene was chosen as the target based on several criteria: 1) sequence alignment using MEGA11 software, 2) analysis of secondary structures via AlleleID, and 3) in silico PCR analysis with suitable primers.

In this study, the amplification efficiency for the US6 target gene was 108%, indicating good linearity and consistent synthetic gene fragments as quantitative PCR (qPCR) standards. For the targeted gene, evaluated using synthetic and clinical DNA standards, the qPCR quantification standards showed no significant difference (p > 0.05) in their linear calibration curves, with coefficients of determination (R²) ≥ 0.98.

Together, these results indicate that PCA-synthesized DNA standards offer a scalable, rapid, sensitive, and implementation-ready alternative for clinical materials for HSV-2 diagnostics, and can be adapted for broader diagnostic targets subject to routine detection.