Journal of

A B S T R A C T
Background and Objectives
The use of positive controls in Real-Time PCR experiments is crucial for ensuring assay accuracy and reliability. Traditionally, such controls are derived from clinical resources. However, in situations where access to biological samples is limited, Polymerase Cycle Assembly (PCA) provides a reliable alternative for creating positive controls. The aim of this study was to evaluate the efficiency of the enzymatic method for producing genomic DNA fragments suitable for molecular biology applications. To the best of our knowledge, while PCA has been primarily employed for assembling sequences, its utility in constructing HSV-2 positive controls has not yet been investigated.
Materials and Methods
In this experimental study, the first step involved designing overlapping DNA fragments targeting the US6 region of the HSV-2 genome, a unique and well-characterized sequence selected as a suitable target. The selection of the US6 region was based on a literature review analysis. The overlapping fragments were evaluated for secondary structures and thermodynamic parameters using AlleleID software. Primer design for the US6 region was performed using MEGA version 11, ensuring specificity to the HSV-2 genome. The evaluation of the genome HSV-2 with the SnapGene software has been carried out. A comprehensive examination of the primers and their products from the secondary structure was conducted using MFold. This software analyses the sequence intended for primer design in terms of its secondary structure.
Results
Alignment analysis revealed a high degree of sequence similarity between HSV-1 and HSV-2. Therefore, primers and overlapping fragments were designed to target a region exhibiting the greatest natural variation. The results demonstrated that the in silico positive controls were comparable to natural controls, and the specificity of designed primers was confirmed.  Using SnapGene software, the size of reproduced DNA segment was estimated to be approximately 70 base pairs, providing a suitable template for future studies.
Conclusions 
The results demonstrate that PCA is an effective and highly reliable method for synthesizing positive controls for molecular diagnostics. Targeting the HSV-2 US6 region ensured specificity and structural integrity of the synthetic fragment.