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.

Degradation of Fuel Cell (FC) components under dynamic loads is one of the biggest bottlenecks in FC commercialization. A novel experimental based model is presented to predict the Catalyst Layer (CL) performance loss under a given cyclic load. It consists of two sub-models: Model 1 computes CL Electro-Chemical Surface Area (ECSA) under an N-cyclic load with the aid of an analogy with fatigue phenomena of carbon steel by using some correction factors. Ostwald ripening of agglomerate particles in the CL is also modeled. Model 1 validation shows good agreements between its outputs and a large number of experiments with maximum 7% error. Model 2 is an already-completed task in an earlier study which uses the agglomerate model to calculate the CL performance for a given ECSA. Combination of Models 1 & 2 predicts the CL performance under a dynamic load. A set of parametric studies was performed to investigate the effects of operating parameters on the Voltage Degradation Rate (VDR). The results show that temperature is the most influential parameter, an increase from 60oC to 80oC leads to 20.26% VDR increase, and pressure is the least effective one, an increase from 2atm to 4atm leads to 1.41% VDR rise.