Rapid, Efficient Synthesis of Double-stranded DNA Standards for Quantitative PCR-Based Detection of HSV-2 using Polymerase Cycling Assembly.
- 1Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran.
- 2Biological Products and Blood Safety Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran.
Abstract
Background
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.Objective
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.Materials and methods
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.Results
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.Conclusion
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.
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BibTeX
@article{zandieh2026rapid,
title = {{Rapid, Efficient Synthesis of Double-stranded DNA Standards for Quantitative PCR-Based Detection of HSV-2 using Polymerase Cycling Assembly.}},
author = {Zahra Sharif Zandieh and Shahram Samiee and Kamran Mousavi Hosseini and Zohreh Sharifi and Amir Teimourpour},
journal = {Iranian journal of biotechnology},
year = {2026},
month = oct,
doi = {10.30498/ijb.2026.530427.4170},
url = {https://europepmc.org/article/MED/42928787},
note = {CC BY-NC},
}
RIS
TY - JOUR
TI - Rapid, Efficient Synthesis of Double-stranded DNA Standards for Quantitative PCR-Based Detection of HSV-2 using Polymerase Cycling Assembly.
AU - Zahra Sharif Zandieh
AU - Shahram Samiee
AU - Kamran Mousavi Hosseini
AU - Zohreh Sharifi
AU - Amir Teimourpour
PY - 2026
DA - 2026/10/01
JO - Iranian journal of biotechnology
DO - 10.30498/ijb.2026.530427.4170
UR - https://europepmc.org/article/MED/42928787
AB - Background 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.Objective 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.Materials and methods 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.Results 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.Conclusion 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.
SN - 1728-3043
SN - 2322-2921
ER -