Volume 22, Issue 2 (Summer 2025)                   bloodj 2025, 22(2): 100-110 | Back to browse issues page

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Sharif Zandieh Z, Samiee S, MousaviHosseini K, Sharifi Z. In Silico Synthesis of HSV-2 Positive Control Using Polymerase Cycling Assembly. bloodj 2025; 22 (2) :100-110
URL: http://bloodjournal.ir/article-1-1572-en.html
In Silico Synthesis of HSV-2 Positive Control Using Polymerase Cycling Assembly
Zahra Sharif Zandieh , Shahram Samiee * , Kamran MousaviHosseini , Zohreh Sharifi
Keywords: oligonucleotides, Real-Time Polymerase Chain Reaction, HSV-2
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References:
  1. Hughes RA, Ellington AD. Synthetic DNA Synthesis and Assembly: Putting the Synthetic in Synthetic Biology. Cold Spring Harb Perspect Biol. 2017 Jan 3;9(1):a023812. [DOI:10.1101/cshperspect.a023812] [PMID] []
  2. Hoose A, Vellacott R, Storch M, Freemont PS, Ryadnov MG. Publisher Correction: DNA synthesis technologies to close the gene writing gap. Nat Rev Chem. 2023 Aug;7(8):590. [DOI:10.1038/s41570-022-00456-9] [PMID] []
  3. Song LF, Deng ZH, Gong ZY, Li LL, Li BZ. Large-Scale de novo Oligonucleotide Synthesis for Whole-Genome Synthesis and Data Storage: Challenges and Opportunities. Front Bioeng Biotechnol. 2021 Jun 22;9:689797. [DOI:10.3389/fbioe.2021.689797] [PMID] []
  4. Cello J, Paul AV, Wimmer E. Chemical synthesis of poliovirus cDNA: generation of infectious virus in the absence of natural template. Science. 2002 Aug 9;297(5583):1016-8. https://doi.org/ 10.1126/science.1072266 [DOI:10.1126/science.1072266] [PMID]
  5. Herpes: HSV1 and HSV2. https://www.hopkinsmedicine.org/health/conditions-and-diseases/herpes-hsv1-and-hsv2#:~:text=In%20the%20case%20of%20HSV,is%20active%20at%20that%20time.
  6. Juhl D, Mosel C, Nawroth F, Funke AM, Dadgar SM, Hagenström H, et al. Detection of herpes simplex virus DNA in plasma of patients with primary but not with recurrent infection: implications for transfusion medicine? Transfus Med. 2010 Feb;20(1):38-47. [DOI:10.1111/j.1365-3148.2009.00951.x] [PMID]
  7. Wong ML, Medrano JF. Real-time PCR for mRNA quantitation. Biotechniques. 2005 Jul;39(1):75-85. [DOI:10.2144/05391RV01] [PMID]
  8. Han X, Beck K, Bürgmann H, Frey B, Stierli B, Frossard A. Synthetic oligonucleotides as quantitative PCR standards for quantifying microbial genes. Front Microbiol. 2023 Oct 24;14:1279041. [DOI:10.3389/fmicb.2023.1279041] [PMID] []
  9. Kadkhodaei S, Rajabi Memari H, Abbasiliasi S, Akhavan Rezaei M, Movahedi A, Joo Shun T, et al. Multiple overlap extension PCR (MOE-PCR): an effective technical shortcut to high throughput synthetic biology. RSC Advances 2016;6(71):66682-66694. [DOI:10.1039/C6RA13172G]
  10. Wilson CC, Wozney KM, Smith CM. Recognizing false positives: synthetic oligonucleotide controls for environmental DNA surveillance. Methods in Ecology and Evolution 2016;7(1):23-29. [DOI:10.1111/2041-210X.12452]
  11. Borst A, Box AT, Fluit AC. False-positive results and contamination in nucleic acid amplification assays: suggestions for a prevent and destroy strategy. Eur J Clin Microbiol Infect Dis. 2004 Apr;23(4):289-99. [DOI:10.1007/s10096-004-1100-1] [PMID]
  12. TerMaat JR, Pienaar E, Whitney SE, Mamedov TG, Subramanian A. Gene synthesis by integrated polymerase chain assembly and PCR amplification using a high-speed thermocycler. J Microbiol Methods. 2009 Dec;79(3):295-300. [DOI:10.1016/j.mimet.2009.09.015] [PMID] []
  13. Chen Z, Zhao K, Tan B, Tong Z, He Z, Luo X, et al. Development of a high specificity typing method for the detection of herpes simplex virus. Front Bioeng Biotechnol. 2022 Aug 17;10:955713. [DOI:10.3389/fbioe.2022.955713] [PMID] []






In Silico Synthesis of HSV-2 Positive Control Using
Polymerase Cycling Assembly
‎
Zahra Sharif Zandieh1, Shahram Samiee1     , Kamran Mousavi Hosseini2     , Zohreh Sharifi2


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

Received: 2025/02/16
Accepted: 2025/04/30
 





    https://doi.org/10.18502/avr.v34i2.18052

    



Citation:
Sharif Zandieh Z, Samiee Sh, Mousavi Hosseini K, Sharifi Z. In Silico Synthesis of HSV-2 Positive Control Using Polymerase Cycling Assembly. J Iran Blood Transfuse. 2025: 22 (2) : 100-110
    



Correspondence: Samiee SH., Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine.
P.O.Box: 14665-1157, Tehran, Iran.
Tel: (+9821); 82052206
E-mail: s.samiee@tmi.ac.ir           

Correspondence: Mousavi Hosseini K., Biological Products and Blood Safety Research Center, High Institute for Research and Education in Transfusion Medicine.
P.O.Box: 14665-1157, Tehran, Iran.
Tel: (+9821); 82052160
E-mail: ka.mousavi@tmi.ac.ir		 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.
Key words: oligonucleotides, Real-Time Polymerase Chain Reaction, HSV-2
Copyright © 2025 Journal of Iranian Blood Transfusion, Published by Blood Transfusion Research Center.
This work is licensed under a Creative Common Attribution-Non Commercial 4.0 International license.




1- Acridine Orange
1- Biological safety cabinet
1- Platelet Concentrate
2- Food and Drug Administration
3- Normal Skin Flora
4- Platelet Rich Plasma-Platelet Concentrate
5- Eosin-Methylene blue
6- Thioglycolate
1- Acridine Orange
1- Biological safety cabinet
1- Platelet Concentrate
2- Food and Drug Administration
3- Normal Skin Flora
4- Platelet Rich Plasma-Platelet Concentrate
5- Eosin-Methylene blue
6- Thioglycolate

 
Type of Study: Research | Subject: Biotechnology
References
1. Hughes RA, Ellington AD. Synthetic DNA Synthesis and Assembly: Putting the Synthetic in Synthetic Biology. Cold Spring Harb Perspect Biol. 2017 Jan 3;9(1):a023812. [DOI:10.1101/cshperspect.a023812] [PMID] []
2. Hoose A, Vellacott R, Storch M, Freemont PS, Ryadnov MG. Publisher Correction: DNA synthesis technologies to close the gene writing gap. Nat Rev Chem. 2023 Aug;7(8):590. [DOI:10.1038/s41570-022-00456-9] [PMID] []
3. Song LF, Deng ZH, Gong ZY, Li LL, Li BZ. Large-Scale de novo Oligonucleotide Synthesis for Whole-Genome Synthesis and Data Storage: Challenges and Opportunities. Front Bioeng Biotechnol. 2021 Jun 22;9:689797. [DOI:10.3389/fbioe.2021.689797] [PMID] []
4. Cello J, Paul AV, Wimmer E. Chemical synthesis of poliovirus cDNA: generation of infectious virus in the absence of natural template. Science. 2002 Aug 9;297(5583):1016-8. https://doi.org/ 10.1126/science.1072266 [DOI:10.1126/science.1072266] [PMID]
5. Herpes: HSV1 and HSV2. https://www.hopkinsmedicine.org/health/conditions-and-diseases/herpes-hsv1-and-hsv2#:~:text=In%20the%20case%20of%20HSV,is%20active%20at%20that%20time.
6. Juhl D, Mosel C, Nawroth F, Funke AM, Dadgar SM, Hagenström H, et al. Detection of herpes simplex virus DNA in plasma of patients with primary but not with recurrent infection: implications for transfusion medicine? Transfus Med. 2010 Feb;20(1):38-47. [DOI:10.1111/j.1365-3148.2009.00951.x] [PMID]
7. Wong ML, Medrano JF. Real-time PCR for mRNA quantitation. Biotechniques. 2005 Jul;39(1):75-85. [DOI:10.2144/05391RV01] [PMID]
8. Han X, Beck K, Bürgmann H, Frey B, Stierli B, Frossard A. Synthetic oligonucleotides as quantitative PCR standards for quantifying microbial genes. Front Microbiol. 2023 Oct 24;14:1279041. [DOI:10.3389/fmicb.2023.1279041] [PMID] []
9. Kadkhodaei S, Rajabi Memari H, Abbasiliasi S, Akhavan Rezaei M, Movahedi A, Joo Shun T, et al. Multiple overlap extension PCR (MOE-PCR): an effective technical shortcut to high throughput synthetic biology. RSC Advances 2016;6(71):66682-66694. [DOI:10.1039/C6RA13172G]
10. Wilson CC, Wozney KM, Smith CM. Recognizing false positives: synthetic oligonucleotide controls for environmental DNA surveillance. Methods in Ecology and Evolution 2016;7(1):23-29. [DOI:10.1111/2041-210X.12452]
11. Borst A, Box AT, Fluit AC. False-positive results and contamination in nucleic acid amplification assays: suggestions for a prevent and destroy strategy. Eur J Clin Microbiol Infect Dis. 2004 Apr;23(4):289-99. [DOI:10.1007/s10096-004-1100-1] [PMID]
12. TerMaat JR, Pienaar E, Whitney SE, Mamedov TG, Subramanian A. Gene synthesis by integrated polymerase chain assembly and PCR amplification using a high-speed thermocycler. J Microbiol Methods. 2009 Dec;79(3):295-300. [DOI:10.1016/j.mimet.2009.09.015] [PMID] []
13. Chen Z, Zhao K, Tan B, Tong Z, He Z, Luo X, et al. Development of a high specificity typing method for the detection of herpes simplex virus. Front Bioeng Biotechnol. 2022 Aug 17;10:955713. [DOI:10.3389/fbioe.2022.955713] [PMID] []
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