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Zandi M, Sharifi Z, Ajorloo M. Design and In-Silico Evaluation of Specific Primers for the HBZ Gene of HTLV-1 to Establish a Semi-Nested PCR Method. Journal of Iranian Blood Transfusion 2025; 22 (1) :19-29
URL: http://bloodjournal.ir/article-1-1567-en.html
Design and In-Silico Evaluation of Specific Primers for the HBZ Gene of HTLV-1 to Establish a Semi-Nested PCR Method
Mehdi Zandi , Zohreh Sharifi * , Mahdi Ajorloo
Keywords: HTLV-1, Nested PCR, Leukemia ​​​​​​​
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Sci J Iran Blood Transfus Organ 2025; 22 (1): 20-30
Original Article
 
Design and In-Silico Evaluation of Specific Primers
for the HBZ Gene of HTLV-1 to Establish a Semi-Nested
PCR Method


Mehdi Zandi1, Zohreh Sharifi1        , Mahdi Ajorloo1


1Biological Products and Blood Safety Resaerch Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran

Received: 2025/01/08
Accepted: 2025/03/09
 





     http://dx.doi.org/10.61186/bloodj.22.1.19
    



Citation:
Zandi M, Sharifi Z, Ajorloo M. Design and In-Silico Evaluation of Specific Primers for the HBZ Gene of HTLV-1 to Establish a Semi-Nested PCR Method. J Iran Blood Transfus. 2025: 22 (1) : 19-29.
    



Correspondence: Sharifi Z, Associate professor of Biological Products and Blood Safety Resaerch Center, High Institute for Research and Education in Transfusion Medicine.
P.O.Box: 14665-1157, Tehran, Iran.
Tel: (+9821) 82052152
E-mail:
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
z.sharifi@tmi.ac.ir  		 A B S T R A C T
Background and Objectives
Human T-cell lymphotropic virus type 1 (HTLV-1) is the only human oncogenic retrovirus associated with adult T-cell leukemia/lymphoma and tropical spastic paralysis. Common serological methods for detecting this virus are ELISA and Western blot. The HBZ gene, encoded by the negative strand of the HTLV-1 provirus, does not undergo continuous mutations, unlike other viral genes. In this study, the HBZ gene of HTLV-1 was used to design specific primers and develop a semi-nested PCR assay.
Materials and Methods
In a cross-sectional study, HBZ gene sequences from various virus isolates were extracted from the NCBI database and aligned using MEGA6 software. Using Gene Runner software, the primer design was performed from the conserved region common to all strains. To evaluate the primers, parameters such as GC content, melting temperature, hairpin, and dimer structures were used calculated by OligoAnalyzer software. To examine the exact binding site of the primers, SnapGene software was used to simulate the PCR reaction and electrophoresis. Primers were screened and synthesized using the BLASTN tool in NCBI. In the laboratory stage, genomic DNA was extracted from blood samples of HTLV-1 positive and healthy individuals and electrophoresed for Semi-nested PCR reaction and evaluation of PCR products.
Results
First, the HBZ protein coding sequences from 43 viral isolates with different geographical origins were aligned, and then primers were designed from the most similar conserved regions. The primers lacked hairpin and dimer structures. The GC percentage was between 45%-60%, and the melting temperature was between 64-67°C. BLAST results showed that the designed primers were 100% capable of identifying the target gene (HBZ), and no overlap with the genomic sequences of other viruses or humans was observed. After genomic DNA extraction and PCR, the length of the desired fragment was 493 bp in the first stage and 106 bp in the second stage, which was consistent with the predicted bioinformatics results.
Conclusions 
The design of HBZ gene primers utilizing bioinformatics software and the successful development of a Semi-nested PCR assay were achieved. The laboratory results confirmed the bioinformatics predictions and demonstrated the effectiveness of this method for the accurate and rapid detection of the virus.
Key words: HTLV-1, Nested PCR, Leukemia
 
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.









 References:
  1. Poiesz BJ, Ruscetti FW, Gazdar AF, Bunn PA, Minna JD, Gallo RC. Detection and isolation of type C retrovirus particles from fresh and cultured lymphocytes of a patient with cutaneous T-cell lymphoma. Proc Natl Acad Sci U S 1980;77(12):7415-9. [DOI:10.1073/pnas.77.12.7415] [PMID]
  2. Baratella M, Forlani G, Raval GU, Tedeschi A, Gout O, Gessain A, et al. Cytoplasmic Localization of HTLV-1 HBZ Protein: A Biomarker of HTLV-1-Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP). PLoS egl Trop Dis 2017;11(1):e0005285. [DOI:10.1371/journal.pntd.0005285] [PMID]
  3. Hedayati-Moghaddam MR. A Systematic Review for Estimation of HTLV-I Infection in the Blood Donors of Iran. Iran J Basic Med Sci. 2013;16(3):196-201.
  4. Proietti FA, Carneiro-Proietti AB, Catalan-Soares BC, Murphy EL. Global epidemiology of HTLV-I infection and associated diseases. Oncogene. 2005;24(39):6058-68. [DOI:10.1038/sj.onc.1208968] [PMID]
  5. Kia V, Forouzandeh Moghadam M, Paryan M, Raz A, Mirab cation of HBV and HTLV-I viruses by Melting curve Samiee S. Simultaneous detection and identifi analysis ofmultiplex Real-time PCR. Molecular and Biochemical Diagnosis Journal 2014;1(1):51-8.
  6. Cánepa C, Salido J, Ruggieri M, Fraile S, Pataccini G,Berini C, et al. Low Proviral Load is Associated with Indeterminate Western Blot Patterns in Human T-Cell Lymphotropic Virus Type 1 Infected Individuals: Could Punctual Mutations be Related? Viruses 2015;7(11):5643-58. [DOI:10.3390/v7112897] [PMID]
  7. Green MR, Sambrook J. Nested Polymerase Chain Reaction (PCR). Cold Spring Harb Protoc. 2019 Feb 1;2019(2). [DOI:10.1101/pdb.prot095182] [PMID]
  8. Caterino-de-Araujo A, Campos KR. Defective particles of human T-lymphotropic virus and negative results in molecular assays. Infect Genet Evol 2021;96:105141. [DOI:10.1016/j.meegid.2021.105141] [PMID]
  9. Begum KT, Wnaiza J, Absar N, Az S. In Silico Primer Design for Accurate Detection of SARS-CoV-2 Delta Variants. JBCG 2022;5:104. [DOI:10.17303/jbcg.2022.5.104]
  10. Gallego S, Mangano A, Gastaldello R, Sen L, Medeot S. Usefulness of a Nested-polymerase chain reaction for molecular diagnosis of human T-cell lymphotropic virus type I/II. Mem Inst Oswaldo Cruz. 2004 Jun;99(4):377-80. [DOI:10.1590/S0074-02762004000400006] [PMID]
  11. Blanco S, Frutos MC, Balangero MC, Gallego SV. Human T-Lymphotropic virus type 1 infection in absence of tax gene: A challenge for molecular diagnosis. Infect Genet Evol 2021;90:104765. [DOI:10.1016/j.meegid.2021.104765] [PMID]
Type of Study: Research | Subject: Virology
References
1. Poiesz BJ, Ruscetti FW, Gazdar AF, Bunn PA, Minna JD, Gallo RC. Detection and isolation of type C retrovirus particles from fresh and cultured lymphocytes of a patient with cutaneous T-cell lymphoma. Proc Natl Acad Sci U S 1980;77(12):7415-9. [DOI:10.1073/pnas.77.12.7415] [PMID] []
2. Baratella M, Forlani G, Raval GU, Tedeschi A, Gout O, Gessain A, et al. Cytoplasmic Localization of HTLV-1 HBZ Protein: A Biomarker of HTLV-1-Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP). PLoS egl Trop Dis 2017;11(1):e0005285. [DOI:10.1371/journal.pntd.0005285] [PMID] []
3. Hedayati-Moghaddam MR. A Systematic Review for Estimation of HTLV-I Infection in the Blood Donors of Iran. Iran J Basic Med Sci. 2013;16(3):196-201.
4. Proietti FA, Carneiro-Proietti AB, Catalan-Soares BC, Murphy EL. Global epidemiology of HTLV-I infection and associated diseases. Oncogene. 2005;24(39):6058-68. [DOI:10.1038/sj.onc.1208968] [PMID]
5. Kia V, Forouzandeh Moghadam M, Paryan M, Raz A, Mirab cation of HBV and HTLV-I viruses by Melting curve Samiee S. Simultaneous detection and identifi analysis ofmultiplex Real-time PCR. Molecular and Biochemical Diagnosis Journal 2014;1(1):51-8.
6. Cánepa C, Salido J, Ruggieri M, Fraile S, Pataccini G,Berini C, et al. Low Proviral Load is Associated with Indeterminate Western Blot Patterns in Human T-Cell Lymphotropic Virus Type 1 Infected Individuals: Could Punctual Mutations be Related? Viruses 2015;7(11):5643-58. [DOI:10.3390/v7112897] [PMID] []
7. Green MR, Sambrook J. Nested Polymerase Chain Reaction (PCR). Cold Spring Harb Protoc. 2019 Feb 1;2019(2). [DOI:10.1101/pdb.prot095182] [PMID]
8. Caterino-de-Araujo A, Campos KR. Defective particles of human T-lymphotropic virus and negative results in molecular assays. Infect Genet Evol 2021;96:105141. [DOI:10.1016/j.meegid.2021.105141] [PMID]
9. Begum KT, Wnaiza J, Absar N, Az S. In Silico Primer Design for Accurate Detection of SARS-CoV-2 Delta Variants. JBCG 2022;5:104. [DOI:10.17303/jbcg.2022.5.104]
10. Gallego S, Mangano A, Gastaldello R, Sen L, Medeot S. Usefulness of a Nested-polymerase chain reaction for molecular diagnosis of human T-cell lymphotropic virus type I/II. Mem Inst Oswaldo Cruz. 2004 Jun;99(4):377-80. [DOI:10.1590/S0074-02762004000400006] [PMID]
11. Blanco S, Frutos MC, Balangero MC, Gallego SV. Human T-Lymphotropic virus type 1 infection in absence of tax gene: A challenge for molecular diagnosis. Infect Genet Evol 2021;90:104765. [DOI:10.1016/j.meegid.2021.104765] [PMID]
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