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Sci J Iran Blood Transfus Organ 2025, 22(1): 19-29 |
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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. Sci J Iran Blood Transfus Organ 2025; 22 (1) :19-29
URL: http://bloodjournal.ir/article-1-1567-en.html
URL: http://bloodjournal.ir/article-1-1567-en.html
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| Abstract (HTML) (531 Views)
Design and In-Silico Evaluation of Specific Primers
for the HBZ Gene of HTLV-1 to Establish a semi-nested
PCR Method
Zandi M.1, Sharifi Z.1, Ajorloo M.1
1Biological Products and Blood Safety Resaerch Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran
Abstract
Background and Objectives
HTLV-1 is the only known oncogenic human retrovirus, linked to ATL and HAM/TSP. Serological methods like ELISA and Western blot have sensitivity limitations, whereas PCR-based techniques offer more reliable detection. The HBZ gene, consistently expressed in ATL and asymptomatic carriers, correlates with proviral load. This study aimed to design specific primers targeting HBZ for a highly sensitive semi-nested PCR assay.
Materials and Methods
In a Cross-Sectional study, the HBZ gene sequence was identified using the NCBI GenBank database. Sequences from various viral isolates were aligned using MEGA6 bioinformatics software. Primers were designed from a conserved region shared by all HTLV-1 strains. Primer design and evaluation were conducted using Primer-BLAST, Primer3, OLIGO Analyze, SnapGene, and Gene Runner software. For the experimental phase, genomic DNA extracted from blood samples underwent a two-step semi-nested PCR. The PCR products were then analyzed using agarose gel electrophoresis.
Results
The primers designed for the HBZ gene successfully enabled the development of a semi-nested PCR assay. In silico predictions of primer performance were validated after synthesis and laboratory testing, with results aligning perfectly with the bioinformatics predictions.
Conclusions
This study demonstrates that the primers designed for the HBZ gene, combined with the semi-nested PCR method, provide an effective means for detecting the HTLV-1 provirus. Both the bioinformatics and laboratory results confirmed the specificity and sensitivity of the method, underscoring its potential for rapid and accurate HTLV-1 detection.
Key words: HTLV-1, Nested PCR, Leukemia
Received: 8 Jan 2025
Accepted: 9 Mar 2025
Correspondence: Sharifi Z., PhD of Virology. 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; Fax: (+9821) 88601555
E-mail: sharifiz@yahoo.com
References:
Full-Text: (138 Views)
|
Sci J Iran Blood Transfus Organ 2025; 22 (1): 20-30 |
|
Original Article
|
for the HBZ Gene of HTLV-1 to Establish a semi-nested
PCR Method
Zandi M.1, Sharifi Z.1, Ajorloo M.1
1Biological Products and Blood Safety Resaerch Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran
Abstract
Background and Objectives
HTLV-1 is the only known oncogenic human retrovirus, linked to ATL and HAM/TSP. Serological methods like ELISA and Western blot have sensitivity limitations, whereas PCR-based techniques offer more reliable detection. The HBZ gene, consistently expressed in ATL and asymptomatic carriers, correlates with proviral load. This study aimed to design specific primers targeting HBZ for a highly sensitive semi-nested PCR assay.
Materials and Methods
In a Cross-Sectional study, the HBZ gene sequence was identified using the NCBI GenBank database. Sequences from various viral isolates were aligned using MEGA6 bioinformatics software. Primers were designed from a conserved region shared by all HTLV-1 strains. Primer design and evaluation were conducted using Primer-BLAST, Primer3, OLIGO Analyze, SnapGene, and Gene Runner software. For the experimental phase, genomic DNA extracted from blood samples underwent a two-step semi-nested PCR. The PCR products were then analyzed using agarose gel electrophoresis.
Results
The primers designed for the HBZ gene successfully enabled the development of a semi-nested PCR assay. In silico predictions of primer performance were validated after synthesis and laboratory testing, with results aligning perfectly with the bioinformatics predictions.
Conclusions
This study demonstrates that the primers designed for the HBZ gene, combined with the semi-nested PCR method, provide an effective means for detecting the HTLV-1 provirus. Both the bioinformatics and laboratory results confirmed the specificity and sensitivity of the method, underscoring its potential for rapid and accurate HTLV-1 detection.
Key words: HTLV-1, Nested PCR, Leukemia
Received: 8 Jan 2025
Accepted: 9 Mar 2025
Correspondence: Sharifi Z., PhD of Virology. 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; Fax: (+9821) 88601555
E-mail: sharifiz@yahoo.com
References:
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- Green MR, Sambrook J. Nested Polymerase Chain Reaction (PCR). Cold Spring Harb Protoc. 2019 Feb 1;2019(2).
- 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.
- 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.
- 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.
- 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.
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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