Volume 21, Issue 1 (Paramedical Sciences and Military Health- Spring- 2026)
Paramedical Sciences and Military Health 2026, 21(1): 14-21 |
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Bagheri A, Dini Talatappeh H, Hosseini Rouzbahani N, Ghorban K, Gholami M, Dadmanesh M et al . Development and Preliminary Validation of a Universal One-Step RT-PCR Assay Targeting the 5′-UTR for Pan-Enterovirus Detection. Paramedical Sciences and Military Health 2026; 21 (1) :14-21
URL: http://jps.ajaums.ac.ir/article-1-525-en.html
URL: http://jps.ajaums.ac.ir/article-1-525-en.html
Ali Bagheri1 
, Hossein Dini Talatappeh2 , Negin Hosseini Rouzbahani2 , Khodayar Ghorban3 , Mohammad Gholami *4 , Maryam Dadmanesh3 , Mahtab Nourifard3
, Hossein Dini Talatappeh2 , Negin Hosseini Rouzbahani2 , Khodayar Ghorban3 , Mohammad Gholami *4 , Maryam Dadmanesh3 , Mahtab Nourifard3
1- Golestan Hospital, Aja University of Medical Sciences, Tehran, Iran.
2- Infectious Diseases Research Center, AJA University of Medical Sciences, Tehran, Iran
3- Infectious Diseases Research Center, AJA University of Medical Sciences, Tehran, Iran.
4- Infectious Diseases Research Center, AJA University of Medical Sciences, Tehran, Iran. ,Mohammadg19@gmail.com
2- Infectious Diseases Research Center, AJA University of Medical Sciences, Tehran, Iran
3- Infectious Diseases Research Center, AJA University of Medical Sciences, Tehran, Iran.
4- Infectious Diseases Research Center, AJA University of Medical Sciences, Tehran, Iran. ,
Abstract: (19 Views)
Introduction: Human enteroviruses (EVs) are a major cause of aseptic meningitis, encephalitis, neonatal sepsis-like illness, and hand-foot-mouth disease that more than 100 of their serotypes classified into 15 species within the Picornaviridae family. Rapid and sensitive detection is critical for clinical management and outbreak control; however, reliance on imported diagnostic kits remains a significant challenge in resource-limited settings such as Iran. This study aimed to design, optimize, and preliminarily validate a universal one-step reverse transcription polymerase chain reaction (RT-PCR) assay targeting the highly conserved 5′-untranslated region (5′-UTR) of the EV genome for broad-spectrum detection of all human EV species.
Materials and Methods: A total of 120 full-length EV genomic sequences (including Enterovirus A–D and representative rhinoviruses) were retrieved from the NCBI GenBank database. Multiple sequence alignment was performed using MEGA X software to identify conserved regions. Universal primers were designed using Oligo7 software and validated in silico via Primer-BLAST against the NCBI nucleotide database. Primers were synthesized by Pishgaman Company. (Tehran, Iran). The assay was optimized using a commercial one-step RT-PCR kit (Biorabite, Iran) and it validated using RNA extracted from oral poliovirus vaccine (Sabin strain) as a positive control. Specificity was assessed by agarose gel electrophoresis and in silico cross-reactivity screening.
Results: The designed primer pair amplified a 110-bp fragment within the 5′-UTR. In silico analysis confirmed 100% coverage for all major EV types without any cross-reactivity to human genomic DNA or unrelated pathogens. Experimental validation yielded a single, sharp band of the expected size on 2% agarose gel using poliovirus RNA; while, there was not any amplification in negative controls.
Conclusion: We report a robust, cost-effective, and indigenous universal RT-PCR assay that is able to detect the diverse human enteroviruses. This platform provides the foundation, domestically produced diagnostic kit which is suitable for clinical and public health applications in Iran and similar resource-limited settings.
Materials and Methods: A total of 120 full-length EV genomic sequences (including Enterovirus A–D and representative rhinoviruses) were retrieved from the NCBI GenBank database. Multiple sequence alignment was performed using MEGA X software to identify conserved regions. Universal primers were designed using Oligo7 software and validated in silico via Primer-BLAST against the NCBI nucleotide database. Primers were synthesized by Pishgaman Company. (Tehran, Iran). The assay was optimized using a commercial one-step RT-PCR kit (Biorabite, Iran) and it validated using RNA extracted from oral poliovirus vaccine (Sabin strain) as a positive control. Specificity was assessed by agarose gel electrophoresis and in silico cross-reactivity screening.
Results: The designed primer pair amplified a 110-bp fragment within the 5′-UTR. In silico analysis confirmed 100% coverage for all major EV types without any cross-reactivity to human genomic DNA or unrelated pathogens. Experimental validation yielded a single, sharp band of the expected size on 2% agarose gel using poliovirus RNA; while, there was not any amplification in negative controls.
Conclusion: We report a robust, cost-effective, and indigenous universal RT-PCR assay that is able to detect the diverse human enteroviruses. This platform provides the foundation, domestically produced diagnostic kit which is suitable for clinical and public health applications in Iran and similar resource-limited settings.
Type of Study: Research |
Subject:
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Received: 2026/05/9 | Accepted: 2026/08/3 | Published: 2026/03/10
Received: 2026/05/9 | Accepted: 2026/08/3 | Published: 2026/03/10
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