Volume 16, Issue 62 (5-2026)
NCMBJ 2026, 16(62): 36-48 |
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Rahmani N, Norouzian D, Tabatabaee Bafroee A S. Comparison of the Effects of Different Master Mixes on the Efficiency of Real-time PCR Technique in Diagnosing Human Papillomavirus (HPV). NCMBJ 2026; 16 (62) :36-48
URL: http://ncmbjpiau.ir/article-1-1818-en.html
URL: http://ncmbjpiau.ir/article-1-1818-en.html
Nasim Institute, Genomics Pole Laboratory, Health Technology Park, Shahid Beheshti University of Medical Sciences
Abstract: (29 Views)
Background and Aim: Awareness of human papillomavirus (HPV) genotypes is crucial for the management and prevention of dysplasia and cervical cancer. HPV comprises over 200 genotypes, approximately 40 of which infect the genital tract. The global prevalence of HPV varies between 1.2% and 25.6% across different regions.
Materials and Methods: In this descriptive-experimental study, 58 clinical samples were collected from women presenting with vaginitis symptoms at Milad Hospital, Tehran. The presence of the HPV-6 genotype was detected using Real-time PCR with specific primers. To optimize the assay, four different master mix formulations containing varying concentrations of Tris-HCl, KCl, and MgCl₂ were designed and compared for their performance in HPV-6 detection.
Results: Eight (13.79%) of the 58 samples tested positive for HPV-6. The master mix containing 10 mM Tris-HCl, 50 mM KCl, and 2 mM MgCl2 demonstrated the best sensitivity and specificity, yielding lower Ct values. Melting curve analysis confirmed the specificity of the PCR products and ruled out primer-dimer and non-specific amplification.
Conclusion: Optimization of master mix components in Real-time PCR can enhance the sensitivity and accuracy of HPV-6 detection, providing a more efficient and cost-effective method for genotype identification. This approach may facilitate the development of improved diagnostic kits and increase patient participation in HPV screening programs, ultimately contributing to better prevention of cervical cancer.
Materials and Methods: In this descriptive-experimental study, 58 clinical samples were collected from women presenting with vaginitis symptoms at Milad Hospital, Tehran. The presence of the HPV-6 genotype was detected using Real-time PCR with specific primers. To optimize the assay, four different master mix formulations containing varying concentrations of Tris-HCl, KCl, and MgCl₂ were designed and compared for their performance in HPV-6 detection.
Results: Eight (13.79%) of the 58 samples tested positive for HPV-6. The master mix containing 10 mM Tris-HCl, 50 mM KCl, and 2 mM MgCl2 demonstrated the best sensitivity and specificity, yielding lower Ct values. Melting curve analysis confirmed the specificity of the PCR products and ruled out primer-dimer and non-specific amplification.
Conclusion: Optimization of master mix components in Real-time PCR can enhance the sensitivity and accuracy of HPV-6 detection, providing a more efficient and cost-effective method for genotype identification. This approach may facilitate the development of improved diagnostic kits and increase patient participation in HPV screening programs, ultimately contributing to better prevention of cervical cancer.
Keywords: Human Papillomavirus (HPV), Real-time PCR, Master Mix, PCR, Molecular Diagnostic, Optimization
Type of Study: Research Article |
Subject:
Genetics
Received: 2026/06/16 | Accepted: 2026/05/31 | Published: 2026/05/31
Received: 2026/06/16 | Accepted: 2026/05/31 | Published: 2026/05/31
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