Design and comparison of PCR-ELISA reaction with other available hybridization methods to identify types 11, 16, and 18 of the human papillomavirus
-
Seyed Mohammad Amin Mousavi-Rad
,
Shohreh Zare Karizi
,
Hamid Sedighian
,
Seyed Ali Mirhosseini
,
Hadi Esmaeili Gouvarchin ghaleh
,
Jafar Amani
Abstract
Background and Objectives: Human papillomavirus (HPV) is a significant etiological agent in cervical cancer. This study aimed to evaluate the performance of PCR-ELISA for detecting HPV genotypes 11, 16, and 18 compared to the conventional hybridization methods.
Materials and Methods: PCR-ELISA was designed and optimized to detect target HPV genotypes using biotin-labeled probes. Sensitivity, specificity and reproducibility were assessed through intra-assay and inter-assay variability tests. Additionally, a cost-benefit analysis was performed to compare PCR-ELISA with RT-PCR and gel electrophoresis.
Results: PCR-ELISA demonstrated high sensitivity (HPV18: 94.92%, HPV16: 98.36%, HPV11: 93.75%) and specificity (100% for all genotypes), with Kappa values ranging from 0.84 to 0.92, indicating strong agreement with the reference standard. Reproducibility analysis showed intra-assay CVs below 5% for most samples and inter-assay CVs within acceptable limits. The cost-benefit analysis revealed significant reductions in reagent and equipment costs compared to RT-PCR, making PCR-ELISA a cost-effective alternative.
Conclusion: PCR-ELISA offers a reliable, sensitive and cost-effective method for HPV detection, particularly in resource-limited settings. Its simplicity and compatibility with existing workflows makes it a promising tool for routine diagnostic applications.
1. McBride AA. Human papillomaviruses: Diversity, infection and host interactions. Nat Rev Microbiol 2022; 20: 95-108.
2. Smith HB (2013). Handbook on human papillomavirus: prevalence, detection and management (Virology Research Progress: Public Health in the 21st Century). Nova Science Pub Inc; 1st edition. https://www.amazon.com/Handbook-Human-Papillomavirus-Prevalence-Management/dp/1626186723
3. Santos-López G, Márquez-Domínguez L, Reyes-Leyva J, Vallejo-Ruiz V. General aspects of structure, classification and replication of human papillomavirus. Rev Med Inst Mex Seguro Soc 2015; 53 Suppl 2: S166-S171.
4. Kashefi F, Alizadeh M, Kalaki NS, Ejlalidiz M, Yazdani S, Shayestehpour M, et al. Molecular Epidemiology of human papilloma virus (HPV) in women’s Cervical samples from Tehran, Iran. Asian Pac J Cancer Biol 2024; 9: 169-174.
5. Galati L, Chiantore MV, Marinaro M, Di Bonito P. Human Oncogenic viruses: Characteristics and prevention Strategies-Lessons learned from human Papillomaviruses. Viruses 2024; 16: 416.
6. Sabatini ME, Chiocca S. Human papillomavirus as a driver of head and neck cancers. Br J Cancer 2020; 122: 306-314.
7. Singh S, Ahmad S, Srivastava AN, Misra JS. A review on role of human Papilloma virus (HPV) in Health-Related diseases. Adv Med Dent Health Sci 2020; 3: 34-40.
8. Niya MHK, Tameshkel FS, Motamed N, Miri SM, Mortazavi H, Ajdarkosh H, et al. Detection of human Papilloma virus (HPV) infection in Colorectal cancer and viral Genome Integration Status. Shiraz E Med J 2022; 23(10): e118289.
9. Qureshi QUA, Qureshi NUAZ, Qureshi PAAA, Abbasi N. Human papilloma virus status among women presenting with vaginal discharge at a tertiary care hospital. J Soc Obstet Gynaecol Pak 2021; 11: 87-90.
10. Hahn A, Podbielski A, Meyer T, Zautner AE, Loderstädt U, Schwarz NG, et al. On detection thresholds–a review on diagnostic approaches in the infectious disease laboratory and the interpretation of their results. Acta Trop 2020; 205: 105377.
11. Rotondo JC, Oton-Gonzalez L, Mazziotta C, Lanzillotti C, Iaquinta MR, Tognon M, et al. Simultaneous detection and viral DNA load quantification of different human papillomavirus types in clinical specimens by the high analytical droplet digital PCR method. Front Microbiol 2020; 11: 591452.
12. Mühr LSA, Guerendiain D, Cuschieri K, Sundström K. Human Papillomavirus detection by Whole-Genome Next-Generation sequencing: Importance of validation and quality assurance procedures. Viruses 2021; 13: 1323.
13. Molijn A, Kleter B, Quint W, van Doorn L-J. Molecular diagnosis of human papillomavirus (HPV) infections. J Clin Virol 2005; 32 Suppl 1: S43-S51.
14. Kingma DW, Allen RA, Caughron SK, Melby M, Moore WE, Gillies EM, et al. Comparison of molecular methods for detection of HPV in oral and oropharyngeal squamous cell carcinoma. Diagn Mol Pathol 2010; 19: 218-223.
15. Borkakoty B, Jakharia A, Singh P, Khan SA (2024). Trends of Diagnostic Methods for Human Viral Diseases. Viral Infectious Diseases Annual Volum 2024. https://www.intechopen.com/chapters/89145
16. Naegele S, Ruiz-Torres DA, Zhao Y, Goss D, Faden DL. Comparing the diagnostic performance of quantitative PCR, digital droplet PCR, and next-generation sequencing liquid biopsies for human Papillomavirus–associated cancers. J Mol Diagn 2024; 26: 179-190.
17. Iftner T, Eberle S, Iftner A, Holz B, Banik N, Quint W, et al. Prevalence of low‐risk and high‐risk types of human papillomavirus and other risk factors for HPV infection in Germany within different age groups in women up to 30 years of age: an epidemiological observational study. J Med Virol 2010; 82: 1928-1939.
18. Boccardo E. New approaches for infective HPV detection, quantification and inactivation: Preventing accidental virus transmission in medical settings. EBioMedicine 2021; 64: 103222.
19. McBride AA. Human papillomaviruses: Diversity, infection and host interactions. Nat Rev Microbiol 2022; 20: 95-108.
20. Sinal SH, Woods CR. Human papillomavirus infections of the genital and respiratory tracts in young children. Semin Pediatr Infect Dis 2005; 16: 306-316.
21. Bagul KA, Khandelwal N, Naphade ME, Panchbudhe SA, Mishra V, Tripathy S, et al. Detection of human Papillomavirus among cervical cancer patients by qualitative polymerase chain reaction on formalin-fixed, paraffin-embedded tissue: A retrospective observational analysis. Nat J Lab Med 2023; 12: PO10-PO13.
22. Bharadwaj M, Hussain S, Tripathi R, Singh N, Mehrotra R (2020). Chapter 6: The clinico-molecular approaches for detection of human papillomavirus. Animal Biotechnology (Scond Edition): Elsevier. p. 103-130. https://www.sciencedirect.com/science/article/abs/pii/B9780128117101000069
23. Comar M, De Seta F, Zanotta N, Del Bue S, Ferrante P (2020). New Diagnostic Approaches to viral Sexually transmitted infections. Sexually Transmitted Infections: Springer. p. 107-148.
24. Mousavi T, Rafiei A, Reza Haghshenas M, Sadeghian-Kiadehi SF, Valadan R. Molecular prevalence and phylogenetic analysis of human papillomavirus in normal cervical samples from northern Iran. Gene Rep 2020; 21: 100958.
25. Sharmin S, Zohura FT, Islam MS, Shimonty A, Khan MA, Parveen R, et al. Mutational profiles of marker genes of cervical carcinoma in Bangladeshi patients. BMC Cancer 2021; 21: 289.
26. Daly P, Doyle S. The development of a competitive PCR–ELISA for the detection of equine herpesvirus-1. J Virol Methods 2003; 107: 237-244.
27. Pereira LQ, Ferreira-Silva MM, Ratkevicius CMA, Gómez-Hérnandez C, De Vito FB, Tanaka SCSV, et al. Identification of Leishmania infantum in blood donors from endemic regions for visceral leishmaniasis. Parasitology 2021; 148: 110-114.
28. Jangpatarapongsa K, Saimuang K, Polpanich D, Thiramanas R, Techakasikornpanich M, Yudech P, et al. Increased sensitivity of enterotoxigenic Escherichia coli detection in stool samples using oligonucleotide immobilized-magnetic nanoparticles. Biotechnol Rep (Amst) 2021; 32: e00677.
29. Khan A, Singh R, Sharma S, Singh V, Sheoran A, Soni A, et al. Diagnosis of osteoarticular tuberculosis by immuno‐PCR assay based on mycobacterial antigen 85 complex detection. Lett Appl Microbiol 2022; 74: 17-26.
30. Zerbini M, Venturoli S, Cricca M, Gallinella G, De Simone P, Costa S, et al. Distribution and viral load of type specific HPVs in different cervical lesions as detected by PCR-ELISA. J Clin Pathol 2001; 54: 377-380.
31. Bartosik M, Moranova L, Izadi N, Strmiskova J, Sebuyoya R, Holcakova J, et al. Advanced technologies towards improved HPV diagnostics. J Med Virol 2024; 96(2): e29409.
32. Musiani M, Venturoli S, Gallinella G, Zerbini M. Qualitative PCR–ELISA protocol for the detection and typing of viral genomes. Nat Protoc 2007; 2: 2502-2510.
2. Smith HB (2013). Handbook on human papillomavirus: prevalence, detection and management (Virology Research Progress: Public Health in the 21st Century). Nova Science Pub Inc; 1st edition. https://www.amazon.com/Handbook-Human-Papillomavirus-Prevalence-Management/dp/1626186723
3. Santos-López G, Márquez-Domínguez L, Reyes-Leyva J, Vallejo-Ruiz V. General aspects of structure, classification and replication of human papillomavirus. Rev Med Inst Mex Seguro Soc 2015; 53 Suppl 2: S166-S171.
4. Kashefi F, Alizadeh M, Kalaki NS, Ejlalidiz M, Yazdani S, Shayestehpour M, et al. Molecular Epidemiology of human papilloma virus (HPV) in women’s Cervical samples from Tehran, Iran. Asian Pac J Cancer Biol 2024; 9: 169-174.
5. Galati L, Chiantore MV, Marinaro M, Di Bonito P. Human Oncogenic viruses: Characteristics and prevention Strategies-Lessons learned from human Papillomaviruses. Viruses 2024; 16: 416.
6. Sabatini ME, Chiocca S. Human papillomavirus as a driver of head and neck cancers. Br J Cancer 2020; 122: 306-314.
7. Singh S, Ahmad S, Srivastava AN, Misra JS. A review on role of human Papilloma virus (HPV) in Health-Related diseases. Adv Med Dent Health Sci 2020; 3: 34-40.
8. Niya MHK, Tameshkel FS, Motamed N, Miri SM, Mortazavi H, Ajdarkosh H, et al. Detection of human Papilloma virus (HPV) infection in Colorectal cancer and viral Genome Integration Status. Shiraz E Med J 2022; 23(10): e118289.
9. Qureshi QUA, Qureshi NUAZ, Qureshi PAAA, Abbasi N. Human papilloma virus status among women presenting with vaginal discharge at a tertiary care hospital. J Soc Obstet Gynaecol Pak 2021; 11: 87-90.
10. Hahn A, Podbielski A, Meyer T, Zautner AE, Loderstädt U, Schwarz NG, et al. On detection thresholds–a review on diagnostic approaches in the infectious disease laboratory and the interpretation of their results. Acta Trop 2020; 205: 105377.
11. Rotondo JC, Oton-Gonzalez L, Mazziotta C, Lanzillotti C, Iaquinta MR, Tognon M, et al. Simultaneous detection and viral DNA load quantification of different human papillomavirus types in clinical specimens by the high analytical droplet digital PCR method. Front Microbiol 2020; 11: 591452.
12. Mühr LSA, Guerendiain D, Cuschieri K, Sundström K. Human Papillomavirus detection by Whole-Genome Next-Generation sequencing: Importance of validation and quality assurance procedures. Viruses 2021; 13: 1323.
13. Molijn A, Kleter B, Quint W, van Doorn L-J. Molecular diagnosis of human papillomavirus (HPV) infections. J Clin Virol 2005; 32 Suppl 1: S43-S51.
14. Kingma DW, Allen RA, Caughron SK, Melby M, Moore WE, Gillies EM, et al. Comparison of molecular methods for detection of HPV in oral and oropharyngeal squamous cell carcinoma. Diagn Mol Pathol 2010; 19: 218-223.
15. Borkakoty B, Jakharia A, Singh P, Khan SA (2024). Trends of Diagnostic Methods for Human Viral Diseases. Viral Infectious Diseases Annual Volum 2024. https://www.intechopen.com/chapters/89145
16. Naegele S, Ruiz-Torres DA, Zhao Y, Goss D, Faden DL. Comparing the diagnostic performance of quantitative PCR, digital droplet PCR, and next-generation sequencing liquid biopsies for human Papillomavirus–associated cancers. J Mol Diagn 2024; 26: 179-190.
17. Iftner T, Eberle S, Iftner A, Holz B, Banik N, Quint W, et al. Prevalence of low‐risk and high‐risk types of human papillomavirus and other risk factors for HPV infection in Germany within different age groups in women up to 30 years of age: an epidemiological observational study. J Med Virol 2010; 82: 1928-1939.
18. Boccardo E. New approaches for infective HPV detection, quantification and inactivation: Preventing accidental virus transmission in medical settings. EBioMedicine 2021; 64: 103222.
19. McBride AA. Human papillomaviruses: Diversity, infection and host interactions. Nat Rev Microbiol 2022; 20: 95-108.
20. Sinal SH, Woods CR. Human papillomavirus infections of the genital and respiratory tracts in young children. Semin Pediatr Infect Dis 2005; 16: 306-316.
21. Bagul KA, Khandelwal N, Naphade ME, Panchbudhe SA, Mishra V, Tripathy S, et al. Detection of human Papillomavirus among cervical cancer patients by qualitative polymerase chain reaction on formalin-fixed, paraffin-embedded tissue: A retrospective observational analysis. Nat J Lab Med 2023; 12: PO10-PO13.
22. Bharadwaj M, Hussain S, Tripathi R, Singh N, Mehrotra R (2020). Chapter 6: The clinico-molecular approaches for detection of human papillomavirus. Animal Biotechnology (Scond Edition): Elsevier. p. 103-130. https://www.sciencedirect.com/science/article/abs/pii/B9780128117101000069
23. Comar M, De Seta F, Zanotta N, Del Bue S, Ferrante P (2020). New Diagnostic Approaches to viral Sexually transmitted infections. Sexually Transmitted Infections: Springer. p. 107-148.
24. Mousavi T, Rafiei A, Reza Haghshenas M, Sadeghian-Kiadehi SF, Valadan R. Molecular prevalence and phylogenetic analysis of human papillomavirus in normal cervical samples from northern Iran. Gene Rep 2020; 21: 100958.
25. Sharmin S, Zohura FT, Islam MS, Shimonty A, Khan MA, Parveen R, et al. Mutational profiles of marker genes of cervical carcinoma in Bangladeshi patients. BMC Cancer 2021; 21: 289.
26. Daly P, Doyle S. The development of a competitive PCR–ELISA for the detection of equine herpesvirus-1. J Virol Methods 2003; 107: 237-244.
27. Pereira LQ, Ferreira-Silva MM, Ratkevicius CMA, Gómez-Hérnandez C, De Vito FB, Tanaka SCSV, et al. Identification of Leishmania infantum in blood donors from endemic regions for visceral leishmaniasis. Parasitology 2021; 148: 110-114.
28. Jangpatarapongsa K, Saimuang K, Polpanich D, Thiramanas R, Techakasikornpanich M, Yudech P, et al. Increased sensitivity of enterotoxigenic Escherichia coli detection in stool samples using oligonucleotide immobilized-magnetic nanoparticles. Biotechnol Rep (Amst) 2021; 32: e00677.
29. Khan A, Singh R, Sharma S, Singh V, Sheoran A, Soni A, et al. Diagnosis of osteoarticular tuberculosis by immuno‐PCR assay based on mycobacterial antigen 85 complex detection. Lett Appl Microbiol 2022; 74: 17-26.
30. Zerbini M, Venturoli S, Cricca M, Gallinella G, De Simone P, Costa S, et al. Distribution and viral load of type specific HPVs in different cervical lesions as detected by PCR-ELISA. J Clin Pathol 2001; 54: 377-380.
31. Bartosik M, Moranova L, Izadi N, Strmiskova J, Sebuyoya R, Holcakova J, et al. Advanced technologies towards improved HPV diagnostics. J Med Virol 2024; 96(2): e29409.
32. Musiani M, Venturoli S, Gallinella G, Zerbini M. Qualitative PCR–ELISA protocol for the detection and typing of viral genomes. Nat Protoc 2007; 2: 2502-2510.
| Files | ||
| Issue | Vol 17 No 3 (2025) | |
| Section | Original Article(s) | |
| DOI | https://doi.org/10.18502/ijm.v17i3.18832 | |
| Keywords | ||
| Human papillomavirus Polymerase chain reaction-Enzyme-linked immunosorbent assay Molecular diagnostics Hybridization techniques HPV DNA detection | ||
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How to Cite
1.
Mousavi-Rad SMA, Zare Karizi S, Sedighian H, Mirhosseini SA, Esmaeili Gouvarchin ghaleh H, Amani J. Design and comparison of PCR-ELISA reaction with other available hybridization methods to identify types 11, 16, and 18 of the human papillomavirus. Iran J Microbiol. 2025;17(3):488-502.
