Development of a Diagnostic Panel to Measure the Viral Load and the Physical Status of the Human Papilloma Virus16 genome using Multiplex Real-Time PCR Method

Authors

DOI:

https://doi.org/10.22100/jkh.v18i2.3048

Keywords:

ویروس پاپیلومای انسانی تیپ 16، بار ویروسی، واکنش زنجیره‌ای پلیمراز در زمان واقعی، کلونینگ مولکولی.

Abstract

Introduction: Higher viral load in women with chronic Human Papillomavirus type 16 (HPV 16) infection and integration of the viral genome into the cell's chromosomes associates with an increased prevalence of premalignant/malignant lesions in the cervix. The present study describes the development of simultaneous measurement of viral load and physical status for the HPV16 genome by the Multiplex Real-Time PCR method.

Methods: In this study, conserved genes (E6 and E2) of HPV16 were extracted from the CaSki cell line and, after PCR amplification, cloned in bacterial plasmid pJET1.2/blunt cloning vector. In addition, recombinant cellular RnaseP gene plasmid was used as an internal amplification control gene.

Results: Based on our findings, the E2 and E6 genes of the HPV16 virus and the cellular RnaseP gene are identified with high efficiency. Besides, the results of the Real-time PCR showed a high R2 coefficient that reflects the linearity of the standard curve. The results of the E2/E6 ratio of CaSki cells (E2/E6 ratio=0.15) indicated the presence of both integrated and episomal forms in this cell line.

Conclusion: According to the pre-determined acceptance criteria, the diagnostic panel designed to measure the viral load and the physical status of the HPV16 genome using the Multiplex Real-Time PCR method has appropriate sensitivity and specificity.

References

Lee SJ, Yang A, Wu TC, Hung CF. Immunotherapy for human papillomavirus-associated disease and cervical cancer: review of clinical and translational research. J Gynecol Oncol 2016;27:e51. doi: 10.3802/jgo.2016.27.e51

Bergmans HE, van Die IM, Hoekstra WP. Transformation in Escherichia coli: stages in the process. J Bacteriol 1981;146:564-70. doi: 10.1128/jb.146.2.564-570.1981

Lefevre J, Hankins C, Pourreaux K, Voyer H, Coutlée F. Real-time PCR assays using internal controls for quantitation of HPV-16 and β-globin DNA in cervicovaginal lavages. J Virol Methods 2003;114:135-44. doi: 10.1016/j.jviromet.2003.09.003

Ho L, Chan SY, Chow V, Chong T, Tay SK, Villa LL, et al. Sequence variants of human papillomavirus type 16 in clinical samples permit verification and extension of epidemiological studies and construction of a phylogenetic tree. J Clin Microbiol 1991;29:1765-72. doi: 10.1128/jcm.29.9.1765-1772.1991

Yamada T, Manos MM, Peto J, Greer CE, Munoz N, Bosch FX, et al. Human papillomavirus type 16 sequence variation in cervical cancers: a worldwide perspective. J Virol 1997;71:2463-72. doi: 10.1128/jvi.71.3.2463-2472.1997

Chang L, He X, Yu G, Wu Y. Effectiveness of HPV 16 viral load and the E2/E6 ratio for the prediction of cervical cancer risk among Chinese women. J Med Virol 2013;85:646-54. doi: 10.1002/jmv.23490

Lefevre J, Hankins C, Money D, Rachlis A, Pourreaux K, Coutlée F. Human Papillomavirus Type 16 Viral Load Is Higher in Human Immunodeficiency Virus-Seropositive Women with High-Grade Squamous Intraepithelial Lesions Than in Those with Normal Cytology Smears. J Clin Microbiol 2004;42:2212-5. doi: 10.1128/JCM.42.5.2212-2215.2004

Peitsaro P, Johansson B, Syrjänen S. Integrated Human Papillomavirus Type 16 Is Frequently Found in Cervical Cancer Precursors as Demonstrated by a Novel Quantitative Real-Time PCR Technique. J Clin Microbiol 2002;40:886-91. doi: 10.1128/JCM.40.3.886-891.2002

Cricca M, Morselli-Labate AM, Venturoli S, Ambretti S, Gentilomi GA, Gallinella G, et al. Viral DNA load, physical status and E2/E6 ratio as markers to grade HPV16 positive women for high-grade cervical lesions. Gynecologic Oncology 2007;106:5.57-49 doi: 10.1016/j.ygyno.2007.05.004

Saunier M, Monnier-Benoit S, Mauny F, Dalstein V, Briolat J, Riethmuller D, et al. Analysis of Human Papillomavirus Type 16 (HPV16) DNA Load and Physical State for Identification of HPV16-Infected Women with High-Grade Lesions or Cervical Carcinoma. J Clin Microbiol 2008;46:3678-85. doi: 10.1128/JCM.01212-08

Khanal S, Shumway BS, Zahin M, Redman RA, Strickley JD, Trainor PJ, et al. Viral DNA integration and methylation of human papillomavirus type 16 in high-grade oral epithelial dysplasia and head and neck squamous cell carcinoma. Oncotarget 2018;9. doi: 10.18632/oncotarget.25754

Meissner JD. Nucleotide sequences and further characterization of human papillomavirus DNA present in the CaSki, SiHa and HeLa cervical carcinoma cell lines. J Gen Virol 1999;80:1725-33. doi: 10.1099/0022-1317-80-7-1725

Momenimovahed Z, Salehiniya H. Cervical cancer in Iran: integrative insights of epidemiological analysis. Biomedicine (Taipei) 2018;8:18. doi: 10.1051/bmdcn/2018080318

Burd EM. Human Papillomavirus and Cervical Cancer.Clin Microbiol Rev 2003;16:1-17. doi: 10.1128/CMR.16.1.1-17.2003

Yang X, Cheng Y, Li C. The role of TLRs in cervical cancer with HPV infection: a review. Signal Transduct Target Ther 2017;2:17055. doi: 10.1038/sigtrans.2017.55

Szymonowicz KA, Chen J. Biological and clinical aspects of HPV-related cancers. Cancer Biol Med 2020;17:864-78. doi: 10.20892/j.issn.2095-3941.2020.0370

Ye H, Song T, Zeng X, Li L, Hou M, Xi M. Association between genital mycoplasmas infection and human papillomavirus infection, abnormal cervical cytopathology, and cervical cancer: a systematic review and meta-analysis. Arch Gynecol Obstet 2018;297:1377-87. doi: 10.1007/s00404-018-4733-5

Liu C, Mann D, Sinha UK, Kokot NC. The molecular mechanisms of increased radiosensitivity of HPV-positive oropharyngeal squamous cell carcinoma (OPSCC): an extensive review. J Otolaryngol 2018;47:59. doi: 10.1186/s40463-018-0302-y

Peitsaro P, Johansson B, Syrjänen SJJocm. Integrated human papillomavirus type 16 is frequently found in cervical cancer precursors as demonstrated by a novel quantitative real-time PCR technique. J Clin Microbiol 2002;40:886-91. doi: 10.1128/JCM.40.3.886-891.2002

Cricca M M-LA, Venturoli S, Ambretti S, Gentilomi GA, Gallinella G, et al. Viral DNA load, physical status and E2/E6 ratioas markers to grade HPV16 positive women for high-grade cervical lesions. Gynecol Oncol 2007;106:549-57. doi: 10.1016/j.ygyno.2007.05.004

Oyervides-Muñoz MA, Pérez-Maya AA, Rodríguez-Gutiérrez HF, Gómez-Macias GS, Fajardo-Ramírez OR, Treviño V, et al. Understanding the HPV integration and its progression to cervical cancer. Infect Genet Evol 2018;61:134-44. doi: 10.1016/j.meegid.2018.03.003

Williams VM, Filippova M, Soto U, Duerksen-Hughes PJ. HPV-DNA integration and carcinogenesis: putative roles for inflammation and oxidative stress. Future Virol 2011;6:45-57. doi: 10.2217/fvl.10.73

Álvarez-Paredes L, Santibañez M, Galiana A, Rodríguez Díaz JC, Parás-Bravo P, Andrada-Becerra ME, et al. Association of Human Papillomavirus Genotype 16 Viral Variant and Viral Load with Cervical High-grade Intraepithelial Lesions. Cancer Prev Res (Phila) 2019;12:547-56. doi: 10.1158/1940-6207.CAPR-18-0397

Tran SL, Puhar A, Ngo-Camus M, Ramarao N. Trypan blue dye enters viable cells incubated with the pore-forming toxin HlyII of Bacillus cereus. PLoS One 2011;6:e22876. doi: 10.1371/journal.pone.0022876

Lorenz TC. Polymerase chain reaction: basic protocol plus troubleshooting and optimization strategies. J Vis Exp 2012:e3998. doi: 10.3791/3998

Downloads

Additional Files

Published

2023-11-26

Issue

Vol 18, No 2:2023

Section

Original Article(s)

License

Creative Commons License

This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.

How to Cite

Development of a Diagnostic Panel to Measure the Viral Load and the Physical Status of the Human Papilloma Virus16 genome using Multiplex Real-Time PCR Method. (2023). Knowledge and Health in Basic Medical Sciences, 18(2), Page:9-19. https://doi.org/10.22100/jkh.v18i2.3048

Most read articles by the same author(s)

<< < 59 60 61 62 63 64 65 66 67 > >>