Korean Journal of Clinical Laboratory Science (대한임상검사과학회지)

Korean Society for Clinical Laboratory Science (대한임상검사과학회)
권호 표지
DOI QR코드

DOI QR Code

Evaluation of Commercial Complementary DNA Synthesis Kits for Detecting Human Papillomavirus

인유두종바이러스 검출을 위한 상용화된 cDNA 합성 키트의 평가

  • Yu, Kwangmin (Department of Biomedical Laboratory Science, College of Health Sciences, Yonsei University) ;
  • Park, Sunyoung (Department of Mechanical Engineering, Yonsei University) ;
  • Chang, Yunhee (Department of Biomedical Laboratory Science, College of Health Sciences, Yonsei University) ;
  • Hwang, Dasom (Department of Biomedical Laboratory Science, College of Health Sciences, Yonsei University) ;
  • Kim, Geehyuk (Ministry of Food and Drug Safety Pharmaceutical Safety Bureau, Osong Health Technology Administration Complex) ;
  • Kim, Jungho (Clinical Vaccine Research Section, International Tuberculosis Research Center) ;
  • Kim, Sunghyun (Department of Clinical Laboratory Science, College of Health Sciences, Catholic University of Pusan) ;
  • Kim, Eun-Joong (Department of Clinical Laboratory Science, Chungbuk Health and Science University) ;
  • Lee, Dongsup (Department of Clinical Laboratory Science, Hyejeon College)
  • 유광민 (연세대학교 임상병리학과) ;
  • 박선영 (연세대학교 기계공학과) ;
  • 장연희 (연세대학교 임상병리학과) ;
  • 황다솜 (연세대학교 임상병리학과) ;
  • 김지혁 (식품의약품안전처 의약품안전국) ;
  • 김정호 (국제결핵연구소 백신연구부) ;
  • 김성현 (부산카톨릭대학교 임상병리학과) ;
  • 김은중 (충북보건과학대학교 임상병리과) ;
  • 이동섭 (혜전대학교 임상병리과)
  • Received : 2019.07.01
  • Accepted : 2019.08.25
  • Published : 2019.09.30
Download PDF
⟨ Previous Next ⟩

Abstract

Cervical cancer is the fourth most common malignant neoplasm in women worldwide. Most cases of cervical cancer are caused by an infection by the human papillomavirus. Molecular diagnostic methods have emerged to detect the HPV for sensitivity, specificity, and objectivity. In particular, real-time PCR has been introduced to acquire a more sensitive target DNA or RNA. RNA extraction and complementary DNA synthesis are proceeded before performing real-time PCR targeting RNA. To identify an adequate and sensitive cDNA synthesis kit, this study evaluated the two commonly used kits for cDNA synthesis. The results show that the $R^2$ and efficiency (%) of the two cDNA synthesis kits were similar in the cervical cancer cell lines. On the other hand, the Takara kit compared to Invitrogen kit showed P<0.001 in the $10^2$ and $10^3$ SiHa cell count. The Takara kit compared to the Invitrogen kit showed P<0.001 in the $10^1$ and $10^2$ HeLa cell count. Furthermore, 8, 4, 2, 1, and 0.5 ml of forty exfoliated cell samples were used to compare the cDNA synthesis kits. The Takara kit compared to the Invitrogen kit showed P<0.01 in 8, 4, and 1 ml and P<0.05 in 0.5 mL. The study was performed to identify the most appropriate cDNA synthesis kit and suggests that a cDNA synthesis kit could affect the real-time PCR results.

자궁경부암은 전 세계적으로 네번째를 차지하는 여성암이다. 자궁경부암의 대부분 원인은 인유두종 바이러스의 감염이다. 인유두종 바이러스를 검출하기 위해 다양한 분자진단학적 방법들이 고안되었다. 분자진단학적 방법 중의 real-time PCR은 목표 DNA 또는 RNA의 정량과 민감도 향상을 목표로 도입되었다. 특히, real-time PCR 과정은 수행 전에 RNA 추출 및 상보적인 DNA 합성 과정이 필요하다. 따라서 본 연구에서는 민감하고 적합한 상보적인 DNA 합성 키트를 알아보기 위해서 상보적인 DNA 합성에 이용되는 두 개의 상용화된 키트를 평가하였다. 자궁경부암 세포주에서 두개의 상보적인 DNA 합성 키트의 $R^2$과 효율성을 비교한 결과 차이가 없었다. 그러나 Invitrogen 키트보다 Takara 키트가 $10^2$ 및 $10^3$ SiHa 세포주에서 P<0.001를 나타내었고 $10^1$ 및 $10^2$ HeLa 세포주에서도 P<0.001를 나타내었다. 이를 통해 Takara 키트가 Invitrogen키트보다 민감도가 높음을 알 수 있었다. 또한 40개의 탈락세포검체의 8, 4, 2, 1 mL을 이용하여 상보적인 DNA 합성 키트를 비교한 결과 Invitrogen 키트보다 Takara 키트가 8, 4, 1 mL에서 P<0.01 및 0.5 mL에서 P<0.05을 나타내어 임상 검체를 이용하였을 때에도 Takara 키트가 Invitrogen 키트보다 민감도가 높음을 알 수 있었다. 본 연구는 적합한 상보적인 DNA 합성 키트를 확인하기 위해 수행되었으며, 상보적인 DNA 합성 키트가 real-time PCR 결과 다양성에 영향을 미친다는 것을 시사하였다.

Keywords

References

  1. Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2018;68:394-424. https://doi.org/10.3322/caac.21492
  2. Walboomers JM, Jacobs MV, Manos MM, Bosch FX, Kummer JA, Shah KV, et al. Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. J Pathol. 1999;189:12-19. https://doi.org/10.1002/(SICI)1096-9896(199909)189:1<12::AID-PATH431>3.0.CO;2-F
  3. Jung KW, Won YJ, Kong HJ, Lee ES; Community of Population-Based Regional Cancer Registries. Cancer statistics in Korea: incidence, mortality, survival, and prevalence in 2015. Cancer Res Treat. 2018;50:303-316. https://doi.org/10.4143/crt.2018.143
  4. Eileen M. Human papillomavirus and cervical cancer. Clin Microbiol Rev. 2003;16:1-17. https://doi.org/10.1128/CMR.16.1.1-17.2003
  5. Maucort-Boulch D, Franceschi S, Plummer M. International correlation between human papillomavirus prevalence and cervical cancer incidence. Cancer Epidemiol Biomarkers Prev. 2008;17:717-720. https://doi.org/10.1158/1055-9965.EPI-07-2691.
  6. Wentzensen N, Arbyn M. HPV-based cervical cancer screening-facts, fiction, and misperceptions. Prev Med. 2017;98: 33-35. https://doi.org/10.1016/j.ypmed.2016.12.040.
  7. Park SM, Lee SK, Kim YS. Inhibition of cervical cancer cell growth by gene silencing of HPV16 E6 induced by short-interfering RNA. Korean J Clin Lab Sci. 2011;43:89-97.
  8. Baptista AD, Simao CX, Santos VCGD, Melgaco JG, Cavalcanti SMB, Fonseca SC, et al. Knowledge of human papillomavirus and Pap test among Brazilian university students. Rev Assoc Med Bras. 2019;65:625-632. https://doi.org/10.1590/1806-9282.65.5.625.
  9. Watson M, Benard V, King J, Crawford A, Saraiya M. National assessment of HPV and Pap tests: Changes in cervical cancer screening, National Health Interview Survey. Prev Med. 2017;100:243-247. https://doi.org/10.1016/j.ypmed.2017.05.004.
  10. Al-Nourhji O, Dermawan JK, Booth CN, Underwood D, Abdul-Karim FW. Role of ThinPrep liquid-based cytology in evaluation of the endocervical canal in patients with abnormal cervical screening. J Am Soc Cytopathol. 2019;8:278-283. https://doi.org/10.1016/j.jasc.2019.05.001.
  11. Nayar R, Wilbur DC. The Pap test and Bethesda 2014. Cancer Cytopathol. 2015;123:271-281. https://doi.org/10.1002/cncy.21521.
  12. Koliopoulos G, Nyaga VN, Santesso N, Bryant A, Martin-Hirsch PP, Mustafa RA, et al. Cytology versus HPV testing for cervical cancer screening in the general population. Cochrane Database Syst Rev. 2017;10:8:CD008587. https://doi.org/10.1002/14651858.CD008587.pub2.
  13. El-Zein M, Richardson L, Franco EL. Cervical cancer screening of HPV vaccinated populations: Cytology, molecular testing, both or none. J Clin Virol. 2016;76(Suppl 1):S62-S68. https://doi.org/10.1016/j.jcv.2015.11.020.
  14. Pruski D, Millert-Kalinska S, Lewek A, Kedzia W. Sensitivity and specificity of HR HPV E6/E7 mRNA test in detecting cervical squamous intraepithelial lesion and cervical cancer. Ginekol Pol. 2019;90:66-71. https://doi.org/10.5603/GP.2019.0011.
  15. Park SY, Yoon HS, Bang HE, Kim Y, Cho SK, Ahn SW, et al. Analytical performance of sensitivity and specificity for rapid multiplex high risk human papillomavirus detection kit: HPV ViroCheck. Korean J Clin Lab Sci. 2017;49:446-454. https://doi.org/10.15324/kjcls.2017.49.4.446
  16. Poljak M, Kocjan BJ, Ostrbenk A, Seme K. Commercially available molecular tests for human papillomaviruses (HPV): 2015 update. J Clin Virol. 2016;76(Suppl 1):3-13. https://doi.org/10.1016/j.jcv.2015.10.023.
  17. Depuydt CE, Boulet GA, Horvath CA, Benoy IH, Vereecken AJ, Bogers JJ. Comparison of MY09/11 consensus PCR and type-specific PCRs in the detection of oncogenic HPV types. J Cell Mol Med. 2007;11:881-891. https://doi.org/10.1111/j.1582-4934.2007.00073.x
  18. Kim GH, Park SY, Wang HY, Kim SH, Park SJ, Yu KM, et al. Distribution of oncogenic human papillomavirus genotypes at high grade cervical lesions above CIN 2 grade with histological diagnosis. Biomed Sci Lett. 2016;22:98-106. https://doi.org/10.15616/BSL.2016.22.3.98
  19. Roberts CC, Swoyer R, Bryan JT, Taddeo FJ. Comparison of real-time multiplex human papillomavirus (HPV) PCR assays with the linear array HPV genotyping PCR assay and influence of DNA extraction method on HPV detection. J Clin Microbiol. 2011;49:1899-1906. https://doi.org/10.1128/JCM.00235-10.
  20. Pasquier C, Saune K, Raymond S, Boisneau J, Courtade M, Izopet J. Comparison of Cobas(R) HPV and $Anyplex^{TM}$ II HPV28 assays for detecting and genotyping human papillomavirus. Diagn Microbiol Infect Dis. 2017;87:25-27. https://doi.org/10.1016/j.diagmicrobio.2016.08.022.
  21. Pan C, Zhou J, Lyu J, Ren X. Development and validation of a multiplex reverse transcript real-time PCR for E6/E7 mRNA detection of high-risk human papillomavirus. J Med Microbiol. 2018;67:1509-1514. https://doi.org/10.1099/jmm.0.000824.
  22. Sahiner F, Kubar A, Yapar M, Sener K, Dede M, Gumral R. Detection of major HPVs by a new multiplex real-time PCR assay using type-specific primers. J Microbiol Methods. 2014;97:44-50. https://doi.org/10.1016/j.mimet.2013.12.012.
  23. Yang H, Li LJ, Xie LX, Luo ZY, Lu M, Lin M, et al. Clinical validation of a novel real-time human papillomavirus assay for simultaneous detection of 14 high-risk HPV type and genotyping HPV type 16 and 18 in China. Arch Virol. 2016;161:449-454. https://doi.org/10.1007/s00705-015-2673-y.
  24. Micalessi IM, Boulet GA, Bogers JJ, Benoy IH, Depuydt CE. High-throughput detection, genotyping and quantification of the human papillomavirus using real-time PCR. Clin Chem Lab Med. 2011;50:655-661. https://doi.org/10.1515/cclm.2011.835.
  25. Harvey RJ, Darlison MG. Random-primed cDNA synthesis facilitates the isolation of multiple 5'-cDNA ends by RACE. Nucleic Acids Res. 1991;19:4002. https://doi.org/10.1093/nar/19.14.4002.
  26. Michaelidou K, Tzovaras A, Missitzis I, Ardavanis A, Scorilas A. The expression of the CEACAM19 gene, a novel member of the CEA family, is associated with breast cancer progression. Int J Oncol. 2013;42:1770-1777. https://doi.org/10.3892/ijo.2013.1860.
  27. Yuan JS, Reed A, Chen F, Stewart CN Jr. Statistical analysis of real-time PCR data. BMC Bioinformatics. 2006;7:85. https://doi.org/10.1186/1471-2105-7-85.
  28. Picard-Meyer E, Peytavin de Garam C, Schereffer JL, Marchal C, Robardet E, Cliquet F. Cross-platform evaluation of commercial real-time SYBR green RT-PCR kits for sensitive and rapid detection of European bat Lyssavirus type 1. Biomed Res Int. 2015;2015:839518. https://doi.org/10.1155/2015/839518.
  29. Kim GH, Cho HM, Lee DS, Park SY, Lee JY, Wang HY, et al. Comparison of FFPE histological versus LBP cytological samples for HPV detection and typing in cervical cancer. Exp Mol Pathol. 2017;102:321-326. https://doi.org/10.1016/j.yexmp.2017.02.015.
  30. Wang HY, Lee DS, Park SY, Kim GH, Kim SH, Han L, et al. Diagnostic performance of HPV E6/E7 mRNA and HPV DNA assays for the detection and screening of oncogenic human papillomavirus infection among woman with cervical lesions in China. Asian Pac J Cancer Prev. 2015;16:7633-7640. https://doi.org/10.7314/APJCP.2015.16.17.7633
  31. Wang HY, Park SY, Lee DS, Kim SH, Kim GH, Park KH, et al. Prevalence of type-specific oncogenic human papillomavirus infection assessed by HPV E6/E7 mRNA among women with high-grade cervical lesions. Int J Infect Dis. 2015;37:135-142. https://doi.org/10.1016/j.ijid.2015.06.018.
  32. Buzard GS, Baker D, Wolcott MJ, Norwood DA, Dauphin LA. Multi-platform comparison of ten commercial master mixes for probe-based real-time polymerase chain reaction detection of bioterrorism threat agents for surge preparedness. Forensic Sci Int. 2012;223:292-297. https://doi.org/10.1016/j.forsciint.2012.10.003.
  33. Wacker MJ, Godard MP. Analysis of one-step and two-step real-time RT-PCR using SuperScript III. J Biomol Tech. 2005;16:266-271.
  34. Jacob F, Guertler R, Naim S, Nixdorf S, Fedier A, Hacker NF, et al. Careful selection of reference genes is required for reliable performance of RT-qPCR in human normal and cancer cell lines. PLoS One. 2013;8:E59180. https://doi.org/10.1371/journal.pone.0059180.
  35. Pfaffl M. Quantification strategies in real-time PCR. In: Bustin S, editor. A-Z of Quantitative PCR. chapter 3. La Jolla, CA, USA: International University Line; 2004; p. 87-112.
  36. Baker CC, Phelps WC, Lindgren V, Braun MJ, Gonda MA, Howley PM. Structural and transcriptional analysis of human papillomavirus type 16 sequences in cervical carcinoma cell lines. J Virol. 1987;61:962-971. https://doi.org/10.1128/JVI.61.4.962-971.1987
  37. Lie AK1, Risberg B, Borge B, Sandstad B, Delabie J, Rimala R, Onsrud M, Thoresen S. DNA- versus RNA-based methods for human papillomavirus detection in cervical neoplasia. Gynecol Oncol. 2005;97:908-915. https://doi.org/10.1016/j.ygyno.2005.02.026.

Cited by

  1. Triglyceride Down-regulates Expression of MSR-1 in PMA-induced THP-1 Macrophages vol.26, pp.3, 2020, https://doi.org/10.15616/bsl.2020.26.3.164
  2. Circular RNA MAT2B promotes migration, invasion and epithelial-mesenchymal transition of non-small cell lung cancer cells by sponging miR-431 vol.20, pp.16, 2019, https://doi.org/10.1080/15384101.2021.1956106