Current Microbiology

, Volume 52, Issue 3, pp 204–209 | Cite as

Oligonucleotide Microarray with RD-PCR Labeling Technique for Detection and Typing of Human Papillomavirus

  • Wei Min
  • Ma Wen-li
  • Zhang Bao
  • Li Ling
  • Sun Zhao-hui
  • Zheng Wen-ling


Currently, screening for high-risk human papillomavirus (HPV) infection remains an important health concern throughout the world, because of the close association between certain types of HPV and cervical cancer. In this study, we explore the possibility of using ∼70mer oligonucleotide microarray for detection and genotyping of HPV. The ∼70mer type-specific oligonucleotide probes of four different types HPV were designed by using biological software Arraydesigner 2.0, which analyzed the whole genome sequences of HPV and selected optimal probes. These probes were synthesized and printed onto the surface of glass slides in order to prepare a low-density microarray. HPV samples were labeled with fluorescence dyes Cy3 using a method of restriction display polymerase chain reaction (RD-PCR). HPV plasmid DNA was restricted with Sau3A I to produce multiple fragments that were ligated to adaptors subsequently and used as PCR template. PCR labeling was performed with the fluorescently labeled universal primer (Cy3-UP) whose sequence is designed according to the adaptor of the RD-PCR approaches. The labeled samples were hybridized with the oligonucleotide microarray. The scanning results showed that HPV DNA hybridized specifically with multiple spots correspondingly to show positive signals, whereas no signals were detected of all the negative and blank controls. These results demonstrated that ∼70mer oligonucleotide microarray can be applied to HPV detection and genotyping. The application of RD-PCR in the sample labeling can increase significantly the sensitivity of the assay and will be especially useful for the discriminate diagnosis of multiple pathogens.



This work is supported by the National Natural Science Foundation (36990032) and the regional Key Project of the Guangzhou City. The HPV plasmids used in this study were kindly provided by Dr. de Villier from Deutsches Krebsforschnngszentrum (Heidelberg, Germany).

Literature Cited

  1. 1.
    Burd EM (2003) Human papillomavirus and cervical cancer. Clin Microbiol Rev 16:1–17CrossRefPubMedGoogle Scholar
  2. 2.
    de Roda Husman AM, Walboomers JM, van den Brule AJ, Meijer CJ, Snijders PJ (1995) The use of general primers GP5 and GP6 elongated at their 3’ ends with adjacent highly conserved sequences improves human papillomavirus detection by PCR. J Gen Virol 76:1057–1062PubMedGoogle Scholar
  3. 3.
    Delrio-Lafreniere SA, Browning MK, McGlennen RC (2004) Low-density addressable array for the detection and typing of the human papillomavirus. Diagn Microbiol Infect Dis 48:23–31CrossRefPubMedGoogle Scholar
  4. 4.
    Gravitt PE, Peyton CL, Alessi TQ, Wheeler CM, Coutlee F, Hildesheim A, Schiffman MH, Scott DR, Apple RJ (2000) Improved amplification of genital human papillomaviruses. J Clin Microbiol 38:357–361PubMedGoogle Scholar
  5. 5.
    Hwang TS, Jeong JK, Park M, Han HS, Choi HK, Park TS (2003) Detection and typing of HPV genotypes in various cervical lesions by HPV oligonucleotide microarray. Gynecol Oncol 90:51–56CrossRefPubMedGoogle Scholar
  6. 6.
    Klaassen CH, Prinsen CF, de Valk HA, Horrevorts AM, Jeunink MA, Thunnissen FB (2004) DNA microarray format for detection and subtyping of human papillomavirus. J Clin Microbiol 42:2152–2160CrossRefPubMedGoogle Scholar
  7. 7.
    Kleter B, van Doorn LJ, ter Schegget J, Schrauwen L, van Krimpen K, Burger M, ter Harmsel B, Quint W (1998) Novel short-fragment PCR assay for highly sensitive broad-spectrum detection of anogenital human papillomaviruses. Am J Pathol 153:1731–1739PubMedGoogle Scholar
  8. 8.
    Li L, Ma WL, Zhu J, Shi R, Liu CH, Chen JK, Zheng WL (2003) A modified restriction display PCR method in sample-labelling of DNA microarray. J Virol Methods 114:71–75CrossRefPubMedGoogle Scholar
  9. 9.
    Ma WL, Zheng WL, James FB, Li BJ (1998) Restriction display: a kind of new technology of differential display. In: Sun ZX (eds) Progression in biochemistry and molecular biology of army. Beijing: Uniform Medical Science Press, pp 113–114Google Scholar
  10. 10.
    Manos MM, Ting T, Wright DK, Lewis AJ, Broker TR, Wolinsky SM (1989) Use of polymerase chain reaction amplification for the detection of genital human papillomaviruses. Cancer Cells 7:209–214Google Scholar
  11. 11.
    Myers G, Lu H, Calef C, Leitner T (1996) Heterogeneity of papillomaviruses. Semin Cancer Biol 7:349–358CrossRefPubMedGoogle Scholar
  12. 12.
    Quint WGV, Scholte G, Van Doorn LJ, Kleeter B, Smits PHM, Lindeman J (2001) Comparative analysis of human papillomavirus infections in cervical scrapes and biopsy specimens by general SPF10 PCR and HPV genotyping. J Pathol 194:51–58CrossRefPubMedGoogle Scholar
  13. 13.
    Spitzer M (1998) Cervical screening adjuncts: recent advances. Am J Obstet Gynecol 179:544–556CrossRefPubMedGoogle Scholar
  14. 14.
    Vernon SD, Unger ER, Williams D (2000) Comparison of human papillomavirus detection and typing by cycle sequencing, line blotting, and hybrid capture. J Clin Microbiol 38:651–655PubMedGoogle Scholar
  15. 15.
    Walboomers JM, Jacobs MV, Manos MM, Bosch FX, Kummer JA, Shah KV, Snijders PJ, Peto J, Meijer CJ, Munoz N (1999) Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. J Pathol 189:12–19CrossRefPubMedGoogle Scholar
  16. 16.
    Zerbini M, Venturoli S, Cricca M, Gallinella G, De Simone P, Costa S, Santini D, Musiani M (2001) Distribution and viral load of type specific HPVs in different cervical lesions as detected by PCR-ELISA. J Clin Pathol 54:377–380CrossRefPubMedGoogle Scholar
  17. 17.
    zur Hausen H (1982) Human genital cancer: synergism between two virus infections and or synergism between a virus infection and initiating events? Lancet 2:1370–1372PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, Inc. 2006

Authors and Affiliations

  • Wei Min
    • 1
  • Ma Wen-li
    • 1
  • Zhang Bao
    • 1
  • Li Ling
    • 1
  • Sun Zhao-hui
    • 1
  • Zheng Wen-ling
    • 2
  1. 1.Institute of Molecular BiologySouthern Medical UniversityP.R. China
  2. 2.Southern China Genomics Research CenterP.R. China

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