Skip to main content

Advertisement

SpringerLink
Log in

Genotyping cytomegalovirus UL97 mutations by high-resolution melting analysis with unlabeled probe

  • Original Article
  • Published:
Archives of Virology Aims and scope Submit manuscript

Abstract

Human cytomegalovirus (CMV) is an opportunistic pathogen, and infections with this virus can be treated with ganciclovir (GCV). Most GCV-resistant clinical CMV isolates contain a mutation in the UL97 gene. Genotypic assays for diagnostic screening of GCV-resistant CMV have been developed. High-resolution melting analysis (HRMA) with unlabeled probe is considered a perfect tool for this purpose. In this study, we have developed an HRMA-based genotypic test for the detection of UL97 mutations. Wild type and M460V/I mutants of UL97 were constructed. HRMA with unlabeled probe was used as a genotyping method for the detection of M460V/I mutations. The melting peaks obtained directly from PCR products did not enable us to distinguish the wild type from M460 mutants. The sensitivity and accuracy of HRMA were dramatically improved by using unlabeled probe. HRMA with unlabeled probe successfully distinguished M460V from M460I and served well for the detection of M460V/I mutations in clinical samples. HRMA with unlabeled probe proves to be a sensitive and cost-effective genotyping method for the detection of M460 mutations.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  1. Baldanti F, Underwood MR, Talarico CL, Simoncini L, Sarasini A, Biron KK, Gerna G (1998) The Cys607–>Tyr change in the UL97 phosphotransferase confers ganciclovir resistance to two human cytomegalovirus strains recovered from two immunocompromised patients. Antimicrob Agents Chemother 42:444–446

    PubMed  CAS  Google Scholar 

  2. Baldanti F, Michel D, Simoncini L, Heuschmid M, Zimmermann A, Minisini R, Schaarschmidt P, Schmid T, Gerna G, Mertens T (2002) Mutations in the UL97 ORF of ganciclovir-resistant clinical cytomegalovirus isolates differentially affect GCV phosphorylation as determined in a recombinant vaccinia virus system. Antiviral Res 54:59–67

    Article  PubMed  CAS  Google Scholar 

  3. Biron KK, Fyfe JA, Stanat SC, Leslie LK, Sorrell JB, Lambe CU, Coen DM (1986) A human cytomegalovirus mutant resistant to the nucleoside analog 9-([2-hydroxy-1-(hydroxymethyl)ethoxy]methyl)guanine (BW B759U) induces reduced levels of BW B759U triphosphate. Proc Natl Acad Sci USA 83:8769–8773

    Article  PubMed  CAS  Google Scholar 

  4. Boeckh M, Fries B, Nichols WG (2004) Recent advances in the prevention of CMV infection and disease after hematopoietic stem cell transplantation. Pediatr Transplant 8(Suppl 5):19–27

    Article  PubMed  Google Scholar 

  5. Boeckh M, Ljungman P (2009) How we treat cytomegalovirus in hematopoietic cell transplant recipients. Blood 113:5711–5719

    Article  PubMed  CAS  Google Scholar 

  6. Chee MS, Bankier AT, Beck S, Bohni R, Brown CM, Cerny R, Horsnell T, Hutchison CA 3rd, Kouzarides T, Martignetti JA et al (1990) Analysis of the protein-coding content of the sequence of human cytomegalovirus strain AD169. Curr Top Microbiol Immunol 154:125–169

    Article  PubMed  CAS  Google Scholar 

  7. Chou S, Erice A, Jordan MC, Vercellotti GM, Michels KR, Talarico CL, Stanat SC, Biron KK (1995) Analysis of the UL97 phosphotransferase coding sequence in clinical cytomegalovirus isolates and identification of mutations conferring ganciclovir resistance. J Infect Dis 171:576–583

    Article  PubMed  CAS  Google Scholar 

  8. Chou S, Guentzel S, Michels KR, Miner RC, Drew WL (1995) Frequency of UL97 phosphotransferase mutations related to ganciclovir resistance in clinical cytomegalovirus isolates. J Infect Dis 172:239–242

    Article  PubMed  CAS  Google Scholar 

  9. Chou S (1999) Antiviral drug resistance in human cytomegalovirus. Transpl Infect Dis 1:105–114

    Article  PubMed  CAS  Google Scholar 

  10. Chou S, Waldemer RH, Senters AE, Michels KS, Kemble GW, Miner RC, Drew WL (2002) Cytomegalovirus UL97 phosphotransferase mutations that affect susceptibility to ganciclovir. J Infect Dis 185:162–169

    Article  PubMed  CAS  Google Scholar 

  11. Chrisp P, Clissold SP (1991) Foscarnet: a review of its antiviral activity, pharmacokinetic properties and therapeutic use in immunocompromised patients with cytomegalovirus retinitis. Drugs 41:104–129

    Article  PubMed  CAS  Google Scholar 

  12. Cihlar T, Chen MS (1996) Identification of enzymes catalyzing two-step phosphorylation of cidofovir and the effect of cytomegalovirus infection on their activities in host cells. Mol Pharmacol 50:1502–1510

    PubMed  CAS  Google Scholar 

  13. Dames S, Pattison DC, Bromley LK, Wittwer CT, Voelkerding KV (2007) Unlabeled probes for the detection and typing of herpes simplex virus. Clin Chem 53:1847–1854

    Article  PubMed  CAS  Google Scholar 

  14. De Leeneer K, Coene I, Poppe B, De Paepe A, Claes K (2009) Genotyping of frequent BRCA1/2 SNPs with unlabeled probes: a supplement to HRMCA mutation scanning, allowing the strong reduction of sequencing burden. J Mol Diagn 11:415–419

    Article  PubMed  Google Scholar 

  15. Drew WL, Ives D, Lalezari JP, Crumpacker C, Follansbee SE, Spector SA, Benson CA, Friedberg DN, Hubbard L, Stempien MJ et al (1995) Oral ganciclovir as maintenance treatment for cytomegalovirus retinitis in patients with AIDS: Syntex Cooperative Oral Ganciclovir Study Group. N Engl J Med 333:615–620

    Article  PubMed  CAS  Google Scholar 

  16. Drew WL (2007) Laboratory diagnosis of cytomegalovirus infection and disease in immunocompromised patients. Curr Opin Infect Dis 20:408–411

    Article  PubMed  CAS  Google Scholar 

  17. Erali M, Palais R, Wittwer C (2008) SNP genotyping by unlabeled probe melting analysis. Methods Mol Biol 429:199–206

    Article  PubMed  CAS  Google Scholar 

  18. Erice A (1999) Resistance of human cytomegalovirus to antiviral drugs. Clin Microbiol Rev 12:286–297

    PubMed  CAS  Google Scholar 

  19. Gerna G, Sarasini A, Percivalle E, Zavattoni M, Baldanti F, Revello MG (1995) Rapid screening for resistance to ganciclovir and foscarnet of primary isolates of human cytomegalovirus from culture-positive blood samples. J Clin Microbiol 33:738–741

    PubMed  CAS  Google Scholar 

  20. Gilbert C, Boivin G (2005) Human cytomegalovirus resistance to antiviral drugs. Antimicrob Agents Chemother 49:873–883

    Article  PubMed  CAS  Google Scholar 

  21. Habalova V, Klimcakova L, Zidzik J, Tkac I (2009) Rapid and cost effective genotyping method for polymorphisms in PPARG, PPARGC1 and TCF7L2 genes. Mol Cell Probes 23:52–54

    Article  PubMed  CAS  Google Scholar 

  22. Hanson MN, Preheim LC, Chou S, Talarico CL, Biron KK, Erice A (1995) Novel mutation in the UL97 gene of a clinical cytomegalovirus strain conferring resistance to ganciclovir. Antimicrob Agents Chemother 39:1204–1205

    PubMed  CAS  Google Scholar 

  23. Kampmann SE, Schindele B, Apelt L, Buhrer C, Garten L, Weizsaecker K, Kruger DH, Ehlers B, Hofmann J (2011) Pyrosequencing allows the detection of emergent ganciclovir resistance mutations after HCMV infection. Med Microbiol Immunol

  24. Landry ML, Stanat S, Biron K, Brambilla D, Britt W, Jokela J, Chou S, Drew WL, Erice A, Gilliam B, Lurain N, Manischewitz J, Miner R, Nokta M, Reichelderfer P, Spector S, Weinberg A, Yen-Lieberman B, Crumpacker C (2000) A standardized plaque reduction assay for determination of drug susceptibilities of cytomegalovirus clinical isolates. Antimicrob Agents Chemother 44:688–692

    Article  PubMed  CAS  Google Scholar 

  25. Liu JB, Zhang Z (2008) Development of SYBR Green I-based real-time PCR assay for detection of drug resistance mutations in cytomegalovirus. J Virol Methods 149:129–135

    Article  PubMed  CAS  Google Scholar 

  26. Martin DF, Sierra-Madero J, Walmsley S, Wolitz RA, Macey K, Georgiou P, Robinson CA, Stempien MJ (2002) A controlled trial of valganciclovir as induction therapy for cytomegalovirus retinitis. N Engl J Med 346:1119–1126

    Article  PubMed  CAS  Google Scholar 

  27. Michel D, Schaarschmidt P, Wunderlich K, Heuschmid M, Simoncini L, Muhlberger D, Zimmermann A, Pavic I, Mertens T (1998) Functional regions of the human cytomegalovirus protein pUL97 involved in nuclear localization and phosphorylation of ganciclovir and pUL97 itself. J Gen Virol 79(Pt 9):2105–2112

    PubMed  CAS  Google Scholar 

  28. Michel D, Mertens T (2004) The UL97 protein kinase of human cytomegalovirus and homologues in other herpesviruses: impact on virus and host. Biochim Biophys Acta 1697:169–180

    PubMed  CAS  Google Scholar 

  29. Montgomery J, Wittwer CT, Palais R, Zhou L (2007) Simultaneous mutation scanning and genotyping by high-resolution DNA melting analysis. Nat Protoc 2:59–66

    Article  PubMed  CAS  Google Scholar 

  30. Noble S, Faulds D (1998) Ganciclovir: an update of its use in the prevention of cytomegalovirus infection and disease in transplant recipients. Drugs 56:115–146

    Article  PubMed  CAS  Google Scholar 

  31. Pepin JM, Simon F, Dussault A, Collin G, Dazza MC, Brun-Vezinet F (1992) Rapid determination of human cytomegalovirus susceptibility to ganciclovir directly from clinical specimen primocultures. J Clin Microbiol 30:2917–2920

    PubMed  CAS  Google Scholar 

  32. Poulson MD, Wittwer CT (2007) Closed-tube genotyping of apolipoprotein E by isolated-probe PCR with multiple unlabeled probes and high-resolution DNA melting analysis. Biotechniques 43:87–91

    Article  PubMed  CAS  Google Scholar 

  33. Prix L, Maierl J, Jahn G, Hamprecht K (1998) A simplified assay for screening of drug resistance of cell-associated cytomegalovirus strains. J Clin Virol 11:29–37

    Article  PubMed  CAS  Google Scholar 

  34. Prix L, Hamprecht K, Holzhuter B, Handgretinger R, Klingebiel T, Jahn G (1999) Comprehensive restriction analysis of the UL97 region allows early detection of ganciclovir-resistant human cytomegalovirus in an immunocompromised child. J Infect Dis 180:491–495

    Article  PubMed  CAS  Google Scholar 

  35. Ronaghi M, Uhlen M, Nyren P (1998) A sequencing method based on real-time pyrophosphate. Science 281:363, 365

    Article  PubMed  CAS  Google Scholar 

  36. Schnepf N, Boiteau N, Petit F, Alain S, Sanson-Le Pors MJ, Mazeron MC (2009) Rapid determination of antiviral drug susceptibility of human cytomegalovirus by real-time PCR. Antiviral Res 81:64–67

    Article  PubMed  CAS  Google Scholar 

  37. Schreiber A, Harter G, Schubert A, Bunjes D, Mertens T, Michel D (2009) Antiviral treatment of cytomegalovirus infection and resistant strains. Expert Opin Pharmacother 10:191–209

    Article  PubMed  CAS  Google Scholar 

  38. Sia IG, Patel R (2000) New strategies for prevention and therapy of cytomegalovirus infection and disease in solid-organ transplant recipients. Clin Microbiol Rev 13:83–121 table of content

    Article  PubMed  CAS  Google Scholar 

  39. Stanat SC, Reardon JE, Erice A, Jordan MC, Drew WL, Biron KK (1991) Ganciclovir-resistant cytomegalovirus clinical isolates: mode of resistance to ganciclovir. Antimicrob Agents Chemother 35:2191–2197

    PubMed  CAS  Google Scholar 

  40. Sullivan V, Talarico CL, Stanat SC, Davis M, Coen DM, Biron KK (1992) A protein kinase homologue controls phosphorylation of ganciclovir in human cytomegalovirus-infected cells. Nature 359:85

    Article  PubMed  CAS  Google Scholar 

  41. Sun HY, Wagener MM, Singh N (2008) Prevention of post-transplant cytomegalovirus disease and related outcomes with valganciclovir: a systematic review. Am J Transplant 8:2111–2118

    Article  PubMed  Google Scholar 

  42. Wittwer CT, Reed GH, Gundry CN, Vandersteen JG, Pryor RJ (2003) High-resolution genotyping by amplicon melting analysis using LCGreen. Clin Chem 49:853–860

    Article  PubMed  CAS  Google Scholar 

  43. Yeo AC, Chan KP, Kumarasinghe G, Yap HK (2005) Rapid detection of codon 460 mutations in the UL97 gene of ganciclovir-resistant cytomegalovirus clinical isolates by real-time PCR using molecular beacons. Mol Cell Probes 19:389–393

    Article  PubMed  CAS  Google Scholar 

  44. Zhou L, Harder TC, Ullmann U, Rautenberg P (1999) Rapid detection by reverse hybridization of mutations in the UL97 gene of human cytomegalovirus conferring resistance to ganciclovir. J Clin Virol 13:53–59

    Article  PubMed  CAS  Google Scholar 

  45. Zhou L, Myers AN, Vandersteen JG, Wang L, Wittwer CT (2004) Closed-tube genotyping with unlabeled oligonucleotide probes and a saturating DNA dye. Clin Chem 50:1328–1335

    Article  PubMed  CAS  Google Scholar 

  46. Zhou L, Wang L, Palais R, Pryor R, Wittwer CT (2005) High-resolution DNA melting analysis for simultaneous mutation scanning and genotyping in solution. Clin Chem 51:1770–1777

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

The study was supported by research grants from the Shenzhen Science and Technology Project. We thank Shenzhen Biomedical Research Support Platform for technical help.

Conflict of interest

None.

Author information

Authors and Affiliations

  1. Department of Clinical Laboratory, Peking University People’s Hospital, No. 11, Xizhimen South Street, Peking, China

    Xiao-Tao Zhao, Chang-Sheng Xia & Zheng Zhang

  2. Department of Laboratory Medicine, Jinshan Hospital, Fudan University, Shanghai, China

    Dan-Qiu Zhou

  3. College of Pharmacy, Ji-Nan University, Guangdong, China

    Shuai Wu, Yue-Wen Chen & Wei Zhang

  4. Shenzhen Key Laboratory for Translational Medicine of Dermatology, Shenzhen-PKU-HKUST Medical Center, Shenzhen, Guangdong, China

    Yong Shao, Jie Zhang, Hong Yang, Jun Wan, Bo Yu & Wei Zhang

  5. Biomedical Research Institute, Shenzhen-PKU-HKUST Medical Center, No. 1120, Lianhua Road, Futian District, Shenzhen, 518036, Guangdong, China

    Yong Shao, Jie Zhang, Ke-Peng Wang, Hong Yang, Jun Wan, Bo Yu & Wei Zhang

  6. Department of Dermatology, Shenzhen Hospital Peking University, Shenzhen, Guangdong, China

    Yong Shao, Jie Zhang & Bo Yu

  7. Division of Life Science, The Hong Kong University of Science and Technology, Hong Kong, China

    Jun Wan

Authors
  1. Xiao-Tao Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Dan-Qiu Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Shuai Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yue-Wen Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yong Shao
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jie Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Chang-Sheng Xia
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Ke-Peng Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Hong Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Jun Wan
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Bo Yu
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Zheng Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Wei Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Bo Yu, Zheng Zhang or Wei Zhang.

Additional information

X.-T. Zhao, D.-Q. Zhou and S. Wu contributed equally to this work. B. Yu, Z. Zhang and W. Zhang contributed equally to this work.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Zhao, XT., Zhou, DQ., Wu, S. et al. Genotyping cytomegalovirus UL97 mutations by high-resolution melting analysis with unlabeled probe. Arch Virol 157, 475–481 (2012). https://doi.org/10.1007/s00705-011-1173-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00705-011-1173-y

Keywords

Search

Navigation