Development of highly efficient protocols for extraction and amplification of cytomegalovirus DNA from dried blood spots for detection and genotyping of polymorphic immunomodulatory genes

English version

Autoři: Christian Berg ^aff001; Martin B. Friis ^aff003; Mette M. Rosenkilde ^aff002; Thomas Benfield ^aff004; Lene Nielsen ^aff003; Hans R. Lüttichau ^aff001; Thomas Sundelin ^aff003
Působiště autorů: Unit for Infectious Diseases, Department of Medicine, Herlev-Gentofte Hospital, University of Copenhagen, Herlev, Denmark ^aff001; Laboratory for Molecular Pharmacology, Department of Biomedical Sciences, Panum Institute, University of Copenhagen, Copenhagen, Denmark ^aff002; Department of Clinical Microbiology, Herlev-Gentofte Hospital, University of Copenhagen, Herlev, Denmark ^aff003; Department of Infectious Diseases, Hvidovre Hospital, University of Copenhagen, Hvidovre, Denmark ^aff004
Vyšlo v časopise: PLoS ONE 14(9)
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.pone.0222053

Souhrn

Congenital cytomegalovirus (CMV) infection is a major cause of birth defects ranging from developmental disorders to stillbirth. Most newborns affected by CMV do not present with symptoms at birth but are at risk of sequelae at later stages of their childhood. Stored dried blood spots (DBS) taken at birth can be used for retrospective diagnosis of hereditary diseases, but detection of pathogens is challenged by potentially low pathogen concentrations in the small blood volume available in a DBS. Here we test four different extraction methods for optimal recovery of CMV DNA from DBS at low to high CMV titers. The recovery efficiencies varied widely between the different extractions (from 3% to 100%) with the most efficient method extracting up to 113-fold more CMV DNA than the least efficient and 8-fold more than the reference protocol. Furthermore, we amplified four immunomodulatory CMV genes from the extracted DNA: the UL40 and UL111A genes which occur as functional knockouts in some circulating CMV strains, and the highly variable UL146 and US28 genes. The PCRs specifically amplified the CMV genes at all tested titers with sufficient quality for sequencing and genotyping. In summary, we here report an extraction method for optimal recovery of CMV DNA from DBSs that can be used for both detection of CMV and for genotyping of polymorphic CMV genes in congenital CMV infection.

Klíčová slova:

DNA – Biology and life sciences – Genetics – Biochemistry – Nucleic acids – Organisms – Engineering and technology – Research and analysis methods – Molecular biology – Molecular biology techniques – Anatomy – Medicine and health sciences – Microbiology – Medical microbiology – Microbial pathogens – Pathology and laboratory medicine – Pathogens – Physiology – Infectious diseases – Viral pathogens – Viruses – Body fluids – Blood – DNA viruses – Herpesviruses – Artificial gene amplification and extension – Polymerase chain reaction – Viral diseases – Equipment – Extraction techniques – Laboratory equipment – Filter paper – Gene amplification – Human cytomegalovirus – DNA extraction – Cytomegalovirus infection

Zdroje

1. Crough T, Khanna R. Immunobiology of human cytomegalovirus: from bench to bedside. Clin Microbiol Rev. 2009;22(1):76–98, Table of Contents. doi: 10.1128/CMR.00034-08 19136435; PubMed Central PMCID: PMC2620639.

2. Dollard SC, Grosse SD, Ross DS. New estimates of the prevalence of neurological and sensory sequelae and mortality associated with congenital cytomegalovirus infection. Rev Med Virol. 2007;17(5):355–63. doi: 10.1002/rmv.544 17542052.

3. Stratton KR, Durch JS, Lawrence RS. Vaccines for the 21st Century: A Tool for Decisionmaking. 2000:165–71. doi: 10.17226/5501

4. Stern-Ginossar N, Weisburd B, Michalski A, Le VT, Hein MY, Huang SX, et al. Decoding human cytomegalovirus. Science. 2012;338(6110):1088–93. doi: 10.1126/science.1227919 23180859; PubMed Central PMCID: PMC3817102.

5. Mocarski ES Jr., Immunomodulation by cytomegaloviruses: manipulative strategies beyond evasion. Trends Microbiol. 2002;10(7):332–9. 12110212.

6. Sijmons S, Thys K, Mbong Ngwese M, Van Damme E, Dvorak J, Van Loock M, et al. High-throughput analysis of human cytomegalovirus genome diversity highlights the widespread occurrence of gene-disrupting mutations and pervasive recombination. J Virol. 2015. doi: 10.1128/JVI.00578-15 25972543; PubMed Central PMCID: PMC4505652.

7. Engel P, Angulo A. Viral immunomodulatory proteins: usurping host genes as a survival strategy. Adv Exp Med Biol. 2012;738:256–76. doi: 10.1007/978-1-4614-1680-7_15 22399384.

8. Wilkinson GW, Tomasec P, Stanton RJ, Armstrong M, Prod'homme V, Aicheler R, et al. Modulation of natural killer cells by human cytomegalovirus. J Clin Virol. 2008;41(3):206–12. doi: 10.1016/j.jcv.2007.10.027 18069056; PubMed Central PMCID: PMC2843162.

9. Dolan A, Cunningham C, Hector RD, Hassan-Walker AF, Lee L, Addison C, et al. Genetic content of wild-type human cytomegalovirus. J Gen Virol. 2004;85(Pt 5):1301–12. 15105547.

10. Kledal TN, Rosenkilde MM, Schwartz TW. Selective recognition of the membrane-bound CX3C chemokine, fractalkine, by the human cytomegalovirus-encoded broad-spectrum receptor US28. FEBS Lett. 1998;441(2):209–14. 9883886.

11. Spiess K, Jeppesen MG, Malmgaard-Clausen M, Krzywkowski K, Dulal K, Cheng T, et al. Rationally designed chemokine-based toxin targeting the viral G protein-coupled receptor US28 potently inhibits cytomegalovirus infection in vivo. Proc Natl Acad Sci U S A. 2015;112(27):8427–32. doi: 10.1073/pnas.1509392112 26080445; PubMed Central PMCID: PMC4500259.

12. Goffard A, Gault E, Rozenberg F, Moret N, Hober D, Deny P. Comparative sequence analysis of US28 gene of human cytomegalovirus strains isolated from HIV-positive patients. Virus Genes. 2006;33(2):175–81. doi: 10.1007/s11262-005-0054-4 16972032.

13. Arav-Boger R, Willoughby RE, Pass RF, Zong JC, Jang WJ, Alcendor D, et al. Polymorphisms of the cytomegalovirus (CMV)-encoded tumor necrosis factor-alpha and beta-chemokine receptors in congenital CMV disease. J Infect Dis. 2002;186(8):1057–64. doi: 10.1086/344238 12355354.

14. Norgaard-Pedersen B, Hougaard DM. Storage policies and use of the Danish Newborn Screening Biobank. J Inherit Metab Dis. 2007;30(4):530–6. doi: 10.1007/s10545-007-0631-x 17632694.

15. Atkinson C, Walter S, Sharland M, Tookey P, Luck S, Peckham C, et al. Use of stored dried blood spots for retrospective diagnosis of congenital CMV. JMedVirol. 2009;81(8):1394–8. doi: 10.1002/jmv.21543

16. Barbi M, Binda S, Primache V, Luraschi C, Corbetta C. Diagnosis of congenital cytomegalovirus infection by detection of viral DNA in dried blood spots. ClinDiagnVirol. 1996;6(1):27–32. 0928019796002280 [pii].

17. Barbi M, Binda S, Primache V, Caroppo S, Dido P, Guidotti P, et al. Cytomegalovirus DNA detection in Guthrie cards: a powerful tool for diagnosing congenital infection. JClinVirol. 2000;17(3):159–65. S1386653200000895 [pii].

18. Binda S, Caroppo S, Dido P, Primache V, Veronesi L, Calvario A, et al. Modification of CMV DNA detection from dried blood spots for diagnosing congenital CMV infection. JClinVirol. 2004;30(3):276–9. doi: 10.1016/j.jcv.2003.11.012 [doi];S138665320300324X [pii].

19. Boppana SB, Ross SA, Novak Z, Shimamura M, Tolan RW Jr., Palmer AL, et al. Dried blood spot real-time polymerase chain reaction assays to screen newborns for congenital cytomegalovirus infection. JAMA. 2010;303(14):1375–82. 303/14/1375 [pii]; doi: 10.1001/jama.2010.423 20388893

20. de Vries JJ, Claas EC, Kroes AC, Vossen AC. Evaluation of DNA extraction methods for dried blood spots in the diagnosis of congenital cytomegalovirus infection. JClinVirol. 2009;46 Suppl 4:S37–S42. S1386-6532(09)00415-6 [pii]; doi: 10.1016/j.jcv.2009.09.001

21. Fischler B, Rodensjo P, Nemeth A, Forsgren M, Lewensohn-Fuchs I. Cytomegalovirus DNA detection on Guthrie cards in patients with neonatal cholestasis. ArchDisChild Fetal Neonatal Ed. 1999;80(2):F130–F4.

22. Inoue N, Koyano S. Evaluation of screening tests for congenital cytomegalovirus infection. PediatrInfectDisJ. 2008;27(2):182–4. doi: 10.1097/INF.0b013e318161a2d5

23. Johansson PJ, Jonsson M, Ahlfors K, Ivarsson SA, Svanberg L, Guthenberg C. Retrospective diagnostics of congenital cytomegalovirus infection performed by polymerase chain reaction in blood stored on filter paper. ScandJInfectDis. 1997;29(5):465–8.

24. Koontz D, Baecher K, Amin M, Nikolova S, Gallagher M, Dollard S. Evaluation of DNA extraction methods for the detection of Cytomegalovirus in dried blood spots. JClinVirol. 2015;66:95–9. S1386-6532(15)00093-1 [pii]; doi: 10.1016/j.jcv.2015.03.015 25866346

25. Scanga L, Chaing S, Powell C, Aylsworth AS, Harrell LJ, Henshaw NG, et al. Diagnosis of human congenital cytomegalovirus infection by amplification of viral DNA from dried blood spots on perinatal cards. JMolDiagn. 2006;8(2):240–5. S1525-1578(10)60724-6 [pii]; doi: 10.2353/jmoldx.2006.050075 16645211

26. Soetens O, Vauloup-Fellous C, Foulon I, Dubreuil P, De SB, Grangeot-Keros L, et al. Evaluation of different cytomegalovirus (CMV) DNA PCR protocols for analysis of dried blood spots from consecutive cases of neonates with congenital CMV infections. JClinMicrobiol. 2008;46(3):943–6. JCM.01391-07 [pii]; doi: 10.1128/JCM.01391-07 18199787

27. Vauloup-Fellous C, Ducroux A, Couloigner V, Marlin S, Picone O, Galimand J, et al. Evaluation of cytomegalovirus (CMV) DNA quantification in dried blood spots: retrospective study of CMV congenital infection. JClinMicrobiol. 2007;45(11):3804–6. JCM.01654-07 [pii]; doi: 10.1128/JCM.01654-07 17898161

28. Vives-Onos I, Codina-Grau MG, Noguera-Julian A, Blazquez-Gamero D, Fortuny C, Baquero-Artigao F, et al. Is Polymerase Chain Reaction in Neonatal Dried Blood Spots Reliable for the Diagnosis of Congenital Cytomegalovirus Infection? Pediatr Infect Dis J. 2018. doi: 10.1097/INF.0000000000002144 30199483.

29. Yamamoto AY, Mussi-Pinhata MM, Pinto PC, Figueiredo LT, Jorge SM. Usefulness of blood and urine samples collected on filter paper in detecting cytomegalovirus by the polymerase chain reaction technique. JVirolMethods. 2001;97(1–2):159–64. S0166093401003470 [pii].

30. Arav-Boger R, Foster CB, Zong JC, Pass RF. Human cytomegalovirus-encoded alpha -chemokines exhibit high sequence variability in congenitally infected newborns. J Infect Dis. 2006;193(6):788–91. doi: 10.1086/500508 16479512.

31. Arista S, De GS, Giammanco GM, Di CP, Iannitto E. Human cytomegalovirus glycoprotein B genotypes in immunocompetent, immunocompromised, and congenitally infected Italian populations. ArchVirol. 2003;148(3):547–54. doi: 10.1007/s00705-002-0941-0

32. Bale JF Jr., Murph JR, Demmler GJ, Dawson J, Miller JE, Petheram SJ. Intrauterine cytomegalovirus infection and glycoprotein B genotypes. JInfectDis. 2000;182(3):933–6. JID000287 [pii]; doi: 10.1086/315770

33. Bale JF Jr., Petheram SJ, Robertson M, Murph JR, Demmler G. Human cytomegalovirus a sequence and UL144 variability in strains from infected children. JMedVirol. 2001;65(1):90–6. doi: 10.1002/jmv.2006 [pii].

34. Barbi M, Binda S, Caroppo S, Primache V, Dido P, Guidotti P, et al. CMV gB genotypes and outcome of vertical transmission: study on dried blood spots of congenitally infected babies. JClinVirol. 2001;21(1):75–9. S1386-6532(00)00188-8 [pii].

35. Heo J, Petheram S, Demmler G, Murph JR, Adler SP, Bale J, et al. Polymorphisms within human cytomegalovirus chemokine (UL146/UL147) and cytokine receptor genes (UL144) are not predictive of sequelae in congenitally infected children. Virology. 2008;378(1):86–96. S0042-6822(08)00313-9 [pii]; doi: 10.1016/j.virol.2008.05.002 18556037

36. Paradowska E, Studzinska M, Nowakowska D, Wilczynski J, Rycel M, Suski P, et al. Distribution of UL144, US28 and UL55 genotypes in Polish newborns with congenital cytomegalovirus infections. Eur J Clin Microbiol Infect Dis. 2012;31(7):1335–45. doi: 10.1007/s10096-011-1447-z 22048843.

37. Paradowska E, Jablonska A, Plociennikowska A, Studzinska M, Suski P, Wisniewska-Ligier M, et al. Cytomegalovirus alpha-chemokine genotypes are associated with clinical manifestations in children with congenital or postnatal infections. Virology. 2014;462–463:207–17. doi: 10.1016/j.virol.2014.06.020 24999045.

38. Paradowska E, Jablonska A, Studzinska M, Kasztelewicz B, Zawilinska B, Wisniewska-Ligier M, et al. Cytomegalovirus glycoprotein H genotype distribution and the relationship with hearing loss in children. JMedVirol. 2014;86(8):1421–7. doi: 10.1002/jmv.23906

39. Paradowska E, Studzinska M, Suski P, Kasztelewicz B, Wisniewska-Ligier M, Zawilinska B, et al. Human cytomegalovirus UL55, UL144, and US28 genotype distribution in infants infected congenitally or postnatally. J Med Virol. 2015;87(10):1737–48. doi: 10.1002/jmv.24222 25926093.

40. Pati SK, Pinninti S, Novak Z, Chowdhury N, Patro RK, Fowler K, et al. Genotypic diversity and mixed infection in newborn disease and hearing loss in congenital cytomegalovirus infection. PediatrInfectDisJ. 2013;32(10):1050–4. doi: 10.1097/INF.0b013e31829bb0b9 23694837

41. Picone O, Costa JM, Chaix ML, Ville Y, Rouzioux C, Leruez-Ville M. Comments on "cytomegalovirus (CMV)-encoded UL144 (truncated tumor necrosis factor receptor) and outcome of congenital CMV infection". JInfectDis. 2007;196(11):1719–20. JID38605 [pii]; doi: 10.1086/522340

42. Pignatelli S, Lazzarotto T, Gatto MR, Dal MP, Landini MP, Faldella G, et al. Cytomegalovirus gN genotypes distribution among congenitally infected newborns and their relationship with symptoms at birth and sequelae. ClinInfectDis. 2010;51(1):33–41. doi: 10.1086/653423

43. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997;25(17):3389–402. doi: 10.1093/nar/25.17.3389 9254694; PubMed Central PMCID: PMC146917.

44. Gohring K, Dietz K, Hartleif S, Jahn G, Hamprecht K. Influence of different extraction methods and PCR techniques on the sensitivity of HCMV-DNA detection in dried blood spot (DBS) filter cards. JClinVirol. 2010;48(4):278–81. S1386-6532(10)00190-3 [pii]; doi: 10.1016/j.jcv.2010.04.011

45. Brytting M, Sundqvist VA, Stalhandske P, Linde A, Wahren B. Cytomegalovirus DNA detection of an immediate early protein gene with nested primer oligonucleotides. J Virol Methods. 1991;32(2–3):127–38. 1651946.

46. Wakefield AJ, Fox JD, Sawyerr AM, Taylor JE, Sweenie CH, Smith M, et al. Detection of herpesvirus DNA in the large intestine of patients with ulcerative colitis and Crohn's disease using the nested polymerase chain reaction. J Med Virol. 1992;38(3):183–90. 1287131.

47. Leruez-Ville M, Ouachee M, Delarue R, Sauget AS, Blanche S, Buzyn A, et al. Monitoring cytomegalovirus infection in adult and pediatric bone marrow transplant recipients by a real-time PCR assay performed with blood plasma. J Clin Microbiol. 2003;41(5):2040–6. doi: 10.1128/JCM.41.5.2040-2046.2003 12734246; PubMed Central PMCID: PMC154722.

Development of highly efficient protocols for extraction and amplification of cytomegalovirus DNA from dried blood spots for detection and genotyping of polymorphic immunomodulatory genes

Souhrn

Klíčová slova:

Zdroje

PLOS One

Aktuální možnosti diagnostiky a léčby litiáz