Nucleic Acid Amplification-Based Diagnostics for Pulmonary Diseases: What Is the Current State and Perspectives of Nucleic Acid Amplification Technologies Used in Diagnostics Associated with Pulmonary Diseases?

  • Oleg Gusev
  • Yoshihide Hayashizaki
  • Kengo UsuiEmail author
Part of the Respiratory Disease Series: Diagnostic Tools and Disease Managements book series (RDSDTDM)


Rapid advances of genomic technologies in medical sciences resulted in growth of identified molecular biomarkers, including those required for proper drug administration and therapy selection in pulmonary diseases. While high-throughput technologies are powerful tool for wide screening, targeted real-time monitoring using nucleic acid amplification is still the most important method for DNA and RNA detection widely employed in clinical diagnostics. In this chapter, we overview the key nucleic acid amplification platforms successfully used in the clinical diagnostics, including that associated with pulmonary diseases, and briefly outline their advantages and pitfalls. We further focus on the specific isothermal amplification technology SmartAmp and Eprobes developed by RIKEN outlining its implementation in quick and robust detection of several clinically important SNP and cancer-associated somatic mutations. Finally, we describe the further potential of expansion of utilization of Eprobes platform for direct protein detection for clinical diagnostic needs.


Nucleic acid amplification Clinical diagnostics Companion diagnostics 


  1. 1.
    Genomes Project C, Auton A, Brooks LD, Durbin RM, Garrison EP, Kang HM, et al. A global reference for human genetic variation. Nature. 2015;526(7571):68–74.CrossRefGoogle Scholar
  2. 2.
    Lele RD. The human genome project: its implications in clinical medicine. J Assoc Physicians India. 2003;51:373–80.PubMedGoogle Scholar
  3. 3.
    Forbes SA, Beare D, Boutselakis H, Bamford S, Bindal N, Tate J, et al. COSMIC: somatic cancer genetics at high-resolution. Nucleic Acids Res. 2017;45(D1):D777–D83.CrossRefPubMedGoogle Scholar
  4. 4.
    Craw P, Balachandran W. Isothermal nucleic acid amplification technologies for point-of-care diagnostics: a critical review. Lab Chip. 2012;12(14):2469–86.CrossRefPubMedGoogle Scholar
  5. 5.
    Spargo CA, Fraiser MS, VanCleve M, Wright DJ, Nycz CM, Spears PA, et al. Detection of M-tuberculosis DNA using thermophilic strand displacement amplification. Mol Cell Probes. 1996;10(4):247–56.CrossRefPubMedGoogle Scholar
  6. 6.
    Compton J. Nucleic acid sequence-based amplification. Nature. 1991;350(6313):91–2.CrossRefGoogle Scholar
  7. 7.
    Vincent M, Xu Y, Kong H. Helicase-dependent isothermal DNA amplification. EMBO Rep. 2004;5(8):795–800.CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, et al. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 2000;28(12)CrossRefGoogle Scholar
  9. 9.
    Piepenburg O, Williams CH, Stemple DL, Armes NADNA. Detection using recombination proteins. PLoS Biol. 2006;4(7):1115–21.CrossRefGoogle Scholar
  10. 10.
    Mitani Y, Lezhava A, Kawai Y, Kikuchi T, Oguchi-Katayama A, Kogo Y, et al. Rapid SNP diagnostics using asymmetric isothermal amplification and a new mismatch-suppression technology. Nat Methods. 2007;4(3):257–62.CrossRefPubMedGoogle Scholar
  11. 11.
    Mitani Y, Lezhava A, Sakurai A, Horikawa A, Nagakura M, Hayashizaki Y, et al. Rapid and cost-effective SNP detection method: application of SmartAmp2 to pharmacogenomics research. Pharmacogenomics. 2009;10(7):1187–97.CrossRefPubMedGoogle Scholar
  12. 12.
    Chang HS, Mizukami K, Yabuki A, Hossain MA, Rahman MM, Uddin MM, et al. A novel rapid genotyping technique for collie eye anomaly: SYBR green-based real-time polymerase chain reaction method applicable to blood and saliva specimens on flinders technology associates filter paper. J Vet Diagn Investig. 2010;22(5):708–15.CrossRefGoogle Scholar
  13. 13.
    Elenitoba-Johnson KSJ, Bohling SD, Wittwer CT, King TC. Multiplex PCR by multicolor fluorimetry and fluorescence melting curve analysis. Nat Med. 2001;7(2):249–53.CrossRefPubMedGoogle Scholar
  14. 14.
    Juskowiak B. Nucleic acid-based fluorescent probes and their analytical potential. Anal Bioanal Chem. 2011;399(9):3157–76.CrossRefPubMedGoogle Scholar
  15. 15.
    Arikawa E, Sun Y, Wang J, Zhou Q, Ning B, Dial SL, et al. Cross-platform comparison of SYBR green real-time PCR with TaqMan PCR, microarrays and other gene expression measurement technologies evaluated in the MicroArray quality control (MAQC) study. BMC Genomics. 2008;9:328.CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Tyagi S, Kramer FR. Molecular beacons: probes that fluoresce upon hybridization. Nat Biotechnol. 1996;14(3):303–8.CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Tapp I, Malmberg L, Rennel E, Wik M, Syvanen AC. Homogeneous scoring of single-nucleotide polymorphisms: comparison of the 5 '-nuclease TaqMan (R) assay and molecular beacon probes (vol 28, pg 732, 2000). BioTechniques. 2000;29(3):546.Google Scholar
  18. 18.
    Whitcombe D, Kelly S, Mann J, Theaker J, Jones C, Little S. Scorpions (TM) primers - a novel method for use in single-tube genotyping. Am J Hum Genet. 1999;65(4):A412.Google Scholar
  19. 19.
    Okamoto A. ECHO probes: a concept of fluorescence control for practical nucleic acid sensing. Chem Soc Rev. 2011;40(12):5815–28.CrossRefPubMedGoogle Scholar
  20. 20.
    Hanami T, Delobel D, Kanamori H, Tanaka Y, Kimura Y, Nakasone A, et al. Eprobe mediated real-time PCR monitoring and melting curve analysis. PLoS One. 2013;8(8)CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Kimura Y, Hanami T, Tanaka Y, de Hoon MJL, Soma T, Harbers M, et al. Effect of thiazole orange doubly labeled thymidine on DNA duplex formation. Biochemistry. 2012;51(31):6056–67.CrossRefPubMedGoogle Scholar
  22. 22.
    Murdock DG, Wallace DC. PNA-mediated PCR clamping. Applications and methods. Methods Mol Biol. 2002;208:145–64.PubMedGoogle Scholar
  23. 23.
    Efrati E, Elkin H, Peerless Y, Sabo E, Ben-Izhak O, Hershkovitz D, LNA-based PCR. Clamping enrichment assay for the identification of KRAS mutations. Cancer Biomark. 2010;8(2):89–94.CrossRefPubMedGoogle Scholar
  24. 24.
    Atsumi J, Hanami T, Enokida Y, Ogawa H, Delobel D, Mitani Y, et al. Eprobe-mediated screening system for somatic mutations in the KRAS locus. Oncol Rep. 2015;33(6):2719–27.CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Mitri Z, Constantine T, O'Regan R. The HER2 receptor in breast cancer: pathophysiology, clinical use, and new advances in therapy. Chemother Res Pract. 2012;2012:743193.PubMedPubMedCentralGoogle Scholar
  26. 26.
    Arcila ME, Chaft JE, Nafa K, Roy-Chowdhuri S, Lau C, Zaidinski M, et al. Prevalence, clinicopathologic associations, and molecular spectrum of ERBB2 (HER2) tyrosine kinase mutations in lung adenocarcinomas. Clin Cancer Res. 2012;18(18):4910–8.CrossRefPubMedGoogle Scholar
  27. 27.
    Mazieres J, Peters S, Lepage B, Cortot AB, Barlesi F, Beau-Faller M, et al. Lung cancer that harbors an HER2 mutation: epidemiologic characteristics and therapeutic perspectives. J Clin Oncol. 2013;31(16):1997–U307.CrossRefPubMedGoogle Scholar
  28. 28.
    Takase Y, Usui K, Shimizu K, Kimura Y, Ichihara T, Ohkawa T, et al. Highly sensitive detection of a HER2 12-base pair duplicated insertion mutation in lung cancer using the Eprobe-PCR method. PLoS One. 2017;12(2):e0171225.CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Enokida Y, Shimizu K, Atsumi J, Lezhava A, Tanaka Y, Kimura Y, et al. Rapid detection of SNP (c.309T > G) in the MDM2 gene by the duplex SmartAmp method. PLoS One. 2013;8(4)CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Bayer PM, Fabian B, Hubl W. Immunofluorescence assays (IFA) and enzyme-linked immunosorbent assays (ELISA) in autoimmune disease diagnostics - technique, benefits, limitations and applications. Scand J Clin Lab Invest Suppl. 2001;61:68–76.CrossRefGoogle Scholar
  31. 31.
    Soderberg O. Detection of proteins and their interactions by proximity-ligation. Chromosom Res. 2007;15:2–3.Google Scholar

Copyright information

© Springer Nature Singapore Pte Ltd. 2018

Authors and Affiliations

  • Oleg Gusev
    • 1
    • 2
    • 3
  • Yoshihide Hayashizaki
    • 1
  • Kengo Usui
    • 4
    Email author
  1. 1.RIKEN Preventive Medicine and Diagnosis Innovation ProgramKanagawaJapan
  2. 2.KFU-RIKEN Translational Genomics UnitRIKEN Innovation CenterKanagawaJapan
  3. 3.Institute of Fundamental Medicine and BiologyKazan Federal UniversityKazanRussia
  4. 4.Genetic Diagnosis Technology UnitRIKEN Center for Integrative Medical SciencesKanagawaJapan

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