Skip to main content
SpringerLink
Log in

A Fluorescence Sensing Method with Reduced DNA Typing and Low-Cost Instrumentation for Detection of Sample Tampering Cases in Urinalysis

  • Original Article
  • Published:
Annals of Biomedical Engineering Aims and scope Submit manuscript

Abstract

This work presents a method to unequivocally detect urine sample tampering in cases where integrity of the sample needs to be verified prior to urinalysis. The technique involves the detection of distinct patterns of a triplex short tandem repeats system in DNA extracted from human urine. The analysis is realized with single-dye fluorescence detection and using a regular smartphone camera. The experimental results had demonstrated the efficacy of the analytical approach to obtaining distinct profiles of amplicons in urine from different sample providers. Reproducibility tests with fresh and stored urine have revealed a maximum variation in the profiles within an interval of 5 to 9%. Cases of urine sample tampering via mixture were simulated in the study, and the experiments have identified patterns of mixed genotypes from dual mixtures of urine samples. Moreover, sample adulteration by mixing a non-human fluid with urine in a volume ratio over 25% can be detected. The low cost of the approach is accompanied by the compatibility of the technique to use with different DNA sample preparation protocols and PCR instrumentation. Furthermore, the possibility of realizing the method in an integrated microchip system open great perspectives to conducting sample integrity tests at the site of urine sample reception and/or at resource-limited settings.

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.

Institutional subscriptions

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6

Similar content being viewed by others

References

  1. Akram, N. A., and S. R. Farooqi. Inferring human phylogenies using three CODIS STR markers (CSF1PO, TPOX and TH01). Int. J. Biol. 2015. https://doi.org/10.5539/ijb.v7n1p1.

    Article  Google Scholar 

  2. Aoki, K., H. Tanaka, and M. Ueki. DNA typing for personal identification of urine after long-term preservation for testing in doping control. Drug Test Anal. 9:1116–1123, 2017.

    Article  CAS  Google Scholar 

  3. Bienvenue, J. M., L. A. Legendre, J. P. Ferrance, and J. P. Landers. An integrated microfluidic device for DNA purification and PCR amplification of STR fragments. Forensic Sci. Int. Gen. 4:178–186, 2010.

    Article  CAS  Google Scholar 

  4. Bijlsma, L., E. Beltrán, C. Boix, J. V. Sancho, and F. Hernández. Improvements in analytical methodology for the determination of frequently consumed illicit drugs in urban wastewater. Anal. Bioanal. Chem. 406:4261–4272, 2014.

    Article  CAS  Google Scholar 

  5. Brito, F. C. A., D. R. B. M. Prata, S. F. P. Martha, and C. G. Bottino. Evaluation of the urinary bladder swabs as a source of DNA for human identification using two different extraction methods. Forensic Sci. Int. Gen. 5:e484–e486, 2015.

    Article  Google Scholar 

  6. Budowle, B., T. R. Moretti, K. M. Keys, B. W. Koons, and J. B. Smerick. Validation studies of the CTT STR multiplex systems. J. Forensic Sci. 42:701–707, 1997.

    CAS  PubMed  Google Scholar 

  7. Castaneto, M. S., A. J. Barnes, M. Concheiro, K. L. Klette, T. A. Martin, and M. A. Huestis. Biochip array technology immunoassay performance and quantitative confirmation of designer piperazines for urine workplace drug testing. Anal. Bioanal. Chem. 407:4639–4648, 2015.

    Article  CAS  Google Scholar 

  8. Castella, V., B. Dimo-Simonin, C. Brandt-Casadevall, N. Robinson, M. Saugy, F. Taroni, and P. Mangin. Forensic identification of urine samples: a comparison between nuclear and mitochondrial DNA markers. Int. J. Legal Med. 120:67–72, 2006.

    Article  CAS  Google Scholar 

  9. de Pancorbo, M., A. Castro, I. Fernández-Fernández, and A. García-Orad. Population genetics and forensic applications Using multiplex PCR (CSF1PO, TPOX, and TH01) loci in the Basque Country. J. Forensic Sci. 43:1181–1187, 1998.

    Article  Google Scholar 

  10. Devesse, L., D. S. Court, and D. Cowan. Determining the authenticity of athlete urine in doping control by DNA analysis. Drug Test Anal. 7:912–918, 2015.

    Article  CAS  Google Scholar 

  11. Dong, T., and N. M. M. Pires. Immunodetection of salivary biomarkers by an optical microfluidic biosensor with polyethylenimine-modified polythiophene-C70 organic photodetectors. Biosens. Bioelectron. 94:321–327, 2017.

    Article  CAS  Google Scholar 

  12. DuVall, J. A., D. Le Roux, B. L. Thompson, C. Birch, D. A. Nelson, J. Li, D. L. Mills, A.-C. Tsuei, M. G. Ensenberger, C. Sprecher, D. R. Storts, B. E. Root, and J. P. Landers. Rapid multiplex DNA amplification on an expensive microdevice for human identification via short tandem repeat analysis. Anal. Chim. Acta 980:41–49, 2017.

    Article  CAS  Google Scholar 

  13. Fu, S. Adulterants in urine drug testing. Adv. Clin. Chem. 76:123–163, 2016.

    Article  CAS  Google Scholar 

  14. Han, J., W. Gan, B. Zhuang, J. Sun, L. Zhao, J. Ye, Y. Liu, C.-X. Li, and P. Liu. A fully integrated microchip system for automated forensic short tandem repeat analysis. Analyst 142:2004–2012, 2017.

    Article  CAS  Google Scholar 

  15. Hochmeister, M. N. Use of the GenePrint™ CSF1PO, TPOX, TH01 multiplex system (CTT) for the analysis of DNA evidence in a serial killer case, profiles in DNA. Promega Corp. 1:3–6, 1997.

    Google Scholar 

  16. Hopwood, A. J., C. Hurth, J. Yang, Z. Cai, N. Moran, J. G. Lee-Edghill, A. Nordquist, R. Lenigk, M. D. Estes, J. P. Haley, C. R. McAlister, X. Chen, C. Brooks, S. Smith, K. Elliott, P. Koumi, F. Zenhausern, and G. Tully. Integrated microfluidic system for rapid forensic DNA analysis: sample collection to DNA profile. Anal. Chem. 82:6991–6999, 2010.

    Article  CAS  Google Scholar 

  17. Jaffee, W. B., E. Trucco, S. Levy, and R. D. Weiss. Is this urine really negative? A systematic review of tampering methods in urine drug screening and testing. J. Subst. Abuse Treat. 33:33–42, 2007.

    Article  Google Scholar 

  18. Karlsen, H., and T. Dong. Smartphone-based rapid screening of urinary biomarkers. IEEE Trans. Biomed. Circuits Syst. 11:455–463, 2017.

    Article  Google Scholar 

  19. Kim, Y. T., D. Lee, H. Y. Heo, J. E. Sim, K. M. Woo, D. H. Kim, S. G. Im, and T. S. Seo. Total integrated slidable and valveless solid phase extraction-polymerase chain reaction-capillary electrophoresis microdevice for mini Y chromosome short tandem repeat genotyping. Biosens. Bioelectron. 78:489–496, 2016.

    Article  CAS  Google Scholar 

  20. Liu, P., J. R. Scherer, S. A. Greenspon, T. N. Chiesl, and R. A. Mathies. Integrated sample cleanup and capillary array electrophoresis microchip for forensic short tandem repeat analysis. Forensic Sci. Int. Gen. 5:484–492, 2011.

    Article  CAS  Google Scholar 

  21. Liu, P., T. S. Seo, N. Beyor, K.-J. Shin, J. R. Scherer, and R. A. Mathies. Integrated portable polymerase chain reaction-capillary electrophoresis microsystem for rapid forensic short tandem repeat typing. Anal. Chem. 79:1881–1889, 2007.

    Article  CAS  Google Scholar 

  22. McNamara-Schroeder, K., C. Olonan, S. Chu, M. C. Montoya, M. Alviri, S. Ginty, and J. J. Love. DNA fingerprint analysis of three short tandem repeat (STR) loci for biochemistry and forensic science laboratory courses. Biochem. Mol. Biol. Edu. 34:378–383, 2006.

    Article  CAS  Google Scholar 

  23. Moretti, T. R., L. I. Moreno, J. B. Smerick, M. L. Pignone, R. Hizon, J. S. Buckleton, J.-A. Bright, and A. J. Onorato. Population data on the expanded CODIS core STR loci for eleven populations of significance for forensic DNA analyses in the United States. Forensic Sci. Int. Gen. 25:175–181, 2016.

    Article  CAS  Google Scholar 

  24. Neste, C. V., F. V. Nieuwerburgh, D. V. Hoofstat, and D. Deforce. Forensic STR analysis using massive parallel sequencing. Forensic Sci. Int. Gen. 6:810–818, 2012.

    Article  Google Scholar 

  25. Schmalzing, D., L. Koutny, A. Adourian, P. Belgrader, P. Matsudaira, and D. Ehrlich. DNA typing in thirty seconds with a microfabricated device. Proc. Natl. Acad. Sci. USA 94:10273–10278, 1997.

    Article  CAS  Google Scholar 

  26. Schumm, J. W. GenePrint™ STR multiplexes: reliability, flexibility and throughput in database and casework: compatible STR analysis, profiles in DNA. Promega Corp. 1:9–13, 1997.

    Google Scholar 

  27. Selbes, Y. S., M. G. Caglayan, M. Eryilmaz, I. H. Boyaci, N. Saglam, A. A. Basaran, and U. Tamer. Surface-enhanced Raman probe for rapid nanoextraction and detection of erythropoietin in urine. Anal. Bioanal. Chem. 498:8447–8456, 2016.

    Article  Google Scholar 

  28. Tachibana, H., M. Hiroaki, S. Shibuya, K. Tsuji, N. Miyagawa, K. Yamanaka, and E. Tamiya. On-chip quantitative detection of pathogen genes by autonomous microfluidic PCR platform. Biosens. Bioelectron. 74:725–730, 2015.

    Article  CAS  Google Scholar 

  29. Thevis, M., H. Geyer, U. Mareck, G. Sigmund, J. Henke, L. Henke, and W. Schanzer. Detection of manipulation in doping control urine sample collection: a multidisciplinary approach to determine identical urine samples. Anal. Bioanal. Chem. 388:1539–1543, 2007.

    Article  CAS  Google Scholar 

  30. Thevis, M., H. Geyer, G. Sigmund, and W. Schanzer. Sports drug testing: analytical aspects of selected cases of suspected, purpoted, and proven urine manipulation. J. Pharm. Biomed. 57:26–32, 2012.

    Article  Google Scholar 

  31. Thevis, M., H. Geyer, L. Tretzel, and W. Schanzer. Sports drug testing using complementary matrices: advantages and limitations. J. Pharm. Biomed. 130:220–230, 2016.

    Article  CAS  Google Scholar 

  32. Thevis, M., O. Krug, H. Geyer, K. Walpurgis, N. Baume, and A. Thomas. Analytical challenges in sports drug testing. Anal. Bioanal. Chem. 410:2275–2281, 2018.

    Article  CAS  Google Scholar 

  33. Tsivou, M., E. Giannadaki, F. Hooghe, K. Roels, W. V. Gansbeke, F. Garribba, E. Lyris, K. Deventer, M. Mazzarino, F. Donati, D. G. Georgakopoulos, P. V. Eenoo, C. G. Georgakopoulos, X. de la Torre, and F. Botrè. Doping control container for urine stabilization: a pilot study. Drug Test Anal. 9:699–712, 2016.

    Article  Google Scholar 

  34. Tsukada, K., K. Takayanagi, H. Asamura, M. Ota, and H. Fukushima. Multiplex short tandem repeat typing in degraded samples using newly designed primers for the TH01, TPOX, CSF1PO, and vWA loci. Legal Med. 4:239–245, 2002.

    Article  CAS  Google Scholar 

  35. Vashist, S. K., O. Mudanyali, M. Schneider, R. Zengerle, and A. Ozcan. Cellphone-based devices for bioanalytical sciences. Anal. Bioanal. Chem. 406:3263–3277, 2014.

    Article  CAS  Google Scholar 

  36. Zhao, X., T. Dong, Z. Yang, N. Pires, and N. Hoivik. Compatible immuno-NASBA LOC device for quantitative detection of waterborne pathogens: design and validation. Lab Chip 12:602–612, 2012.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

The authors acknowledge the funds from the National Natural Science Foundation of China (Grant Nos. 61650410655 and 61550110253). Furthermore, the work is also a major result of the research efforts conducted in the NFR-FORNY SENS-U project (Proj. No. 268481), with the aim to develop smartphone-based methods and devices for personalized urinalysis. Chongqing Xiji Hospital is acknowledged for the support to the experiments. The authors would like to specially thank Zhongqiang Li for his prodigious work on previous INNOMED URUS project report, served as part of the basis to new FORNY SENS-U project. Design and experimental concepts were partially motivated from the aforementioned report prepared by Zhongqiang Li to whom we are heartily thankful. Thanks are also given to lab assistance and discussions from Luis F.B.A. da Silva and Birgitte Hønsvall. The support from RFF Oslofjordfondet (Proj. No. 285575), Science and Technology Research Program of Chongqing Education Commission (Grant No. KJZD-K201800802) and Chongqing Research Program of Basic Research and Frontier Technology (Proj. Nos. cstc2017jcyjA1842, cstc2018jcyjA4046) are also acknowledged.

Conflict of interest

The authors declare that they have no conflict of interest.

Author information

Authors and Affiliations

  1. Chongqing Key Laboratory of Micro-Nano Systems and Smart Transduction, Collaborative Innovation Center on Micro-Nano Transduction and Intelligent Eco-Internet of Things, Chongqing Key Laboratory of Colleges and Universities on Micro-Nano Systems Technology and Smart Transducing, National Research Base of Intelligent Manufacturing Service, Chongqing Technology and Business University, Nan’an District, Chongqing, 400067, China

    Nuno M. M. Pires, Zhaochu Yang & Simão M. B. Santos

  2. Department of Microsystems – IMS, Faculty of Technology, Natural Sciences and Maritime Sciences, University of South-Eastern Norway-USN, P.O. Box 235, 3603, Kongsberg, Norway

    Nuno M. M. Pires, Tao Dong & Simão M. B. Santos

Authors
  1. Nuno M. M. Pires
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tao Dong
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Zhaochu Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Simão M. B. Santos
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Tao Dong.

Additional information

Associate Editor Stefan M. Duma oversaw the review of this article.

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (PDF 253 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Pires, N.M.M., Dong, T., Yang, Z. et al. A Fluorescence Sensing Method with Reduced DNA Typing and Low-Cost Instrumentation for Detection of Sample Tampering Cases in Urinalysis. Ann Biomed Eng 48, 644–654 (2020). https://doi.org/10.1007/s10439-019-02386-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10439-019-02386-y

Keywords

Search

Navigation