Single Fluorescence Channel-based Multiplex Detection of Avian Influenza Virus by Quantitative PCR with Intercalating Dye (original) (raw)

Abstract

Since its invention in 1985 the polymerase chain reaction (PCR) has become a well-established method for amplification and detection of segments of double-stranded DNA. Incorporation of fluorogenic probe or DNA intercalating dyes (such as SYBR Green) into the PCR mixture allowed real-time reaction monitoring and extraction of quantitative information (qPCR). Probes with different excitation spectra enable multiplex qPCR of several DNA segments using multi-channel optical detection systems. Here we show multiplex qPCR using an economical EvaGreen-based system with single optical channel detection. Previously reported non quantitative multiplex realtime PCR techniques based on intercalating dyes were conducted once the PCR is completed by performing melting curve analysis (MCA). The technique presented in this paper is both qualitative and quantitative as it provides information about the presence of multiple DNA strands as well as the number of starting copies in the tested sample. Besides important internal control, multiplex qPCR also allows detecting concentrations of more than one DNA strand within the same sample. Detection of the avian influenza virus H7N9 by PCR is a well established method. Multiplex qPCR greatly enhances its specificity as it is capable of distinguishing both haemagglutinin (HA) and neuraminidase (NA) genes as well as their ratio.

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References (20)

  1. Saiki, R. K. et al. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230, 1350-1354 (1985).
  2. Saiki, R. K. et al. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239, 487-491 (1988).
  3. Higuchi, R., Fockler, C., Dollinger, G. & Watson, R. Kinetic PCR analysis: real-time monitoring of DNA amplification reactions. Biotechnology (N Y) 11, 1026-1030 (1993).
  4. Lee, L. G., Connell, C. R. & Bloch, W. Allelic discrimination by nick-translation PCR with fluorogenic probes. Nucleic acids research 21, 3761-3766 (1993).
  5. Kubista, M. et al. The real-time polymerase chain reaction. Molecular aspects of medicine 27, 95-125, doi: 10.1016/j. mam.2005.12.007 (2006).
  6. Cheng, J., Shoffner, M. A., Mitchelson, K. R., Kricka, L. J. & Wilding, P. Analysis of ligase chain reaction products amplified in a silicon-glass chip using capillary electrophoresis. Journal of chromatography. A 732, 151-158 (1996).
  7. Huang, Q. et al. Multicolor combinatorial probe coding for real-time PCR. PloS one 6, e16033, doi: 10.1371/journal.pone.0016033 (2011).
  8. Fixman, M. & Freire, J. J. Theory of DNA melting curves. Biopolymers 16, 2693-2704, doi: 10.1002/bip.1977.360161209 (1977).
  9. Andersson, A. et al. Paired multiplex reverse-transcriptase polymerase chain reaction (PMRT-PCR) analysis as a rapid and accurate diagnostic tool for the detection of MLL fusion genes in hematologic malignancies. Leukemia 15, 1293-1300 (2001).
  10. Gubala, A. J. Multiplex real-time PCR detection of Vibrio cholerae. Journal of microbiological methods 65, 278-293, doi: 10.1016/j. mimet.2005.07.017 (2006).
  11. Wittwer, C. T., Herrmann, M. G., Moss, A. A. & Rasmussen, R. P. Continuous fluorescence monitoring of rapid cycle DNA amplification. BioTechniques 22, 130-131, 134-138 (1997).
  12. Ririe, K. M., Rasmussen, R. P. & Wittwer, C. T. Product differentiation by analysis of DNA melting curves during the polymerase chain reaction. Analytical biochemistry 245, 154-160, doi: 10.1006/abio.1996.9916 (1997).
  13. Neuzil, P., Pipper, J. & Hsieh, T. M. Disposable real-time microPCR device: lab-on-a-chip at a low cost. Molecular bioSystems 2, 292-298, doi: 10.1039/b605957k (2006).
  14. Neuzil, P., Karasek, K., Sun, W.-X. & Manz, A. Nanoliter-sized Overheated Reactor. Applied Physics Letters 106, 024104, doi: 10.1063/1.4905851 (2015).
  15. Corman, V. M. E. M., Landt, O., Bleicker, T., Brünink, S., Eschbach-Bludau, M., Matrosovich, M., Becker, S., Drosten, C. Specific detection by real-time reverse-transcription reaction assays of a novel avian influenza A(H7N9) strain associated with human spillover infections in China. Euro Surveill. 18 (2013).
  16. Mao, F., Leung, W.-Y. & Xin, X. Characterization of EvaGreen and the implication of its physicochemical properties for qPCR applications. BMC Biotechnology 7, 76 (2007).
  17. Pasay, C. et al. High-resolution melt analysis for the detection of a mutation associated with permethrin resistance in a population of scabies mites. Medical and Veterinary Entomology 22, 82-88, doi: 10.1111/j.1365-2915.2008.00716.x (2008).
  18. Neuzil, P., Cheng, F., Soon, J. B., Qian, L. L. & Reboud, J. Non-contact fluorescent bleaching-independent method for temperature measurement in microfluidic systems based on DNA melting curves. Lab on a chip 10, 2818-2821, doi: 10.1039/c005243d (2010).
  19. Pipper, J. et al. Catching bird flu in a droplet. Nat Med 13, 1259-1263, doi: 10.1038/Nm1634 (2007).
  20. Pipper, J., Zhang, Y., Neuzil, P. & Hsieh, T. M. Clockwork PCR including sample preparation. Angew Chem Int Ed Engl 47, 3900-3904, doi: 10.1002/anie.200705016 (2008).