Advertisement

Applied Biochemistry and Biotechnology

, Volume 187, Issue 1, pp 323–337 | Cite as

Optimal Conditions for the Asymmetric Polymerase Chain Reaction for Detecting Food Pathogenic Bacteria Using a Personal SPR Sensor

  • Haruka Nagai
  • Kanji Tomioka
  • Shiro OkumuraEmail author
Article

Abstract

We have been developing quick and simple system for detecting food-poisoning bacteria using a combination of an asymmetric PCR and a portable surface plasmon resonance (SPR) sensor. The system would be suitable for point-of-care detection of food-poisoning bacteria in the field of food industry. In this study, we established a novel method for quantifying the amplified forward (F) and reverse (R) chains of Staphylococcus aureus separately by high-performance liquid chromatography (HPLC). The concentration of single-stranded DNA amplicon excessively amplified, which is crucial for the system, could be calculated as the difference between those of the F- and R-chains. For the R-chain, a correction based on the F-chain concentration in the sample was used to obtain a more accurate value, because the determination of the R-chain concentration was affected by that of the coexisting F-chain. The concentration values were also determined by fluorescence imaging for electrophoresis gels of amplicons with FITC- or Cy5-conjugated primers, and they were in good agreement with the values by the HPLC. The measured concentration of the single-strand F-chain correlated well with the value of the SPR response against the probe that was a complementary sequence of the F-chain, immobilized on the sensor chip of the SPR sensor.

Keywords

Surface plasmon resonance Food-poisoning bacteria HPLC Asymmetric PCR Single-strand DNA 

Abbreviations

Buffer A

10 mM HEPES-NaOH buffer, pH 7.4, containing 1 mM EDTA, 0.05% polyoxyethylene (20) sorbitan monolaurate, and 150 mM NaCl

Buffer B

10 mM HEPES-NaOH buffer, pH 7.4, containing 1 mM EDTA, 0.05% polyoxyethylene (20) sorbitan monolaurate, and 1000 mM NaCl

dNTP

Deoxynucleotide

dsDNA

Double-strand DNA

EDTA

Ethylenediaminetetraacetic acid

F-chain

Forward chain

FITC

Fluoresceinisothiocyanate

HEB

Hybridization enhancement blocker

HEPES

4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid

HPLC

High-performance liquid chromatography

LM

Listeria monocytogenes

nucA

Thermostable nuclease

ODS

Octa decyl silyl

OPC

Oligonucleotide purification cartridge

R-chain

Reverse chain

RU

Resonance unit

SA

Staphylococcus aureus

SAM

Self-assembled monolayer

SPR

Surface plasmon resonance

ssDNA

Single-strand DNA

TAE

Tris-acetate-EDTA buffer

TE

Tris-EDTA buffer

Notes

Acknowledgments

We thank Mr. Munehiro Iwakura and Mr. Shinya Azuma of Kyushu Keisokki Co., Ltd. (Fukuoka, Japan) for providing the compact SPR sensor used in this research. We thank Philip Creed, PhD, from Edanz Group (www.edanzediting.com/ac) for editing a draft of this manuscript.

Compliance with Ethical Standards

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Supplementary material

12010_2018_2819_Fig6_ESM.png (98 kb)
Supplementary Fig. 1

Molar concentrations of primers used and the sum of the determined value of amplicons and the primers remaining. The asymmetric PCR products were examined using a total concentration of 1 μM primers with the various sets of forward: reverse primers at ratios from 1:1 to 79:1. The concentrations of the amplified F- and R-chains in the PCR products and the remaining forward and reverse primers were determined by HPLC. (a) The sum of amplified F-chains and remaining F-primers were compared with the concentration of forward primers used in the PCR. (b) The concentrations of the R-primer used were also compared with the sum of the remaining R-primer and R-chain. The HPLC measurements were made three times. The error bars indicate the standard deviation. (PNG 98 kb)

12010_2018_2819_MOESM1_ESM.eps (636 kb)
High resolution image (EPS 635 kb)
12010_2018_2819_Fig7_ESM.png (501 kb)
Supplementary Fig. 2

Electrophoretic images of the asymmetric PCR products with fluorescence primers. The asymmetric PCR products with various primer ratios were determined by acrylamide gel electrophoresis. The FITC conjugated forward primer and F-chain derived from the primer were detected by a fluorescence scanner (Typhoon 9200) with a 526-nm filter (shown in green). The Cy5 conjugated reverse primer and R-chain derived from the primer were detected with a 655–685-nm filter (shown in red). (b) The two images were merged. The gel was then stained with SYBR Green and the total gel image was captured by UV irradiation (a). The orange bands in (b) indicated by a black arrow were 138 bp double stranded target DNA. The green bands indicated by a white arrow were the single stranded F-chain of the target DNA. All bands less than 50 bp are the remaining primers. (PNG 500 kb)

12010_2018_2819_MOESM2_ESM.eps (2 mb)
High resolution image (EPS 2062 kb)

References

  1. 1.
    Japan Food Hygiene Association. (2015). Standard methods of analysis in food safety regulation (9th ed.). Tokyo: Japan Food Hygiene Association. Retrieved from http://iss.ndl.go.jp/books/R100000002-I026300447-00
  2. 2.
    Law, J. W. F., Mutalib, N. S. A., Chan, K. G., & Lee, L. H. (2014). Rapid metho ds for the detection of foodborne bacterial pathogens: Principles, applications, advantages and limitations. Frontiers in Microbiology, 5(DEC), 1–19.  https://doi.org/10.3389/fmicb.2014.00770.
  3. 3.
    Bhardwaj, N., Bhardwaj, S. K., Nayak, M. K., Mehta, J., Kim, K. H., & Deep, A. (2017). Fluorescent nanobiosensors for the targeted detection of foodborne bacteria. TrAC - Trends in Analytical Chemistry, 97, 120–135.  https://doi.org/10.1016/j.trac.2017.09.010.CrossRefGoogle Scholar
  4. 4.
    Malhotra, B. D., Srivastava, S., Ali, M. A., & Singh, C. (2014). Nanomaterial-based biosensors for food toxin detection. Applied Biochemistry and Biotechnology, 174(3), 880–896.  https://doi.org/10.1007/s12010-014-0993-0.CrossRefGoogle Scholar
  5. 5.
    Liu, X., & Zhang, X. (2015). Aptamer-based Technology for Food Analysis. Applied Biochemistry and Biotechnology, 175(1), 603–624.  https://doi.org/10.1007/s12010-014-1289-0.CrossRefGoogle Scholar
  6. 6.
    Park, H. C., Baig, I. A., Lee, S. C., Moon, J. Y., & Yoon, M. Y. (2014). Development of ssDNA aptamers for the sensitive detection of Salmonella typhimurium and Salmonella enteritidis. Applied Biochemistry and Biotechnology, 174(2), 793–802.  https://doi.org/10.1007/s12010-014-1103-z.CrossRefGoogle Scholar
  7. 7.
    Zhang, Y., Zhu, L., Zhang, Y., He, P., & Wang, Q. (2018). Simultaneous detection of three foodborne pathogenic bacteria in food samples by microchip capillary electrophoresis in combination with polymerase chain reaction. Journal of Chromatography A, 1555, 100–105.  https://doi.org/10.1016/j.chroma.2018.04.058.CrossRefGoogle Scholar
  8. 8.
    Malorny, B., Huehn, S., Dieckmann, R., Krämer, N., & Helmuth, R. (2009). Food analytical methods. Food analytical methods (Vol. 2). Springer. Retrieved from https://www.cabdirect.org/cabdirect/abstract/20093201005
  9. 9.
    Agarwal, A., Makker, A., & Goel, S. K. (2002). Application of the PCR technique for a rapid, specific and sensitive detection of salmonella spp. In foods. Molecular and Cellular Probes, 16(4), 243–250.  https://doi.org/10.1006/mcpr.2002.0418.CrossRefGoogle Scholar
  10. 10.
    Li, Y., Zhuang, S., & Mustapha, A. (2005). Application of a multiplex PCR for the simultaneous detection of Escherichia coli O157:H7, salmonella and Shigella in raw and ready-to-eat meat products. Meat Science, 71(2), 402–406.  https://doi.org/10.1016/j.meatsci.2005.04.013.CrossRefGoogle Scholar
  11. 11.
    Homola, J. (2008). Surface plasmon resonance sensors for detection of chemical and biological species. Chemical Reviews, 108(2), 462–493.  https://doi.org/10.1021/cr068107d.CrossRefGoogle Scholar
  12. 12.
    Wang, X.-W., Zhang, L., Jin, L.-Q., Jin, M., Shen, Z.-Q., An, S., et al. (2007). Development and application of an oligonucleotide microarray for the detection of food-borne bacterial pathogens. Applied Microbiology and Biotechnology, 76(1), 225–233.  https://doi.org/10.1007/s00253-007-0993-x.CrossRefGoogle Scholar
  13. 13.
    Kobayashi, H., Kubota, J., Fujihara, K., Honjoh, K., Iio, M., Fujiki, N., et al. (2009). Simultaneous enrichment of Salmonella spp., Escherichia coli O157:H7, Vibrio parahaemolyticus, Staphylococcus aureus, Bacillus cereus, and Listeria monocytogenes by single broth and screening of the pathogens by multiplex real-time PCR. Food Science and Technology Research, 15(4), 427–438.  https://doi.org/10.3136/fstr.15.427.CrossRefGoogle Scholar
  14. 14.
    Kim, D.-K., Kerman, K., Saito, M., Sathuluri, R. R., Endo, T., Yamamura, S., et al. (2007). Label-free DNA biosensor based on localized surface Plasmon resonance coupled with interferometry. Analytical Chemistry, 79(5), 1855–1864.  https://doi.org/10.1021/ac061909o.CrossRefGoogle Scholar
  15. 15.
    Singh, A., Verma, H. N., & Arora, K. (2014). Surface Plasmon resonance based label-free detection of salmonella using DNA self assembly. Applied Biochemistry and Biotechnology, 175(3), 1330–1343.  https://doi.org/10.1007/s12010-014-1319-y.CrossRefGoogle Scholar
  16. 16.
    Okumura, S., Kuroda, R., & Inouye, K. (2014). Single nucleotide polymorphism typing with a surface Plasmon resonance-based sensor using hybridization enhancement blockers. Applied Biochemistry and Biotechnology, 174(2), 494–505.  https://doi.org/10.1007/s12010-014-1072-2.CrossRefGoogle Scholar
  17. 17.
    Pattnaik, P. (2005). Surface Plasmon resonance: Applications in understanding receptor–ligand interaction. Applied Biochemistry and Biotechnology, 126(2), 079–092.  https://doi.org/10.1385/ABAB:126:2:079.CrossRefGoogle Scholar
  18. 18.
    Miura, K. (2001). Imaging and detection technologies for image analysis in electrophoresis. ELECTROPHORESIS, 22(5), 801–813.  https://doi.org/10.1002/1522-2683()22:5<801::AID-ELPS801>3.0.CO;2-X.CrossRefGoogle Scholar
  19. 19.
    Katz, E. D. (1996). Quantitation and purification of polymerase chain reaction products by high-performance liquid chromatography. Molecular Biotechnology, 6(1), 79–86.CrossRefGoogle Scholar
  20. 20.
    Devaney, J. M., Pettit, E., Kaler, S. G., Vallone, P. M., Butler, J. M., & Marino, M. A. (2001). Genotyping of two mutations in the HFE gene using single-base extension and high-performance liquid chromatography. Analytical Chemistry, 73(3), 620–624.  https://doi.org/10.1021/ac000912j.CrossRefGoogle Scholar
  21. 21.
    Devaney, J. M., Girard, J. E., Marino, M. A., Road, F., & Suite, E. (2000). DNA microsatellite analysis using ion-pair reversed-phase high-performance liquid chromatography. Analytical Chemistry, 72(4), 858–864.  https://doi.org/10.1021/ac9908896.CrossRefGoogle Scholar
  22. 22.
    Marimuthu, C., Tang, T.-H., Tominaga, J., Tan, S.-C., & Gopinath, S. C. B. (2012). Single-stranded DNA (ssDNA) production in DNA aptamer generation. The Analyst, 137(6), 1307.  https://doi.org/10.1039/c2an15905h.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Biochemistry and Applied ChemistryNational Institute of TechnologyFukuokaJapan
  2. 2.Biotechnology and Food Research InstituteFukuoka Industrial Technology CenterFukuokaJapan

Personalised recommendations