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


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.


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


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




Double-strand DNA


Ethylenediaminetetraacetic acid


Forward chain




Hybridization enhancement blocker


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


High-performance liquid chromatography


Listeria monocytogenes


Thermostable nuclease


Octa decyl silyl


Oligonucleotide purification cartridge


Reverse chain


Resonance unit


Staphylococcus aureus


Self-assembled monolayer


Surface plasmon resonance


Single-strand DNA


Tris-acetate-EDTA buffer


Tris-EDTA buffer



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 ( 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)


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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

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