Optimal Conditions for the Asymmetric Polymerase Chain Reaction for Detecting Food Pathogenic Bacteria Using a Personal SPR Sensor
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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.
KeywordsSurface 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
Hybridization enhancement blocker
High-performance liquid chromatography
Octa decyl silyl
Oligonucleotide purification cartridge
Surface plasmon resonance
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.
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