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

• Published in: Applied biochemistry and biotechnology Vol. 187; no. 1; pp. 323 - 337
• Main Authors: Nagai, Haruka, Tomioka, Kanji, Okumura, Shiro
• Format: Journal Article
• Language: English
• Published: New York Springer US 01.01.2019; Springer Nature B.V
• Subjects: Amplification; Bacteria; Base Sequence; Biochemistry; Biosensing Techniques; Biotechnology; Chemistry; Chemistry and Materials Science; Chromatography, High Pressure Liquid - methods; Deoxyribonucleic acid; DNA; DNA Primers; DNA, Bacterial - isolation & purification; DNA, Single-Stranded - genetics; Electrophoresis; Electrophoresis, Polyacrylamide Gel; Fluorescence; Food; Food chains; Food contamination; Food Industry; Food Microbiology; Food poisoning; Food processing industry; Gels; Genes, Bacterial; High performance liquid chromatography; Liquid chromatography; Microbiological Techniques; Optical Imaging; Point-of-Care Systems; Poisoning; Polymerase chain reaction; Polymerase Chain Reaction - methods; Primers; Sensors; Single-stranded DNA; Staphylococcus aureus - genetics; Staphylococcus aureus - isolation & purification; Surface plasmon resonance; Surface Plasmon Resonance - instrumentation; Single-strand DNA; Surface plasmon resonance; Asymmetric PCR; HPLC; Food-poisoning bacteria
• DOI: 10.1007/s12010-018-2819-y
• ISSN: 0273-2289

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.