Studies For Detection Of Ochratoxin A Based On Polyketide Synthase Gene By SPR Biosensor

Ochratoxin A (OTA), whose toxicity is less only than that of aflatoxin B1(AFB1), is a kind of mycotoxin. It can contaminate foodstuffs and consequently threaten human health through food chain. Surface plasmon resonance (SPR) is a kind of novel food analyte detection techniques, which has been proven to have advantages of rapid, label-free, high-sensitive, high specific detection and low detection limit. Qualitative and quantitative analysis can be performed in real-time by using SPR biosensor.The25-base oligonucleotide sequence of polyketide synthase (PKS) gene, which was expressed during early stage of OTA synthesis, was detected respectively using direct assay and gold nanoparticles as sensing membrane assay based on the dual-channel SPR biosensor (One channel is as the reference channel to subtract background interference), thus OTA could be detected indirectly. At the same time, influence of6-mercapto-1-hexanol (MCH) blocking on SPR signal was also studied. The main contents were as follows:(1). Aspergillus ochraceus3.4520(Aspergillus ochraceus NRRL3174) was cultured by Czapek's liquid medium. The enzyme linked immunosorbent test result shows that the strain can produce OTA. After extracting the total DNAof Aspergillus ochraceus3.4520and PCR-specific amplification, the homology comparison between the PCR products and PKS gene sequences of Aspergillus ochraceus NRRL3174in Genbank showed that the sequence of the PCR products and the sequences of PKS gene of Aspergillus ochraceus NRRL3174were highly homologous.(2). A25-base oligonucleotide sequence of polyketide synthase (PKS) gene were chosen to be immobilised onto the SPR Au chip by adding a SH-to the5'end. The minimum detection concentration of the fully complementary oligonucleotide sequence was12.5nmol/L by using dual-channel SPR biosensor. In order to test the specificity of probe, two mismatch oligonucleotides were designed as a negative control. And the results showed that the mismatch oligonucleotide only hybridizes with parts of the probe, the SPR response was significantly lower than that of the fully complementary oligonucleotide. The more the number of base mismatch was, the lower the SPR response showed.(3). The experiment studied that MCH as a blocking solution affect SPR detection. The result showed that DNA probe could orientate more relative orderly when MCH as a blocking solution, thus it benefited the DNA hybridization. In addition, it could reduce the non-specific adsorption, and then increase the hybridization efficiency. Though the minimum detection concentration was the same as direct detection as12.5nmol/L, the SPR response signal increased a little. At the same time, the sample was successfully detected, which indicated that this method is feasible.(4). Gold nanoparticles (15nm) were prepared by the reduction of citrate sodium, and charactered by UV-Vis spectrophotometer and TEM. Gold nanoparticles as the sensing film of the SPR biochip using the dual-channel SPR biosensor detected PKS gene-specific25base sequence. The refractive index of gold nanoparticles is large. Moreover, surface plasmons of gold nanoparticles coupled with that of gold film surface plasmons. By applying gold nanoparticles (15nm) for signal enhancement, the minimum detection concentration dropped from12.5nmol/L to0.25nmol/L, and the sensitivity was50times higher than that of direct detection. The experiment showed that SPR signal could be dramatically enhanced with gold nanoparticles as sensing membrane instead of as label, and the operation was simple.