Jingjing Shen

Date of Award

8-2010

Level of Access Assigned by Author

Campus-Only Thesis

Degree Name

Master of Science (MS)

Department

Food Science and Human Nutrition

Advisor

Vivian C.H. Wu

Second Committee Member

Benildo G. de los Reyes

Third Committee Member

Chih-Sheng Lin

Abstract

Escherichia coli O157:H7 has been a major foodborne pathogen associated with numerous cases of fatal foodborne diseases. Therefore, there is a great need to develop an efficient method to detect this bacterium. The objective of this study was to develop a label-free DNA based colorimetric method based on gold nanoparticles (AuNPs) sandwich hybridization for rapid detection of E. coli O157:H7.

Specific probes [P1, P2; 30mer oligonucleotides with or without -SH, and with or without 12-dT] were selected for detection of E. coli O157:H7 gene eaeA. The probes were immobilized onto AuNPs surfaces. A 104 bp DNA fragment within the E. coli O157:H7 eaeA gene was amplified by PCR via the use of primer pair (E157eae/F and E157eae/R). Then the 104 bp E. coli O157:H7 target sequences were mixed with probe-functionalized AuNPs for sandwich hybridization. The detection is based on aggregation of probe-functionalized AuNPs due to sandwich hybridization of probes and the complementary target sequence. The aggregation was controlled by temperature and salt concentration of hybridization reaction and observed by color reaction.

To optimize the immobilization efficiency of probes on AuNPs, different concentrations of probes and salt solutions after immobilization were compared. Hybridization efficiency was evaluated under different hybridization temperatures. Salt solutions at various concentrations were tested for distinctive color changes after sandwich hybridization. Sensitivity study was conducted and the developed assay was for detection of E. coli O157:H7 in inoculated food samples.

It was found that the control of salt concentration plays an important role in hybridization and detection efficiency. The optimal concentration of probes with and without 12-dT was 0.5 and 0.25 μM, respectively. The optimal salt concentration after probe immobilization was 0.1 M. The highest sandwich hybridization efficiency was obtained at 50°C, and 1 M salt solution added after hybridization brought the best color differentiation among targets, non-targets, and blank. The PCR products amplified from the concentration of 3.2 x 10° CFU/ml of E. coli 0157:H7 were detectable by the developed assay. The sensitivity for the inoculated blueberry, ground beef and spinach samples was 5.2 x 10°CFU/g, 1.8 x 101 CFU/g and 8.6 x 10°CFU/g, respectively.

In conclusion, the optimized optical AuNPs sandwich hybridization assay can be used as a rapid and simple detection method for E. coli O157:H7 and the results can be read by naked eyes without needing an optical instrument.

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