Preparation of target DNA for the biosensors of Escherichia coli O157:H7

碩士 === 大同大學 === 生物工程研究所 === 92 === Rapid detection and characterization of food borne pathogens such as Escherichia coli O157:H7 is crucial for epidemiological investigations and food safety surveillance. As an alternative to conventional technologies, the nucleic acid biosensors for detecting and...

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Bibliographic Details
Main Authors: Ming-Jung Wu, 吳明蓉
Other Authors: Ming-Tse Lin
Format: Others
Language:en_US
Published: 2004
Online Access:http://ndltd.ncl.edu.tw/handle/69634945465697364219
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Summary:碩士 === 大同大學 === 生物工程研究所 === 92 === Rapid detection and characterization of food borne pathogens such as Escherichia coli O157:H7 is crucial for epidemiological investigations and food safety surveillance. As an alternative to conventional technologies, the nucleic acid biosensors for detecting and genotyping of E. coli O157:H7 were examined the sensitivity and specificity. At first, primer sets for the pathogenic and sero-type genes, eaeA, hlyA, StxI, StxII, O157, and H7, were synthesized and evidenced the specificities in detection of E. coli O157:H7. Therefore, the biosensors were fixed with oligonucleotide probes of 30mer complementary to a virulence loci intimin, eaeA. Target DNA was amplified from disrupted cells directly or from purified DNA via single polymerase chain reaction, and PCR products were hybridized to the biosensors without further modification or purification. In order to improve the sensitivity and shorten the detection time for the identification of E. coli O157:H7 in food obtained from markets, we designed a series of experiments about sample preparation. The E. coli O157:H7 was diluted appropriately and then added 100 CFU/ml into LB or BHI medium for enrichment. The E. coli O157:H7 was directly disrupted by boiling and released its genomic DNA for PCR amplification. The specific PCR products could be amplified from the boiled supernatant after culture more than 6 hr. Co-culture the 101 to 104 E. coli O157:H7 with 106 E. coli K12 organisms per ml, the specific PCR products were also amplified from the boiled media cultivated for 4 hr which was not interfered by E. coli K12. Pure cultures of E. coli O157:H7 (101 to 104 CFU/ml) could be detected within 7 h including 4 h for incubation in LB broth and others for cell disruption, PCR, and electrophoresis. Our data show that the high level of K12 in medium did not impede the detection of low levels of O157:H7. The minimum detectable numbers of cells present in the initial inoculum was 101 CFU/ml after 4-h enrichment. The amplified PCR products could be detected by hybridization with the immobilized probes on the chips of quartz crystal microbalance and optic fiber.