臺灣博碩士論文加值系統

快速偵測與定性諸如大腸桿菌O157:H7之食品致病菌，對於流行病學研究與食品安全檢測是相當重要。核苷酸生物感測器為對於大腸桿菌O157:H7之偵測與基因型判定，具有敏感性與專一性的另一種方便的技術。
首先針對致病性與血清型基因eaeA, hlyA, StxI, StxII, O157與H7之引子組合進行設計合成，並証明對大腸桿菌O157:H7具專一性之偵測。之後於生物感測器上固定互補於致病基因eaeA 之30 個核苷酸長之探針，及自破碎細胞或純化之基因體DNA，藉由聚合酶連鎖反應進行合成，以增生目標DNA，此PCR產物並不需經過修飾或純化即可雜交至生物感測器上。
為能提高存在於市售食品中大腸桿菌O157:H7之檢測敏感度與縮短偵測時間，我們設計一系列的樣本製備實驗。收集並適當稀釋大腸桿菌O157:H7，然後以100 CFU/ml之數量接種於LB或BHI培養基中進行增殖，然後大腸桿菌O157:H7以煮沸方式破碎菌體並釋出基因體DNA以進行PCR合成反應，而自培養至少6小時後經煮沸處理的上清液，可合成具專一性的PCR產物。
大腸桿菌O157:H7(101至104 CFU/ml) 與大腸桿菌K12共同培養4小時而經煮沸處理之LB培養基，亦能夠增生出具專一性的PCR產物，而不受大腸桿菌K12干擾。
大腸桿菌O157:H7 (101至104 CFU/ml) 的純系培養，能在包含4小時培養與其他如細胞破碎、PCR反應及電泳等步驟操作，而於7小時內成功偵測。與高濃度大腸桿菌K12共同培養並不會影響對於低量大腸桿菌O157:H7之偵測，經4小時增殖後的最低之可偵測量可達到101 CFU/ ml。增生後之PCR產物，可與固定於石英晶體微天平晶片和光纖上之探針進行雜交而被偵測。

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.

ABSTRACT I
中文摘要 III
CONTENT V
LIST OF TABLES IX
LIST OF FIGURES X
NOMENCLATURE XIII
CHAPTER 1 INTRODUCTION 1
1.1 Genosensors 2
1.1.1 Direct sequencing 2
1.1.2 DNA hybridization 2
1.1.3 Electrochemical devices 5
1.1.4 Optical devices 7
1.1.5 Acoustic wave devices 9
1.1.6 DNA microchips 13
1.2 Enterohemorrhagic Escherichia coli O157:H7 15
1.2.1 Introduction of Escherichia coli O157:H7 16
1.2.2 E. coli O157:H7 spread 18
1.2.3 Illness caused by E. coli O157:H7 19
1.2.4 E. coli O157:H7 infection diagnosed 22
1.2.5 E. coli O157:H7 in Apple cider 22
1.3 Methods to detect E. coli O157:H7 24
1.3.1 Polymerase chain reaction (PCR) 25
1.3.2 Infrared spectroscopy 27
1.3.3 Bioluminescence 27
1.3.4 Immunomagnetic flow cytometry 28
1.3.5 Time-resolved fluorometry (TRF) 28
1.4 Sample preparation methods for PCR detection 29
1.5 The framework of this monographic study 30
CHAPTER 2 MATERIALS AND METHODS 32
2.1 Materials 32
2.1.1 Chemicals and reagents 32
2.1.2 Bacteria strains 33
2.2 Instruments and Software 33
2.2.1 Instruments 33
2.2.2 Software for DNA sequence analysis 35
2.3 Designs for Primers and oligonucleotide probes 36
2.4 Sample preparation 37
2.4.1. Bacterial strains and their culture conditions 37
2.4.2. Preparation of purified bacteria DNA 38
2.4.3 Cell disruption by boiling method 38
2.4.4 Comparison of enrichment media 39
2.4.5 Preparation of E. coli mixtures 40
2.5 Detection by polymerase chain reaction 40
2.5.1 Polymerase chain reaction 40
2.5.2 Study for optimal annealing temperature 41
2.5.3 Primers specificity 42
2.6 Optical fiber 42
2.6.1 Coating of fibers 42
2.6.2 Detection by fiber-optic biosensor 44
2.7 Liquid phase quartz crystal microbalance 44
2.7.1 Preparation of QCM chip 44
2.7.2 QCM detection 45
CHAPTER 3 RESULTS AND DISCUSSION 47
3.1 Primer design 47
3.2 Detection of E. coli O157:H7 by PCR 48
3.2.1 Polymerase chain reaction of E. coli O157:H7 48
3.2.2 The specificity of primers 58
3.2.3 PCR from crude DNA released by boiling method 58
3.3 Culture enrichment 62

3.3.1 Comparison of enrichment media 62
3.3.2 Detection in mixing culture 68
3.4 Detection of fiber-optic biosensor for E. coli O157:H7 72
3.5 Detection of QCM for E. coli O157:H7 76
CHAPTER 4 CONCLUSIONS
CHAPTER 5 REFERENCES 81

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