臺灣博碩士論文加值系統

針對犬貓泌尿道感染以及血液感染，現今仍以細菌培養為目前臨床上診斷的黃金標準。而培養及菌種鑑定時間至少約需72小時，且在血液檢體上的敏感性甚低，不但降低了臨床醫師進行細菌培養的意願，且因只能盲目地使用經驗性抗生素，又可能造成抗生素的濫用。因此，在臨床上急需更快速且具高敏感性之診斷方法。目前微生物演化分類上最被廣泛使用的親源標記分子為rRNA基因，其中以16S rRNA基因長短適中，最常應用於細菌之分類與鑑定，它由許多高度變異區（variable）及保留區（conserved）之片段所組成，其變異區具有序列多樣性，可分為v1到v9區段，因研究顯示v2, v3及v6這些片段鑑定菌種的能力幾乎與全長的16S rRNA基因片段能力相當，適合作為菌種鑑定所用。因此本實驗擬利用多數細菌共有的16S rRNA v3片段所製成之universal primer，以聚合酶鏈鎖反應方式增幅細菌DNA，先針對過去在本院曾經培養過的細菌進行測試，接下來則收集臨床上已知或懷疑有感染的待測犬貓尿液、血液檢體及其他體液（如腹水或胸水），以此法增幅細菌DNA，並針對陽性樣本做細菌菌種之鑑定，與細菌培養的結果作對照細作為此方法之敏感性及特異性指標。希望能建立懷疑病患有細菌感染時快速確診的方法，期望能提升臨床醫師在選擇使用抗生素與否上的正確率及治療感染的成功率，並快速鑑別感染源菌種，另外也希望此法可以有效縮短給予抗生素之時間，未來進一步達成減少抗生素濫用及抗藥性菌種產生之遠程目標。

Regarding to urinary tract and bloodstream infections in dogs and cats, bacterial culture is the gold standard to confirm the diagnosis. As the duration of culture and strain identification need 72 hours at least, the clinicians often use antibiotics before the cultural results; consequently the problems of antibiotic abuse and resistance increase. Thus the less time and higher sensitivity diagnostic methods were indeed needed. The 16S rRNA gene is used for phylogenetic studies as it is highly conserved between different species of bacteria. In addition to highly conserved sites, 16S rRNA gene sequences also contain hyper-variable regions that can provide species-specific signature sequences useful for identification of bacteria, which can be divided into v1 to v9 regions. Those species-specific sequences within a given hyper-variable region constitute useful targets for diagnostic assays and other scientific investigations. In this study, universal PCR primers that can amplify 16S rRNA hyper-variable regions from a large number of different bacterial species were designed. Also, minimum detectable concentration with serial dilution method to determine PCR sensitivity and specificity was determined and applied for the cases with bacterial culture of urine, blood and other body fluids such as ascites and pleural effusions at the same time. We aimed to identify infectious pathogens quickly to achieve the goal of early diagnosis and proper treatment without antibiotic abuse in the near future.

口試委員會審定書…………………..…………………………..…………………….i
致謝………………..…………………………………..………………………………ii
中文摘要……………………………………………………………………………..iii
ABSTRACT…………………………………………………………………………..iv
CONTENTS…………………………………………………………………………..v
LIST OF FIGURES…………………………………………………………………..ix
LIST OF TABLES…………………………………………………………………….x
Chapter 1 Introduction……………………………………………………………1
Chapter 2 Literature review………………………………………………………5
2.1 Urinary Tract Disease……………………………………………………….5
2.1.1 Uncomplicated and Complicated Urinary Tract Infection…………...6
2.1.2 Upper and Lower Urinary Tract Infection…………………………...7
2.2 Bloodstream Infection (BSI)………………………………………………...9
2.2.1 Systemic Inflammatory Response Syndrome (SIRS)………………..9
2.2.2 Sepsis, Septic shock and Multiple Organ Dysfunction Syndrome
(MODS)……………………………………………………………..10
2.3 Traditional Diagnostic Methods……………………………………………11
2.3.1 Diagnosis of Uncomplicated UTI…………………………………..11
2.3.2 Diagnosis of Complicated UTI …………………………………….14
2.3.3 Diagnosis of SIRS…………………………………………………..15
2.3.4 Diagnosis of Sepsis and Septic shock……..………………………..16
2.4 Novel Diagnostic Methods…………………………………………………19
2.4.1 Lateral Flow Immunoassay…………………………………………19
2.4.2 Flow Cytometry…………………………………………………….21
2.4.3 Mass Spectroscopy………………………………………………….22
2.5 Introduction and Characteristics of 16S rRNA gene……………………….23
2.6 16S rRNA in Veterinary Medicine Researches…………………………….28
2.7 Selection of Universal Primer……………………………………………...30
Chapter 3 Materials and Methods……………………………………………….32
3.1 Patients and samples collection…………………………………………….32
3.1.1 Patient and control groups ………………………………………….32
3.1.2 Laboratory measurements, sample preparation and storage………..36
3.2 Preparation of polymerase chain reaction (PCR)………...………….……..40
3.2.1 Extraction of bacterial genomic DNA from different clinical
samples…………………………………………………………….40
3.2.2 Selection of universal PCR primer………………………………….44
3.3 PCR reaction conditions of clinical samples……………………………….45
3.4 Identification of bacterial v3 gene segment………………………………..46
Chapter 4 Statistical methods……………………………………………………47
Chapter 5 Results…………………………………………………………………49
5.1 Patients and Samples Collection…………………………………………...49
5.2 Evaluation of spiked bacterial strain by bacterial culture and PCR………..52
5.3 Evaluation of clinical samples by bacterial culture and PCR……...…..…..54
5.3.1 The correlation between PCR results and bacterial culture for urine
samples…………………………………………………………….54
5.3.2 PCR results of whole blood samples……………..…………………61
5.3.3 The correlation between PCR results and bacterial culture for ascites
and pleural effusion samples………………………………………62
5.4 Evaluation of the Urine Samples…………………………………………...65
5.4.1 The correlation between signalment and culture results of the Urine
samples…………………………………………………………….65
5.4.2 The correlation between signalment and PCR results of the Urine
samples…………………………………………………………….68
5.4.3 The Comorbidities of Complicated UTIs in canine and feline
groups……………………………………………………………...70
5.4.4 The correlation between Urinalysis and culture results of the Urine
samples…………………………………………………………….72
5.4.5 The correlation between Urinalysis and PCR results of the Urine
samples…………………………………………………………….75
5.4.6 The correlation factors in canine and feline with UTIs……………..77
5.5 Evaluation of the Blood Samples…………………………………………..80
5.5.1 The correlation between control and SIRS or Sepsis-suspected
samples…………………………………………………………….80
5.5.2 The Signalment difference between control and SIRS/Sepsis animals
of the Blood samples………………………………………………82
5.6 Evaluation of the Ascites and Pleural effusion Samples…………………...83
5.6.1 Cytology analysis of the Ascites and Pleural effusions samples…...83
5.6.2 The Signalment of the canine and feline with Ascites and Pleural
effusion…………………………………………………………….84
Chapter 6 Discussion……………………………………………………………..86
Chapter 7 Conclusion…………………………………………………………….98
Reference…………………………………………………………………………….99

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