臺灣博碩士論文加值系統

隱球菌症(Cryptococosis)是由具有莢膜之致腫瘤型隱球菌(Cryptococcus neoformans)所引起，主要引發人類的隱球菌性腦膜炎，常發生於免疫力有缺陷的病患，如後天免疫不全症候群(AIDS)、器官移植、癌症的患者。隱球菌在分類學上可以分為五種血清型A、B、C、D、AD及三種變異型grubii、neoformans、gattii。為了探討不同來源之隱球菌的分佈情形，以及比較所分離之隱球菌株在基因分型上有何差異及關聯性，本研究以sunflower seed agar及cornmeal-tween80 caffeic acid agar進行分離，再以API 20C AUX及Crypto Check Iatron RM 304-K kit進行隱球菌之鑑定及血清型之分型；分離樣本來源則分別由鳥糞、小動物之鼻腔拭子及不同植物來源進行隱球菌之分離，另外，尚有自人類臨床分離之隱球菌。共採集322個鳥糞檢體、10個狗的鼻腔拭子及32個肯氏蒲桃(Syzygium cumini)樣本；鳥類糞便中有25個檢體可分離出隱球菌，分離率約7.8％；在狗鼻腔拭子則分離出1株隱球菌，分離率為10％，而植物來源則尚未分離出隱球菌。將分離所得之隱球菌分別進行CAP59基因定序、利用PCR-RFLP搭配限制酵素Ava I分析磷酸脂酶B基因(Phospholipase B1, PLB1)以及使用M13進行PCR-fingerprinting；以CAP59基因序列進行親緣演化樹分析，可以將所分離到的菌株分成三個不同的變異型相符合，而所分離之隱球菌皆屬於gattii變異型，在此族群中又可以區分為三個組別，發現人類分離株的親緣關係與禽類糞便分離株是較為接近的；使用PCR-RFLP可以將所有的隱球菌株區分為四種不同的型別，分別為P1、P2、P3、P4，其中P3、P4可與Latouche等人在2003年所進行之PCR-RFLP互相比對，其餘的菌株皆無法比對出來，顯示台灣所分離出來的隱球菌株與作者所分離的菌株在PLB1基因上有所差異；採用PCR-fingerprinting的方式，以M13進行PCR反應，發現可將隱球菌區分為11種不同的型別，其中狗的PCR-fingerprinting型別與其中一種的禽類來源分離株的型別是相符的，由此可以推測這兩種來源的分離株是具有相關性的，而人類的臨床分離株方面，可觀察到編號1117與其他三株人類分離株的指紋圖譜有所不同，而人類分離株的指紋圖譜與本研究所分離到其他的隱球菌株之指紋圖譜並不相同，因此在分型結果上與本研究所分離的隱球菌株之指紋圖譜並不相符；而比較所使用的三種分型方法，發現PCR-fingerprinting具較佳的分型能力，但容易因PCR條件的差異而得到不同的結果，未來可以考慮針對這些菌株再利用分型能力較好的Pulsed-Field Gel Electrophoresis (PFGE)來分型，以找出其間的關連性，以提供防疫單位作為疾病防疫的參考。

Cryptococcus neoformans is an encapsulated fungal organism that can cause disease in apparently immunocompetent, as well as immunocompromised hosts, such as chemotherapy, acquired immunodeficiency syndrome (AIDS) and organ transplantation. There are three varieties (grubii, neoformans, gattii) and five serotypes (A, B, C, D, AD) of this organism. In order to study the distribution and the genotype of Crytpococcus neoformans from different sources.. Sunflower seed agar and Cornmeal Tween 80 Caffeic acid agar were used for Cryptococcus neoformans isolation. To clarify the relationship between isolates from human, dog, plants, and avian in central Taiwan, the methods of serotyping, CAP59 sequencing, PCR-RFLP, and PCR-fingerprintig were applied to explore the genetic structure of the organism and understand the molecular epidemiology of clinical and environmental isolates. The prevalence of C. neoformans in avian excreta, dog nasal swabs and plants were 7.8, 10, 0% (25/322, 1/10, 0/32). CAP59 gene sequencing could differentiate C. neoformans into three groups and these groups were corresponding to three varieties. In contract, PCR-RFLP of PLB1 method could differentiate all isolations into four types and there were no association between dog, avian, and human isolates. Application of PCR-fingerprinting could differentiate all the strains into eleven types, and the dog strain has close relation to avian strains. In addition, the PCR-fingerprinting patterns of human were unique in this study. The source of human strains need further study to elucidate the relationship between different sources.

中文摘要............................................ I
英文摘要............................................ III
目次................................................ IV
表次................................................ VII
圖次................................................ VIII
第一章 緒言......................................... 01
第二章 文獻探討..................................... 02
第一節 致腫瘤隱球菌(Cryptococcus neoformans)之介紹.. 02
一、致腫瘤隱球菌之歷史及命名.................. 02
二、致腫瘤隱球菌之分類.......................... 02
三、致腫瘤隱球菌之生物學特性................... 04
(一) 致腫瘤隱球菌之診斷.................... 04
(二) 致腫瘤隱球菌之型態特色................ 04
四、致腫瘤隱球菌之分佈.......................... 04
第二節 人類及動物之隱球菌症......................... 05
一、人類之隱球菌症.............................. 05
二、動物之隱球菌症.............................. 06
第三節 隱球菌之毒力因子............................. 06
一、37℃生長的能力.............................. 06
二、黑色素(melanin) ............................ 07
三、莢膜(Capsule) .............................. 07
四、產生D-甘露醇(D-mannitol)的能力.............. 08
五、磷脂酶B1 (Phospholipase B1) ................ 09
六、隱球菌之交配型............................. 09
第四節 致腫瘤隱球菌之流行病學...................... 09
一、人類之流行病學............................. 09
(一) 易感染族群分布....................... 09
(二) 血清型分布情形........................ 10
(三) 台灣地區致腫瘤隱球菌血清型之分布...... 11
二、動物之流行病學............................. 11
三、植物之流行病學.............................. 12
第五節 致腫瘤隱球菌之分型方法...................... 13
一、電泳核型分析................................ 14
二、基因定序.................................... 14
三、PCR-restriction fragment length polymorphism
(PCR-RFLP).................................. 15
四、PCR-fingerprinting.......................... 15
第三章 材料與方法..................................... 17
實驗材料............................................. 17
第一節 隱球菌株及DNA來源............................ 17
一、標準菌株.................................... 17
二、人類隱球菌株來源............................ 17
三、檢體來源.................................... 17
(一) 鳥類糞便檢體之收集.................... 17
(二) 小動物鼻腔拭子之收集.................. 17
(三) 植物之檢體收集........................ 18
第二節 本實驗所應用之培養基及鑑定套組............... 18
一、致腫瘤隱球菌分離及繼代之培養基.............. 18
二、致腫瘤隱球菌之鑑定.......................... 18
三、致腫瘤隱球菌鑑定血清型之套組................ 18
第三節 核酸萃取、聚合酶連鎖反應(Polymerase Chain
Reaction, PCR)純化及電泳之試劑............... 18
ㄧ、細菌之基因去氧核醣核酸(Genomic DNA)萃取試劑. 18
二、聚合酶連鎖反應用試劑........................ 19
(一) CAP59基因增幅引子..................... 19
(二) PLB1基因增幅引子...................... 19
(三) PCR-fingerprinting引子................ 19
三、電泳用試劑.................................. 19
四、PCR產物純化套組............................. 20
實驗方法............................................. 20
第一節 致腫瘤隱球菌之分離、鑑定及保存.............. 20
一、糞便及植物檢體中致腫瘤隱球菌之分離.......... 20
二、鼻腔拭子中致腫瘤隱球菌之分離................ 20
三、致腫瘤隱球菌之鑑定.......................... 21
(一) 37℃生長的能力........................ 21
(二) 尿素酶試驗............................ 21
(三) API ID 20C AUX鑑定套組操作方法........ 21
四、致腫瘤隱球菌之保存.......................... 21
第二節 致腫瘤隱球菌血清型別之鑑定................... 22
第三節 致腫瘤隱球菌之CAP59定序分析.................. 22
一、致腫瘤隱球菌DNA之萃取....................... 22
二、致腫瘤隱球菌CAP59基因之增幅................. 23
三、CAP59核酸序列之鑑定與比對................... 23
第四節 致腫瘤隱球菌之磷脂酶B基因(Phospholipase B,
PLB1) PCR-Restriction Fragment Lenth Polymorphism
(PCR-RFLP)分析............................... 24
一、致腫瘤隱球菌磷脂酶B基因之增幅............... 23
二、致腫瘤隱球菌磷脂酶B基因之限制酵素反應....... 23
第五節 致腫瘤隱球菌之M13 PCR-fingerprinting型別分析. 26
第四章 結果........................................... 25
第一節 鳥類、動物及植物之致腫瘤隱球菌分離結果....... 25
一、鳥類糞便之分離結果.......................... 25
二、動物及植物之分離結果........................ 25
三、人類臨床分離株.............................. 25
第二節 血清型分型結果............................... 25
第三節 CAP59分型結果................................ 26
第四節 PCR-RFLP分型結果............................. 26
第五節 M13 fingerpringting分型結果.................. 26
第五章 討論........................................... 39
參考文獻.............................................. 45
附錄 培養基與試藥之製備方法.......................... 53

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