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研究生:吳碧真
論文名稱:利用即時反轉錄聚合酶鏈鎖反應與高解析熔解分析法於福馬林固定石蠟包埋切片上 同時診斷並分型鯨豚麻疹病毒
論文名稱(外文):Simultaneous Diagnosis and Genotyping of Cetacean Morbillivirus from Formalin-fixed Paraffin-embedded Sections Using qRT-PCR and High Resolution Melting Assay
指導教授:詹昆衛詹昆衛引用關係楊瑋誠
學位類別:碩士
校院名稱:國立嘉義大學
系所名稱:獸醫學系研究所
學門:獸醫學門
學類:獸醫學類
論文種類:學術論文
畢業學年度:102
中文關鍵詞:鯨豚麻疹病毒福馬林固定石蠟包埋組織樣本即時反轉錄聚合酶鏈鎖反應高解析熔解分析法回溯性研究
外文關鍵詞:Cetacean morbillivirusformalin-fixed paraffin-embedded samplesreal-time reverse transcription polymerase chain reactionhigh resolution meltingthe retrospective study
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鯨豚麻疹病毒為鯨豚最重要病原之一,感染鯨豚麻疹病毒之鯨豚會有嚴重的呼吸道、中樞神經系統疾病,以及因免疫抑制的問題,造成二次性感染,例如細菌或黴菌性感染。鯨豚麻疹病毒過去20幾年,與在美國、西班牙及義大利國家海岸的某些品種的鯨豚族群高死亡率事件顯示是有所相關的。至今,西太平洋的台灣海域,僅限一篇於西元1999年一隻小抹香鯨感染鯨豚麻疹病毒的報告。其他洲例如亞洲,大洋洲因為鯨豚麻疹病毒感染的海豚擱淺事件是零星被報導的。在這些大陸對於此病毒感染海豚方面的資訊仍然不足。此外,過往文獻曾提及在病理學檢查之無明顯鯨豚麻疹病毒感染特徵的切片,其樣本卻在反轉錄聚合酶鏈鎖反應得到了陽性結果。這表示,過往病例僅以病理學檢查結果確診,很可能會誤診鯨豚麻疹病毒。在低病毒量持續感染病例,或者使用不夠敏感的診斷方法的情況下,亦會造成偽陰性。為釐清鯨豚麻疹病毒在全球真正的分布及可能的帶原者,回溯性的研究鯨豚麻疹病毒是重要的。然而,在這回溯性研究中,在取得及保存方面,以新鮮樣本做為核酸來源比福馬林固定石蠟包埋組織樣本來說是較為侷限的。在這研究中,發展了以高效率、高敏感性及高通量的即時反轉錄聚合酶鏈鎖反應,結合可分型的高解析溶解分析法,去應用在易取得的樣本─福馬林固定石蠟包埋組織樣本。此法其結果顯示,存放19年的福馬林固定石蠟包埋組織樣本,仍有足夠的核酸 (the CT value of ß-actin = 24.41)。本實驗所設計的引子,可利用即時反轉錄聚合酶鏈鎖反應成功地增幅福馬林固定石蠟包埋組織樣本中的鯨豚麻疹病毒之高保留區域─磷酸蛋白基因。接著,高解析熔解分析法分型了鯨豚麻疹病毒的分株─海豚麻疹病毒與領航鯨麻疹病毒。此些研究結果顯示,這個高效率又快速及可分型的診斷方法被成功建立,將來應可使用在回溯性研究上。
Cetacean morbillivirus (CeMV) is considered one of the most important viral pathogens in cetaceans. In infected cetaceans, the virus causes serious respiratory, central nervous system disease and immuosuppression which lead to serious secondary bacterial and fungal infections. CeMV was shown to be associated with high mortality events of the population of several cetacean species along the coast of the United States, Spain, Italy in the past three decades. To date, there has been only one report of CeMV infection in a pygmy sperm whale (Kogia breviceps) in the Western Pacific Ocean around Taiwan in 1999. Straned cetacean events resulting from CeMV infection in other continents including Asia and Oceania also were sporadic reoprted. The information of CeMV infection is still lack in cetaceans around these continents. In addition, there were no typical lesions resulting from CeMV infection on pathological sections in previous study, but these case were positive by reverse transcription polymerase chain reaction (RT-PCR). It revealed that it could have misdiagnosis of CeMV infection if researchers made a diagnosis only relying on the results of pathological examination on retrospective study. It also may bring risks of false negative results in several conditions such as applying unsensitive detection method and the cases with a persistent infection with low copies of virus. To clarify the real distribution of CeMV and possible carriers, the retrospective study of CeMV is important. However, fresh tissues as nucleic acid sources on retrospective study are more limited than FFPE samples on the acquisition and preservation. In this study, a high efficient, sensitive and throughput real-time RT-PCR (qRT-PCR) combines with high resolution melting (HRM) for genotyping was developed to the easy obtained formalin-fixed paraffin-embedded (FFPE) samples. The results of this study show the stored 19-year FFPE sample included sufficient amount of nucleic acid (the CT value of ß-actin = 24.41). The designed specific primers for CeMV could successfully amplify the highly conserved sequence within the phosphoprotein gene from FFPE samples to diagnose CeMV using qRT-PCR. Furthermore, the HRM could distinguish PWMV and DMV, belonged two strains of CeMV. The results of this experiment imply that the developed method with a high efficiency for genotyping of CeMV strains was successfully established and could be applied to the retrospective study.
中文摘要 I
Abstract III
Content V
Introduction 1
Paper review 3
Morbillivirus 3
Cetacean morbillivirus (CeMV) 4
CeMV epidemiology 5
Pathology 8
Molecular Diagnostics 9
Materials and Methods 13
1. Housekeeping genes for RNA quality evaluation 13
1.1 Primers design 13
1.2 Primers selection for reverse transcription (RT) 14
1.3 qRT-PCR of FFPE samples for different formalin fixed time 15
1.4 qRT-PCR of FFPE samples for different storage times 15
2 Target genes for CeMV detection 16
2.1 Primers design 16
2.2 Sensitivity of the qRT-PCR assays 16
2.3 qRT-PCR and HRM of positive samples 17
2.4 TA-cloning for sequencing 18
Results 20
qRT-PCR of HKGs in cetaceans 20
Primers selection for RT 20
The RNA quality of FFPE samples for different formalin fixed time 21
The RNA quality of FFPE samples for different storage time 21
Pathological examination 21
Sensitivity of the qRT-PCR assays with P1 and P2 primers 22
The qRT-PCR and HRM of positive samples 22
Discussion 24
Conclusion 31
Reference 32
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