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標題: 組織培養豬瘟疫苗與LPC疫苗分子檢測方法之建立
The Development of Molecular Identification on Cell Culture and LPC Classical Swine Fever Vaccine
作者: 簡茂盛
關鍵字: 生物技術, 畜牧獸醫類
摘要: 豬瘟(classical swine fever; CSF)是一種豬隻高度傳染性病毒性疾病,臨床症狀以高熱、全身之出血病灶及白血球減少症為主,並會造成急性、慢性或持續性的感染,引起豬隻的發病死亡,因而對養豬戶造成重大經濟損失。豬瘟病毒係屬於為 Flaviviridae 病毒科中之 Pestivirus 病毒屬,豬瘟病毒為具封套外膜之RNA病毒,故在疫苗的製程中易有耗損,因而容易降低疫苗之免疫保護效力。目前台灣對豬瘟的預防是以施打兔化豬瘟疫苗或組織培養豬瘟活毒疫苗為主,這二株疫苗都是源自於LPC疫苗株。然而目前對市售兔化豬瘟組織或組織活毒疫苗的病毒力價及效力檢測上,除了檢定標準規定之動物接種保護試驗外,本計畫擬以間接螢光染色(indirect fluorescence antibody, IFA)與RT-PCR方法進行疫苗之檢測及半定量分析。此外,將開發以即時定量PCR (real-time quantitative PCR)進行偵測疫苗內實際病毒病毒力價,本計畫也將嘗試以Antigen-ELISA法同時配合進行分析病毒抗原,並針對可能迷入如circovirus type-I or II之病毒,以多組不同之特異性引子,進行監測疫苗是否遭受其他病毒之污染,以確保豬瘟疫苗的製成品質。而經由上述方法能進一步確認疫苗中有效病毒力價後,期能將開發之診斷方法技術轉移至國內疫苗廠商,以做為疫苗品質管制上之篩選依據。
Classical swine fever (CSF) is a highly contagious viral disease of swine with clinical signs of fever, encephalitis, massive hemorrhage, leuckopenia and high mortality. The duration of CSF infection can happen acute, chronic or even become persistent in the farm and induce severe economic loss in swine production. The causative agent of classical swine fever is a member of the Pestivirus genus of Flaviviridae, have envelope and a signle positive strand RNA with a genome of approximately 12.3kb. LPC strain of CSFV has been used as a live vaccine for control CSF in Taiwan since 1950's. However, the processing procedures of vaccine will usually destroy virus particle and decreasing the effective titer. The aim of this study is to develop a standard procedure for detecting the titer of CSFV and also to prevent the contamination in vaccine by cellular and molecular diagnosis including the detection of viral antigen, isolation of virus, and genome analysis. For detecting the viral antigen, the porcine kidney (PK-15) cell line or primary swine testis culture will be applied for indirect fluorescent antibody (IFA) test. Moreover, based on the reverse transcrption and polymerase chain reaction (RT-PCR) from NS5B region, the quantitative real-time PCR will be utilized according to the fluorescent signal produced proportionally during amplification of CSFV genome for detection the titer of virus. The sensitivity of real-time PCR method should allow the acquisitioned data to define CSFV titer and provide a rapid detection to make certain of vaccine quality. Furthermore, a multiplex PCR assay will be established to detect the porcine circovirus type I and II for avoiding the contamination and residing persistently in the PK cell line that might influence the efficacy of LPC or cell culture CSF vaccine.
其他識別: 91農科-3.1.3-檢-B1(8)
Appears in Collections:獸醫病理生物學所



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