最佳化之平行即時聚合酶鏈鎖反應以診斷經由精液傳播之豬繁殖性疾病

Establishment of Optimized Parallel Real-time PCR for the Diagnosis of Semen-Transmitted Swine Reproductive Diseases

陳怡廷(Yi-Ting Chen)

指導教授 ： 龐飛

共同指導教授 ： 蕭世烜 ； 鄭謙仁(Chian-Ren Jeng)

國立臺灣大學/獸醫專業學院/獸醫學研究所/碩士(2011年)

https://doi.org/10.6342/NTU.2011.02974

摘要

隨著人工授精 (artificial insemination; AI) 技術的引進，豬隻繁殖障礙的流行病有了重大改變。AI有防止疾病傳播、加速畜群改良以及降低生產成本等功能，然而文獻顯示由於公豬精液的病原檢測做得不夠徹底，導致疾病傳播造成龐大的經濟損失。在現今大量使用AI的情況下，為確保精液品質，需於運送前3-4小時即完成其品質的確定，故時間上的迫切性相當重要。本研究的目的為針對9種常見藉由精液傳播且影響經濟效益之重要病原，包括假性狂犬病病毒 (pseudorabies virus, PRV)、豬小病毒 (porcine parvovirus, PPV)、豬第二型環狀病毒 (porcine circovirus type 2, PCV2)、日本腦炎病毒 (Japanese encephalitis virus, JEV）、豬生殖與呼吸綜合症病毒 (porcine reproductive and respiratory syndrome virus, PRRSV)、古典豬瘟病毒 (classical swine fever virus, CSFV)、弓蟲 (Toxoplasma gondii)、鉤端螺旋體 (Leptospira spp.) 及豬布氏桿菌 (Brucella suis)，建立一套具快速檢測特質的平行即時聚合酶鏈鎖反應 (multiplexed parallel real-time polymerase chain reaction, multiplexed parallel real-time PCR) 方法，以達上述精液品質確認所需。本研究使用Clontech® (Clontech laboratories, Inc., USA) 的試劑及Mastercycler® ep realplex (Eppendorf, Germany) 檢測系統，在以下條件進行反應，SYBR Adventage® qPCR premix檢測為95℃30秒，95℃5秒及60℃30秒，共45個循環；one-step qRT-PCR SYBR kit檢測為48℃20分鐘，95℃3分鐘，95℃15秒及60℃1分鐘，共45個循環，以及QTaq DNA polymerase mix檢測為95℃3分鐘，95℃15秒及60℃1分鐘，共45個循環；QTaq one-step qRT-PCR kit檢測為48℃20分鐘，95℃3分鐘，95℃15秒及60℃1分鐘，共45個循環，建立上述9種病原之平行方式同步偵測技術；其中包括以SYBR Green作為螢光訊號的來源，配合real-time PCR產物裂解溫度 (melting temperature; Tm) 的不同，進行引子對可行性的確認及單一病原偵測技術的建立，進而再以TaqMan在增幅片段上加入特定探針 (probe)，利用不同波長的螢光偵測，發展可同時檢測多種病原之快速檢測方法，並針對其特異性和靈敏度進行分析，以確認其檢測之穩定性及再現性。本研究針對上述9種病原所建立之方法，SYBR Green系統偵測靈敏度以PPV和Leptospira spp.的17.6及19.6 copies/μL為最佳，其餘為101~2 copies/μL，而TaqMan系統以JEV的3.6 copies/μL為最佳，其餘為101 copies/μL。上述方法之建立，僅需耗時3~4小時即可快速檢測豬隻精液以確保其品質，進而達到豬隻繁殖障礙疾病的防控，提升配種受胎率和經濟產能，以而降低豬場和國家的經濟損失。

關鍵字

平行即時聚合酶鏈鎖反應 ； 豬隻繁殖障礙疾病 ； 人工授精 ； SYBR Green ； TaqMan

並列摘要

The epidemiology of swine reproductive diseases has changed significantly following the introduction of artificial insemination (AI). The advantages of AI include prevention of disease transmission, breeding improvement, cost reduction, and overcoming the impediment in natural mating. However, it has also been shown more serious disease spreading and economic loss can occur due to incomplete pathogen inspection in the semen used for AI. Under the current high demanding of AI in pig industry, it is required that each semen sample used for AI needs to be thoroughly examined 3-4 hours before shipping to ensure its quality. Therefore, the speed for semen examination is critical. In the present study, a high throughput multiplexed, parallel real-time polymerase chain reaction (rt-PCR) was established to simultaneously detect 9 important reproductive pathogens, including pseudorabies virus (PRV), parvovirus (PPV), porcine circovirus type 2 (PCV2), Japanese encephalitis virus (JEV), porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), Toxoplasma gondii, Leptospira spp., and Brucella suis, in swine semen. Reagents of Clontech® (Clontech laboratories, Inc., USA) and Mastercycler® ep realplex (Eppendorf, Germany) were used for the establishment of the parallel rt-PCR for the 9 pathogens, under the following conditions: initial denaturation at 95 ℃ for 30 sec, 45 cycles of denaturation at 95 ℃ for 5 sec, and annealing and extension at 60 ℃ for 30 sec of SYBR Adventage® qPCR premix; initial reverse transcription at 48 ℃for 20 min, followed by initial denaturation at 95 ℃ for 3 min, 45 cycles of denaturation at 95℃ for 15 sec, and annealing and extension at 60 ℃for 1 min of one-step qRT-PCR SYBR kit; furthermore, initial denaturation at 95 ℃for 3 min, 45 cycles of denaturation at 95 ℃ for 15 sec, and annealing and extension at 60 ℃ for 1 min of QTaq DNA polymerase mix; initial reverse transcription at 48 ℃for 20 min, followed by initial denaturation at 95 ℃ for 3 min, 45 cycles of denaturation at 95℃ for 15 sec, and annealing and extension at 60 ℃for 1 min of QTaq one-step qRT-PCR kit. Two fluorescence detection methods for monitoring rt-PCR were included. The dsDNA-binding dye SYBR Green I was used along with the analysis of the melting temperature (Tm) of the amplified products for primer validation and singleplex condition optimization. TaqMan probes were then designed so that they could anneal within a DNA region amplified by a specific set of primers to develop a rapid multiplexed, parallel rt-PCR for the detection of the 9 above mentioned pathogens simultaneously. Furthermore, analysis of the specificity and sensitivity to validate the stability and reproducibility of each assay was performed. The optimal detection limit for SYBR Green was 17.6 copies/μL for PPV, 19.6 copies/μL for Leptospira spp., and 101~2copies/μL for others. The optimal detection limit for TaqMan was 3.6 copies/μL for JEV and 101copies/μL for others. The established multiplexed parallel rt-PCR could check the 9 target pathogens simultaneously and rapidly which could ensure the completion of quality examination of semen within 3-4 hours and prevent the spread of reproductive pathogens via semen in the future. The improved semen quality control for AI will effectively enhance the reproduction performance in pig industry and reduce the associated economic loss.

並列關鍵字

parallel real-time polymerase chain reaction ； pig reproductive disorders ； artificial insemination ； SYBR Green ； TaqMan

參考文獻

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