建立偵測常見豬隻呼吸道病毒性疾病即時聚合酶鍊鎖反應技術

Establishment of Real-time (RT-)PCR for Common Swine Respiratory Viral Disease Detection

蕭君倪(Chun-Ni Hsiao)

指導教授 ： 龐飛

共同指導教授 ： 鄭謙仁 ； 蕭世烜

國立臺灣大學/獸醫專業學院/獸醫學研究所/碩士(2010年)

https://doi.org/10.6342/NTU.2010.02189

摘要

Porcine circovirus (PCV2)、porcine reproductive and respiratory syndrome virus (PRRSV)、swine influenza virus (SIV)、Torque teno virus (TTV)、porcine parvovirus (PPV) 以及classical swine fever virus (CSFV)為六種豬隻常見的混合性病毒感染，患豬臨床上呈現呼吸道症狀或繁殖障礙，導致疾病診斷的困難與經濟損失。即時聚合酶鏈鎖反應(real-time PCR)的分子診斷技術是藉由調整PCR反應中聚合酶、引子及探針的濃度及運用電腦軟體分析結果，同步判定檢體中是否有病原的存在（定性）及其濃度（定量）；且可在不同探針上標定不同波長的螢光物質，進一步建立於單一樣本的同一PCR反應管中同時偵測多重病原的通用反應條件。本研究利用ClontechR (Clontech laboratories, Inc., USA)所生產的試劑於MastercyclerR ep realplex (Eppendorf, Germany)建立上述六種病毒之多種偵測技術：SYBR Green real-time (RT-)PCR，其中PCV2、PPV及TTV1的偵測敏感度分別為2.27 ×101~10、1.76 × 101~10及4.47 × 100~9 copies/μL，PRRSV及CSFV的最低偵測敏感度則皆為101 TCID50/mL，並可進一步做為multiplex parallel SYBR Green real-time (RT-)PCR的使用；singleplex TaqMan real-time (RT-)PCR，PCV2、PPV及TTV1可進行定量的敏感度分別為2.2 ×101、1.57 × 101及4.47 × 100 copies/μL；PRRSV及CSFV的最低敏感度則分別為100 及101 TCID50/mL，而SIV相對於conventional RT-PCR也有較佳的敏感性，且自採樣檢體的萃取至偵測病毒感染，可在3~6小時內完成。另外，我們亦藉由調整引子及探針的濃度最佳化反應試劑的條件，嘗試建立duplex TaqMan real-time (RT-)PCR以便同步偵測PCV2與PPV或PRRSV與SIV；且若不經最佳化調整，在特定比例的濃度範圍以上，也可進行triplex TaqMan real-time (RT-)PCR來同步偵測各管中三種DNA或RNA病毒。以上技術的建立，可更快速及敏感的進行區別診斷，有助於豬隻疾病的防疫與監控工作。

關鍵字

即時聚合酶鏈鎖反應 ； 豬隻呼吸道病毒性疾病 ； SYBR Green即時聚合酶鏈鎖反應 ； TaqMan即時聚合酶鏈鎖反應,同步偵測 ； 平行偵測 ； 多重病原診斷

並列摘要

Porcine circovirus (PCV2)、porcine reproductive and respiratory syndrome virus (PRRSV)、swine influenza virus (SIV)、Torque teno virus (TTV)、porcine parvovirus (PPV) and classical swine fever virus (CSFV) are the major pathogens of combined infection pigs. The combined infection not only causes serious economic loss but also increases the diagnostic difficulty. Real-time polymerase chain reaction (real-time PCR) is a powerful molecular technology for pathogen detection and quantification via a mathematical and computerizable way. The further development of multiplex real-time PCR can simultaneously analyze several targets in a single reaction, which also reduced sample volume, testing time, and cost. In the present study, we established the SYBR Green real-time (RT-)PCR for each of the above mentioned six important swine viral pathogens. The detection limit of the DNA/RNA assays are 22.7 copies/μL for PCV2, 17.6 copies/μl for PPV, 4.47 copies/μl for TTV1 and 101 TCID50/mL for PRRSV and CSFV. Besides, we also build singleplex TaqMan real-time (RT-)PCR. The detection limit of the DNA/RNA assays are 22 copies/μL for PCV2、15.7 copies/μl for PPV, 4.47 copies/μl for TTV1, 100 TCID50/mL for PRRSV, and 101 TCID50/mL for CSFV. Furthermore, the conditions of duplex TaqMan real-time (RT-)PCR for PCV2/PPV and PRRSV/SIV by decreasing concentrations of probe-primers and the detection limits of triplex TaqMan real-time (RT-)PCR for PCV2/PPV/TTV1 and PRRSV/SIV/CSF without optimization were both evaluated. These assays established may provide a more rapid and sensitive methodologies for swine disease diagnosis in the field.

並列關鍵字

Swine viral disease diagnosis ； SYBR Green real-time (RT-)PCR ； TaqMan real-time (RT-)PCR ； Multiplex ； Parallel

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