以聚合酶鏈反應、DNA序列及原位雜交方法確認台灣地區豬環狀病毒之感染

Identification of Porcine Circovirus Infection in Taiwan by Using Polymerase Chain Reaction, Analysis of DNA Sequences and In Situ Hybridization

張甘楠(Gan-Nan Chang) ； 蘇柏栩(Bor-Sheu Su) ； 邱振豪(Jenn-How Chiou) ； 劉宏仁(Hung-Jen Llu) ； 蔡信雄(Shinn-Shong Tasi)

《臺灣獸醫學雜誌》 28卷3期 (2002/09) Pp. 186-194

https://doi.org/10.7009/TVJ.200209.0186

摘要

豬環狀病毒（Porcine circovirus, PCV）之感染，主要造成豬斷奶後多系統消耗性症候群（Postweaning Multisystemic Wasting Syndrome, PMWS），為豬隻重要新病，在本省亦有本病發生，但尚無正式報告。本病以感染保育豬與肉豬為主，造成患豬體重銳減、精神沉鬱、呼吸困難、黃疸、間質性肺炎、間質性肝炎、間質性腎炎及全身淋巴腺病或皮腺炎。為進一步了解台灣地區豬環狀病毒感染之情形，首先將臨床症狀上、組織病理學上，有懷疑豬隻感染環狀病毒時，從石蠟切片刮下組織抽取DNA做為模板（Template），以一對特異性引子（Primer），在適當的條件下進行聚合酶鏈反應（PCR）增幅豬環狀病毒DNA片段，其分子量大小約為428 bp。將此DNA片段利用限制酶切割後進行限制酶片段長度之多型性分析（PCRRFLP）進一步確認確為豬環狀病毒之第二型（PCV-2）。並將此DNA與pGEM-T載體（Vector）進行選殖，選殖後進行基因定序（Sequences）分析其DNA序列，並與GenBank內PCV之DNA序列比對結果亦為PCV-2，本試驗所選殖之DNA序列與法國株AF055394相似性達99%與台灣株AF166528及AF154679相似性達95%以上。又經原位雜交方法結果獲知在患豬之扁桃腺、肺門淋巴結及腸間膜淋巴結均呈陽性反應。可知目前台灣發生豬環狀病毒感染屬於PCV-2。

關鍵字

環狀病毒 ； 多系統消耗性症候群 ； 聚合酶鏈反應 ； 原位雜交

並列摘要

Postweaning multisystemic wasting syndrome (PMWS) caused by porcine circovirus type 2 (PCV-2) was reported in weaned pigs in Taiwan. The infected pigs were suffering from loss weight, dyspnea, icterus, interstitial pneumonia, hepatitis, nephritis, dermatitis and depletion of lymphocytes in lymph nodes. Typical syncytial cells were observed in many lymphoid organs such as tonsiI, lymph node, mesenteric lymph nodes and spleen of the infected pigs. Polymerase chain reaction (PCR) was employed for the diagnosis of porcine circovirus type 1 (PCV-1) and PCV-2 when a pair universal primers were used. The amplicon of PCV-2 was 428bp (NPUST-2Kl) and sequenced. Moreover, the restrict ion fragment length polymorphism was observed when analyzed with Hae lll & Hha l. Only Hae lll could digest PCV-1 other than PCV-2. Although Hha I could digest both PCV-1 and PCV-2, it could demonstrate fragment length polymorphism when analyzed by agarose gel electrophoresis in terms of differential diagnosis. However, in situ hybridization test was also carried out and the resulting data showed positive reaction in the tonsil, hilus lymph node of lungs and mesenteric lymph nodes when the NPUST-2K1 (PCV-2) was used as probe. The DNA sequences of the NPUST-2K1 belong to PCV-2 in this study and showed the similarity more than 95% to France strain (AF055394), Canada strain (AF055392) and Taiwan strains (AF166528, AF154679).

並列關鍵字

Circovirus ； PCV ； PMWS ； PCR ； In situ hybridization