豬緊迫綜合症之基因型檢測技術探討

Study on the Genotyping of Porcine Stress Syndrome

鄭益謙(Ivan-Chen Cheng) ； 劉名允(Min-Yun Liu) ； 劉學陶(Hsueh-Tao Liu) ； 黎漢龍(Han-Long Li)

《中華民國獸醫學會雜誌》 23卷3期 (1997/06) Pp. 274-282

https://doi.org/10.7009/JTCSVS.199706.0274

摘要

豬緊迫綜合症（Porcine stress syndrome, PSS）為調節骨骼肌細胞內鈣離子濃度平衡之ryanodine receptor基因之C1843突變為T1843所導致。目前已有合併利用聚酶鏈反應、（Polymerase chain reaction, PCR）和限制酶片段長度多型性（Restriction fragment length polymorphism, RFLP）原理發展之檢測技術可以鑑定豬之PSS基因型，代替傳統使用鹵乙烷檢測法（Halothane challenge test, HCT）。此一基因型檢測所用樣品為抗凝之血液，再純化其白血球的去氧核醣核酸做為進行PCR之模版。本文將現行操作步驟做以下修改，如以去離子水加入血樣後加熱，利用細胞內外壓力不等張及溫度效應以製備PCR模版，配合一對自行設計之引子（Primer）直接進行PCR，得到363鹼基對長的產物，做為後續檢測材料；為便利保存、運輸，同時以血液乾燥於濾紙上的採樣直接PCR進行基因型檢測試驗，修改後之基因型檢測結果與目前所用者完全相同。本文並以746頭8週齡豬採血進行基因型檢測，與HCT結果做比較；共發現23頭豬（3.1％）判定HCT陽性，但PSS基因型檢測有36頭T1843純合子，其中的19頭為HCT判定陰性170頭PSS基因型雜合子中有6頭HCT判定陽性，佔HCT判定陽性豬之26％（6／23）；故基因型檢測技術不僅比HCT準確，且可分辨雜合子與PSS基因型純合子。

關鍵字

豬緊迫綜合症 ； 基因型檢測 ； 聚合酶鏈反應 ； 鹵乙烷檢測法

並列摘要

Porcine Stress Syndrome (PSS) is caused by the point mutation, C1843 to T1843, of ryanodine receptor responsible for the regulation of calcium-release channel of skeletal muscle sarcoplasmic reticulum. There has been a technique of molecular genetics, polymerase chain reaction (PCR) combining with restriction fragment length polymorphism (RFLP) developed to identify the PSS genotype of pig which was typed by halothane challenge test (HCT) before. The DNA purified from white blood cell of anticoagulant blood was utilized as the template of PCR for the genotyping technique. The typing procedures established were modified in our test to improve the typing efficiency as follows: first, blood samples were mixed with deionized water and heated to release the DNA directly as the template of PCR by osmotic and thermal effects. Secondly, the unique PCR products sized 363 b.p. defined by the two chosen primers were obtained as the material for testing the genotype of PSS. Thirdly, DNA processed from the dried blood on filter, a convenient way for sample preservation and shipping between the field and laboratory, was used to carry the PCR and genotyping directly. The PSS typing results from our modified procedures was identical with those obtained by the conventional technique. We also compared the results of genotyping from the blood with those of HCT of 746 piglets at the age of 8 weeks. Although thirty-six homozygous for T1843 by genotyping was identified, there were only 23 piglets (3.1%) determined as HCT positive. There were 19 individuals among the 36 judged as HCT negative. Six out of the 170 piglets genotyped heterozygous for PSS were determined as HCT positive. The six were the 26% (6/23) of HCT positive piglets. To identify the PSS genotype, the technique presented here is more accurate than HCT, also can clearly distinguish the heterozygous pigs from the PSS homozygotes which can't be done by HCT.

並列關鍵字

PSS ； Genotyping ； PCR ； HCT