應用聚合酶鏈鎖反應技術與限制酶切割多型性檢測緊迫敏感豬

The Use of Polymerase Chain Reaction and Restriction Fragment Length Polymorphism to Detect the Gene for Porcine Stress Syndrome

陳銘正(Ming-Cheng Chen) ； 林佳靜(Chai-Ching Lin) ； 李盈漢(Yieng-Han Lee) ； 吳伊倫(Yi-Luen Wu) ； 李易儒(Yi-Ru Lee) ； 林瑞毓(Ruei-Yu Lin) ； 張俊達(Chiun-Da Chan) ； 王祥宇(Hsiang-Yu Wang)

《宜蘭技術學報》 4期 (2000/06) Pp. 19-25

https://doi.org/10.6179/nit.2000.04.12

摘要

本試驗之目的乃建立聚合酶鏈鎖反應（polymerase chain reaction, PCR）與限制酶切割多型性（restriction fragment length polymorphism, RFLP）技術，檢測豬鈣離子釋放管道（calcium release channel, CRC）基因是否突變，俾快速鑑定豬緊迫敏感徵候群（porcine stress syndrome, PSS）基因。豬血液與精液樣品萃取之基因組DNA（genomic DNA, gDNA）做為PCR之模板，以CRC基因之1614-1633鹼基序列為上游引子，另以1957-1976鹼基序列之互補序列為下游引子，經聚合酶鏈鎖反應後得363鹼基對（base pair, bp）之DNA片段。以Hha I限制酶切割此片段，經電泳後可由限制酶分切片段區分緊迫敏感基因為純合子或雜合子。正常純合子、雜合子、PSS純合子三者之DNA片段長度組合分別為（231，132 bp）、（363，231，132 bp）與（363 bp）。採樣分析宜蘭某豬場豬隻CRC遺傳型，發現正常純合子、雜合子與PSS純合子之豬隻分別佔60%、35%與5%，估計緊迫敏感基因頻率為22.5%。應用PCR技術可以快速判定PSS豬隻，且可準確分辨基因型為雜合子或純合子。

關鍵字

聚合酶鏈鎖反應 ； 鈣離子釋放管道 ； 豬緊迫敏感徵候群

並列摘要

Pigs with mutated CRC gene have genetic disorder which can result in porcine stress syndrome (PSS). The purpose of this study was to establish polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) techniques for detecting mutation of calcium release channel (CRC) gene among pigs. All genomic DNA extracted from pig blood and semen samples were provided as PCR templates. The forward primer used in PCR amplification was corresponding to nucleotides 1614 to 1633, whereas the reverse primer was complementary to nucleotides 1957 to 1976 of CRC gene. A 363-bp nucleotide fragment was amplified by PCR method. Being restricted with Hha I and gel electrophoresis, the CRC genotypes of pigs were determined by their DNA fragment patterns. The fragment length consisted of (231, 132 bp), (363, 231, 132 bp) and (363 bp) represented normal homozygous, heterozygous and PSS homozygous genotypes, respectively. By analyzing CRC genotypes of pigs sampled from one of the farms located in I-Lan, it was found that there were 60 %, 35 % and 5 % of pigs determined to be normal homozygous, heterozygous and PSS homozygous genotypes. The PSS gene frequency was 22.5 % The PCR method for amplifying CRC gene could be used for rapid1y detecting the PSS and distinguishing heterozygous from homozygous genotype of normal pigs.

並列關鍵字

Polymerase chain reaction ； Calcium release channel ； Porcine stress syndrome