林德育 黃鈺嘉 陳若菁 楊德威 吳明哲 張秀鑾
傳統乳牛遺傳疾病的遺傳型鑑定是由牛隻個體血樣、精液或乳樣萃取 DNA後，利用已知特異性引子藉由PCR增幅特定DNA片段，再以限制脢處理後，由電泳所呈現的結果來判定。然而，對於一些頻率較低的遺傳疾病的篩檢，若以個體一一檢測，則將費時費力又耗費經費，如果能藉由總乳或混合精液來進行全場牛隻突變基因的檢測將可快速而經濟地對特定遺傳疾病進行篩檢。本試驗針對先天性下痢致死基因(CD18)，將已萃取的總乳或混合樣本 DNA，先以限制脢於特定位置將正常型 DNA截斷，以提高於後續特異性引子PCR增幅步驟中，雜合型 DNA的競爭能力。於PCR增幅後，再以限制脢作第二次處理，截切殘留的正常型特定DNA片段，而後經電泳所呈現的結果來判定樣本中是否含有雜合型或有病型的個體。以現階段台灣乳牛頭數約十萬頭，酪農戶約一千戶計算，平均每戶僅約有50~60頭牛供榨乳。因此，本試驗以牛淋巴球黏力缺失症雜合型與正常型牛隻之牛血與牛乳依不同比例(1:9、1:24、1:49與1:99)混合後萃取DNA隻進行檢測。結果顯示，無論是牛血樣或乳樣，既使在1:99混合樣品亦能測出在均能測出牛淋巴球黏力缺失症突變基因特異性片段。此種混合樣本的檢測方法，可應用於監控族群中突變或稀有的重要基因，藉總乳或混合血樣全面篩檢後，再進一步檢測可疑的家族或個體，以節省成本與時間。
DETECTION OF GENOTYPES OF BOVINE LEUKOCYTE
DEFICIENCY SYNDROME USING MIXED MILK SAMPLE
D. Y. Lin, Y. C. Huang, Z. C. Chen, T. W. Yang, M. J. Wu and H. L. Chang
Taiwan Livestock Research Institute, Council of Agriculture
Conventional genetic disease test was based on individual blood, semen or milk samples. Starting with DNA extraction from the raw sample, specificity primers were used to amplify DNA fragments by PCR. Finally, the PCR product was digested by restriction enzyme and diagnosed the genotype using the electrophoresis prints. Because defect gene was rare in general, individual genetic test will consume intolerable money and time for population screen. If carriers in bulk tank milk or other mixed samples can be detected, genetic screen will reduce total cost tremendously. Bovine Leukocyte Adhesion Deficiency(BLAD) is a serious genetic defect of Taiwan dairy cattle. In this study, milk or blood from identified genotypes(BL, TL and BLAD) were mixed by proposed ratio. DNA was digested by restriction enzyme before PCR procedure. The purpose is to cut the normal DNA into fragment to decline the primer competition with the rare mutant DNA. Then, following conventional procedures, PCR and 2nd restriction enzyme treatment(for cutting residual normal DNA into short fragments), carriers in sample will be discovered by electrophoresis prints. Milk samples and whole blood of both genotypes (carrier BL and normal TL), were mixed from 1:9, 1:24, 1:49 to 1:99 and then extracted the DNA and followed with genotyping procedures. Because there were only about 100,000 dairy cows and 1000 dairy farmers in Taiwan, bulk tank milk of each farm was mixed from 50 to 60 cows in average. Results showed the method can detect the exist of the carriers(BL) in all mixed samples, up to 1:99. This modified method can be used for rare gene detection and genetic disease control program. Tests may concentrate on suspected family and individuals only, farms with carriers, and total cost will be decreased notably.
Key Words: DNA mixes, Bulk tank milk, Bovine leukocyte adhesion deficiency syndrome.