[目的]本试验旨在建立肌肉生成抑制素基因(myostatin,MSTN)敲除猪的快速检测方法,为该品系转基因猪及其产品的检测提供技术支持.[方法]根据猪β2-微球蛋白基因(β2-microglobulin,B2M)设计猪内源性基因引物,标记基因新霉素磷酸转移酶基因(neomycin phosphotransferaseⅡ,NPTⅡ)和测定的边界序列设计特异性引物,对引物的量进行调整,优化反应条件.对PCR的敏感性和特异性进行检测,并应用建立的方法对随机选取的32份猪肺和脾脏组织样品进行检测,均仅扩增出B2M条带.[结果]该方法的最佳退火温度为56 ℃,最低能检测出含1% MSTN基因敲除基因组成分的样本,对32份组织样本仅扩增出B2M条带.[结论]建立的多重PCR方法敏感、特异,在一个反应中能检测多个基因片段,可用于MSTN基因敲除猪的检测,为此品系转基因动物检测标准的建立提供依据.[Objectives]To provide technical supports for the detecting of myostatin gene(MSTN)knockout pigs and their derived food,a rapid multiplex PCR technique was established in this study. [Methods]Three pairs of specificity primers were designed according to the sequences of β2-microglobulin in pig,neomycin phosphotransferaseⅡ and the flanking sequences of the myostatin knockout pigs,respectively. The system of the multiplex PCR reaction was optimized by adjusting the amount of primers. The sensitivity of the technique was evaluated with transgenic pig samples. The bovine,sheep,mouse and untransgenic pig samples were used for analysing the specificity of the method. The spleen and lung samples of pig(n=32)were collected,and tested using the method established. [Results]The optimized conditions were an annealing temperature at 56 ℃. The method could detect pig samples containing 1% MSTN knockout pig genome. There is only B2M amplified bands in test of 32 samples. [Conclusions]The results showed that the multiplex PCR technique established in the study had high sensitivity and specificity,in which only one reaction was necessary to detect multiple target fragments. It could be reliably used to identify with myostatin knockout pigs. This work lays a foundation for the establishment of detecting standard on the transgenic animals
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