猪应激综合征HRM检测方法的建立与应用

发布时间：2018-06-06 19:05

本文选题：猪应激综合症 + 高分辨率熔解曲线　；参考：《山西农业大学》2015年硕士论文

【摘要】：目的：氟烷基因(Hal)是导致猪发生应激综合症(porcine stress syndrome, PSS)的一个主效基因,猪应激综合症能够直接影响到猪的肉质,应激敏感的个体在运输过程、驱赶甚至配种等应激因子的作用下会突发恶性高热症甚至导致死亡,另外,应激敏感个体猪在经过屠宰后会生成白肌肉,导致肉质变差,从而给养猪户带来一定的经济损失。本实验的目的,就是建立一种简易、快捷、准确、自动化程度高的检测Hal的方法。方法：本实验首先根据GenBank中PSS RYR1基因序列,结合突变位点,合成C/C型和T/T型两种标准质粒,将C/C、T/T型质粒等比例混匀后,即可得C/T型质粒。然后以构建的质粒为标准,建立HRM小片段和非标记性探针法两种检测Hal的高分辨率熔解曲线(High Resolution Melting, HRM)方法。用建立的两种方法对300头猪进行检测。为了验证这两种检测方法的准确性,用目前最常用的PCR-RFLP方法也对这300头猪进行了检测。结果：①成功的构建了三种标准质粒②成功的构建了HRM小片段法,检测结果显示300份样品中均没有氟烷基因的携带者。③成功的构建了HRM非标记性探针法,检测结果显示300份样品中没有氟烷基因的携带者。④PCR-RFLP检测结果显示300份样品中没有氟烷基因的携带者。结论：三种方法的检测结果一致,即用高分辨率熔解曲线检测Hal是可行的。
[Abstract]:Objective: halothane gene is a major gene responsible for porcine stress syndrome (PSSs) in pigs. Porcine stress syndrome can directly affect the meat quality of pigs, and stress-sensitive individuals are involved in the transport process. Under the action of stress factors, such as driving out and even mating, it may burst into malignant hyperpyrexia or even lead to death. In addition, after slaughtering, stress-sensitive individual pigs will produce white muscles, resulting in poor meat quality, which will bring certain economic losses to pig farmers. The purpose of this experiment is to establish a simple, fast, accurate and automatic method for detecting Hal. Methods: according to the sequence of PSS RYR1 gene in GenBank, two standard plasmids of C / C type and T / T type were synthesized according to the mutation site. The C / T type plasmids were obtained by mixing the C / C / T / T type plasmids. Then, using the constructed plasmids as the standard, two high resolution melting curve (HRM) methods for detecting Hal by HRM small fragment and unlabeled probe method were established. The established two methods were used to detect 300 pigs. In order to verify the accuracy of the two methods, 300 pigs were tested with the most commonly used PCR-RFLP method. Results 1 successfully constructed three standard plasmids 2 and constructed HRM small fragment method successfully. The results showed that 3. 3 carriers without halothane gene in 300 samples successfully constructed HRM unlabeled probe method. The results showed that 300 samples had no Halothane gene carriers. 4 PCR-RFLP analysis showed that there was no Halothane gene carrier in 300 samples. Conclusion: it is feasible to detect Hal by high resolution melting curve.
【学位授予单位】：山西农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.28

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