【作者】 韦栋平；

【导师】 陆芹章； 罗廷荣；

【作者基本信息】 广西大学 ， 预防兽医学， 2001， 硕士

【摘要】 本研究引用HCV特异性引物A1/A2直接对来自广西部分地区58份疑似猪瘟病料 进行检测，其中34份为阳性，阳性率为58.32%。再根据已发表的猪瘟病毒核酸序列， 设计并合成4对EP1/EP4、EP2/EP3、EP3/EP7和EP5/EP6，以引物EP1/EP4和EP2/EP3或 EP3/EP7和EP5/EP6配对，应用RT-PCR技术分两段扩增猪瘟病毒全E2基因，并对反 应条件进行优化。结果显示：引物EP1/EP4和EP2/EP3只能扩增猪瘟兔化弱毒(HCV) 和石门系强毒而不能扩增广西流行株：EP3/EP7和EP5/EP6则可成功扩增出广西流行株 和另外两个毒株。反应条件中，反转录所需的总RNA量为10μ1(约20μg);PCR扩增 时，cDNA用量为12.5μl，正、负引物各用 50 Pmol，EP3/EP7和EPS/EP6引物对分别以 57℃t和61℃的退火温度PCR扩增35个循环，PCR产物量和扩增特异性句达到最佳水 平。 猪瘟病毒广西流行株E2基因的成功扩增和最佳反应条件的建立为今后E2基因的克 隆与序列分析及其更深入的研究奠定了实验基础。更多还原

【Abstract】 In this study, Hog Cholera Virus (HCV) specific primers Al/A2 was used to detect 58 clinical samples from some places in Guangxi with reverse transcrition-olymerase chain reaction (RT- CR). The results showed that 34 samples were positive and the positive rates were 58.62%. According to the published nucleotide sequence of genome of HCV, 4 pairs of specific primers, EP1/EP4, EP2/EP3, EP3/EPland EPS/EP6, were designed and synthesized. Primers EP1/EP4 and EP2/EP3 were matched, so the primers EP3/EP7 and EP5/EP6. The E2 gene fragment of HCV Guangxi prevalence strain was amplified in two parts with RT- CR method and the reaction conditions were optimized. The results demonstrated that Hog cholera virus lapinized Chinese strain (HCLV) and Shimen strain could be amplified with EP1/EP4 and EP2/EP3, but Guangxi prevalence strain not, while EP3/EP7 and EP5/EP6 could successfully amplify Guangxi prevalence strain, HCLV and Shimen strain. The reaction conditions could be optimized by under methods. lOul of the total RNA and 12. 5u1 of cDNA were used in reverse transcrition and PCR respectively, positive and negative primers were SOpmol respectively, EP3/EP7 and EP5/EP6 were denaturated at 570Cand 61 0C respectively with 35 cycles. Then both the product of PCR and its amplifing specificity amounted to the highest level. The successful amplification of E2 gene of Guangxi prevalence strain and the optimum reaction conditions play an important role in the further research of cloning and sequence analyzing on E2 gene.更多还原