化学修饰法制备B组轮状病毒生物素核酸探针及应用研究

Studies on the Preparation and Application of Biotin-labelled Nucleic Acid Probe for Group B Rotavirus

用亚硫酸氢钠—乙二胺溶液对B组轮状病毒的ssRNA胞嘧啶修饰和转氨基作用，与生物素e—氨基己酸N—羟基琥珀酰亚胺酯反应，制备探针与样品在硝酸纤维膜上进行点杂交，经亲合素—碱性磷酸酶系统显色，可测出106.25－212.5pg的RNA靶序列，特异性试验表明：B组生物素探针只能与B组核酸杂交，而与无关核酸无杂交信号，用B组探针检测45份仔猪腹泻粪样，检出B组阳性5份，检出率11．1％，明显高于PAGE法，实验结果表明，生物素核酸探针制备简单并且有特异、灵敏、稳定的优点，可用于各组轮状病毒的检测。

The biotin - labelled probe for group B rotavirus involve the trans-animation of unpaired cytosine residuesin ssRNA with sodium bisulfite and ethyenedamine. Primary amino on the cytosine derivatives are reacted withbiotinyl-e-aminocaproic acid N - hydroxysuccinimide ester. The probe was hybridized with the nitrocellulosefilter-bond RNA of group B rotavirus. The 106. 25 - 212. 5pg target sequence could be ditected by the probe.Probe of groupB can only recongnize group B ssRNA.The 45 fecal samples from diarrheal piglet detected by the probe resulting 5 positive to group B (11. 11%).It was more sensitive than PAGE detection. The study indicated that biotin-labeled probes have possessed sev-eral advantage 5 such as high sensitivity, specificity and stability. The labeling method is simpler,quicker andless expensive than radioacteve albeling method. Biotin - labeled probe can be widely applied to diagnostic micro-biologyand basic research.