轮状病毒的生物素核酸探针及分子杂交的研究

Reseach of Biotin-labeled Nucleic Acids as Hybridization Probes for Rotaviruses

用亚硫酸氢钠-乙二胺溶液对轮状病毒的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,制备探针与结合在硝基纤维膜上的核酸样品杂交,然后用亲和素和生物素化碱性磷酸酶温育,2-萘酚磷酸和固蓝B盐显色,阳性反应呈蓝紫色;轮状病毒生物素核酸探针可检测20～39pgRNA靶序列。SA_(11)或NCDV探针可识别同源的SA_(11)和NCDV核酸序列,也能识别同属A群的Wa株,羊轮状病毒S_1株的核酸序列。检测141份猪、羊和人腹泻粪样,杂交阳性118份,并对几株细胞培养物进行了杂交试验。结果表明,该法制备的生物素探针具有稳定、特异、灵敏等特点,较其他化学方法制备生物素探针简单,取材容易,价廉,可以广泛应用于各种微生物的基础研究及其所致疾病的临床诊断。

The biotin-labeled probes for rotaviruses involve the transamination of unpaired cytosine residues in ssRNA with sodium bisulfite and ethyenedamine. Primary amino groups on the cytosine derivatives are reacted with biotinyl-e-aminocaproic acid N-hydroxysuccinimide ester. The probes was hybridized with the nitrocellulose filter-bond RNA and incubated in the solution of avidin and biootinyl-aminocaproic acid alkaline phsphatase; The colorimetric substrates are 2-Naphthyl phosphate Na salt and fast blue B salt. The positive results appeared in blue-purple color. The 20-39 pg sequence of the target could be detected by the probes. Both SA11 and NCDV probes could recognize the RNA sequence of SA11 ,NCDV, Wa and S1 rotaviruses ;The samples of 141 porcine, sheep and children diarrhoeal faeces and the cultured cells were detected by the probes. The results showel that this method is simpler and less expensive than other methods for the preparation of biotin-labeled probes ; It should lead to the wider application of hybridization techniques in diagnostic microbiology and basic research in infectious agents.