用生物素标记核酸探针检测鸭瘟病毒DNA的研究

DETECTION OF DUCK PLAGUE VIRUS DNA WITH A BIOTIN-LABELLED PROBE

用生物素-11-脱氧尿苷三磷酸(Biotin-11-dUTP),通过缺口平移法标记提纯的鸭瘟病毒 DNA,制备生物素化鸭瘟病毒 DNA 全基因组探针;以斑点杂交检测固定在硝酸纤维膜上的样品鸭瘟病毒 DNA 同源序列,杂交后用亲和素-碱性磷酸酶孵育,底物显色,阳性反应呈蓝紫色斑点。试验结果表明,生物素标记核酸探针可检出10pg 提纯的鸭瘟病毒DNA,并检出稀释10~5倍和肝组织鸭瘟病毒 DNA;对鸡马立克氏病毒 DNA,鸡痘病毒DNA 和噬菌体 DNA 无杂交反应;对鸭瘟病毒弱毒 DNA 产生微弱杂交。该技术具有快速、敏感、特异和无放射污染等优点。

A new technique for detection of DNA using duck plague virus(DPV) DNA genome probe labelled with biotin-11-dUTP by Nick-Translation Beaction was described.The homologous sequences of DPV sample were detected on the nitrocellulose membrane(NC,M)through dot hybridization by the DNA probe.After hybridization, the NCM was incubated with avidin-alkaline phosphatase that transformed its substrate to show the colour,the positive results were observed to be blue-violet dots.The results showed that the biotinylated DNA probe could detect purified homologous DNA target at 10 pg level and could detect DPV DNA in 10~5-fold dilution of the liver tissue.It did not hybridize with MDA DNA,avian poxvirus DNA,bacteriophage λ DNA,and avirulent DPV DNA.This technique has many advantages including rapidity,sensitivity, specificity and no radioactive pollution.