摘要
用生物素-11-dUTP通过缺口平移将克隆于pST-B5和pST-B14质粒中的马铃薯纺锤块茎类病毒(Potato spindle tuber viroid,PSTV)cDNA进行标记,制备了生物素标记的PSTV-cDNA探针,在硝酸纤维素膜上进行核酸斑点杂交(Nucleic acid spot hybridization,NASH),检测了提纯的PSTV-RNA,带有PSTV全序列的质粒及感染PSTV的马铃薯植株提取液,前两者可检出的最低含量分别为8pg/斑点和0.35pg/斑点。以生物素标记的pST-B14和pST-B5为探针检测感染PSTV的马铃薯植株提取汁液。可测出的最高稀释倍数分别为126和162~243。
Potato spindle taber viroid ( PSTV ) cDNA cloned in pST-B5 and pST-B14 plasmids were labelled with biotin-11-dUTP by nick translation. The biotin-labelled PSTV cDNA probes have been used for the detection of purified PSTV-RNA, plasmids with PSTV insert and PSTV-infected potato extracts by nucleic acid spot hybridization. Using pST-B14 as probe, the minimal amount of purified PSTV-RNA detected was 8 pg/test spot. Using pST-B5 as probe, the minimal amount of pST-B5 DNA detected was 0.35 pg/test spot. The highest dilution of PSTV-infected potato extract which gives visible signal was 1/125 with pST-B14 probe and 1/162-243 with pST-B5 probe.In view of the fact that the biotinylated cDNA probes have a long shelf-life ( can be stored in solution at -20℃ for at least 1 year) and do not need special facilities for isotope work, they are more suitable for routine use in testing seed potatoes for the detection of PSTV.
出处
《病毒学报》
CAS
CSCD
北大核心
1989年第1期72-76,共5页
Chinese Journal of Virology
基金
国际马铃薯中心
内蒙自然科学基金
关键词
马铃薯类病毒
生物素标记
cDNA探测
Biotin-labelled cDNA probe Potato spindle tuber viroid Nucleic acid spot hybridization