用生物素标记的cDNA探针检测马铃薯纺锤块茎类病毒

DETECTION OF POTATO SPINDLE TUBER VIROID(PSTV) WITH BIOTIN-LABELLED PSTV cDNA PROBE

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.