摘要
采用随机引物法制备地高辛素、生物素和^(32)P标记产毒性大肠杆菌Ⅰ型不耐热肠毒素(LT_1)DNA探针。地高辛素、生物素和^(32)P LT_1探针分别在DNA斑点杂交试验中检测到0.1pg、1pg和0.1pg的质粒DNA,在菌落原位杂交试验中检测到12、120和12个细菌/ml,在粪便斑点杂交试验中检测到24、240和24个细菌/ml。采用RNA酶A和蛋白酶K消化处理滤膜,消除了假阳性,使得地高辛素、生物素LT_1探针的特异性与^(32)PLT_1探针完全一致。检测143株自腹泻病人分离的大肠杆菌,14株(9.79％)阳性,3种探针结果相同。
Type I heat-labile toxin(LT1) DNA probes were labeled with digoxigenin, biotin, and 32P by random priming. Digoxigenin labeled, biotinylated, and 32P LTj probe detected 0.1 Pg, 1 Pg. and 0.1 Pg of EWD299 DNA in DNA blot hybridization; 12, 120 and 12 bacteria per millilitre of normal saline in bacteria in situ hybridization; 24, 240, and 24 bacteria per millilitre of stool suspension in stool blot hybridization after exposure to indicator dyes for 4 hours or X-ray film for 72 hours. The treatment of nitrocellulose membrane filters with RNase A and proteinase K eliminated the possibility of false-positivity and made the specificity of digoxigenin labeled and biotinylated LT: probes as good as 32P
LTI probe. 9.79% (14/143) Escherichia coli from diarrheal patients were detected by these three probes.
出处
《中山医科大学学报》
CSCD
1990年第1期38-42,共5页
Academic Journal of Sun Yat-sen University of Medical Sciences
