16S～23SrDNA间隔区序列在分枝杆菌分类鉴定中的应用研究

STUDIES ON APPLICATION FOR CLASSIFICATION AND IDENTIFICATION IN MYCOBACTERIUM BY ANALYSIS OF PCR AMPLIFICATION OF 16S～23S RIBOSOMAL DNA SPACER SEQUENCES

采用聚合酶链反应 (PCR)技术对分枝杆菌 1 6S～ 2 3SrDNA间隔区序列进行扩增 ,其产物经聚丙烯酰胺凝胶电泳 (PAGE) ,并通过计算机聚类分析 ,在基因水平上评价其对分枝杆菌分类与鉴定的意义。退火温度 45℃时 ,PCR扩增的敏感性为 50 0fg/μL ,而 50℃时的敏感性为5pg/μL。已通过对 2 2种分枝杆菌和 9种非分枝杆菌的特异性实验 ,结果表明 :扩增条带多集中在 30 0～ 60 0bp之间 ,多数受试快速生长分枝杆菌和非分枝杆菌扩增条带多且分子量较大 ,缓慢生长分枝杆菌扩增条带相对较少 ,分子量较小。PAGE和计算机聚类分析结果显示 ,分枝杆菌种间条带特异性的相似性系数均小于 70 % ,且绝大多数菌种间小于 50 %。可将被试菌株鉴定到种的水平。PAGE和聚类分析是分枝杆菌分类鉴定的一种快速、有效的方法。

The 16S\|23S ribosomal DNA spacer sequence of \%mycobacterium\% were amplified by PCR. The products were visualized by PAGE, and evaluate the possibility for classification and identification of mycobacterium at gene level. The sensitivity of PCR in annealing temperature 45℃ was 500fg/μL, whereas 50℃ was 5pg/μL. The results showed that: the amplified bands ranging from 300 to 600bp, most of rapid\|growing \%Mycobacterium\% and \%Nonmycobacterium\% tested have more bands and the bands molecular weights were larger than slow\|growing \%Mycobacterium\%. The relativity of mycobacterium <70%, most of them <50%. This experimental method might be rapid and effective for differentiation of \%Mycobacterium\% at species level.