套式结核菌基因扩增试验的建立及初步应用

Detection of mycobateriuin tulierculosis using a nested-primer gene amplification assay

根据结核菌（Ｍｔｂ）之ｇｒｏＥＬ基因序列中发现的有特异性顺序设计的外引物及内引物建立了套式Ｍｔｂ基因扩增试验（Ｍｔｂ－ＮＰＧＡＡ）。用顺序１０倍稀释Ｍｔｂ菌悬液法显示Ｍｔｂ－ＮＰＧＡＡ敏感度达１条菌／ｍｌ，并能完全重复。在试验用的１３株分支杆菌中，在３４４ｂｐ处Ｍｔｂ出现了阳性扩增带。方法引物可以合成，试剂及仪器全部国产，整个实验６～８小时可以完成，这些结果表明Ｍｔｂ－ＮＰＧＡＡ是一种高度敏感、特异和能获得满意结果的和有望普及推广的Ｍｔｂ检测技术。当用ＮＰＧＡＡ检测以往皮肤病人皮损分离的抗酸菌培养物时，这些培养物显示与标推Ｍｔｂ有相同阳性ＤＮＡ扩增模型。此外尚对菌体ＤＮＡ释放条件，抗酸菌分离培养标本处理等问题作了讨论。

A nestedprimer gene arnplification assay(NPGAA) was established by using two pairs ofprimers, an outside pair of primers and an inside pairof primers, sequences of which were from M. tuberculosis(Mtb) gro EL genes. The outside pair of primersshould amplify a 576bp piece of the Mtb gro EL genethat contains sites for the inside pair of priirters, whichshould amplify a 344bp piece. The results showsNPGAA'S detectable limitation was 1 organismlmland no amplification products were produced fromDNAs of other mycobacterium species tested in thisstudy. For detecting Mtb, the entire NPGAA, fromsample preparation to data analysis, can be completedwithin 68 hours. When identifying AFB cultures iso-size patterns were obtained.