Ibrahim A, Norlander L, Macellaro A, Sjöstedt A
Department of Microbiology, National Defence Research Establishment, Umeå, Sweden.
Eur J Epidemiol. 1997 Apr;13(3):329-34. doi: 10.1023/a:1007385104687.
A previously published sequence of the 23S rRNA gene of Coxiella burnetii has been reported to contain an intervening sequence of 444 base pairs (bp). The sequence information on the intervening sequence and the 23S rRNA gene was exploited to develop a specific PCR-based assay for C. burnetii. A primer set was designed that amplified a 477-bp fragment encompassing part of the intervening sequence and part of the 23S rDNA. From all of nine C. burnetii strains tested, a fragment of the expected size was amplified. As predicted from the published sequence, restriction endonuclease digestion of the PCR product from the Coxiella strains with RsaI produced two distinct fragments approximately 210- and 270-bp in size. The PCR-based method showed a detection limit of 10(2) bacteria as determined by visualization of the amplicon on an agarose gel. When experimentally infected blood was analyzed, the detection limit was 10(3) bacteria. No visible amplicons were observed when 41 bacterial strains, representing 29 species other than C. burnetii, were tested. The presence of the DNA in all bacterial samples was confirmed by amplification of a 350-bp fragment of the 16S rDNA using two universal primers. The described method proved to be specific for C. burnetii and may become a rapid and sensitive diagnostic assay for C. burnetii. The results also demonstrate that the intervening sequence within the 23S rRNA gene is generally found among isolates of C. burnetii.
据报道，先前发表的伯纳特柯克斯体23S rRNA基因序列包含一段444个碱基对（bp）的间隔序列。利用该间隔序列和23S rRNA基因的序列信息，开发了一种基于PCR的伯纳特柯克斯体特异性检测方法。设计了一组引物，可扩增出一个477 bp的片段，该片段包含部分间隔序列和部分23S rDNA。在所检测的全部9株伯纳特柯克斯体菌株中，均扩增出了预期大小的片段。正如从已发表序列所预测的那样，用RsaI对来自柯克斯体菌株的PCR产物进行限制性内切酶消化，产生了两个大小约为210 bp和270 bp的不同片段。通过在琼脂糖凝胶上观察扩增子确定，基于PCR的方法检测限为10²个细菌。对实验感染的血液进行分析时，检测限为10³个细菌。当检测代表除伯纳特柯克斯体之外的29个物种的41株细菌菌株时，未观察到可见的扩增子。使用两个通用引物扩增16S rDNA的350 bp片段，证实了所有细菌样本中均存在DNA。所描述的方法被证明对伯纳特柯克斯体具有特异性，可能成为一种快速、灵敏的伯纳特柯克斯体诊断检测方法。结果还表明，23S rRNA基因内的间隔序列通常存在于伯纳特柯克斯体分离株中。
