Masala Speranza, Molicotti Paola, Bua Alessandra, Zumbo Antonella, Delogu Giovanni, Sechi Leonardo A, Zanetti Stefania
Department of Biomedical Sciences, University of Sassari.
New Microbiol. 2010 Apr;33(2):155-62.
An evaluation of the utility of rep PCR typing compared to the 15 loci discriminatory set of MIRU-VNTR was undertaken. Twenty-nine isolates of Mycobacterium tuberculosis from patients were examined. Genomic DNA was extracted from the isolates by standard method. The number of copies of tandem repeats of the 15 MIRU-VNTR loci was determined by PCR amplification and agarose gel electrophoresis of the amplicons. M. tuberculosis outbreak-related strains were distinguished from other isolates. MIRU-VNTR typing identified 4 major clusters of strains. The same isolates clustered together after RFLP typing, but rep-PCR identified only 3 of them. The concordance between RFLP and MIRU-VNTR typing was complete, with the exception of two isolates with identical RFLP patterns that differed in the number of tandem repeat copies at two MIRU-VNTR alleles. A further isolate, even sharing the same RFLP pattern, differed by one repeat from the rest of its cluster. We also tested the use of an automated rep-PCR for clinical laboratory applications but it failed to identify the link between two pairs of epidemiologically related strains clustered by the other 2 techniques. For superior discrimination, ease of comparison of results and lower cost, MIRU-VNTR typing should be the favored PCR-based typing tool.
对重复聚合酶链反应（rep PCR）分型与15个位点的MIRU-VNTR鉴别组相比的效用进行了评估。检查了29株来自患者的结核分枝杆菌分离株。通过标准方法从分离株中提取基因组DNA。通过PCR扩增和扩增产物的琼脂糖凝胶电泳确定15个MIRU-VNTR位点串联重复的拷贝数。区分了与结核分枝杆菌暴发相关的菌株和其他分离株。MIRU-VNTR分型鉴定出4个主要菌株簇。在限制性片段长度多态性（RFLP）分型后，相同的分离株聚集在一起，但rep-PCR仅鉴定出其中3个。RFLP和MIRU-VNTR分型之间的一致性是完全的，但有两个具有相同RFLP模式的分离株在两个MIRU-VNTR等位基因的串联重复拷贝数上有所不同。另一个分离株，即使具有相同的RFLP模式，与其簇中的其他分离株在一个重复上也有所不同。我们还测试了自动rep-PCR在临床实验室应用中的使用，但它未能识别通过其他两种技术聚类的两对流行病学相关菌株之间的联系。为了获得更好的鉴别能力、便于结果比较和降低成本，MIRU-VNTR分型应该是首选的基于PCR的分型工具。
