Use of tuf sequences for genus-specific PCR detection and phylogenetic analysis of 28 streptococcal species
Use of tuf sequences for genus-specific PCR detection and phylogenetic analysis of 28 streptococcal species
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DOI：
10.1128/jcm.42.8.3686-3695.2004
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发表时间：
2004-08-01
影响因子：
9.4
通讯作者：
Bergeron, MG
中科院分区：
文献类型：
--
作者：
Picard, FJ;Ke, DB;Bergeron, MG
一个761 bp的部分tuf基因（编码延长因子涂）从28个临床相关的链球菌种，通过测序扩增子产生的使用宽范围PCR引物。这些tuf序列用于选择链球菌特异性PCR引物并进行系统发育分析。使用102种不同的细菌物种（包括28种链球菌）验证了PCR检测的特异性。从所有链球菌物种纯化的基因组DNA被有效地检测到，而没有扩增的DNA从74个nonstreptococcal细菌物种测试的72。与肠球菌和乳酸乳球菌的DNA存在交叉扩增。然而，与任何链球菌tuf序列相比，这两个物种的761-bp tuf部分的15 - 31%核苷酸序列差异提供了足够的序列差异，以允许开发对链球菌特异性的内部探针。链球菌特异性测定对所有28种测试的链球菌物种都是高度敏感的（即，每个PCR的检测限为1至10个基因组拷贝）。的tuf序列数据也被用来进行广泛的系统发育分析，这是一般在协议的基础上确定的16 S rRNA基因数据的系统发育。然而，tuf基因在链球菌种水平上提供了更好的区分，这对于鉴定非常密切相关的种特别有用。总之，tuf似乎更适合于16 S核糖体RNA基因的发展诊断检测和鉴定链球菌种，因为它的物种特异性遗传分化水平较高。
A 761-bp portion of the tuf gene (encoding the elongation factor Tu) from 28 clinically relevant streptococcal species was obtained by sequencing amplicons generated using broad-range PCR primers. These tuf sequences were used to select Streptococcus-specific PCR primers and to perform phylogenetic analysis. The specificity of the PCR assay was verified using 102 different bacterial species, including the 28 streptococcal species. Genomic DNA purified from all streptococcal species was efficiently detected, whereas there was no amplification with DNA from 72 of the 74 nonstreptococcal bacterial species tested. There was cross-amplification with DNAs from Enterococcus durans and Lactococcus lactis. However, the 15 to 31% nucleotide sequence divergence in the 761-bp tuf portion of these two species compared to any streptococcal tuf sequence provides ample sequence divergence to allow the development of internal probes specific to streptococci. The Streptococcus-specific assay was highly sensitive for all 28 streptococcal species tested (i.e., detection limit of 1 to 10 genome copies per PCR). The tuf sequence data was also used to perform extensive phylogenetic analysis, which was generally in agreement with phylogeny determined on the basis of 16S rRNA gene data. However, the tuf gene provided a better discrimination at the streptococcal species level that should be particularly useful for the identification of very closely related species. In conclusion, tuf appears more suitable than the 16S ribosomal RNA gene for the development of diagnostic assays for the detection and identification of streptococcal species because of its higher level of species-specific genetic divergence.