Development of a real-time fluorescence resonance energy transfer PCR to detect Arcobacter species
Development of a real-time fluorescence resonance energy transfer PCR to detect Arcobacter species
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DOI：
10.1128/jcm.00256-07
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发表时间：
2007-09-01
影响因子：
9.4
通讯作者：
Menard, Armelle
中科院分区：
文献类型：
--
作者：
Abdelbaqi, Khalil;Buissonniere, Alice;Menard, Armelle
针对喹诺酮类药物耐药决定区以外的促旋酶A亚基基因的实时PCR方法已被开发用于弓形杆菌属的检测。利用荧光共振能量转移技术，通过探针杂交和熔解曲线分析进行种属鉴定。弓形杆菌属物种之间的区分是直接的，因为相应的熔点显示出显著差异，对于布氏弓形杆菌、嗜冷弓形杆菌、食源弓形杆菌和硝化弓形杆菌型菌株，特征性熔化温度分别为63.5 ° C、58.4 ° C、60.6 ° C和51.8 ° C。使用通过表型方法和16S rRNA基因测序鉴定的来自人类临床和兽医标本的106株弓形杆菌分离株的纯培养物证实了本试验的特异性。然后使用该测定筛选从腹泻患者获得的345份临床粪便样品。该测定法检测到A. Butzleri在这些临床样本中的四个（1.2%）中。通过靶向16S rRNA基因的常规PCR方法以及随后的PCR产物测序证实了这些结果。总之，该实时测定检测并区分纯培养物以及粪便基质的竞争微生物群中的弓形杆菌属物种。由于仅使用一种双探针，并且在一次试验中鉴定了多种弓形杆菌属，因此该测定是经济的。
A real-time PCR targeting the gyrase A subunit gene outside the quinolone resistance-determining region has been developed to detect Arcobacter species. The species identification was done by probe hybridization and melting curve analysis, using fluorescence resonance energy transfer technology. Discrimination between Arcobacter species was straightforward, as the corresponding melting points showed significant differences with the characteristic melting temperatures of 63.5 degrees C, 58.4 degrees C, 60.6 degrees C, and 51.8 degrees C for the Arcobacter butzleri, Arcobacter cryaerophilus, Arcobacter cibarius, and Arcobacter nitrofigilis type strains, respectively. The specificity of this assay was confirmed with pure cultures of 106 Arcobacter isolates from human clinical and veterinary specimens identified by phenotypic methods and 16S rRNA gene sequencing. The assay was then used to screen 345 clinical stool samples obtained from patients with diarrhea. The assay detected A. butzleri in four of these clinical samples (1.2%). These results were confirmed by a conventional PCR method targeting the 16S rRNA gene with subsequent sequencing of the PCR product. In conclusion, this real-time assay detects and differentiates Arcobacter species in pure culture as well as in the competing microbiota of the stool matrix. The assay is economical since only one biprobe is used and multiple Arcobacter species are identified in a single test.