Quantitative multiplex real-time PCR for detecting class 1, 2 and 3 integrons
Quantitative multiplex real-time PCR for detecting class 1, 2 and 3 integrons
复制标题
DOI：
10.1093/jac/dkq167
复制
发表时间：
2010-08-01
影响因子：
5.2
通讯作者：
Ploy, M. C.
中科院分区：
文献类型：
--
作者：
Barraud, O.;Baclet, M. C.;Ploy, M. C.
整合子是细菌的遗传元件，可以捕获和表达包含在移动磁带中的基因。整合子在革兰氏阴性菌中被广泛发现，是抗生素耐药性的标志。我们开发了一种特异性和敏感性的基于Taqman（(R)）探针的实时PCR方法，使用三种不同的引物-探针对同时检测三种主要类型的整合子。通过检测三个靶点（每个整合子类的整合酶基因）的混合物，在10到10(8)个拷贝范围内评估敏感性。用含整合子和不含整合子的对照菌株进行特异性测定。然后将该方法应用于临床样品。PCR方法具有特异性，对所有三个基因的敏感性均为10(2)个拷贝，无论它们各自的数量如何。该方法的定量范围从10(3)到10(7)拷贝，并且能够直接检测生物样品中的整合子。我们开发了一种快速、定量、特异和敏感的方法，可用于初步筛选革兰氏阴性分离株或临床样本，以发现可能的多药耐药性。
Integrons are bacterial genetic elements that can capture and express genes contained in mobile cassettes. Integrons have been described worldwide in Gram-negative bacteria and are a marker of antibiotic resistance. We developed a specific and sensitive Taqman((R)) probe-based real-time PCR method with three different primer-probe pairs for simultaneous detection of the three main classes of integron.Sensitivity was assessed by testing mixtures of the three targets (intI integrase genes of each integron class) ranging from 10 to 10(8) copies. Specificity was determined with a panel of integron-containing and integron-free control strains. The method was then applied to clinical samples.The PCR method was specific and had a sensitivity of 10(2) copies for all three genes, regardless of their respective quantities. The method was quantitative from 10(3) to 10(7) copies, and was able to detect integrons directly in biological samples.We have developed a rapid, quantitative, specific and sensitive method that could prove useful for initial screening of Gram-negative isolates, or clinical samples, for likely multidrug resistance.