Fan Wei, Hamilton Tacita, Webster-Sesay S, Nikolich Mikeljon P, Lindler Luther E
Division of Bacterial and Rickettsial Diseases, Walter Reed Army Institute of Research, Silver Spring, MD 20910, USA.
Mol Cell Probes. 2007 Aug;21(4):245-56. doi: 10.1016/j.mcp.2006.12.005. Epub 2007 Jan 25.
In an effort to find a rapid, efficient, and reliable method for screening and classifying large numbers of tetracycline-resistant bacterial isolates, we developed a multiplex, real-time PCR assay using SYBR Green I and the Roche LightCycler. The assay can rapidly identify eight genes encoding tetracycline resistance efflux pumps including tet(A), tet(B), tet(C), tet(D), tet(E), tet(G), tet(H) and tet(J). Primers were selected for PCR amplification of these eight tetracycline resistance determinant (tet) genes commonly found in Gram-negative organisms. We combined primer pairs together to make a single-tube multiplex PCR reaction followed by melting curve analysis. Amplification of the expected tet gene products was confirmed by both agarose gel electrophoresis and DNA sequence analysis. Based on melting temperature differences, we could identify the different classes of tet genes. To test the multiplex PCR, the assay was used on 107 tetracycline-resistant clinical isolates of various Gram-negative organisms isolated in several locations around the world. About 49.5% of those strains carried a tet(A) gene, 35.5% carried a tet(B), 7.5% carried a tet(J), 5.6% carried a tet(C) and 1.9% carried a tet(D) gene. DNA sequence analysis of the amplicons confirmed that the specificity of the test was 100%. The sensitivity of the multiplex test varied from 10 to 1000 CFU per PCR reaction. Our real time PCR assay utilizing SYBR Green I and melting point analysis on the Lightcycler system showed not only a high confidence level in differentiation of the classes of tet genes but also precise reproducibility. Our multiplex PCR tet gene class identification assay offers a significant savings of time and labor in the analysis of large numbers of clinical strains compared with assays using individual gene PCR or traditional phenotype methods.
为了找到一种快速、高效且可靠的方法来筛选和分类大量耐四环素细菌分离株，我们开发了一种使用SYBR Green I和罗氏LightCycler的多重实时PCR检测方法。该检测方法可以快速鉴定编码四环素抗性外排泵的八个基因，包括tet(A)、tet(B)、tet(C)、tet(D)、tet(E)、tet(G)、tet(H)和tet(J)。选择引物用于PCR扩增革兰氏阴性菌中常见的这八个四环素抗性决定簇（tet）基因。我们将引物对组合在一起进行单管多重PCR反应，随后进行熔解曲线分析。通过琼脂糖凝胶电泳和DNA序列分析确认了预期tet基因产物的扩增。基于熔解温度差异，我们可以鉴定不同类别的tet基因。为了测试多重PCR，该检测方法应用于从世界各地不同地点分离的107株耐四环素临床革兰氏阴性菌分离株。这些菌株中约49.5%携带tet(A)基因，35.5%携带tet(B)基因，7.5%携带tet(J)基因，5.6%携带tet(C)基因，1.9%携带tet(D)基因。扩增子的DNA序列分析证实该检测的特异性为100%。多重检测的灵敏度为每个PCR反应10至1000 CFU。我们利用SYBR Green I和Lightcycler系统上的熔点分析的实时PCR检测方法不仅在区分tet基因类别方面具有高置信度，而且具有精确的可重复性。与使用单个基因PCR或传统表型方法的检测相比，我们的多重PCR tet基因类别鉴定检测方法在分析大量临床菌株时显著节省了时间和劳动力。
