A novel approach to eliminate detection of contaminating Staphylococcal species introduced during clinical testing
A novel approach to eliminate detection of contaminating Staphylococcal species introduced during clinical testing
复制标题
DOI：
10.1371/journal.pone.0171915
复制
发表时间：
2017-02-22
期刊：
影响因子：
3.7
通讯作者：
Jenison, Robert
中科院分区：
文献类型：
--
作者：
Ao, Wanyuan;Clifford, Adrianne;Jenison, Robert
我们在这里描述了一种策略，可以区分真正存在于临床样品中的葡萄球菌属物种与测试过程中引入的污染葡萄球菌属物种。污染性葡萄球菌属在PCR试剂中的含量较低，并在实验室人员中定植。为了消除污染物的检测，我们描述了一种方法，该方法利用向解旋酶依赖性等温扩增反应中添加足够量的非靶葡萄球菌细胞（琥珀葡萄球菌或蝇葡萄球菌）或合成寡核苷酸模板，以消耗葡萄球菌特异性tuf和mecA基因引物，从而将污染葡萄球菌扩增抑制至检测限以下。抑制模板DNA设计为与检测试剂中使用的引物完全同源，但内部序列与检测目标葡萄球菌菌种无关。抑制剂的输入量由本文所述的数学模型确定，并且被证明完全抑制葡萄球菌的污染水平，同时不会对适当的临床测定检测限产生负面影响。我们已经应用这种方法来提高检测葡萄球菌属的特异性存在于阳性血培养使用基于芯片的阵列，产生肉眼可见的结果。
We describe here a strategy that can distinguish between Staphylococcus species truly present in a clinical sample from contaminating Staphylococcus species introduced during the testing process. Contaminating Staphylococcus species are present at low levels in PCR reagents and colonize lab personnel. To eliminate detection of contaminants, we describe an approach that utilizes addition of sufficient quantities of either non-target Staphylococcal cells (Staphylococcus succinus or Staphylococcus muscae) or synthetic oligonucleotide templates to helicase dependent isothermal amplification reactions to consume Staphylococcus-specific tuf and mecA gene primers such that contaminating Staphylococcus amplification is suppressed to below assay limits of detection. The suppressor template DNA is designed with perfect homology to the primers used in the assay but an internal sequence that is unrelated to the Staphylococcal species targeted for detection. Input amount of the suppressor is determined by a mathematical model described herein and is demonstrated to completely suppress contaminating levels of Staphylococcus while not negatively impacting the appropriate clinical assay limit of detection. We have applied this approach to improve the specificity of detection of Staphylococcus species present in positive blood cultures using a chip-based array that produces results visible to the unaided eye.