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首页> 外文期刊>Journal of Clinical Microbiology >Use of Quantitative Real-Time PCR To Study the Kinetics of Extracellular DNA Released from Candida albicans, with Implications for Diagnosis of Invasive Candidiasis
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Use of Quantitative Real-Time PCR To Study the Kinetics of Extracellular DNA Released from Candida albicans, with Implications for Diagnosis of Invasive Candidiasis

机译：利用定量实时PCR研究白色念珠菌释放的细胞外DNA的动力学，对诊断浸润性念珠菌病有重要意义

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Quantitative real-time PCR (qPCR) is considered one of the most sensitive methods to detect low levels of DNA from pathogens in clinical samples. To improve the design of qPCR for the detection of deeply invasive candidiasis, we sought to develop a more comprehensive understanding of the kinetics of DNA released from Candida albicans in vitro and in vivo. We developed a C. albicans-specific assay targeting the rRNA gene complex and studied the kinetics of DNA released from C. albicans alone, in the presence of human blood monocytes (H-MNCs), and in the bloodstream of rabbits with experimental disseminated candidiasis. The analytical qPCR assay was highly specific and sensitive (10 fg). Cells of C. albicans incubated in Hanks balanced salt solution (±10% bovine serum albumin [BSA]) or RPMI (±10% BSA) showed a significant release of DNA at T equal to 24 h compared to T equal to 0 h (P ≤ 0.01). C. albicans incubated with H-MNCs exhibited a greater release of DNA than C. albicans cells alone over 24 h (P = 0.0001). Rabbits with disseminated candidiasis showed a steady increase of detectable DNA levels in plasma as disease progressed. Plasma cultures showed minimal growth of C. albicans, demonstrating that DNA extracted from plasma reflected fungal cell-free DNA. In summary, these studies of the kinetics of DNA release by C. albicans collectively demonstrate that cell-free fungal DNA is released into the bloodstream of hosts with disseminated candidiasis, that phagocytic cells may play an active role in increasing this release over time, and that plasma is a suitable blood fraction for the detection of C. albicans DNA.
机译：实时定量PCR（qPCR）被认为是检测临床样品中病原体中低水平DNA的最灵敏方法之一。为了改进qPCR的设计，以检测深侵袭性念珠菌病，我们寻求对白念珠菌在体外和体内释放的DNA动力学有更全面的了解。我们开发了 C。以白色念珠菌为靶标的rRNA基因复合物特异性检测方法，研究了 C释放的DNA动力学。在人血单核细胞（H-MNC）存在的情况下，以及在实验性传播念珠菌病的兔子的血流中单独使用白色念珠菌。 qPCR分析方法具有很高的特异性和敏感性（10 fg）。 C的单元格。在汉克斯（Hanks）平衡盐溶液（±10％牛血清白蛋白[BSA]）或RPMI（±10％BSA）中孵育的白色念珠菌在 T 处的DNA释放量等于24小时， T 等于0小时（ P ≤0.01）。 C。 H-MNCs培养的白色念珠菌比 C释放出更多的DNA。 24小时内单独的白色念珠菌细胞（ P = 0.0001）。随着疾病的进展，散播性念珠菌病的兔子血浆中可检测的DNA水平稳定增加。血浆培养物显示 C的生长最少。 ，表明从血浆中提取的DNA反映了无真菌细胞的DNA。总之，这些关于 C释放DNA动力学的研究。 albicans 共同证明无细胞真菌DNA释放到散播念珠菌病宿主的血液中，吞噬细胞可能随着时间的流逝在增加这种释放中起积极作用，血浆是检测血液的合适血液成分的 C。白色念珠菌 DNA。

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