Early detection and identification of commonly encountered Candida species from simulated blood cultures by using a real-time PCR-based assay
Early detection and identification of commonly encountered Candida species from simulated blood cultures by using a real-time PCR-based assay
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DOI：
10.1016/s1525-1578(10)60498-9
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发表时间：
2004-05-01
影响因子：
4.1
通讯作者：
Crokaert, F
中科院分区：
文献类型：
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作者：
Maaroufi, Y;De Bruyne, JM;Crokaert, F
在最近的一项研究中，使用基于实时pcr的方法检测临床血液样本中的念珠菌种类（Maaroufi et al ., journal of clinical microbiology, 2003,41: 3293-3298）。在本研究中，我们评估了该方法作为BACTEC血培养系统的辅助方法在模拟低念珠菌阳性和阴性的早期检测中的效率。我们首先在体外建立了最常见的念珠菌种的接种量与这些微生物的阳性时间之间的相关性。然后，随着培养时间的增加，从最终平均接种念珠菌量为3.18菌落形成单位(CFU)/培养瓶（范围为1至6 CFU）的血培养瓶中收集等份，提取DNA并提交TaqMan-based PCR检测。为了优化这一实验，我们评估了在DNA提取物中添加0.5%牛血清白蛋白（BSA）的效果，发现它降低了抑制剂的作用。使用对所测念珠菌种类的特异性探针，在最低培养周转时间（TAT）为3.11 +/- 1.24小时后，从BACTEC系统收集的血培养液PCR检测呈阳性。在PCR反应混合物中加入BSA可提高TAT（1.84 +/- 0.41小时）。因此，将正常生长的培养瓶中的DNA“扩增”与PCR的DNA扩增相结合，可能是促进低念珠菌早期诊断的可靠工具。
In a recent study, Candida species in clinical blood samples were detected using a real-time PCR-based method (Maaroufi et al, J Clin Microbiol 2003, 41: 3293-3298). For the present study, we evaluated the efficiency of this method as an adjunct to the BACTEC blood culture system to early detection of positivity and negativity of simulated low candidemias. We first established an in vitro correlation between the inoculum of the most frequently encountered Candida species and the time to positivity of these microorganisms. Then, aliquots from blood culture bottles infected with a final average candidal inoculum of 3.18 colony-forming units (CFU)/culturc bottle (range, I to 6 CFU) were collected at increasing incubation times, and DNA was extracted and submitted to the TaqMan-based PCR assay. To optimize this assay, we evaluated the effect of adding 0.5% bovine serum albumin (BSA) to DNA extracts and found that it decreased the effects of inhibitors. Using specific probes for the tested Candida species, the PCR assay was positive on blood culture aliquots collected from the BACTEC system after a minimum culture turnaround time (TAT) of 3.11 +/- 1.24 hours. Addition of BSA to PCR reaction mixtures improves the TAT (1.84 +/- 0.41 hours). Hence, the combination of DNA "amplification" in the culture bottles by normal growth with an additional DNA amplification by PCR might be a reliable tool facilitating the early diagnosis of low candidemias.