A Rapid and Automated Sample-to-Result Candida auris Real-Time PCR Assay for High-Throughput Testing of Surveillance Samples with the BD Max Open System
A Rapid and Automated Sample-to-Result Candida auris Real-Time PCR Assay for High-Throughput Testing of Surveillance Samples with the BD Max Open System
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DOI：
10.1128/jcm.00630-19
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发表时间：
2019-10-01
影响因子：
9.4
通讯作者：
Chaturvedi, V
中科院分区：
文献类型：
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作者：
Leach, L.;Russell, A.;Chaturvedi, V
耐多药酵母菌病原体耳念珠菌继续在全世界引起暴发和临床病例聚集。在此之前，我们开发了一种实时PCR检测C。从监测样品中分离的耳螨（L. Leach，Y. zhu和S. Chaturvedi，J Clin Microbiol 56：e01223-17，2018，https：//doi.org/10.1128/JCM.01223-17）。该检测方法在正在进行的C.纽约市爆发了耳道感染。为了便于在其他实验室实施该检测，我们开发了一种自动化的样品到结果实时C。使用BD Max开放系统的耳PCR测定。我们优化了样品提取在三个不同的温度和四个孵育期。使用八个患者样本池确定灵敏度，使用四个C分支计算特异性。耳和密切和远亲酵母。对两个患者样本池进行三次独立浸提和四次重复检测，获得了试验精密度。BD Max最佳测定条件如下：在75 ° C下提取DNA 20分钟，并使用PerfeCTa多重定量PCR（qPCR）ToughMix。该方法的检测限（LOD）为1 C。耳内菌落形成单位/PCR。我们检测到了C的所有四个分支。与其他酵母菌无交叉反应。在检测的110份患者监测样本中，有50份对C呈阳性。与手动检测结果相比，BD Max系统的临床灵敏度为96%，准确度为94%。BD Max检测允许高通量C.在12小时的工作日内对180份监测样本进行耳筛查。
The multidrug-resistant yeast pathogen Candida auris continues to cause outbreaks and clusters of clinical cases worldwide. Previously, we developed a real-time PCR assay for the detection of C. auris from surveillance samples (L. Leach, Y. Zhu, and S. Chaturvedi, J Clin Microbiol 56:e01223-17, 2018, https://doi.org/10.1128/JCM.01223-17). The assay played a crucial role in the ongoing investigations of the C. auris outbreak in New York City. To ease the implementation of the assay in other laboratories, we developed an automated sample-to-result real-time C. auris PCR assay using the BD Max open system. We optimized sample extraction at three different temperatures and four incubation periods. Sensitivity was determined using eight pools of patient samples, and specificity was calculated using four clades of C. auris and closely and distantly related yeasts. Three independent extractions and testing of two patient sample pools in quadruplicate yielded assay precision. BD Max optimum assay conditions were as follows: DNA extraction at 75 degrees C for 20 min and the use of PerfeCTa multiplex quantitative PCR (qPCR) ToughMix. The limit of detection (LOD) of the assay was one C. auris CFU/PCR. We detected all four clades of C. auris without cross-reactivity to other yeasts. Of the 110 patient surveillance samples tested, 50 were positive for C. auris using the BD Max system with 96% clinical sensitivity and 94% accuracy compared to the results of the manual assay. The BD Max assay allows high-throughput C. auris screening of 180 surveillance samples in a 12-h workday.