Henderson Kelley C, Sheppard Edward S, Rivera-Betancourt Omar E, Choi Joo-Young, Dluhy Richard A, Thurman Kathleen A, Winchell Jonas M, Krause Duncan C
Department of Microbiology, University of Georgia, Athens, GA, USA.
Analyst. 2014 Dec 21;139(24):6426-34. doi: 10.1039/c4an01141d.
Mycoplasma pneumoniae is a cell wall-less bacterial pathogen of the human respiratory tract that accounts for up to 20% of community-acquired pneumonia. At present, the standard for detection and genotyping is quantitative polymerase chain reaction (qPCR), which can exhibit excellent sensitivity but lacks standardization and has limited practicality for widespread, point-of-care use. We previously described a Ag nanorod array-surface enhanced Raman spectroscopy (NA-SERS) biosensing platform capable of detecting M. pneumoniae in simulated and true clinical throat swab samples with statistically significant specificity and sensitivity. We report here that differences in sample preparation influence the integrity of mycoplasma cells for NA-SERS analysis, which in turn impacts the resulting spectra. We have established a multivariate detection limit (MDL) using NA-SERS for M. pneumoniae intact-cell sample preparations. Using an adaptation of International Union of Pure and Applied Chemistry (IUPAC)-recommended methods for analyzing multivariate data sets, we found that qPCR had roughly 10× better detection limits than NA-SERS when expressed in CFU ml(-1) and DNA concentration (fg). However, the NA-SERS MDL for intact M. pneumoniae was 5.3 ± 1.0 genome equivalents (cells per μl). By comparison, qPCR of a parallel set of samples yielded a limit of detection of 2.5 ± 0.25 cells per μl. Therefore, for certain standard metrics NA-SERS provides a multivariate detection limit for M. pneumoniae that is essentially identical to that determined via qPCR.
肺炎支原体是人类呼吸道的一种无细胞壁细菌病原体，占社区获得性肺炎的比例高达20%。目前，检测和基因分型的标准是定量聚合酶链反应（qPCR），它具有出色的灵敏度，但缺乏标准化，在广泛的即时检测应用中实用性有限。我们之前描述了一种银纳米棒阵列表面增强拉曼光谱（NA-SERS）生物传感平台，该平台能够在模拟和真实临床咽拭子样本中检测肺炎支原体，具有统计学上显著的特异性和灵敏度。我们在此报告，样本制备的差异会影响用于NA-SERS分析的支原体细胞的完整性，进而影响所得光谱。我们已经为肺炎支原体完整细胞样本制备建立了使用NA-SERS的多变量检测限（MDL）。通过采用国际纯粹与应用化学联合会（IUPAC）推荐的分析多变量数据集的方法，我们发现当以CFU ml(-1)和DNA浓度（fg）表示时，qPCR的检测限比NA-SERS大约高10倍。然而，完整肺炎支原体的NA-SERS MDL为5.3±1.0基因组当量（每微升细胞数）。相比之下，一组平行样本的qPCR检测限为每微升2.5±0.25个细胞。因此，对于某些标准指标，NA-SERS为肺炎支原体提供的多变量检测限与通过qPCR确定的检测限基本相同。
