Medical Research Council Unit The Gambia at the London School of Hygiene & Tropical Medicine, Banjul, The Gambia.
J. Craig Venter Institute, La Jolla, CA, United States of America.
PLoS One. 2023 Aug 3;18(8):e0289557. doi: 10.1371/journal.pone.0289557. eCollection 2023.
Several important human pathogens that cause life-threatening infections are asymptomatically carried in the Nasopharynx/Oropharynx (NP/OP). DNA extraction is a prerequisite for most culture-independent techniques used to identify pathogens in the NP/OP. However, components of DNA extraction kits differ thereby giving rise to differences in performance. We compared the DNA concentration and the detection of three pathogens in the NP/OP using the discontinued DNeasy PowerSoil Kit (Kit DP) and the DNeasy PowerLyzer PowerSoil Kit (Kit DPP).
DNA was extracted from the same set of 103 NP/OP samples using the two kits. DNA concentration was measured using the Qubit 2.0 Fluorometer. Real-time Polymerase Chain reaction (RT-PCR) was done using the QuantStudio 7-flex system to detect three pathogens: S. pneumoniae, H. influenzae, and N. meningitidis. Bland-Altman statistics and plots were used to determine the threshold cycle (Ct) value agreement for the two kits.
The average DNA concentration from kit DPP was higher than Kit DP; 1235.6 ng/ml (SD = 1368.3) vs 884.9 ng/ml (SD = 1095.3), p = 0.002. Using a Ct value cutoff of 40 for positivity, the concordance for the presence of S. pneumoniae was 82% (84/102); 94%(96/103) for N. meningitidis and 92%(95/103) for H. influenzae. Kit DP proportionately resulted in higher Ct values than Kit DPP for all pathogens. The Ct value bias of measurement for S. pneumoniae was +2.4 (95% CI, 1.9-3.0), +1.4 (95% CI, 0.9-1.9) for N. meningitidis and +1.4 (95% CI, 0.2-2.5) for H. influenzae.
The higher DNA concentration obtained using kit DPP could increase the chances of recovering low abundant bacteria. The PCR results were reproducible for more than 90% of the samples for the gram-negative H. influenzae and N. meningitidis. Ct value variations of the kits must be taken into consideration when comparing studies that have used the two kits.
几种重要的引起危及生命的感染的人类病原体无症状地存在于鼻咽/口咽（NP/OP）中。DNA 提取是用于在 NP/OP 中鉴定病原体的大多数非培养依赖技术的前提。然而，DNA 提取试剂盒的成分不同，因此性能存在差异。我们比较了使用已停产的 DNeasy PowerSoil 试剂盒（试剂盒 DP）和 DNeasy PowerLyzer PowerSoil 试剂盒（试剂盒 DPP）从同一组 103 个 NP/OP 样本中提取的 DNA 浓度和三种病原体的检测。
使用两种试剂盒从相同的 103 个 NP/OP 样本中提取 DNA。使用 Qubit 2.0 荧光计测量 DNA 浓度。使用 QuantStudio 7-flex 系统进行实时聚合酶链反应（RT-PCR），以检测三种病原体：肺炎链球菌、流感嗜血杆菌和脑膜炎奈瑟菌。Bland-Altman 统计和图用于确定两种试剂盒的阈值循环（Ct）值的一致性。
试剂盒 DPP 的平均 DNA 浓度高于试剂盒 DP；1235.6ng/ml（SD=1368.3）比 884.9ng/ml（SD=1095.3），p=0.002。使用 40 的 Ct 值作为阳性的截止值，肺炎链球菌的一致性为 82%（84/102）；94%（96/103）为脑膜炎奈瑟菌，92%（95/103）为流感嗜血杆菌。对于所有病原体，试剂盒 DP 相对于试剂盒 DPP 的 Ct 值呈比例升高。肺炎链球菌的测量 Ct 值偏倚为+2.4（95%CI，1.9-3.0），脑膜炎奈瑟菌为+1.4（95%CI，0.9-1.9），流感嗜血杆菌为+1.4（95%CI，0.2-2.5）。
使用试剂盒 DPP 获得的更高 DNA 浓度可能会增加恢复低丰度细菌的机会。革兰氏阴性菌流感嗜血杆菌和脑膜炎奈瑟菌的超过 90%的样本的 PCR 结果具有可重复性。当比较使用两种试剂盒的研究时，必须考虑试剂盒的 Ct 值变化。
