比较两种用于鼻咽/口咽样本的 DNA 提取试剂盒的 DNA 浓度和细菌病原体 PCR 检测结果。

Comparison of DNA concentration and bacterial pathogen PCR detection when using two DNA extraction kits for nasopharyngeal/oropharyngeal samples.

机构信息

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.

出版信息

PLoS One. 2023 Aug 3;18(8):e0289557. doi: 10.1371/journal.pone.0289557. eCollection 2023.

DOI:10.1371/journal.pone.0289557

PMID:37535692

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC10399880/

Abstract

INTRODUCTION

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).

METHODS

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.

RESULTS

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.

CONCLUSION

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.

摘要

简介

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。

方法

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。

结果

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）。