开发两种多重 PCR 检测方法,用于快速检测脓毒血症患儿体内的 11 种革兰氏阴性细菌。

IF 3.6 Q1 TROPICAL MEDICINE Tropical Medicine and Health Pub Date : 2024-06-05 DOI:10.1186/s41182-024-00606-3
Gabriel Miringu, Abednego Musyoki, Betty Muriithi, Ernest Wandera, Dan Waithiru, Erick Odoyo, Hisashi Shoji, Nelson Menza, Yoshio Ichinose
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引用次数: 0

摘要

目的:本研究旨在开发一种多重 PCR 检测方法,用于同时检测败血症的主要革兰氏阴性病原菌,并评估其性能:方法:针对 11 种细菌菌株开发了多重 PCR(mPCR)检测方法。使用已知的临床分离菌株和标准菌株确认了菌种特异性引物。针对每个引物的目标细菌基因进行梯度 PCR,以确定其最佳扩增条件。通过将细菌 DNA 浓度调整为 100 纳克/微升,并用无 DNAse 水将其稀释 10 倍至 10 pg/微升,评估了两种检测方法的最低 DNA 检测浓度。通过对 60 份临床血液样本进行检测,确定了 mPCR 检测方法的诊断准确性:结果:开发出两种 mPCR 检测方法。确定了最佳引物退火温度为 55 °C,并将其用于最终扩增条件。这些检测方法可检测到所有目标细菌,最低 DNA 检测浓度为 100 pg。虽然不能直接从全血中检测到病原体,但在培养 4 小时和 8 小时后,分别从培养液中检测到 41% (5/12)和 100% (12/12)的细菌。与培养相比,该检测方法还能发现沙门氏菌属和肺炎克雷伯菌合并感染以及额外的病原体(1 个大肠杆菌和 2 个肺炎克雷伯菌)。mPCR 的灵敏度和特异性分别为 100.0%(71.7-100.0)和 98.0%(90.7-99.0)。ROC曲线下面积为1.00(1.00-1.00):mPCR 检测作为一种快速诊断败血症的工具,与传统的血液培养方法相比具有巨大的潜力。值得注意的是,它能识别更多的分离株,检测合并感染,并能以高灵敏度有效检测低细菌 DNA 负荷,这意味着它在提高败血症诊断效率方面具有重要价值。
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Development of two multiplex PCR assays for rapid detection of eleven Gram-negative bacteria in children with septicemia.

Aim: This study aimed to develop a multiplex PCR assay for simultaneous detection of major Gram-negative etiologies of septicemia and evaluate its performance.

Methods: Multiplex PCR (mPCR) assays were developed targeting 11 bacterial strains. Species-specific primers were confirmed using known clinical isolates and standard strains. Gradient PCR was performed on each primer against its target bacterial gene to determine its optimal amplification condition. The minimum detectable DNA concentration of the two assays was evaluated by adjusting bacterial DNA concentration to 100 ng/μL and, tenfold serially diluting it up to 10 pg/μL with DNAse-free water. The diagnostic accuracy of mPCR assays was established by subjecting the assays to 60 clinical blood samples.

Results: Two mPCR assays were developed. Optimal primer annealing temperature of 55 °C was established and utilized in the final amplification conditions. The assays detected all targeted bacteria, with a 100 pg minimum detectable DNA concentration. Pathogens were not detected directly from whole blood, but after 4 h and 8 h of incubation, 41% (5/12) and 100% (12/12) of the bacteria were detected in culture fluids, respectively. The assays also identified Salmonella spp. and Klebsiella pneumoniae co-infections and extra pathogens (1 E. coli and 2 K. pneumoniae) compared with culture. The sensitivity and specificity of the mPCR were 100.0% (71.7-100.0) and 98.0% (90.7-99.0), respectively. The area under the ROC curve was 1.00 (1.00-1.00).

Conclusions: The mPCR assays demonstrated substantial potential as a rapid tool for septicemia diagnosis alongside the traditional blood culture method. Notably, it was able to identify additional isolates, detect co-infections, and efficiently detect low bacterial DNA loads with high sensitivity, implying its value in enhancing efficiency of diagnosis of septicemia.

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来源期刊
Tropical Medicine and Health
Tropical Medicine and Health TROPICAL MEDICINE-
CiteScore
7.00
自引率
2.20%
发文量
90
审稿时长
11 weeks
期刊最新文献
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