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Abstract：
OBJECTIVE: 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.
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.
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因并评估其性能。
方法：开发了针对11种细菌菌株的多重PCR（mPCR）测定法。使用已知的临床分离株和标准菌株确认物种特异性引物。对每个引物针对其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浓度调节至100ng/μL来评估两种测定的最小可检测DNA浓度，用无DNA酶的水将其连续稀释至10μg/μL。mPCR测定的诊断准确性是通过对60个临床血液样品进行测定来建立的。
结果:开发了两种mPCR测定法。建立了55°C的最佳引物退火温度，并将其用于最终的扩增条件。化验检测到所有目标细菌，具有100pg的最小可检测DNA浓度。未从全血直接检测到病原体,但在孵育4小时和8小时后，在培养液中检测到41%(5/12)和100%(12/12)的细菌，分别。该测定还鉴定了沙门氏菌。与培养物相比，肺炎克雷伯菌共感染和额外病原体（1个大肠杆菌和2个肺炎克雷伯菌）。mPCR的敏感性和特异性分别为100.0%(71.7-100.0)和98.0%(90.7-99.0),分别。ROC曲线下面积为1.00(1.00-1.00)。
结论：mPCR检测与传统的血培养方法一样，显示出作为败血症诊断的快速工具的巨大潜力。值得注意的是,它能够识别更多的分离株，检测共感染，并以高灵敏度有效检测低细菌DNA负荷，提示其在提高败血症诊断效率方面的价值。
方法：开发了针对11种细菌菌株的多重PCR（mPCR）测定法。使用已知的临床分离株和标准菌株确认物种特异性引物。对每个引物针对其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浓度调节至100ng/μL来评估两种测定的最小可检测DNA浓度，用无DNA酶的水将其连续稀释至10μg/μL。mPCR测定的诊断准确性是通过对60个临床血液样品进行测定来建立的。
结果:开发了两种mPCR测定法。建立了55°C的最佳引物退火温度，并将其用于最终的扩增条件。化验检测到所有目标细菌，具有100pg的最小可检测DNA浓度。未从全血直接检测到病原体,但在孵育4小时和8小时后，在培养液中检测到41%(5/12)和100%(12/12)的细菌，分别。该测定还鉴定了沙门氏菌。与培养物相比，肺炎克雷伯菌共感染和额外病原体（1个大肠杆菌和2个肺炎克雷伯菌）。mPCR的敏感性和特异性分别为100.0%(71.7-100.0)和98.0%(90.7-99.0),分别。ROC曲线下面积为1.00(1.00-1.00)。
结论：mPCR检测与传统的血培养方法一样，显示出作为败血症诊断的快速工具的巨大潜力。值得注意的是,它能够识别更多的分离株，检测共感染，并以高灵敏度有效检测低细菌DNA负荷，提示其在提高败血症诊断效率方面的价值。
