Liu Chang-Feng, Shi Xin-Ping, Chen Yun, Jin Ye, Zhang Bing
Department of General Surgery, Tongde Hospital of Zhejiang Province, Hangzhou, Zhejiang Province, China.
Department of Clinical Laboratory, Tongde Hospital of Zhejiang Province, Hangzhou, Zhejiang Province, China.
J Clin Lab Anal. 2018 Feb;32(2). doi: 10.1002/jcla.22256. Epub 2017 May 17.
The survival rate of septic patients mainly depends on a rapid and reliable diagnosis. A rapid, broad range, specific and sensitive quantitative diagnostic test is the urgent need. Thus, we developed a TaqMan-Based Multiplex real-time PCR assays to identify bloodstream pathogens within a few hours.
Primers and TaqMan probes were designed to be complementary to conserved regions in the 16S rDNA gene of different kinds of bacteria. To evaluate accurately, sensitively, and specifically, the known bacteria samples (Standard strains, whole blood samples) are determined by TaqMan-Based Multiplex real-time PCR. In addition, 30 blood samples taken from patients with clinical symptoms of sepsis were tested by TaqMan-Based Multiplex real-time PCR and blood culture.
The mean frequency of positive for Multiplex real-time PCR was 96% at a concentration of 100 CFU/mL, and it was 100% at a concentration greater than 1000 CFU/mL. All the known blood samples and Standard strains were detected positively by TaqMan-Based Multiplex PCR, no PCR products were detected when DNAs from other bacterium were used in the multiplex assay. Among the 30 patients with clinical symptoms of sepsis, 18 patients were confirmed positive by Multiplex real-time PCR and seven patients were confirmed positive by blood culture.
TaqMan-Based Multiplex real-time PCR assay with highly sensitivity, specificity and broad detection range, is a rapid and accurate method in the detection of bacterial pathogens of sepsis and should have a promising usage in the diagnosis of sepsis.
脓毒症患者的存活率主要取决于快速且可靠的诊断。一种快速、检测范围广、特异且灵敏的定量诊断测试是迫切需求。因此，我们开发了一种基于TaqMan的多重实时PCR检测方法，可在数小时内鉴定血流中的病原体。
设计引物和TaqMan探针，使其与不同种类细菌的16S rDNA基因中的保守区域互补。为了准确、灵敏且特异的评估，通过基于TaqMan的多重实时PCR对已知细菌样本（标准菌株、全血样本）进行检测。此外，对30例有脓毒症临床症状患者的血样进行基于TaqMan的多重实时PCR检测和血培养。
多重实时PCR在浓度为100 CFU/mL时的平均阳性检出率为96%，浓度大于1000 CFU/mL时为100%。所有已知血样和标准菌株均通过基于TaqMan的多重PCR检测呈阳性，当在多重检测中使用其他细菌的DNA时未检测到PCR产物。在30例有脓毒症临床症状的患者中，18例通过多重实时PCR确诊为阳性，7例通过血培养确诊为阳性。
基于TaqMan的多重实时PCR检测方法具有高灵敏度、特异性和广泛的检测范围，是一种快速、准确检测脓毒症细菌病原体的方法，在脓毒症诊断中应具有广阔的应用前景。
