实时荧光定量PCR快速检测无菌性体液革兰阳/阴性细菌感染

目的　探讨将实时荧光定量PCR（qPCR）检测细菌16S rRNA基因用于快速判断无菌性体液革兰阳/阴性细菌感染，并评价其分析性能。方法　以细菌16S rRNA基因为目标设计通用引物、通用探针及革兰阴性（GN）菌探针，对14种标准菌株、20种临床菌株、白色念珠菌、乙型肝炎病毒(HBV)、人基因组及阴性对照进行qPCR检测，评价该方法引物和探针的通用性和特异性及检测限。以104 cfu/ml菌悬液、阴性对照的循环域（Ct）值99%置信区间下限分别作为尿路、其他无菌性体液细菌感染判定的分界值。用271例临床无菌性体液标本评价qPCR法的临床效能。结果　通用探针-qPCR，所有实验菌株检测为阳性。GN探针-qPCR，所有实验革兰阴性菌株检测为阳性。通用探针的检测限为1×101～1×102 cfu/ml。GN探针的检测限可低至1.0×101 cfu/ml。判定尿路、其他无菌性体液细菌感染的分界值分别为23.76、29.37。结论　应用细菌16S rRNA基因通用探针和GN探针的qPCR方法通用性好、特异性强、检测限低，可快速检测临床无菌性体液常见细菌感染，为临床提供准确、可靠的病原学诊断依据。

Real-time fluorescence quantitative PCR for rapid detection of gram-negative or gram-positive bacterial infection in sterile body fluids

Objective To investigate the detection of bacterial 16S rRNA gene by real-time fluorescence quantitative PCR (qPCR) for rapid determination of gram-positive/negative bacterial infection in aseptic body fluids and evaluate its analytical performance. Methods Universal primers, universal probes and gram-negative(GN) probes were designed based on the bacterial 16S rRNA gene. qPCR was performed on 14 standard strains, 20 clinical isolations, Candida albicans, HBV, human genome and negative control to evaluate the versatility and specificity of the primers and probes and the limit of detection. The lower limit of the 99% confidence interval of Ct value detected by 104 cfu/ml bacterial suspension and negative control was used as the cut-off value for the determination of bacterial infection in the urinary tract and other aseptic body fluids bacterial infections. The clinical efficacy of the qPCR method was evaluated using 271 clinical aseptic fluid samples. Results Bacterial DNA was positive for all tested strains in universal probe-qPCR. The bacterial DNA test results of all the tested gram-negative strains were positive in GN probe-qPCR. The detection limit of the universal probe is 1x101 to 1x102 cfu/ml, and the detection limit of GN probe can be as low as 1.0x101 cfu/ml. The cutoff values of urinary tract and other aseptic body fluids bacterial infections were 23.76 and 29.37 respectively. Conclusion The qPCR with universal probes and GN probes based on the bacterial 16S rRNA gene is characterized by its high specificity, good versatility and low limitation which can rapidly detect common bacterial infections in clinical aseptic body fluids and provide accurate and reliable etiological diagnosis basis for clinical application.