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目的探讨新生儿败血症的快速可靠诊断方法,以提高临床检测细菌的速度及准确性。方法以细菌16S rRNA 基因为靶序列,设计通用引物及 TaqMan 探针,建立细菌16S rRNA 基因实时荧光定量 PCR 反应体系;选择临床引起新生儿败血症的常见菌如金黄色葡萄球菌、表皮葡萄球菌和大肠埃希菌等进行浓度梯度实验;对临床疑为新生儿败血症的830例感染性疾病的新生儿抽取静脉血分别做细菌16S rRNA 基因荧光定量 PCR 和血培养检测。结果临床常见分离株金黄色葡萄球菌、表皮葡萄球菌、大肠埃希菌荧光定量 PCR 检测结果均为阳性;巨细胞病毒、EB 病毒、乙肝病毒,新型隐球菌及白色念珠菌,人基因组 DNA 及空白对照均为阴性。细菌荧光定量 PCR 最低能检测到3个细菌,其荧光定量 CT 值为37.90;对临床疑为感染性疾病的830例患儿标本中,荧光定量 PCR 检测血标本阳性率5.18%(43/830),血培养阳性率2.41%(20/830),前者明显高于后者,差异具有统计学意义,P<0.01,30例非感染性疾病患儿血标本荧光定量 PCR 检测及细菌培养均为阴性。若以血培养作为对照,荧光定量 PCR 方法的诊断敏感性为100%,特异性为97.16%,正确诊断指数为0.972。结论建立了细菌16S rRNA 基因荧光定量 PCR 诊断新生儿败血症方法。其检测快速、简便,为新生儿败血症提供早期、敏感的病原学诊断依据。
Objective To explore a rapid and reliable diagnostic method for neonatal sepsis to improve the speed and accuracy of clinical detection of bacteria. Methods The bacterial 16S rRNA gene was used as target sequence to design universal primers and TaqMan probe. The 16S rRNA gene real-time PCR system was established. The common bacteria causing clinical neonatal sepsis, such as Staphylococcus aureus, Staphylococcus epidermidis and large intestine Escherichia coli and other concentration gradient experiments; clinically suspected neonatal sepsis in 830 cases of infectious disease neonates were venous blood bacterial 16S rRNA gene PCR and blood culture were detected. Results The common clinical isolates Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli fluorescence quantitative PCR test results were positive; cytomegalovirus, Epstein-Barr virus, hepatitis C virus, Cryptococcus neoformans and Candida albicans, human genomic DNA and blank Controls were negative. The bacterial fluorescence quantitative PCR could detect 3 bacteria at a minimum, and the fluorescence quantitative CT value was 37.90. Among the 830 children with clinically suspected infectious diseases, the positive rate of fluorescence quantitative PCR was 5.18% (43/830) , Blood culture positive rate of 2.41% (20/830), the former was significantly higher than the latter, the difference was statistically significant, P <0.01, 30 cases of non-infectious disease in blood samples of fluorescent quantitative PCR detection and bacterial culture were negative . If the blood culture as a control, the fluorescence quantitative PCR diagnostic sensitivity of 100%, specificity was 97.16%, the correct diagnostic index was 0.972. Conclusion A bacterial quantitative 16S rRNA gene PCR method for the diagnosis of neonatal sepsis was established. The test is fast and easy, providing early and sensitive etiological diagnosis basis for neonatal sepsis.