脑脊液细菌16S rRNA荧光定量PCR快速诊断儿童化脓性脑膜炎

A rapid diagnosis of purulent meningitis in children by 16S rRNA FQ-PCR in CSF

目的探讨儿童化脓性脑膜炎(化脑)新的快速诊断方法。方法2003-08—2005-12采用16SrRNA荧光定量法对浙江大学儿童医院49例临床疑似化脑患儿脑脊液(CSF)的细菌DNA进行测定;监测化脑患儿脑脊液细菌DNA拷贝数,同期进行CSF细菌培养的对照。结果(1)荧光定量PCR(FQ-PCR)检测49份脑脊液标本发现17份阳性,阳性率为34.7%(17/49),明显高于脑脊液培养的阳性率10.2%(5/49),差异具有显著性(P<0.01)。(2)对17份FQ-PCR阳性标本进一步测定细菌DNA的拷贝数,发现患儿病情与其DNA拷贝数呈正相关,与其Ct值(指每个反应管内的荧光信号到达设定的阈值时所经历的循环数)呈负相关,Ct值越低,脑脊液细菌DNA拷贝数越高,患儿的预后越差。(3)FQ-PCR、CSF细菌培养同时阳性的仅为5例。(4)对2例脑脊液FQ-PCR的产物测序,Ct值17.9的测序提示为大肠埃希菌,符合CSF细菌培养结果;Ct值31.8的,测序未果。结论荧光定量PCR特异性强、敏感性高,需标本量少,是早期快速诊断儿童化脑的可靠方法,具有较大的应用价值。

Objective To explore a new technique of rapid diagnosis of purulent meningitis in children. Methods Cerebrospinal fluid（CSF） specimens from 49 cases of suspected purulent meningitis were detected by 16S rRNA FQ- PCR in the Children Hospital Affiliated to Zhejiang University from August ,2003 to December ,2005. The copy numbers of bacterial DNA in CSF were detected, and the bacterial cultures of CSF were done as controls at the same time. Results （ 1 ） Of the 49 cases, 17 were positive by FQ- PCR,the positive rate（34.7% ） （17/49） was significantly higher than that of CSF culture 10. 2% （5/49） （ P 〈0. 01）. （2）The copy numbers of bacterial DNA in CSF were determined among the seventeen FQ- PCR positive specimens, and they were found to have positive correlation with the state of illness in children;the number of Ct found negative correlation with the illness. The lower the Ct numbers, the higher the DNA copy numbers, and the prognosis of children would be worse. （3）Of the 49 samples, only 5 cases were both positive by FQ-PCR and routing bacterial culture of CSF. （4） Two products of FQ-PCR in CSF were sequenced. Bacterium coli were shown in the sequence result of Ct 17. 9;it corresponded with the result of CSF culture. Sequencing failed in Ct 31.8. Conclusion FQ-PCR with higher specificity and sensitivity, demanding less CSF,is rapid and reliable for diagnosing purulent meningitis in children. So it has considerable value of application.