16S rRNA基因芯片杂交技术快速诊断新生儿化脓性脑膜炎临床研究

Rapid diagnosis of purulent meningitis in neonates by 16SrRNA microarrays

目的探讨新生儿化脓性脑膜炎(以下简称化脑)新的快速诊断方法。方法2003年8至2006年2月浙江大学医学院附属儿童医院采用脑脊液细菌16S rRNA基因芯片技术对24例临床上疑似化脑患儿脑脊液(CSF)的细菌DNA进行测定,同期进行与CSF细菌培养的对照。结果(1)49株细菌PCR扩增产物基因芯片杂交结果显示,33株G+菌其G+探针和通用探针均阳性,G-探针阴性;16株G-菌中,G-探针和通用探针均阳性,G+探针阴性,均能与相应特异的探针发生杂交。(2)大肠埃希菌DNA的PCR产物其16S rRNA基因芯片的最小检出量为1pg,约等于10个拷贝数,相当于2个细菌。(3)16S rRNA基因芯片检测24份脑脊液标本发现11份阳性,阳性率为45.83%(11/24),明显高于脑脊液培养的阳性率12.50%(3/24),差异有统计学意义(P<0.01)。结论脑脊液细菌16S rRNA基因芯片检测技术特异性强、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 neonates. Methods Cerebrospinal fluid（CSF） specimens from 24 cases of suspected purulent meningitis were detected by 16SrRNA Microarrays in the Children＇ s Hospital affilated to Zhejiang University from August 2003 to February, 2006 ; the bacterial cultures of CSF were done as controls at the same time. Results （ 1 ） The PCR amplified products of 49 strains were tested by the gene chip hybridization, including 33 Gram-positve and 16 Gram-negative strains. Using the amplified DNA of the 33 Gram-positive bacteria as templets, there were positive findings for the two universal probes and the one Gram-positve probe. The Gram-negative probes and universal probes showed positive results when 16 Gram-negative bacteria were used as templates. Each strain specifically hybridized with its own probe. （2） 1pg（10-12g） （10 copies） were detected by 165 rRNA gene chip hybridization at least in the PCR amplified products of Escherichia coli-enteric bacterium, equaled to 2 bacteria nearly. （3） Of the 24 cases, 11 were positive by 16S rRNA Microarrays, the positive rate 45.83% （11/24） being significantly higher than that of CSF culture （12.50%） （3/24） （P 〈 0.01 ）. Conclusion 16SrRNA Microarrays with higher specificity and sensitivity, demanding less CSF, is rapid and reliable for diagnosing purulent meningitis in neonates. So it has considerable value of application.