应用荧光定量PCR检测细菌性脑膜炎病原体DNA的研究

Studies using fluorescence quantitative PCR to detect DNA of pathogens responsible for bacterial meningitis pathogen

目的应用荧光定量PCR检测细菌性脑膜炎病原体DNA,并对脑膜炎奈瑟菌进行基因分群。方法提取脑膜炎患者脑脊液和血标本中待检菌DNA,采用荧光定量PCR扩增ctrA、bexA、lytA基因,对ctrA扩增阳性标本及部分流脑菌株进行基因分群。结果 685份脑脊液标本中19份检出脑膜炎奈瑟菌、8份检出肺炎链球菌、2份检出b型流感嗜血杆菌DNA基因片段;2份血清标本脑膜炎奈瑟菌DNA基因检测均为阳性。对ctrA基因扩增阳性标本进行A、B、C、W135、X及Y分群,有18份为C群,3份为B群;部分健康人群携带的流脑菌株有14份为B群,2份为C群,1份为X群。结论荧光定量PCR灵敏性高,检测快速,可用于细菌性脑膜炎病原体的检测、鉴别及对脑膜炎奈瑟菌的分群。

Objectives To use real-time fluorescence quantitative PCR to detect the pathogens responsible for bacterial meningitis and to identify the serogroups of Neisseria meningitidis.Methods Bacterial DNA was extracted from 685 samples of cerebral spinal fluid（CSF） and 2 blood samples.Species-specific genes（ctrA for N.meningitidis,bexA for Haemophilus influenzae,and lytA for Streptococcus pneumoniae） were detected from the extracted DNA with real-time PCR,and ctrA-positive specimens were serogrouped.Results Of the 685 CSF samples,19 were positive for ctrA,8 were positive for lytA,and 2 were positive for bexA.Both of the two blood samples were positive for ctrA.Of the 21 samples positive for ctrA,18 were serogroup C and 3 were serogroup B.Of the 17 N.meningitidis strains isolated from healthy carriers,14 were serogroup B,2 were serogroup C,and 1was serogroup X.Conclusion Real-time PCR was sensitive and rapid.This method can be used to detect pathogens in clinical specimens of bacterial meningitis and identify the serogroup of N.meningitides.