儿童呼吸道易感病原菌多重PCR检测方法的建立

目的建立儿童呼吸道5种易感病原菌多重PCR(Multiplex PCR,mPCR)检测方法,并应用该方法分析北京地区儿童呼吸道易感病原菌菌群分布情况。方法分别设计针对肺炎链球菌(Streptococcus pneumoniae,SP)自溶素基因(ply)、肺炎克雷伯杆菌(Klebsiella pneumoniae,KP)16S核糖体RNA基因(16S rRNA)、金黄色葡萄球菌(Staphylococcus aureus,SA)热核酸酶基因(nuc)、表皮葡萄球菌(Staphylococcus epidermidis,SE)特异性保守基因(SeS)、流感嗜血杆菌(Haemophilus influenzae,HI)16S rRNA基因的特异性引物,建立mPCR检测方法;验证该方法的特异性及敏感性;并采用该方法对北京地区128例呼吸道感染患儿和80名健康儿童咽拭子或痰标本进行检测,并与传统培养法检测结果进行比对。结果 mPCR法能对同一样品中5种病原菌的DNA模板同时进行PCR扩增;该方法检测5种病原菌之间及与呼吸道其他易感病原之间无交叉反应,敏感性可达5×10-10μg/μl;mPCR法检测128例呼吸道感染患儿的菌群分布情况为:HI 24.22%、SP 15.63%、KP 4.69%,SA 2.34%,未检出SE,与培养法相比,两种检测方法的总符合率为78.91%;感染患儿与健康儿童相比,HI和SP的感染率差异有统计学意义(P<0.01)。结论建立了从呼吸道标本中直接检测多病原儿童易感病原菌的mPCR检测方法,该方法特异性强、敏感性高,为临床正确诊断和治疗细菌感染性疾病提供了参考。

Development of a multiplex PCR assay for simultaneous detection of five major pathogenic bacteria of respiratory diseases in children

Objective To develop a multiplex PCR assay for simultaneous detection of five major pathogenic bacteria of respiratory diseases in children and use for analysis of flora distribution of the pathogenic bacteria in Beijing region.Methods Specific primers were designed according to the ply gene of Streptococcus pneumoniae(SP),16S rRNA gene of Klebsiella pneumoniae(KP),nuc gene of Staphylococcus aureus(SA),SeS gene of Staphylococcus epidermidis(SE)and 16S rRNA gene of Haemophilus influenzae(HI),based on which a multiple PCR(mPCR)method was developed and verified for specificity and sensitivity.Pharynx swabs or sputum specimens from 128 children with respiratory infection and 80 healthy children were determined by the developed method,of which the results were compared with those by traditional culture method.Results The DNA templates of five pathogenic bacteria in the same sample were simultaneously amplified by the developed mPCR method.No cross reactions were observed between the five bacteria and other respiratory pathogens.The sensitivity of the developed mPCR method was 5 × 10-10 μg / μl.Of the 128 clinical samples,22.42% were positive for HI,15.63% for SP,4.69% for KP,and 2.34% for SA,while no SE was detected.The total coincidence rate of detection results by the developed mPCR method and traditional culture method was 78.91%.The infection rates of HI and SP showed significant difference in the children with respiratory infection and healthy children(P < 0.01).Conclusion A multiplex PCR assay for simultaneous detection of five major pathogenic bacteria of respiratory diseases in children was developed,which showed high specificity and sensitivity.It provided a reference for diagnosis and treatment of bacterial infection associated diseases in clinic.