细菌性肺炎快速诊断基因芯片的初步构建

Construction of gene chip and its application in bacterium pneumonia

目的根据细菌16SrRNA基因特点设计常见病原菌的特异性探针,采用酶显色技术构建基因芯片,探讨其临床应用的可能性。方法选取肺炎链球菌、流感嗜血杆菌及铜绿假单胞菌等8种细菌性肺炎常见的病原菌的标准菌株作为研究对象,并选择12份患者的痰液标本进行检测。在16SrRNA基因保守区设计PCR反应的通用引物及革兰阳性菌、革兰阴性菌的通用探针,利用可变区的差异设计合成特异性探针,构建基因芯片。利用细菌16S rRNA基因设计的PCR通用引物进行扩增,所有8种细菌均获得350bp的扩增产物。以地高辛标记特异性探针,构建完成可用于8种常见病原菌检测的基因芯片,结果 8种标准菌株基因芯片检测均取得了预期效果,对12份痰标本中常规培养阳性7份,其对应芯片检测结果均成阳性,5份常规培养阴性的标本中,芯片结果提示阳性的有3份,其中1份为嗜肺军团菌,2份为使用抗生素后的患者标本。结论设计合成的PCR通用引物对扩增细菌的16SrRNA基因具有较高的特异性及灵敏度。构建的基因芯片可用于常见细菌性肺炎病原菌的检测鉴定,且对抗生素使用后的临床标本及苛养菌有一定的诊断价值。本研究所获得基因芯片对于细菌性肺炎的检测具有简单、快速、特异性及敏感性高的特点。

Objective According to the characteristics of bacterial 16S rRNA gene,design specific probe to common pathogenic bacteria.Use of chromogenic enzyme gene chip technology and explore its clinical application.Methods Select Streptococcus pneumoniae,Haemophilus influenzae,and Pseudomonas aeruginosa eight kinds of bacterial pneumonia common standard strains of pathogens as the research obj ect,and select the 1 2 patients with sputum specimens for testing.Universal primers designed to conserved regions of the 16S rRNA gene PCR reactions and Gram-positive bacteria,Gram-negative bacteria universal probe,using the variable region of design differences SYNTHESIS specific probe, constructed gene chip.The use of bacterial 16S rRNA gene PCR universal primers designed to amplify all eight species of bacteria were obtained amplification product of 350 bp.Digoxin labeled with specific probes can be used to complete construction of eight common pathogen detection microarray.Results 8 standard strains of gene chips have achieved the desired effect.We detected 1 2 sample of sputum,and 7 cases culture-positive which corresponds to the chip test results were positive.In 5 samples with negative culture results,there remained 3 positive in gene chips,1 identified as Legionella pneumophila and 2 cases with antibiotics treatments earlier.Conclusion Currency primer of 16S rRNA has attributes of high specificity and sensitivity.Gene chip we constructed can be applicated to detect common clinic pathogen of bacterical pneumonia and clinic specimens,even those antibiotic use after clinical specimens and fastidious bacteria have certain diagnostic value.In this study,the gene chip for the detection of bacterial pneumonia is simple,fast,high specificity and sensitivity.