CAJ | 학술논문

目的：根据细菌16 S rRNA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16 S rRNA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350 bp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16 S rRNA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。
목적：근거세균16 S rRNA기인특점설계상견병원균적특이성탐침，채용매현색기술구건기인심편，탐토기림상응용적가능성。방법선취폐염련구균、류감기혈간균급동록가단포균등8충세균성폐염상견적병원균적표준균주작위연구대상，병선택12빈환자적담액표본진행검측。재16 S rRNA기인보수구설계 PCR반응적통용인물급혁란양성균、혁란음성균적통용탐침，이용가변구적차이설계합성특이성탐침，구건기인심편。이용세균16S rRNA기인설계적PCR통용인물진행확증，소유8충세균균획득350 bp적확증산물。이지고신표기특이성탐침，구건완성가용우8충상견병원균검측적기인심편，결과8충표준균주기인심편검측균취득료예기효과，대12빈담표본중상규배양양성7빈，기대응심편검측결과균성양성，5빈상규배양음성적표본중，심편결과제시양성적유3빈，기중1빈위기폐군단균，2빈위사용항생소후적환자표본。결론설계합성적 PCR통용인물대확증세균적16 S rRNA기인구유교고적특이성급령민도。구건적기인심편가용우상견세균성폐염병원균적검측감정，차대항생소사용후적림상표본급가양균유일정적진단개치。본연구소획득기인심편대우세균성폐염적검측구유간단、쾌속、특이성급민감성고적특점。
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 Se-lect Streptococcus pneumoniae,Haemophilus influenzae,and Pseudomonas aeruginosa eight kinds of bacterial pneumoni-a 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 bacte-ria,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 re-sults 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 patho-gen of bacterical pneumonia and clinic specimens,even those antibiotic use after clinical specimens and fastidious bacte-ria have certain diagnostic value.In this study,the gene chip for the detection of bacterial pneumonia is simple,fast,high specificity and sensitivity.