论文标题:多重半套式聚合酶链反应检测脑脊液中细菌16S rRNA基因
Studies on the preparation and quality standard of Xinnaotong Capsule
论文作者 闫志勇
论文导师 王斌,论文学位 硕士,论文专业 病原生物学
论文单位 青岛大学,点击次数 203,论文页数 55页File Size3170k
2001-04-01论文网 http://www.lw23.com/lunwen_1184442/ 聚合酶链反应;细菌;16SrRNA; 基因;脑脊液
polymerase chain reaction;bacteria;16SrRNA;Gene;CSF
目的 建立多重半套式聚合酶链反应（multiplex semi-nested PCR） 快速检测脑脊液标本中常见病原菌的方法。 方法 通过对多数病原菌16S rRNA基因保守区和变异区的序列分析 ， 设计通用引物和革兰阴性菌及革兰阳性菌的特异性引物分别作为外侧 和内侧引物，建立了对不同细菌的16S rRNA基因进行多重半套式PCR 扩增方法；对该反应体系的敏感性和特异性进行了检测；并用该方法和 常规细菌分离培养法同时对62份临床脑脊液标本进行检测并进行了比较。 结果 革兰阳性菌和革兰阴性菌经扩增后均有1032 bp长度的DNA片段产生，同时 革兰阳性菌有一336 bp的特异性产物，革兰阴性菌有一127 bp的特异性产物； 而白色念珠菌、人乳头瘤病毒和人基因组DNA经用相同方法扩增后均未见特异性带产生； 该方法最低可检测出8 CFU/mL的大肠埃希菌；以常规细菌学分离培养为参照，对62份脑 脊液标本的检测结果表明，该方法敏感性为93.8%，特异性为95.7%，阳性预测值88.2%， 阴性预测值达97.8%。结论 该方法检测脑脊液感染的常见病原菌具有特异 、敏感、快速等优点， 具有较好的临床推广应用价值。
Objective To design and establish a new method to dectect patho-genic bacterial 16S rRNA gene in cerebrospinal fluid (CSF) using mutiplex semi nested PCR. Method Acoording to the analysis of the conservative and variable regions in bacterial 16S rRNA genes , we designed universal primers for all bacteria and specific primers for most gram-positive and gram-negtive bacteria . All primers were added into the same reaction systems successively of a two-step PCR assay to amplify the differient bacterial 16S rRNA gene in 62 CSF samples, and compared with common culture method；The sesitivity and the specificity were detected at the same time. Results Both E. coli and S. aureus amplified a 1032bp DNA fragment; In addtion,specific fragments of 336bp and 127bp were amplified in S.aureus and E.coli respectively ；HPV，C. albicans ，huaman genomic DNA，and water had no specific amplicon. The detection limit for E.coli was 8CFU/ml. 62 CSF samples were decteced by both the multiplex semi-nested PCR and conventional bacteriologic method ,the comparision revealed sensitivity,specificity, positive and negative predictive values of 93.8%,95.7%,88.2% and 97.8% res-pctively for PCR. Conclusion The result suggests that the multiplex semi-nested PCR we established is a more sesitive,specific and rapid method for clinical laboratroy to detect bacterial pathogenes.
|
