PCR快速检测细菌16S rRNA基因的初步研究

目的：建立检测细菌16SrRNA基因的PCR方法。方法:以细菌16S rRNA基因为靶序列。利用计算机软件primer5．0，Bioedit设计2条引物-pml.,pm2并建立相应的PCR方法。采用该PCR方法扩增实验室保留菌株的16srRNA基因,以人类外周血白细胞基因组DNA、HBV—DNA阳性血清以及白色念珠菌为对照。检测该实验方法的特异性;采用倍比稀释的方法进行该实验方法的敏感性实验。结果：用引物pml，pm2对17个不同菌株进行PCR扩增。均得到371bp左右长度的DNA片段。特异性实验表明,此通用引物与人类基因组DNA、真菌及病毒无交叉反应。敏感性实验表明。用pml，pm2引物进行的PCR扩增可检测出20CFU／ml,结论：16S rRNA基因PCR检测方法具有特异、敏感、快速的特点。

Primary study on rapid detection of bacteria by use of polymerase chain reaction targeted at 16S rRNA gene

Objective:To establish a method of polymerase chain reaction(PCR) to detect the existence of bacterial 16S rRNA gene.Methods:With the help of computer software , primer5.0 and Bioedit, we designed a pair of primer targeted at the highly conserved 16S rRNA gene sequence-pm1 and pm2. 16S rRNA gene fragments from different bacterial species were amplified with polymerase chain reaction.By using human genome DNA from peripheral blood white cells ,HBV-DNA positive sera and Calbicans as comparison , the specificity of pm1 and pm2 was tested.Sensitivity test was done by the method of gradual dilution.Results:By using the primers-pm1 and pm2,371bp DNA fragments were amplified from all the 17 different species. No signal was observed when human genome DNA, C.albicans and HBV-DNA positive serum were used as templates. Sensitivity test showed that PCR amplification done by primer mp1 and pm2 could detect bacteria 20CFU/ml.Conclusions:The method is rapid and highly specific and sensitive in detecting the existence of bacterial 16S rRNA gene.