Identification Of Bacteroides By PCR And Study Of Their Bio-information

Objective:The objective of this study is to design universal primers and probe to identify bacteroides by polymerase chain reaction (PCR) technology, and establish gem; evolvement trees of bacteroides through mulriple comparing sequences of 16S rRNA gene of bacteroides by ClustalW1.83 and treeview software in order to analyse the evolvement differencement in different origin bacteroides and between isolated strains and standard strains.Methods:1. Universal primers and probe were designed based on the conservative domain of 16S rRNA gene sequences of bacteroides to be often found in paients. 2. Routine PCR and FQ-PCR were developed to identify bacteroides with B.fragilis, B.thetaiotaomicron, B.vulgatus, B.ovatus (13 strains) as positive control and Aerobes of 40 strains, Escherichia coli, B.longum, Fusobacterium, G~+ cocci, G~+ bacillus, HBV-DNA genome and Monocyte as negative control. 3. Gene evolvement tree was established based on the detected 16S rRNA gene sequences and corresponding standard 16S rRNA gene sequences. 4. Better method to make standard sample was searched for by comparing the detected results of FQ-PCR with different standard samples. 5. The results of identification PCR and phenotype for bacteroides were analysed.Results:1. B.fragilis, B.thetaiotaomicron, B.vulgatus, B.ovatuswre can be detected by this routine PCR and this FQ-PCR, however, Aerobes of 40 strains, Escherichia coli, B.longum, Fusobacterium, G~+ cocci, G~+ bacillus, HBV-DNA genome and Monocyte not. The routine PCR and FQ-PCR are satisfied with high sensitivity. 100 bacteria genome DNA /μl can be detected by this routine PCR, 10 bacteria genome DNA/μl could by this FQ-PCR.2. CV values in the group to obtain by repeating the samples of 10~4 bacteria genome DNA /μl, 10~5 bacteria genome DNA/μl are 0.704%, 0.74% respectively, CV values of different groups are 3.49%, 1.19%.3. The two FQ-PCR systems were developed. One used the method of directly counting bacteria make standard sample, the other used produce of routine PCR asstandard sample. The sample of 10 bacteria genome DNA were detected by the abovetwo FQ-PCR systems, the results were 763.8 bacteria genome DNA/μl,112.9 bacteria genome DNA /μl respectively.4. The phenotype results of three strains are not consentaneous with their identification result by PCR in the study.5. The results of established gene tree and Blast show that there are a few differences each other among these 16S rRNA gene sequences of isolated stains and in between 16S rRNA gene sequences of isolated stains and corresponding standard 16S rRNA gene sequences from GenBank.Conclusion:1. The developed routine PCR and FQ-PCR systems are satisfied with high Sensitivity (routine PCR can detect 100 bacteria genom/μl e and FQ-PCR can detect 10 bacteria genome/μl)and specificity.2. There is excellent reproducibility about the FQ-PCR, their CV values are in 5% .3. The detection result by the FQ-PCR system using bacteria genome as standard sample is closer to the actual than using produce of routine PCR.5. In some time, the phenotype result was wrong.5. There are a few differences for 16S rRNA gene sequences of the same strains fromdifferent origins.