| Background and ObjectiveSepticemia is a kind of disease with systemic infection, which may cause death. Single or compound bacteria all can induce septicemia. Some cases of septicemia have infective shock and migration focus. Bacterial culture, methods of histology and immunology, and polymerase chain reaction have been used in the identification of bacteria. However, all of these methods have shortcomings. Now, automatic bacteria-culture machine is used broadly in clinic. But it takes a long time to identify bacteria by using this kind of machine, and sometimes pseudo-positive or pseudo-negative results were obtained. Moreover, some kinds of bacteria which are difficult to culture, like influenza bacilli or anaerobe, cannot be identified by using this kind of machine.As a sensitive method with high effectiveness, the technology of gene microarray will be a revolution in life science research. The technology can analyze the gene expression in karyocyte systemically. With the implementation of human genome project and continuing clone of new genes, the technology of gene microarray will be renewed continuously and used in research of medicine, pharmacology, and life science.In our study, we designed universal primers for polymerase chain reaction and specific probes for different bacteria based on the technology principle of gene microarray. Comparison of gene sequences and phyligenetic analysis were included in this study. The objective was to construct diagnostic gene microarray for common nosogenetic bacteria causing septicemia. The gene microarray will be used in clinic for quick identification of bacteria.Materials and Methods(1) The DNA of different bacteria was exit acted by using phenol/chloro-form/isoamyl alcohol method. The DNA sequencing was used to determine the purity of the DNA extracted.(2) Accord to the highly conserved region of bacteria, the 16S rRNA region, we designed a pair of universal primers. The 5 end of reversed primer was labeled with Cy5. Symmetry polymerase chain reaction was performed with the reversed chain in superiority.( 3 ) Based on the results of DNA sequencing, by using the Blast and Clust-alW software, we compared the sequences and constructed the phyligenetic tree of 16S rRNA region of bacteria. A group of oligonucleotide probes for 17 kinds of bacteria were designed according to the comparison and analysis results. The 5'end of every probe was modified with amino. We re - suspended the freezing and dried probes with carbonate buffer ( pH 9,0.1 mol/L) to 100 |xmol/L.(4) The cover slides were soaked in chromic acid solution for more than 12 hours, then washed with water, soaked in sodium hydroxide solution, then in acetone. The surface of the slides were modified with arm molecule, and then put in roaster of 80°C to fix the arm molecule. After the modification of arm molecule with aldehyde, the slides were ready for use.( 5 ) The forward primer of the same concentration was used as positive control, and put in the 384 -pore plate with the other 18 probes. With the BioRo-botic machine, the probes were grilled onto the surface of the slides. The mi-croarrays were designed before the grilling. The slides were hydrated for using after the grilling.(6) The oligonucleotide microarrays were cleaned with water and hybridized with the products of polymerase chain reaction. The products were diluted with hybridization buffer already. The hybridization was under temperature of 70*^0 for 90 minutes.( 7 ) After the hybridization, the slides were gradient washed with 1 x SSC/ 0.1 % SDS, 0. 5 x SSC/0. 1 % SDS, 0. 1 x SSC, and ddH2O which were all preheated to 60TI. By using the Laser Cofocal Scanner and CCD imaging soft-ware, we analyzed the hybridization results on the dried slides.Results(1) The results of polymerase chain reaction were examined through poly-acrylamide gel electrophoresis. The target sequence was about 308 bp, which was identified with the expectation. We obtained complete sequencing results in seventeen of 21 kinds of bacteria. The specific probes can be designed based on the sequencing results.( 2 ) According to the sequence and ClustalW analysis, 4 parts of sequence appeared in 16S rRNA region of all 17 kinds of bacteria. The 4 parts of sequence were dispersed. Between 15 pairs of bacteria, scores between 2 sequences were more than 90. We should emphasize on these 15 pairs of bacteria during the designation of probes. The phyligenetic tree directly demonstrated the concordance scoring.(3 ) The polymerase chain reaction products of E. streptococcus and Stenotrophomonas maltophilia were not specifically hybridized with the special probes. However, the products of other bacteria were all hybridized with special probes. At the same time, hybridization with other non - specific probe was also observed. Cross - hybridization were shown. The quantitive analysis for brightness of hybridization were performed, and we found that the brightness of hybridization between target sequence and specific probe was very near to, and sometimes even equal to the brightness of positive probes. The repeated rate of hybridization was high (100% ) , but the specificity was not so good.ConclusionThe 16S rRNA region of bacteria gene was highly conserved. The universal primers can be designed for polymerase chain reaction of all bacteria.Usually, the phyligenetic tree analysis was used to evaluate the variation and relative relationships of gene or protein in evolution. When the pathogen was not separated successfully, we can study the possible source of the pathogen through the phyligenetic tree analysis, which must be based on the gene sequence of the pathogen. In this study, we designed specific probes for different... |
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