| Septicoemia is a kind of the common disease in clinical practice. Effective treatment of bacterial infections often requires the rapid and accurate detection and identification of bacteria in blood. Among the procedures currently used in clinical laboratories, the most sensitive method of detecting bacteria is growth in a culture. Culturing requires commonly seven days, which will delay clinical therapy. Because cul-turing is affected by many elements, positive rate is not high. Poly-merase chain reaction (PCR) technologies is one of the molecular diagnosis method that has been developed since 1983. It has proven to be accurate and quick methods for identifying the presence of microbi-al pathogen. But the genetic complexity of this non-target DNA may also generate the amplification of a false positive'product when a primer anneals to a mismatched sequence, especially as the number of cycles increases. And the specificity of the test is often inadequate, which will limit its applicable field.Using the gene chip can resolve the problems. Gene chip has become one of the most powerful tools in the area of disease diagnostics. Microarray have been able to detect both the presence of bacterium species and subspecies. Because microarray technology allows the simultaneous analysis of thousands of probe information within a single experiment, the gene chip technology can detect the common pathogenic bacteria quickly and accurately.MaterialsThe bacteria strains used in this study were obtained from micro-biology department in China Medical University and detective section in Shenyang Army General Hospital.MethodsIn order to detect the common pathogenic bacteria quickly and accurately, a rapid experimental procedure based upon the gene chip technology has been set up. It is composed of four steps. The first step is the DNA isolation. The DNA was isolated from bacteria by the lysozyme - sodium dodelylsulfate ( SDS ) - proteinase K - phenol -chloroform method. The second step is the universal 16SrDNA amplification. The locations of the universal primers F482 and R789 within the 16SrRNA gene are indicated in table 1. After that, oligonucleotide probes of 60 kinds of bacteria were designed by using of some bioinfor-matics softwares. Then they were synthesized. At last an oligonucleotide microarry was designed and made in the lab.ResultAs shown in Figl, all of the DNAs from 10 kinds of species were amplified well and PCR product which is 308bp was fluorescently labeled by random primer with Cy5-dUTP. The labeled sample was hybridized to the oligonucleotide arrays and A sequence - specific interaction with the target DNA for each probe were calculated by measuring the ratio of fluorescence intensity of the sequence - specific probe and the mismatch control probe. A series of specific hybridization maps corresponding to each kind of bacterium are obtained. The results of these experiments indicate that 40C and 2 hours offer the best combination of sensitivity and specificity for most gene probes.Coupling PCR technology with the hybridization of the resulting amplification products on microarrays fields an extremely powerful detection system. Quality control in this technology is critical for its efficient application. Many methods have set up for monitoring the fabrication steps from oligonucleotid probe printing to data analysis. Hybridization conditions such as temperatures, time , and the length and consistency of probes were studied. We have developed several design methods for high density probe-array, which might be improve the hybridization. The stability and reproducibility of the system was evaluated by the results of self - comparision experiments and repeated experiments.ConclusionCoupling PCR technology with the hybridization of the resulting amplification products on microarrays fields an extremely powerful detection system. Microarray technology allows the simultaneous analysis of thousands of probe information within a single experiment. It can detect the common pathoge... |
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