| Staphylococcus aureus is one of the most widely distributed pathogens. It secretes many poisonous force factors which lead to pneumonia, wound infection and food poisoning, etc., which accounts for 10% of nosocomial infection cases. In recent years, the wide use of antibiotics leads to the appearance of a large number of Methicillin-resistant Staphylococcus aureus (MRSA), which makes it one of the world's top three infection persistent ailment including HIV/AIDS and Hepatitis B. The traditional test methods have the advantages of simple operation and handy equipment. However, it's time consumption and poor sensitivity. With the wide application of Molecular biology technology and Proteomics technology, the S. aureus identification methods have been transformed into such modern technologies, from traditional ones, as PCR, Hybridization probe, Gene chip and MS, which test the existence of pathogens more accurately and sensitively from gene and protein level. Firstly, we used PCR-RFLP (PCR-Restriction fragment length polymorphism analysis) to identify clinical common staphylococcus. Secondly, based on the differences in proteomics, SELDI-TOF-MS was used to detect the protein peaks of clinical common staphylococcus and control bacteria. Thirdly, significantly different peaks of staphylococcus were screened. At last, classification tree model was established to evaluate the clinical value of S. aureus identification.ObjectiveAt present, it is hot of identifying bacteria on the molecular level in clinical laboratory of microbiological research. This study used PCR-RFLP to identify the S. aureus; besides, proteomics technology was applied to screen the protein biomarkers of S. aureus. Then the classification tree model is developed and the clinical value of this pattern in S. aureus appraisal is discussed.Methods1.Four methods were performed to extract bacterial DNA from staphylococcus aureus (S.aureus) in order to obtain optimal DNA extraction scheme for S.aureus.2.16S rDNA of S. aureus was amplified by PCR, then PCR products were sequenced by commercial company, and bacteria were identified by analysis the similarity between the obtained sequences and sequences from GenBank.3. DNA sequence of tuf gene in staphylococci was obtained from GenBank, and the enzyme site and enzyme patterns were analyzed by DNA sequence analysis software. PCR amplification condition of tuf gene was optimized, and tuf of staphylococci was amplified with the optimized condition, after PCR products were digested with two restriction enzymes AluI and Hinf, the bacteria were identified according to the agarose gel electrophoresis results.4.Different laser intensities were selected to determine the best parameters of protein profiles detected by SELDI.5. S.aureus was incubated for 24h,48h,72h respectively and was detected by SELDI to observe the influence of culture time on protein profiles.6. To observe the reproducibility of bacteria spectrum,3 S. aureus strains were detected in 20 single test.7.60 S. aureus and 84 control strains were assigned into training set.56 S. aureus and 75 control bacteria were assigned into testing set. The protein profiles of training set and testing set were detected by SELDI. The significant protein peaks of S. aureus were screened by Biomarker Wizard Software. Biomarker Patterns Software was used to develop the classification tree model of S. aureus.8. The classification tree model was blindly tested with testing set, including 56 S. aureus and 75 control bacteria.Results1.In the four DNA extraction methods, only modified NaOH method could successfully extract bacterial DNA from S. aureus.2.16S rDNA was amplified in all S. aureus strains, and the sequence of PCR products had 99.5% similarity with that in GenBank database.3.The results of DNA analysis software showed that tuf gene of Staphylococci had more than two enzyme sites including Alul and Hinfl. PCR results expressed that 668bp PCR products could be found in different staphylococci under optimized reaction condition. The products of the two enzymes could distinguish S. aureus from other staphylococci.4.There was a little difference of protein profiles measured with various laser intensities.The best protein profiles was observed when laser intensity was 190.5.After culture for 24h,48h,72h, the bacterial spectra of S. aureus detected by SELDI-TOF-MS showed similar peak profiles,and several protein markers were stably expressed at different times.6.S.aureus strains spectra were detected in 20 single test. The coefficient variation of six characteristic peaks was no more than 0.05%, demonstrating that the peak profiles of S. aureus obtained from SELDI-TOF-MS had good reproducibility.7. The results of SELDI showed that all the S. aureus had similar protein profiles.75 protein peaks were detected between 2,000 and 30,000 m/z in bacteria, among which 47 peaks were significantly different between S. aureus and controls (P<0.001). Two of them(m/z at 13,113,4,458) were the most distinct protein peaks. Classification tree model of S. aureus was developed by Biomarker Patterns Software.8. The result of blinded validation revealed that the sensitivity and specificity of the model were both 100%.Conclusion:Our study has successfully established a PCR-RFLP method which can distinguish different Staphylococci. At the same time different protein profiles of a variety of bacteria has been conducted by using SELDI, and a classification &identification model for the proteins of S. aureus has been established by using Biomarker Patterns software, which lays a foundation for rapid identification of S. aureus. |
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