The Study Of The Rapid Detection Of Salmonella Paratyphi A By Seldi-Tof-Ms

Objective: To establish a protein fingerprint database of Salmonellaparatyphi A by surface enhanced laser desorption&ionizationtime-of-flight mass spectrometry（SELDI-TOF-MS） and to verify thediagnosis efficiency with molecular biology techniques for providing apracticable tool for rapid identification of the bacteria.Methods:1.36clinical bacterial isolates were collected and studied.2.All the strains were identified by16S rRNA gene sequencing.3. Bacterialproteins were detected by SELDI-TOF-MS. Protein fingerprints wereanalyzed by ProteinChip and Biomarker Wizard software. Thereproducibility of SELDI-TOF-MS and the stability of the proteinexpression of bacteria from each species were analyzed based on theprotein fingerprints.4. The reproducibility of the method were evaluatedthrough the several detections of the same bacterial protein and the analysisof protein fingerprint differences.5. The bacterial protein expressionstability was analysis by the comparison of protein fingerprint differencesof different strains of bacteria.6. The effect on bacterial protein fingerprintcaused by culture time was validated through the comparison of differences of bacterial protein fingerprint with different culture time.7.132strainswere divided into training set and test set, respectively, meanwhile, theexpressions of protein were detected and the protein fingerprints wereanalysis. The results were established the classification tree identificationmodel by using BioMarker Patterns software, and conducted a blindedvalidation.Results:1. The sequenced results of PCR products were show, theconsistency of the target fragment DNA sequences and the strains genesequence alignment in GenBank is greater than or equal to98.5%, as sameas the traditional microbial identification results.2.104protein peaks werecaptured,90protein peaks were statistically significant (P＜0.01) in themolecular weight of3000～20000Da range.3. The bacterial proteinsbetween holes have well repeatability, and reproducibility between chipsdetection is well, meanwhile, the repeat detections on the same strain ofbacteria have similar protein fingerprint.4. The protein fingerprinting ofdifferent strains of bacteria is stable, and the coefficient of variation of totalprotein peak molecular weight is less than0.5%.5. Different culture timehad little effect on bacterial protein fingerprinting.6. Salmonella paratyphiA classification tree model was established by selected protein peaks whosemass-to-charge ratio (M/Z) was10061.7, and sensitivity and specificity ofSalmonella paratyphi A diagnosis was100%.Conclusion: The classification tree model of Salmonella paratyphi A can be established by using AU protein chip and SELDI-TOF-MStechnology, and it can be used for rapid identification of Salmonellaparatyphi A.