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Rapid Identification of Staphylococcus Aureus with Molecular Biology and Proteomics Technology

Author: QiWei
Tutor: HuangWenFang
School: Luzhou Medical College
Course: Internal Medicine
Keywords: Staphylococcus aureus Polymerase chain reaction - restriction fragment length polymorphism analysis Surface enhanced laser desorption ionization time-of-flight mass spectrometry Decision tree classification diagnostic model Protein markers
CLC: R440
Type: Master's thesis
Year: 2011
Downloads: 179
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Abstract

Staphylococcus aureus is the most widely distributed pathogen secretes a variety of virulence factors, can lead to pneumonia, wound infections and food poisoning and other serious diseases, accounting for 10% of hospital infections. In recent years, with extensive use of antibiotics, resulting in a large number of methicillin-resistant Staphylococcus aureus (MRSA), making it the world's three tied with AIDS, hepatitis B infection ills. The traditional detection method is easy to operate, and the advantages of simple equipment required, but there are time-consuming, sensitivity of the shortcomings of poor. With the wide application of molecular biology and proteomics technology, the detection of Staphylococcus aureus transition from traditional methods to the modern technology-based PCR, hybridization probes, gene chips and MS from gene and protein The level is more accurate, more sensitive detection of the presence of pathogens. The study first PCR-RFLP (polymerase chain reaction - restriction fragment length polymorphism analysis) identified common clinical Staphylococcus aureus, and then take advantage of the differences in the methods of proteomics, mass spectrometry using SELDI-TOF-MS detection golden yellow aureus and other bacterial protein fingerprint of the control group, and filter out the Staphylococcus aureus-specific expression of protein markers, decision tree prediction model to explore its for Staphylococcus aureus clinical value of laboratory diagnosis. The purpose of identification of the bacteria at the molecular level is one of the clinical laboratory field of microbiology. In this study, the use of the polymerase chain reaction - restriction fragment length polymorphism analysis techniques and differences proteomics technology to the rapid identification of common clinical Staphylococcus aureus, laid the foundation for the study of the clinical microbiology identification methods. Method 1. Using four DNA extraction methods Staphylococcus aureus DNA screening suitable DNA extraction solution. 2.PCR amplification of the 16S rDNA of Staphylococcus aureus, the products were sequenced, the results with GenBank than be identified. 3 Get aureus tuf gene sequence analysis of the tuf gene restriction sites and digestion mode via software. To optimize the reaction conditions of the PCR detection tuf gene aureus tuf gene was amplified by PCR, the product was purified by restriction enzymes AluI, HinfI digestion, agarose gel electrophoresis analysis of the cleavage products after the digestion pattern of Staphylococcus aureus identification. 4 different laser intensity to explore and establish the conditions of SELDI-TOF-MS detection of bacterial protein fingerprint. 5. Collect Staphylococcus aureus cultured for 24h, 48h, 72h, SELDI-TOF-MS detection of protein fingerprint analysis of the incubation time on protein fingerprint detection. 6. 3 Staphylococcus aureus duplicate detection 20 times to analyze the reproducibility of SELDI-TOF-MS detection of bacterial protein fingerprint. 7 Select 60 Staphylococcus aureus and 84 control bacteria for the training set, 56 Staphylococcus aureus bacteria and 75 controls bacteria validation set, using SELDI-TOF-MS detection training set and validation set bacterial protein fingerprints. Biomarker Wizard software screening Staphylococcus aureus characteristic protein established by Biomarker Patterns Software Staphylococcus aureus decision tree diagnostic model. 8. Verify concentrated 56 Staphylococcus aureus and 75 blinded validation control bacterial protein fingerprint diagnostic model. The results of four DNA extraction methods, only improved NaOH method can successfully extract Staphylococcus aureus DNA. All Staphylococcus aureus can amplify 16SrDNA PCR amplification products were sequenced and results in GenBank Staphylococcus aureus sequence comparison similarity is greater than 99.5%. The 3 software analysis found aureus tuf gene having a plurality of restriction sites such as AluI, HinfI point t ore gene after AluI, HinfI digestion of the product has a characteristic, can be different staphylococcal separate. Was found that different staphylococci 668bp of the product could be amplified under optimized PCR conditions. PCR products after AluI, HinfI digestion, agarose gel electrophoresis results showed that the digestion results consistent with the theory, and can differentiate between Staphylococcus aureus. 4 different laser intensity, the bacterial protein fingerprinting slightly different laser intensity to 190 test results. Staphylococcus aureus was cultured 24h, 48h and 72h, SELDI-TOF-MS mass spectrometry detection found that the incubation time for bacterial protein expression, also found that stable expression of multiple protein markers in different time . 6 Staphylococcus aureus duplicate detection 20, and 6 characterized in the molecular weight of the protein peak coefficient of variation is ≤ 0.05% Step SELDI-TOF-MS detection aureus characteristic protein having a good reproducibility. 7. SELDI-TOF-MS detection results show that all Staphylococcus aureus protein fingerprint, the molecular weight of 3 within the range 000-20 000Da A total of 75 protein peaks, of which 47 protein peak difference was statistically Significance (P lt; 0.001), wherein the mass to charge ratio (M / Z) 13 113,4 458 2 protein the most obvious differences. Biomarker Patterns software to establish a Staphylococcus aureus decision tree classification and identification of model. 8 The the blinded validation results show that the classification and identification of models of Staphylococcus aureus diagnostic sensitivity and specificity were 100%. Conclusion This study has successfully established a PCR-RFLP method to distinguish between different aureus. SELDI-TOF-MS technology to build a variety of bacterial protein fingerprint Biomarker Patterns software to build a model of a Staphylococcus aureus protein Decision Tree classification and identification, creating a new model on a molecular level, in order to rapid diagnostic identification of Staphylococcus aureus.

Rapid Identification of Staphylococcus Aureus with Molecular Biology and Proteomics Technology

Abstract

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