Help coming up with an experimental plan only... PLEASE help! 4:30? 4 Search Int
ID: 300479 • Letter: H
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Help coming up with an experimental plan only... PLEASE help!
4:30? 4 Search Introduction Problem Statement: Enterobacteriaceae are a family of pathogens that are commonly found in the human intestine and can be spread easily from person to person. Since 2000, the spread of Carbapenemase- Producing Carbapenum-Resistant Enterobacteriaceae (CP-CRE) has been on the rise worldwide, and they are difficult to treat and have high mortality rates. As a result, there is a need for a method of rapidly and reliably identifying CP-CREs in clinical samples Current tests for rapid identification of CP-CREs generally only identify the presence or absence of such organisms. Optimizing a technique to both rapidly and reliably identify the presence of such organisms and identify the species of Enterobacteriaceae present should allow for a more rapid, appropriate and focused course of antibiotic therapy Hypothesis: Developing a protocol to test blood samples directly using matrix-assisted laser desorption/ ionization-time of flight (MALDI-TOF) will allow for the presence and species of Enterobacteriaceae to be identified in a time frame comparable with other techniques used to rapidly identify the presence of CP-CRE in clinical samples. Aim(s):This project has three primary goals 1. Identify and remove the barriers to successfully analyzing noncultured blood samples with MALDI-TOF. 2. Demonstrate that MALDI-TOF can identify Enterobacteriaceae species in noncultured blood samples (as defined by obtaining a score of greater than 1.70) with a success rate of greater than 90%. Reduce the total time of MALDI-TOF analysis for blood samples, including required all pre- processing, to less than two (2) hours. 3. Why This Experiment Will Succeed: MALDI-TOF Mass Spectrometry has been shown to be equally reliable when identifying thousands of bacterial isolates. When compared to the standard 16s sequencing technique it was identical. It is much faster and can identify multiple species at once with the same efficacy Experiment Essential Data: XXExplanation / Answer
Experimental plan: MALDI TOF is one of the most accurate, reliable and the time saving techniques for the identification of the microorganisms based on ribosomal proteins directly from any sample like blood, urine etc.
It is a revolution that we are able to identify the pathogens directly from blood sample through MALDI as it is non redundant, precise and reduced the result outcome time and so that we can start the treatment as soon as possible after the detection of pathogens.
The experimental plan will be as follows:
Blood cultures: Blood will be collected and allowed to grow after inoculation in BacT Alert charcoal free bottles. Then after the positive alarm of culture growth from the machine, the gram staining will be performed.
1ml of this positive culture will be dispensed in tube containing lysis buffer and centrifuged. The pellet was washed and extracted with ethanol-formic acid extraction, followed by spotting of this extract on the MALDI TPF target. This will be overlaid by the matrix and analysed with the machine like biotyper.
A score of > 1.7 indicates the genus while >2.0 will considered valid for species identification.
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