Need a simple experimental plan.. how would you run this experiment? 4:30? 4 Sea
ID: 300445 • Letter: N
Question
Need a simple experimental plan.. how would you run this experiment?
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
1) The experimental plan for MALDI-TOF varies with the type of instrument used and also the manufacturer.But, have given below the BRUKER manufacturer protocol for the MALDI-TOF Mass spectrpscopy in step wise
a) Sample Preperation:
Chemicals and materials required
Acetonitrile (ACN) 0.1%
Trifluoroacetic acid (0.1% TFA) in water
A direct smear method with a 70% formic acid overlay is used for preparing aerobic and anaerobic bacteria samples.
Preparation of sample solution Dissolve samples (whole contents of tubes 4-10) in each case in 125 µL 0.1% TFA solvent and shake for several seconds.
b)Preparation of matrix solution :
Produce a mix of ACN and 0.1% TFA in a volume ratio 1:2. Solve HCCA, SA or 2,5-DHB in this solvent up to saturation at room temperature. Assist the solution process by help of ultrasonic device. Spin down excess matrix in a centrifuge (5 min at 14000 rpm) and use only the homogeneous transparent phase.
Tube Sample Matrix
4 Peptide calibration standard -HCCA, 2,5-DHB Matrix
5 Protein calibration standard I- HCCA, SA, 2,5-DHB Matrix
6 Protein calibration standard II- SA, 2,5-DHB Matrix
7 Tryptic digest of bovine serum albumin -HCCA, 2,5-DHB Matrix
8 Angiotensin II -HCCA, 2,5-DHB Matrix
9 ACTH, clip 18-39 - HCCA, 2,5-DHB Matrix
10 Somatostatin 28 - HCCA, 2,5-DHB Matrix
c)Preparation onto MALDI-TOF-Target
According to the dried droplet method, mix equal volumes of sample and matrix solution (HCCA, SA or 2,5-DHB). Apply 1 µL onto a standard steel target and dry at room temperature.
Related Questions
drjack9650@gmail.com
Navigate
Integrity-first tutoring: explanations and feedback only — we do not complete graded work. Learn more.