Trials and methods Flashcards
What is the purpose of culture based methods?
isolate the target microbes to study their metabolism,
morphology or strain typing
How do non-culturing methods work?
Use genetic material to identify,
classify and quantify the target microbes
What method do you use if you want to investigate what a probiotic drink from a supermarket contains?
Culturing
What method do you use if you want to see how the microbiome has changed as a result of consuming a probiotic?
16S sequencing
What method do you use if you want to test if a probiotic has live bacteria in it?
Culturing
If the aim is related to live bacteria which method should come to mind?
If the aim is related to live microorganisms, culturing can be
the 1st option. There are non-culture based quantification
methods for live microbes as well.
If the aim does not require live bacteria which method should come to mind?
If the aim does not require alive microbes, genetic material
based methods should come to mind in first place.
How would you quantify Enterobacteriaceae in
the contaminated food?
Take a food sample, make a dilution serie and
incoluate VRBD (Violet Red Bile Dextrose) agar
plates, incubate at aerobic conditions at 37 °C for
24 h.
Calculate the amount using dilution factor. For
example at 10-8, 5 colonies are found. Then it
should be 5x10^8 in the un-diluted inoculation.
If you took 10 g of contaminated food from 200 g
of the food, then the total amount of
Enterobacteiraceae should be 1x10^10 CFU/g
What is the purpose of End point PCR?
presence/absence of the target, verify the correct size for cloning
To see if it is there or not
What is the purpose of nested PCR?
1st PCR amplicon is used as template for 2nd PCR, increased sensitivity, false positive risk
What is the purpose of Multiplex PCR?
Use multiple sets of primers/probes to target multiple region in a single PCR reaction
What is the purpose of real time PCR?
Quantification (absolute/relative, standard curve/housekeeping gene)
What is the purpose of Reverse transcription PCR (RT-PCR)?
convert mRNA to cDNA then same as real time PCR
What are the principles (3 steps) of PCR?
- Denaturation - Open up the double helix structure (heating 94-96 °C)
– Annealing – primers/oligonucleotides bind on your separated DNA strands (50-60 °C)
– Elongation – DNA polymerase help dNTPs (nucleotides added in the solution) find the
complementary strand and extend (72 °C)
What is SYBR green?
Non-specific flourescent DNA dye with high affinity for double stranded DNA
Number of cycles required to reach a flourescence threshold
What is TAQMAN?
Fluorescently labeled oligonucleotide probes
A probe is designed with a fluorescent dye
reporter in one end (5 ́) and a quencher in the
other end (3 ́)
* During new DNA strand synthesis,the Taq DNA
polymerase comes into contact with the probe
and cleavesit
* The dye emitslight when not in contact with the
quencher
The light is measured and translated to amounts of
copied strands
What are the pros of QPCR?
Can quantitatively detect both
bacteria (total), bacterial groups, and
specific genes based on primers
Small amount of template
Highly sensitive
Affordable cost
What are the cons of QPCR?
SYBR green bind to any dsDNA
(e.g. primer timer, non-specific
reaction products)
Not possible to distinguish
between live and dead cells
When you want to target bacteria you use 16S, what do you use for yeast?
18S
What is first generation sequencing?
Sanger sequencing
4 PCR reactions
piece it together
time consuming and cannot scale up
What is 2nd generation sequencing?
454 Pyrosequencing
Ion torrent
Illumina
Oxford Nanopore
What are the pros and cons of culturing?
Pros: easy and cheap
Cons: Many microbes unculturable and it is time consuming
What are the pros and cons of PCR/Sanger sequencing?
Pros: It is fast and cheap and can target unculturable microbes.
Cons: Contamination and false positives. Time consuming and expensive if the samples are large
What are the pros and cons of NGS?
Pros: Can identify rare taxa, can handle large datasets
Cons: Expensive for small scale use, the data handling is not easy
