Lab 15 Yogurt Microbial Analysis Part 2 Flashcards
1
Q
Enumeration can occur via traditional spread plating on selective media or …
A
quantitative PCR (qPCR)
2
Q
What is qPCR?
A
- A widely used technique for detection and quantification of DNA or RNA sequences in a sample based on the polymerase chain reaction principle, but includes additional steps that allow measurement of the amount of target nucleic acid present in the sample.
3
Q
What is the basic principle of qPCR?
A
- To amplify a specific region of DNA or RNA using primers, a DNA polymerase enzyme, and nucleotides.
- The amount of amplified product is then measured in real-time using a fluorescent signal generated by binding of a fluorescent dye to a double-stranded DNA.
- During the amplification process, the fluorescent signal increases in proportion to the amount of DNA product generated.
4
Q
What carries out the detection of the signal in qPCR?
A
- A qPCR machine which monitors the fluorescence (RFU = relative fluorescence units) at each cycle of the amplification process.
5
Q
What is the cycle threshold?
A
- Cycle threshold (Ct) is defined as the cycle number at which the fluorescence signal reaches a predetermined threshold level.
- The lower the Ct value, the more DNA originally present in a sample.
- The amount of DNA or RNA in the initial sample can be determined by comparing the Ct value obtained from the sample with a standard curve generated from known quantities of a control DNA sample.
6
Q
qPCR enumeration offers several advantages over traditional plating methods. Describe these advantages.
A
- Results can be obtained within a matter of hours instead of over several days required for plating methods.
- qPCR primers are highly specific to target species, while selective media used in plating methods are only moderately selective.
- Harsh additives in selective media can suppress microbial growth leading to an underestimation of the number of microbes present, whereas qPCR provides more accurate and precise results.
7
Q
After optimization, what is the main limitation of qPCR?
A
- Inability to differentiate between viable and non-viable microbes.
- Additional measures, such as culture-based techniques or viability staining, should be employed to confirm the presence of viable organisms.
8
Q
How do commercially available DNA extraction kits work?
Why are these kits common?
A
- By lysing bacterial cells present in a food sample and then purifying the DNA by removing all other inorganic and organic material.
- Convenient and cost-effective compard to preparing the solutions manually.
9
Q
What does a PCR master mix include? [5]
A
- Forward and reverse primers specifically designed to attach to a genomic region in the target organism.
- Nucleotides required for elongation of the primers.
- DNA polymerase to attach the nucleotides
- Magnesium chloride, which is a cofactor for DNA polymerase.
- Fluorescent dye that binds to double-stranded DNA and enables its quantification
10
Q
What are the steps in DNA extraction?
A
- Collect cells
- Lyse cells to release DNA
- Remove inhibitors
- Bind DNA to membrane
- Wash DNA
- Elute and collect purified DNA
11
Q
How is DNA quality & concentration evaluated?
A
- Good practice to check DNA quality and concentration by evaluating absorbance of the sample at 260 and 280 nm.
- DNA absorbs wavelengths of 260 nm
- Protein, phenol, and other contaminates absorb wavelengths close to 280 nm
- Ideally ~1.8 ratio of 260:280
- nanodrop commonly used.
12
Q
Describe amplification by PCR.
A
- Nucleic acid based assay
- Amplifies a sequence of DNA or RNA (usually 100 to 1000 bp in length)
- Sequence of interest is amplified by a factor of 2^N, where N is the number of PCR cycles
13
Q
What is the first step of PCR?
A
- Double stranded DNA is heated to form single stranded DNA
- 95 C
- Called ‘denaturing’ the DNA
14
Q
What is the second step of PCR?
A
- Single stranded DNA is cooled to between 50 and 65 C.
- Primers hybridize (bind/anneal) to ssDNA
- Temperature depends on how much C+G is in the primers.
15
Q
Why does temperature in the second step of PCR depend on how much C+G is in the primers?
A
- C-G form 3 hydogen bonds
- A-T form 2 hydrogen bonds
- More C+G = higher annealing temperature
16
Q
What is the third step of PCR?
A
- Polymerase binds to DNA-primer complex and begins adding bases
- Performs best at 72 C
- Can survive the 95 C denaturation temperature
17
Q
Describe a qPCR experiment for Bifidobacterium, Streptococcus, and Lactobacillus.
A
Note the different temperatures required.
18
Q
What is the output from a qPCR machne?
A
- Plot of relative fluorescence units (RFU) verus number of PCR cycles
- Master mix has a dye (SYBR Green) that binds to dsDNA; more dsDNA = more fluorescence
- Ct value = the cycle at which the fluorescence signal extends above the threshold level (background noise)
- If you also run serial dilutions of a known concentration of microbial DNA, you can quantify the amount of a microbe in a product.
- Need a standard curve for each microbe of interest (Ct versus log (concentration))
19
Q
What is required to quantify the amount of microorganism in a product?
A
- If you run serial dilutions of a known concentration of microbe
- Need standard curve for each microbe of interest (Ct versus log(concentration))
- 10-fold dilutions should have Ct values 3.3 cycles apart.
20
Q
Why are melting curves relevant in PCR analysis?
A
- After PCR cycles are done, the machine conducts a melting analysis where amplified dsDNA is melted apart
- Tm is the temperature at which 50% of the amplified dsDNA has been melted.
- Thus, if primers accidentally amplified something other than the target sequence, more than one melting curve will be visible!
- Use the Tm to confirm the positive controls to confirm you amplified the correct DNA sequence.
21
Q
What is Tm?
A
- The temperature at which 50% of the amplified dsDNA has been melted.
- It depends on the length and G+C content of a DNA sequence.
- Use to confirm that the correct DNA sequence was amplified.
- If primers amplified something other than the target sequence, more than one melting curve will be visible!
