Total bacteria must be less than 600,000 colony/ml; E.coli. must be less than 10
ID: 227408 • Letter: T
Question
Total bacteria must be less than 600,000 colony/ml;
E.coli. must be less than 10,000 colony/ml;
Thermotolerant bacteria must be less than 1000 colony/ml;
1. If use qPCR technique to examine these bacteria in beverage, please answer questions below.
1.1 Please describe more detail about the methodology and experimental design to do determine bacteria in beverage. If you have sample from 2 group
1.2 If finished the qPCR experiment and the result are shown in table. Please draw the qPCR realtime graph showing the result and show the standard curve to calculate the number of each bacteria.(Please write some details )
Amount of Bacteria in raw milk
group 1
(colony/ml)
group 2
(colony/ml)
Total bacteria
200,000
800,000
Coliforms bacteria
3,000
100,000
Thermotolerant bacteria
500
2000
Amount of Bacteria in raw milk
group 1
(colony/ml)
group 2
(colony/ml)
Total bacteria
200,000
800,000
Coliforms bacteria
3,000
100,000
Thermotolerant bacteria
500
2000
Fluorescent intensityExplanation / Answer
methodology of q pcr - when performing qPCR, a fluorescent reporter dye is used as an indirect measure of the amount of nucleic acid present during each amplification cycle. The increase in fluorescent signal is directly proportional to the quantity of exponentially accumulating PCR product molecules (amplicons) produced during the repeating phases of the reaction (see Polymerase Chain Reaction). Reporter molecules are categorized as; double-stranded DNA (dsDNA) binding dyes, dyes conjugated to primers, or additional dye-conjugated oligonucleotides, referred to as probes (see Quantitative PCR and Digital PCR Detection Methods).
The use of a dsDNA-binding dye, such as SYBR® Green I, represents the simplest form of detection chemistry. When free in solution or with only single-stranded DNA (ssDNA) present, SYBR Green I dye emits light at low signal intensity. As the PCR progresses and the quantity of dsDNA increases, more dye binds to the amplicons and hence, the signal intensity increases.
Alternatively, a probe (or combination of two depending on the detection chemistry) can add a level of detection specificity beyond the dsDNA-binding dye, since it binds to a specific region of the template that is located between the primers. The most commonly used probe format is the Dual-Labeled Probe (DLP; also referred to as a Hydrolysis or TaqMan® Probe). The DLP is an oligonucleotide with a 5’ fluorescent label, e.g., 6-FAM™ and a 3’ quenching molecule, such as one of the dark quenchers e.g., BHQ®1 or OQ™ (see Quantitative PCR and Digital PCR Detection Methods). These probes are designed to hybridize to the template between the two primers and are used in conjunction with a DNA polymerase that has inherent 5’ to 3’ exonuclease activity. When the DLP is free in solution, the signal intensity is low because the reporter dye is in close proximity to the quencher moiety. As more of the template is produced during the reaction, more probes hybridize to the template, which in turn are cleaved by the 5’ to 3’ exonuclease activity of the advancing DNA polymerase. The fluorescent signal intensity increases as the 5’ reporter dye is released into solution. The use of probes labeled with different reporter dyes allows for the simultaneous detection and quantification of multiple targets in a single (multiplex) reaction.
A typical qPCR run consists of repeated cycles of alternating temperature incubations, see Cycling Procedure 3.1. This profile is often used when dsDNA-binding dyes, Molecular Beacons, or Scorpion® Probes are the chosen detection chemistries for qPCR. Primer extension is most efficient at 72 °C because this is the optimal temperature for processivity of most DNA polymerases. At 72 °C, polymerization occurs at a rate of approximately 100 bases per second. However, there is still processivity at lower temperatures that is sufficient to amplify shorter templates. qPCR amplicons are typically shorter (<200 bases) than conventional PCR products, thus extension is often combined with annealing in a single step at 60 °C when working with Dual-Labeled Probles.
qPCR Requirements / experimental setup
1- Instruments
Many qPCR instruments have been designed to support a specific range of applications, e.g., contrast the capability of the ABi 7900 high throughput instrument using automatic loading of 384-well plates with the Illumina produced and marketed Eco instrument that supports a single 48-well plate. Therefore, the most suitable instrument meets the needs of the research. It is desirable to select an instrument with user friendly software that performs the most desirable functions and has flexibility in terms of data output so that it can be easily manipulated in downstream statistical analysis software packages. This reduces the time required to train personnel and therefore to begin generating results. Additional features that are required include a PCR block that is absolutely uniform (an absolute maximum deviation of 1 Cq = 2-fold across 96 wells of replication) and an optical system that excites and detects emission as sensitively and as evenly as possible across a wide range of wavelengths. This allows for a wide choice of fluorophores and enables multiplexing. Other features to consider are the operating costs associated with specific consumables, e.g., if a standard microtitre plate is not used for reactions and also the convenience of loading plates/tubes that are non-standard format.
2-Template
Very few copies of target nucleic acid (equivalent to about 100 pg of gDNA or cDNA) are required to initiate qPCR. To minimize contamination with reaction inhibitors, the starting template amount should be kept to the minimum required to achieve accurate quantification. When the starting material is RNA, primer design and DNase I treatment will reduce signals that may be generated from gDNA contamination.
Primers-
Whether using a dsDNA-binding dye or a probe-based detection chemistry, designing high-quality primers is one of the most critical pre-experimental steps in qPCR. For a detailed discussion of assay design, see PCR/qPCR/dPCR Assay Design.
Probe(s)-
When using probes as the amplicon detection mechanism, primer dimers and nonspecific products are not detected but should be avoided because they can lower reaction efficiency. To maximize sensitivity and specificity, the appropriate probe type for the application should be selected and any required modifications, such as Locked Nucleic Acid® (LNA®), included (see qPCR Detection Chemistry).
dNTPs-
Standard PCR/qPCR master mixes contain dATP, dCTP, dGTP and dTTP. However, some mixes are available that replace dTTP with dUTP. Products from previous reactions run with dUTP will contain uracil instead of thymine. These are then susceptible to cleavage by Uracil-DNA-Glycosylase (UNG). Therefore, prior incubation of subsequent reactions with UNG prevents carryover contamination between reactions. To be effective, all PCRs in the laboratory must use dUTP.
Magnesium-
Magnesium chloride (MgCl2) is necessary for reverse transcriptase, Taq DNA polymerase and Taq DNA 5’ to 3’ exonuclease activity. Optimum Mg2+ concentrations for reactions containing DLPs are usually between 3–6 mM. The majority of master mixes contain MgCl2, however, it is sometimes necessary to optimize the concentration, so an additional tube of pure MgCl2is typically included with the master mix product (see Assay Optimization and Validation). In some cases, a reaction mix that does not contain MgCl2 may be required so that a low concentration can be used, e.g., when using Scorpions® Probe detection.
Reverse Transcriptase-
A reverse transcriptase enzyme that provides high yields of cDNA, while retaining activity at high temperature, is critical to the success of RT-qPCR. Performance at high temperatures helps to ensure that regions of RNA with significant secondary structure are destabilized and accessible for hybridization and subsequent amplification. When performing one-step RTqPCR, high-temperature performance allows the use of genespecific primers with high melting temperatures (Tm), which increases reaction specificity. When performing two step protocols, it is important to ensure that the enzyme results in a linear and proportional yield of cDNA from RNA (see Reverse Transcription for details of RT evaluation).
Taq DNA Polymerase-
As with selecting the most appropriate reverse transcriptase for the RT, selection of the appropriate polymerase enzyme is vital. A fundamental problem with natural Taq DNA polymerase is that the enzyme has residual activity at low temperature. Nonspecific primer binding leads to nonspecific product formation as a result of this residual polymerase activity. Antibody-blocked or chemically blocked Taq DNA polymerases (‘hot start’) help to rectify this situation by preventing enzyme activity until the high-temperature, denaturation step begins.
Buffer-
Buffers or reaction master mixes, typically contain dNTPs, a Taq DNA polymerase, MgCl2 and stabilizers. SYBR Green I dye, ROX™, fluorescein and inert loading dyes may also be included (see Loading Control Dyes), depending on the detection chemistry, instrument and reaction requirements. The PCR buffer components and stabilizers are typically proprietary to the manufacturer. If purchased separately, maximum flexibility is possible, since each ingredient can be optimized individually in the reaction. However, in contrast, while purchasing the ingredients together as a master mix reduces flexibility, it increases batch consistency and convenience while reducing the number of pipetting steps and hence, the chances of error and contamination.
Loading Control Dyes-
Some real-time PCR thermal cyclers require a loading dye such as ROX to be included into each reaction to control for variability in the optical system and to normalize differences in signal intensity. Likewise, some thermal cyclers require an initial fluorescein signal to create a virtual background when working with SYBR Green I dye assays (which have very low background). These may be supplied in the master mix or as separate components so that the appropriate concentration can be used.