However, it may be prudent to titrate the manipulated reagent. Changing the magnesium concentration is one of the easiest reagents to manipulate with perhaps the greatest impact on the stringency of PCR. The 10 X PCR buffer solutions may contain 15 mM MgCl 2 , which is enough for a typical PCR reaction, or it may be added separately at a concentration optimized for a particular reaction.
If the desired amplicon is below bp and long non-specific products are forming, specificity may be improved by titrating KCl, increasing the concentration in 10 mM increments up to mM. Thus, choosing an appropriate enzyme can be helpful for obtaining desired amplicon products.
The addition of a 3' adenine has become a useful strategy for cloning PCR products into TA vectors whit 3' thymine overhangs. However, if fidelity is more important an enzyme such as Pfu may be a better choice.
Several manufactures have an array of specific DNA polymerases designed for specialized needs. Take a look at the reaction conditions and characteristics of the desired amplicon, and then match the PCR experiment with the appropriate DNA polymerase.
Most manufactures have tables that aid DNA polymerase selection by listing characteristics such as fidelity, yield, speed, optimal target lengths, and whether it is useful for G-C rich amplification or hot start PCR. Optimal target molecules are between 10 4 to 10 7 molecules and may be calculated as was described in the notes above. Additive reagents may yield results when all else fails.
Understanding the reagents and what they are used for is critical in determining which reagents may be most effective in the acquisition of the desired PCR product.
Adding reagents to the reaction is complicated by the fact that manipulation of one reagent may impact the usable concentration of another reagent. In addition to the reagents listed below, proprietary commercially available additives are available from many biotechnology companies.
Formamide final reaction concentration of 1. Formamide also has been shown to be an enhancer for G-C rich templates. As the amplicon or template DNA is denatured, it will often form secondary structures such as hairpin loops. Betaine final reaction concentration of 0. Non ionic detergents function to suppress secondary structure formation and help stabilize the DNA polymerase.
Non ionic detergents such as Triton X, Tween 20, or NP may be used at reaction concentrations of 0. The presence of non ionic detergents decreases PCR stringency, potentially leading to spurious product formation. However, their use will also neutralize the inhibitory affects of SDS, an occasional contaminant of DNA extraction protocols. Hot start PCR is a versatile modification in which the initial denaturation time is increased dramatically Table 4. This modification can be incorporated with or without other modifications to cycling conditions.
Moreover, it is often used in conjunction with additives for temperamental amplicon formation. In fact, hot start PCR is increasingly included as a regular aspect of general cycling conditions. Hot start has been demonstrated to increase amplicon yield, while increasing the specificity and fidelity of the reaction.
The rationale behind hot start PCR is to eliminate primer-dimer and non-specific priming that may result as a consequence of setting up the reaction below the T m.
In general, the DNA polymerase is withheld from the reaction during the initial, elongated, denaturing time. Although other components of the reaction are sometimes omitted instead of the DNA polymerase, here we will focus on the DNA polymerase. There are several methods which allow the DNA polymerase to remain inactive or physically separated until the initial denaturation period has completed, including the use of a solid wax barrier, anti-DNA polymerase antibodies, and accessory proteins.
Alternatively, the DNA polymerase may simply be added to the reaction after the initial denaturation cycle is complete. The concept is to design two phases of cycling conditions Table 5. The first phase employs successively lower annealing temperatures every second cycle traditionally 1. The function of the first phase should alleviate mispriming, conferring a 4-fold advantage to the correct product.
Thus, after 10 cycles, a fold advantage would yield copies of the correct product over any spurious priming. This would allow the correct product a fold advantage over false priming products. The concept takes into account a relatively new feature associated with modern thermal cyclers, which allows adjustment of the ramp speed as well as the cooling rate.
The ramp speed is lowered to 2. Nested PCR is a powerful tool used to eliminate spurious products. The use of nested primers is particularly helpful when there are several paralogous genes in a single genome or when there is low copy number of a target sequence within a heterogeneous population of orthologous sequences. The basic procedure involves two sets of primers that amplify a single region of DNA.
The outer primers straddle the segment of interest and are used to generate PCR products that are often non-specific in 20 to 30 cycles. Other PCR protocols are more specialized and go beyond the scope of this paper. The results incorporate several troubleshooting strategies to demonstrate the effect of various reagents and conditions on the reaction.
Genes from the budding yeast Saccharomyces cerevisiae and from an uncharacterized Mycobacteriophage were amplified in these experiments. The standard 3-step PCR protocol outlined in Table 2 was employed for all three experiments described below.
The working stocks were prepared as follows. Since the S. This phage DNA is about 67 Kb. Thus, 1 ng contains 2. The working stocks were then used to generate the Master Mix solutions outlined in Table 7. Experiments varied cycling conditions as described below. No MgCl 2 was present in the original PCR buffer and had to be supplemented at the concentrations indicated with a range tested from 0.
The recommended concentration provided by the manufacturer was 1. Perhaps surprisingly, the necessary concentration needed for product formation in this experiment exceeded this amount. A different DNA template was used for the experiment presented in Figure 3b. As shown in Figure 3b , amplification of the desired PCR product requires at least 2.
Notice that in the experiments presented in Figures 3A and 3B , a discrete band was obtained using the cycling conditions thought to be optimal based on primer annealing temperatures.
For the third experiment presented in Figure 3c , three changes were made to the cycling conditions used to amplify the yeast GAL3 gene. Second, the extension time was extended to 1 minute and 30 seconds. Third, the number of cycles was increased from 30 to 35 times. The purpose was to demonstrate the effects of sub-optimal amplification conditions i. As shown in Figure 3c , what was a discrete band in Figure 3a , becomes a smear of non-specific products under these sub-optimal cycling conditions.
These results also demonstrate that when both the cycling conditions are correctly designed and the reagents are at optimal concentrations, the PCR experiment produces a discreet amplicon corresponding to the expected size.
The results show the importance of performing PCR experiments at a sufficiently high stringency e. Moreover, the experiments indicate that changing one parameter can influence another parameter, thus affecting the reaction outcome.
The Master Mix depicted in the above table is calculated for 11 reactions plus 2 extra reactions to accommodate pipette transfer loss ensuring there is enough to aliquot to each reaction tube. Table 7. Figure 1. Note that primers do not always anneal at the extreme ends and may form smaller loop structures.
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The reliability of the calibration curve in enabling quantification is then determined by the spacing of the serial dilutions. If the Log 10 of the concentration or copy number of each standard is plotted against its C q value Figure 1 , the E can be derived from the regression equation describing the linear function:. The intercept shows the C q value when one copy would be theoretically detected Kubista et al.
The concentration or amount of target nucleic acid in unknown samples is then calculated according to the C q value through Equation 5. Figure 1. Model calibration curve with the regression equation characterized by the slope and intercept and regression coefficient.
From the definitions above it is evident that C q values are instrumental readings, and must be recalculated to values with specific units, e. However, referral to C q values in scientific papers is widespread and interpretations based on C q values can lead to misleading conclusions.
Concentrations in qPCR are expressed in the logarithmic scale Figure 1 and C q differences between fold serial dilutions are theoretically always 3. Therefore, although the numerical difference between C q 20 and 35 is rather negligible, the difference in real numbers copies, ng is almost five orders of magnitude Log This feature must be reflected in the subsequent calculations.
For example, the coefficient of variation CV, ratio between standard deviation and mean calculated from the C q values and real numbers results in profoundly different results. The same applies for any statistical tests where C q values are used, even for cases where the logarithm of C q values is used for the normalization of data before the statistical evaluation.
The correct procedure should include initial recalculation to real numbers followed by logarithmic transformation. With the increasing amount of sequencing data available, it is literally possible to design qPCR assays for every microorganism groups and subgroups of microorganisms, etc. The main advantages of qPCR are that it provides fast and high-throughput detection and quantification of target DNA sequences in different matrices.
The lower time of amplification is facilitated by the simultaneous amplification and visualization of newly formed DNA amplicons.
Moreover, qPCR is safer in terms of avoiding cross contaminations because no further manipulation with samples is required after the amplification. Other advantages of qPCR include a wide dynamic range for quantification 7—8 Log 10 and the multiplexing of amplification of several targets into a single reaction Klein, The multiplexing option is essential for detection and quantification in diagnostic qPCR assays that rely on the inclusion of internal amplification controls Yang and Rothman, ; Kubista et al.
Therefore, although qPCR-based typing tests are faster, their results should be correlated with phenotypic and biochemical tests Levin, ; Osei Sekyere et al. As for the microbial diagnostics, there are different considerations in detecting and quantifying viral, bacterial, and parasitic agents. This is because detection of important clinical and veterinary viruses using culture methods is time-consuming or impossible, while ELISA tests are not universally available and suffer from comparatively low sensitivity and specificity.
Moreover, determination of the viral load by RT -qPCR is used as an indicator of the response to antiviral therapies Watzinger et al. The situation is similar in the case of intestinal protozoan diagnostics Rijsman et al.
The gold standard technique for the detection of protozoan agents, the microscopic examination of feces, is laborious, time-consuming, and requires specifically trained personnel.
Therefore, qPCR is now emerging as a powerful tool in the routine detection, quantification, and typing of intestinal parasitic protozoa. In contrast to viral and protozoan detection and quantification, many bacteria of clinical, veterinary, and food safety significance, can be cultured.
For this reason, culture is considered as the gold standard in bacterial detection and quantification. However, in cases when critical and timely intervention for infectious disease is required, the traditional, slow, and multistep culture techniques cannot provide results in a reasonable time.
This limitation is compounded by the necessity of culturing fastidious pathogens and additional testing species determination, identification of virulence factors, and antimicrobial resistance. In food safety, all international standards for food quality rely on the determination of pathogenic microorganisms using traditional culture methods. However, there are limitations with respect to the sensitivity of assays based on qPCR. As culture methods rely on the multiplication of bacteria during the pre-culture steps pre-enrichment , samples for DNA isolation usually initially contain very low numbers of target bacteria Rodriguez-Lazaro et al.
This limitation leads to the most important disadvantage of qPCR, which is its inherent incapability of distinguishing between live and dead cells.
The usage of qPCR itself is therefore limited to the typing of bacterial strains, identification of antimicrobial resistance, detection, and possibly quantification in non-processed and raw food.
It is important to note that processed food can still contain amplifiable DNA even if all the potentially pathogenic bacteria in food are devitalized and the foodstuff is microbiologically safe for consumption Rodriguez-Lazaro et al. To overcome this problem, a pre-enrichment of sample in culture media could be placed prior to the qPCR.
This step may include non-selective enrichment in buffered peptone water or specific selective media for the respective bacterium. The extraction of the DNA from the culture media is easier than that from the food samples, which are much more heterogeneous in terms of composition Margot et al. Although qPCR itself cannot distinguish among viable and dead cells attempts have been made to adapt qPCR for viability detection. It was shown that RNA has low stability and should be degraded in dead cells within minutes.
However, the correlation of cell viability with the persistence of nucleic acid species must be well characterized for a particular situation before an appropriate amplification-based analytical method can be adopted as a surrogate for more traditional culture techniques Birch et al.
Moreover, difficulties connected with RNA isolation from samples like food, feces or environmental samples can provide false-negative results especially when low numbers of target cells are expected. In these methods, the criterion for viability determination is membrane integrity. Metabolically active cells regardless of their cultivability with full membrane integrity keep the dyes outside the cells and are therefore considered as viable. However, if plasma membrane integrity is compromised, the dyes penetrate the cells, or react with the DNA outside of dead cells.
The labeled DNA is then not available for the amplification by qPCR and the difference between treated and untreated cells provides information about the proportion of viable cells in the sample.
The limitation of this method is the necessity to have the cells in a light-transparent matrix, e. Therefore, samples of insufficient light transparency do not permit the application of these dyes. Moreover, another topic we want to just to mention here is the generation and use of standards required for the calibration curves.
In general, two are the most diffused approaches for the generation of calibration curves. One employs dilutions of target genomic nucleic acid and the other plasmid standards. Both strategies can lead to a final quantification of the target, but plasmids containing specific target sequences offer the advantages of easy production, stability, and cheapness. On the other hand, in principle, PCR efficiency obtained by plasmid standards sometimes could differ compared to the efficiency obtained using genomic standard, which instead, for organisms fastidious to growth, could be isolated only starting from a given matrix, and thus susceptible to degradation and losses Chaouachi et al.
This parameter in qPCR refers to the specificity of primers for target of interest. Analytical specificity consists of two concepts: inclusivity describes the ability of the method to detect a wide range of targets with defined relatedness e.
Another definition describes inclusivity as the strains or isolates of the target analyte s that the method can detect Anonymous, ISO and other standards recommend that inclusivity should be determined on 20—50 well-defined certified strains of the target organism Anonymous, , , , a ; Broeders et al.
On the other hand, exclusivity describes the ability of the method to distinguish the target from similar but genetically distinct non-targets.
In other words, exclusivity can also be defined as the lack of interference from a relevant range of non-target strains, which are potentially cross-reactive Anonymous, , , , a.
The desirable number of positive samples in exclusivity testing is zero Johnson et al. Many official documents have discussed theories and procedures for the correct definition of the LOD for different methods. A general consensus was reached around the definition of the LOD as the lowest amount of analyte, which can be detected with more than a stated percentage of confidence, but, not necessarily quantified as an exact value Anonymous, , , In this regard, the confidence level obtained or requested for the definition of LOD can reflect the number of replicates both technical and experimental needed by the assay in order to reach the requested level of confidence e.
It is clear that the more replicates are tested, the narrower will be the interval of confidence. Another definition describes the LOD as the lowest concentration level that can be determined as statistically different from a blank at a specified level of confidence.
This value should be determined from the analysis of sample blanks and samples at levels near the expected LOD Anonymous, a. However, it should be noted that LOD definitions described above were reported for chemical methods, and are not perfectly suited for PCR methods Burns and Valdivia,
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