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The polymerase chain reaction has come a long way since its discovery. Many modifications have come their way, which have been used in different aspects of the scientific world. One such discovery is real-time PCR. Quantitative PCR and qualitative PCR all come in the category of real-time polymerase chain reaction or rt pcr. They are known as the well-established method in the detection of microbial agents in clinical, veterinary, and food safety areas.
It is seen that the basic polymerase chain reaction is simple than the qPCR techniques. But while performing the real-time qPCR procedure, the researchers and scientists have to be specific about its technicalities. These technicalities include:
All these parameters should be kept in mind while performing the real-time pcr (qPCR) in the areas of microbial detections.
Real-time pcr is the most substantial milestone in polymerase chain reaction utilization applications. The introduction of the monitoring of pcr amplification gave the idea of the real-time pcr. Here, the monitoring work is done through DNA fluorescence which indicates the presence of DNA. In real-time pcr, fluorescence is measured after each pcr cycle. The intensity of fluorescence is recorded in the monitor screen which indicates the momentary amount of DNA amplicons in the sample of pcr. Fluorescent dyes give out the fluorescence at a specific time in each pcr cycle. At the initial steps of pcr, the fluorescence is low but can be distinguishable from the background.
The fluorescence level corresponds directly to the initial number of template DNA used in the Pcr sample for real-time qPCR. This point at which the fluorescence is bright is called the quantification point and the cycle as the quantification cycle. However, different companies and pcr machines use their terminology for the quantification point and cycles. In the whole process of real-time qPCR, a calibration curve is constructed which shows all serially diluted standard samples of DNA. The graph is useful in measuring the intensity of the DNA samples produced in the pcr procedure.
Real-time pcr should not be confused with the reverse transcriptase pcr, as both are different terms. The qPCR or real-time pcr does not use any reverse transcription pcr enzymes, while the rt pcr does use it. Other types of PCR are also different from real-time qPCR.
This advancement of using the real-time pcr has helped in the detection of the presence of microbes in the samples. The real-time pcr gives out two strategies of visualization using the amplified DNA fragments :
These two approaches are the base of the real-time qPCR processes which detect the pathogen in the samples. In most cases, the fluorescently labeled oligonucleotide probes are used which give out the fluorescence for pathogen or microbial detection. These probes are called primer dimers that are used with the steps of pcr.
Moreover, the real-time qPCR is also useful in providing the semi-quantitative results out of the target DNA samples. This semi-quantitative pcr step does not use any standard point for amplification. Instead, it uses a reference material for the amplification process. The results of real-time pcr can be expressed in lower multiplies with the reference points. In microbial detection and quantification, real-time PCR is used extensively.
To execute the steps of pcr, some parameters are kept in mind. These parameters govern almost all types of pcr. Following are the QPCR parameters used in the real-time qPCR :
This parameter refers to the specificity of primers used in the pcr technique. The primers are used for targeting the sample DNA used. It uses the two concepts for inclusivity in the real-time pcr:
The primers used in this process should be of a high degree of specificity. So, that the results can be detected in the process of qPCR. Here, the inclusivity of the process determines the 20-50 well-defined strains of the target organism.
On the opposite side, exclusivity means the ability of the qPCR method to distinguish between the target DNA from the similar but different non-target DNA. The analytical specificity is mandatory for the real-time pcr reactions. This ensures the perfect results out of the pcr procedures.
The analytical sensitivity is defined as the lowest amount of analyte that can be detected with more than a stated percentage of confidence. This value is not necessarily quantified as an exact value. In this case, the confidence level obtained reflects the number of replicates in both technical as well as experimental. These numbers are needed to reach the obtained or requested level of confidence. With this, it is clear that more replicates are tested in the process of real-time qPCR.
In another definition of the LOD, it is the lowest concentration level that can be evaluated statistically different from a blank at a specified level of confidence. All the confidence levels are analyzed from the analysis of sample blanks and samples near the expected LOD. Here, the LOD levels are reported through chemical methods. The blank samples taken in the qPCR steps should never be positive, as they can cause hindrances in the confidence level analyzed.
A different conservative approach is considered in the light of LOD value. It is the minimum concentration of nucleic acid or number of cells, which gives out a positive PCR result in all replicates in the qPCR. The majority of the reaction gives out the replicates per the 95% level of confidence. Here, the multiple aliquots of a specific matrix are used which are diluted with serial dilutions of the target organisms. The microbial detection is done by setting a proper LOD value for the qPCR procedure.
The limit of quantification is defined as the smallest amount of analyte that can be analyzed and quantified with a defined precision and accuracy during the real-time qPCR reaction. In this case, the measurement is done through defined accuracy under the experimental condition of the method under validations in the real-time PCR. It should be ensured that the LOQ should not be lower than the LOD value while performing the qPCR reactions in the microbial detection. In executing the pcr steps, the LOQ is determined as the LOD, on replicates of spiked samples. Here, the assessment is quantitative.
All the above-mentioned parameters are critical to microbial detection in the real-time pcr process. Real-time pcr or qPCR is called a sensitive reaction which requires more attention than the conventional polymerase chain reaction. The LOD and LOQ values should be per the primers used in the process, to give out the microbial detection in the DNA sample. PCR machines also have the machinery for real-time PCR, to test the samples for microbial or pathogen detection.