One of the most common and effective methods for the quantification, typing and detection of microbial agents is Real-Time PCR(or qPCR). These are primarily used for testing in terms of veterinary and clinical diagnostic and food safety.
While the method of PCR is quite effective and simple, some considerations should be made while performing this test to avoid inaccurate results. This means that the user of this technology should be well versed with the principle of PCR, definitions and terminology, interpretation and data presentation challenges, limitations as well as the areas of microbial diagnosis.
What Are The Principles For Detection?
A laboratory can use semi-skirted PCR plates to assist in your qPCR experiment. Because it is important for a lab worker performing PCR testing to be aware of each aspect of qPCR, including the optimization, validation, and design, here is a basic guide for PCR testing.
Read ahead to find out the basic parameters of qPCR testing.
1.  Analytical Specificity:
This involves defining the specificity of all participants that are a part of the target. In analytical specificity, both inclusivity and exclusivity are determined. The former identifies the ability of a clinical application to detect a number of targets that are related to each other. It can also be defined as the type of strains that is collected from the target of interest. (A Basic Guide to Real Time PCR in Microbial Diagnostics: Definitions, Parameters, and Everything, 2017)
Exclusivity determines whether the particular method is able to distinguish the target from genetically different but physically similar non-targets. It means that it revolves around the lowest percentage of interference from a non-target strain range that is relevant to the sample. The positive test samples that are required to perform exclusivity testing is zero.
2.  Limit Of Detection(LOD):
After much debate on the definition of LOD, a unified decision came forth which defined the limit of detection as the smallest analyte amount. It can be identified with more than one percent of confidence, but is unlikely to produce an exact value of quantification.
So we can say that it measures the number of technical and experimental replicates required in an assay for reaching a requested level of confidence, example, 90%. With the increase in the tested number of replicates, the interval of confidence also becomes narrow. We can also say that LOD defines the lowest level of concentration that is possible to be determined as statistically distinguished from a blank available at a specific confidence level. Remember that a blank sample never comes positive in PCR.
3.  Limit Of Quantification(LOQ):
LOQ is the smallest number of analytes which is quantified and computed with detailed accuracy and precision. It shall be analyzed under experimental conditions validated by a specific method. We can also claim that LOQ defines the lowest analyte concentration or amount that can be determined quantitatively with a defined level of uncertainty that is under the acceptable limit. Additionally, all the previous definitions point out to the idea that LOQ should never be lower than LOD in any PCR application.
4.  Efficiency Of Amplification(E):
The efficiency of PCR should fall between the range of 0 to 1(0-100%) when E is equal to 1. This means that in each cycle, the number of DNA amplicons formed will double. However, efficiency in PCR is a bit challenging to obtain repeatedly. The parameter can, however, be calculated through the estimated slope of calibration. This means that the range 90-105% is the more practical range to achieve for amplification efficiency.
5.  Accuracy:
The parameters mentioned above define a way to compare new qPCR methods with older reference materials and methods. Accuracy in a test is the length of conformity of the resultant value obtained from a specified procedure.
We can also say that accuracy is the attained level of agreement achieved between measured values and the reference. When performing a qPCR test, one must consider some things to achieve the highest level of accuracy. This will ensure that your resultant value is the closest to the actual value.
To sum it up, PCR principles are quite simple, but each requires its own methods of quantification and detection. qPCR helps obtain accurate results in certain cases, especially if the correct method and material are used to perform the testing. If your lab needs to upgrade its PCR testing gear, head over to Molecular Biology Products. MBP INC is efficiently stocked with all sorts of laboratory equipment and sample handling devices.
