What is Real-Time PCR?
Usually, in conventional PCR, the detection of the amplified DNA product occurs in an end-point analysis. However, in real-time PCR, the amount of amplification product is calculated along with the progression of the reaction in real-time. Other than that, product quantification is also measured after each cycle.
What is the qPCR workflow?
The qPCR workflow has specific steps that are based only on a real-time PCR system. In the first step, amplification reactions take place. The system is set up with PCR reagents and custom primers. With the use of real-time PCR instruments, reactions are accommodated, and the collected data is analyzed via the proprietary instrument software.
What are the main advantages of real-time PCR?
One of the main advantages of real-time PCR against conventional PCR is that it allows you to observe the initial number of template DNA copies. If you compare the results of both these systems, you will observe that real-time PCR will offer more accuracy and high sensitivity over a wide dynamic range.
Mainly, there are two types of Real-time PCR results:
- Qualitative results where there is an absence or presence of a sequence.
- Quantitative results where the copy numbers are analyzed.
The qPCR analysis is another name for quantitative real-time PCR. To some extent, PCR is semiquantitative. On top of that, real-time qPCR can be subjected to evaluation in the absence of gel electrophoresis that leads to increased throughput and decreased bench time. As real-time qPCR reactions are closely observed in unified systems, the chances of the sample getting contaminated are reduced to almost 10%. As a result, the requirement for post-amplification which is otherwise a crucial step in PCR tests is eliminated.
What are the applications of Real-Time PCR and qPCR assays?
In the field of genetics, the tests that take place in laboratories are sensitive to handle, manage, and integrate. One needs to implement consistent and persistent technology to carry out their research. If you talk about qPCR tests, these assays have become the number one choice for analysis and determination of rapid and critical elements concerning nucleic acids testings. In the modern life sciences department, qPCR assays have diverse applications such as the detection of genetically modified organisms in food, gene expression, and cancer phenotyping.
In new-age research laboratories, qPCR assays are widely acknowledged for their efficiency in the quantitative measurement of processes like gene copy numbers or detection of mutant genes. Researchers are now combining the technology of reverse-transcription PCR (RT-PCR) with qPCR assays to precisely monitor quantitative changes in gene expression. Examples of such procedures are gene expression technology that is a need during drug treatment and in determining environmental conditions. All of these take place in monitoring the alteration in cellular mRNA levels. (BIO-RAD, 2021)
What is the Applied Biosystems 7500 Real-Time PCR System?
- The 7500 Real-Time PCR System is one of a kind qPCR system that is playing a pivotal role in the field of biosciences. In simple words, it is a versatile and leading-edge platform offering advanced capabilities and a modern pathway in high-speed thermal cycling. Additionally, it also reduces your run time for quantitative real-time PCR applications. The 7500 real-time PCR system delivers results in a minimum period of 35 minutes.
- The five-color detection systems propose superior flexibility, so the users can perform several applications such as gene expression analysis, internal positive controls, and SNP genotyping.
- Applied Biosystems 7500 Real-Time PCR system possesses a design that is made up of small instruments. This smart architecture permits the user to place the instrument easily in the laboratory, even if they can not afford much space.
- Dynamic range
The 7500 system has 9 logs of linear dynamic range.
It can detect 10 copies of templates in a 50 microliter solution for a single Tapman assay. The confidence rate is 99.7%.
The usual run time is one hour and 50 minutes for a 40 cycle PCR reaction if the user is using a Tapman RNase Instrument Verification Plate.
Thermal cycling system
It consists of Peltier-based and a 96-well block.
The features are CCD camera, Five-excitation, and Five-emission filter.
Quantitative PCR run time
Generally, it is < 2 hours.
- Standard Optical 96-well plates
- Optical Fast 96-well plates
- 8-strip 0.2mL tubes
- Optical Adhesive Covers
- 0.2mL tubes
- Optical Adhesive Covers
- Optical Flat Caps
20-100 microliters. (Applied Biosystems, 2004)
What are the applications of Applied Biosynthesis 7500 Real-Time PCR?
The following are the new-age applications of Applied Biosynthesis 7500 Real-Time PCR:
- Fluorescence detection.
- Sequence detection software.
- Installation specifications.
- Computer specifications.
- Reagents and disposables.
The role of PCR tests in the outbreak of Covid-19
As the novel coronavirus is spreading the Covid-19 around the world, scientists and researchers from all around the world are trying to find different ways to contain and limit the spread of the aggressive virus. FAO and other renowned organizations are offering their support, expertise, and knowledge to help affected countries fight the virus with the help of real-time reverse transcription-polymerase chain reactions (qPCR and RT-PCR). These methods are one the fastest and reliable laboratory methods that are playing a major role in the detection, tracking, and research of the COVID-19 virus.
In the majority of countries, the qPCR technology is being used in the following way to isolate the victims and monitor the spread of the coronavirus.
During testing, a sample is collected from different parts of the body where the COVID-19 virus might gather. These areas are an individual’s nose or throat. Afterwards, the sample is treated with several chemical reactions to isolate molecules like proteins and fats. What follows is the separation of RNA. If the person is COVID-positive, the RNA will be a mix of the individual’s genetic material and the virus’ RNA as well. After subjecting the extracted RNA with reverse transcription, further DNA fragments are added by the doctors. In case, the virus is present, these additional fragments will attach themselves to specific target areas. The health professionals identify the marker strands and ensure that the coronavirus exists in the patient’s body. (Jawerth, 2020).
Applied Biosystems. (2004). Applied Biosystems 7500 Real-Time PCR System and Applied Biosystems 7500 Fast Real-Time PCR System. Real-Time PCR Systems. https://www.gene-quantification.de/abi7500.pdf
BIO-RAD. (2021). What is Real-Time PCR? BIO-RAD. https://www.bio-rad.com/en-pk/applications-technologies/what-real-time-pcr-qpcr?ID=LUSO4W8UU
Jawerth, N. (2020, June). How is the COVID-19 virus detected using real-time RT–PCR? IAEA Bulletin, 61(2). https://www.iaea.org/bulletin/infectious-diseases/how-is-the-covid-19-virus-detected-using-real-time-rt-pcr#:~:text=Compared%20to%20other%20available%20virus,of%20the%20COVID-19%20virus.