Cell culture has proven effective in the identification process of infectious pathogens and remains the goal standard for identification despite it numerous shortcomings such as the fact that it is time consuming. That is, having a turnaround time ranging anywhere from days to weeks depending on the type of pathogen isolated. It also comes with high rates of contamination and there are some microorganisms that cannot be cultivated making this technique virtually impossible to identify them. Apart from culture techniques, other methods such as serological assay, polymerase chain reaction can be used to identify infectious agents.
There is need for a rapid, accurate and sensitive identification technique so as to ensure appropriate treatment and improve patient care. Nucleic acid based detection and amplification-based assays has proving to be the most accurate and quickest identification technique and process. These techniques provide results within two to three hours compared to conventional microbial culture. In this article, we shall be discussing about polymerase chain reaction abbreviated PCR.
What is Polymerase chain reaction?
Polymerase chain reaction is a method widely used to proliferate or rapidly make millions to billions of copies of a specific DNA sample. This makes it possible for scientist to multiply a very small sample of DNA and amplify it to a large number of identical copies of the sample in order to detail study it through repeated polymerase cycle. PCR was invented by Dr Kary Mullis in 1983. The first PCR diagnosis assay (the AMPLICOR Chlmaydia trachomatis test) was FDA approved is the United States of America. Since then, PCR techniques has been frequently used in clinical laboratories. It requires two set of primers complementary to the both ends of the DNA template and a thermostable DNA polymerase.
Components of Polymerase Chain Reaction (PCR)
The following are component of PCR
- DNA template
- Deoxyribonucleoside triphosphates (dNTPs)
- PCR buffer
- Primers (forward and reverse)
- Taq polymerase
Steps in PCR technique
This is where the double-stranded DNA is separated and single strands of DNA are formed.
The reaction temperature is reduced to allow the complementary base pairing and annealing of the primer.
This involves the Taq polymerase, which is a thermostable DNA polymerase. The DNA polymerase adds nucleotides to the primer and extends the complementary strand in the 5’-3’ direction.
Types of Polymerase Chain Reactions
There are several types of PCR based on several modifications to enhance the utility in diagnostic settings based on their applications. The most common includes;
- Real-Time PCR (quantitative PCR or qPCR)
- Reverse-Transcriptase (RT-PCR)
- Multiplex PCR
- Nested PCR
- High-fidelity PCR
- Fast PCR
- Hot-start PCR
- GC-rich PCR
- Long-range PCR
- Arbitrarily primed polymerase chain reaction (AP-PCR)
Applications of PCR
- Cloning genes
- Detection of antimicrobial resistance
- Detection of mutation (investigation of genetic diseases)
- Direct detection of microorganisms in patient specimens
- Genetic fingerprinting (forensic application/paternity testing)
- Identification and characterization of infectious agents
- Identification of microorganisms grown in culture
- Investigation of strain relatedness of a pathogen of interest
- PCR sequencing
Traditional PCR technique is a non-automated semi quantitative process. It detect and estimate the amount of amplified PCR product at end point of several PCR cycles. The concentration of nucleic acids is determined by comparing the amplified band intensity on an electrophoresis gel to a known standard. Unfortunately it has some shortcoming:
- It has a low sensitivity, poor precision and accuracy.
- The use of an agarose gels for detection is time consuming and also leads to poor resolution of bands on gel which makes it difficult to detect fivefold changes on the gel.
- It is limited to amplification of nucleic acids for sequencing, cloning, and genotyping
- Size-based discrimination is possible
- There is no numerical expression of results and post-PCR processing is required
Real Time PCR
Real time PCR is automated and quantitative. It detect PCR product while in progress and quantify DNA and RNA as the reaction proceeds. It can detect as little as a twofold change in the generated copies and eliminates the need for agarose gel and post PCR processing. Compared to end point or traditional PCR, RT-PCR is far accurate and reliable and is the rise in most clinical laboratories.