PCR-ELISA: A Powerful Tool in Molecular Detection and Diagnosis

Introduction

In the early 1990s, DNA studies surged in interest with Friedrich Miescher's identification and isolation of DNA, coupled with James D. Watson and Francis Crick's groundbreaking discovery of the double helix structure in 1953. This period marked the introduction of key molecular techniques, including gel electrophoresis, understanding the DNA double helix, and Kary Mullis' invention of polymerase chain reaction (PCR) in 1983, a groundbreaking and widely used life sciences technique. Despite PCR's potency, its application requires a robust detection tool. Gel electrophoresis, a common method for detecting PCR products, has limitations such as a low detection threshold, only indicating the presence or absence of a specific gene. Real-time PCR emerged as a popular detection method.

In the late 1980s, a surge of interest in immunodetection of DNA occurred. Various approaches, including Coutlée et al.'s study on DNA immunodetection using biotinylated RNA probes, were published. This led to subsequent studies on DNA immunodetection using enzyme-linked immunosorbent assay (ELISA) techniques, eventually giving rise to the polymerase chain reaction-enzyme-linked immunosorbent assay (PCR-ELISA). Combining PCR and ELISA into a unified analytical technique, PCR-ELISA allows the detection of nucleic acids instead of proteins, expanding the capabilities of traditional ELISA.

Fig. 1 Illustration of the 3-step PCR-ELISA method (Sue M.J., et al. 2014).

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PCR-ELISA

PCR-ELISA, or polymerase chain reaction enzyme-linked immunosorbent assay, is an advanced molecular diagnostic technique that has gained significant attention in the field of molecular detection and diagnosis. This powerful analytical methodology combines the reliability of PCR (used for amplification of specific DNA sequences) with the sensitivity of ELISA (used for detecting specific antigens). It provides an increased specificity and sensitivity for the detection and quantification of specific nucleic acid sequences.

PCR-ELISA technique uses the principle of PCR, wherein a specific segment of DNA is replicated numerous times to generate thousands to millions of copies of a particular DNA sequence. To improve the accuracy of detection and diagnosis, it utilizes biotinylated primer during the PCR. Once amplified, the biotinylated PCR product can be detected using enzyme conjugated avidin or streptavidin and specific ELISA substrate through a colorimetric reaction which could be read directly in a microtiter plate reader.

Advantages of PCR-ELISA

Sensitivity: PCR-ELISA stands out for its heightened sensitivity, enabling the detection of even minute amounts of target nucleic acids in a given sample. This attribute proves particularly beneficial for the early identification of infections or diseases, especially when the concentration of the target sequence is at a low level.

Specificity: The technique excels in specificity, precisely identifying a particular nucleic acid sequence within intricate sample compositions. This specificity is achieved through the utilization of sequence-specific primers and probes. These molecular tools exclusively amplify and detect the target sequence of interest, ensuring accuracy and reliability in results.

Quantification: PCR-ELISA facilitates the quantification of target nucleic acids in a sample. This quantitative aspect provides valuable insights into the concentration of the target sequence, offering essential information for monitoring disease progression and evaluating treatment efficacy.

High Throughput: As a high-throughput methodology, PCR-ELISA has the capability to process multiple samples simultaneously. This characteristic makes it an ideal choice for efficiently screening large numbers of samples, whether in a clinical or research setting.

Flexibility: PCR-ELISA exhibits remarkable adaptability, accommodating the detection of a diverse array of target sequences, including DNA, RNA, and viral genomes. This versatility empowers the technique to identify various pathogens and genetic mutations, broadening its applications in diagnostics and research endeavors.

Applications of PCR-ELISA

In infectious disease diagnostics, PCR-ELISA is effective in detecting and quantifying the viral load in patients with human immunodeficiency virus (HIV), hepatitis B virus, and hepatitis C virus. Similarly, it can detect bacterial infections like tuberculosis and parasitic infections like malaria, even in the early stages of the diseases. It also distinguishes between viable and non-viable pathogens, helping to monitor the effectiveness of antimicrobial treatment.

In the field of genetic testing, PCR-ELISA facilitates the detection of genetic mutations and variations. This is pivotal in identifying individuals at risk of genetic disorders or assessing the efficacy of therapeutic interventions. The technique's ability to amplify specific DNA fragments ensures the reliable detection of genetic markers, contributing to advancements in personalized medicine.

PCR-ELISA is extensively employed in environmental monitoring and food safety. It enables the detection of microbial contaminants in food products or environmental samples. By targeting specific DNA or RNA sequences, it provides a highly sensitive and specific tool for identifying the presence of pathogens, ensuring the safety of consumables and the environment.

Forensic science benefits from PCR-ELISA in DNA profiling and analysis. The method's precision in amplifying and detecting specific DNA sequences aids in the identification of individuals, crucial in criminal investigations and paternity testing. The sensitivity of PCR-ELISA allows for the analysis of minute amounts of DNA, even from degraded or challenging samples.

In the pharmaceutical industry, PCR-ELISA plays a pivotal role in drug development and quality control. It is employed for the detection and quantification of nucleic acid contaminants in biopharmaceutical products. This ensures the purity and safety of pharmaceuticals, meeting regulatory standards and assuring product quality.

Furthermore, PCR-ELISA is integral in research applications, enabling the study of gene expression, regulation, and function. It facilitates the quantification of specific nucleic acid sequences, providing insights into biological processes and mechanisms. Researchers use PCR-ELISA to unravel the complexities of diseases, contributing to the development of novel therapeutic approaches.

In conclusion, PCR-ELISA's broad range of applications underscores its significance in various scientific and medical disciplines. Its ability to combine the precision of PCR with the specificity of ELISA makes it a valuable tool for diagnostics, genetic testing, environmental monitoring, forensic science, pharmaceuticals, and research. As technology continues to advance, PCR-ELISA is likely to evolve and find even more applications in the ever-expanding landscape of life sciences.

References

1. Sue M.J., et al. Application of PCR-ELISA in molecular diagnosis. BioMed Research International. 2014, 2014.
2. Arya N., Kaur A. A review paper on application of PCR-ELISA in the molecular diagnosis. International Journal of Innovative Research in Engineering & Management. 2022;9(1):341-4.