The Polymerase Chain Reaction (PCR) is a laboratory technology used to amplify small DNA segments from millions to billions for analysis. Think of it like photocopying a document to create multiple identical copies. During the COVID-19 pandemic, PCR became a familiar term, as it was widely used to detect SARS-CoV-2, the virus responsible for COVID-19. However, its benefits extend far beyond this. PCR plays a vital role in healthcare, environmental research, and biotechnology.

Over time, PCR technology has evolved to deliver more consistent and accurate results. One of its groundbreaking innovations is quantitative PCR (qPCR) or real-time PCR (RT-PCR). Unlike traditional PCR, which only detects target DNA at the end of the process, qPCR can detect and quantify target DNA in real time during the reaction. This is possible due to fluorescent dyes or probes that emit signals as DNA is amplified.

The primary difference between traditional PCR and qPCR lies in their accuracy and functionality. Traditional PCR acts like a traffic light, indicating whether the target DNA is present (green for yes, red for no). In contrast, qPCR is like a calculator, providing precise numerical data on how much target DNA is present. Additionally, qPCR's ability to detect diseases at an early stage—even before symptoms appear—has made it indispensable in healthcare.

The Broad Benefits of qPCR Technology

In healthcare, qPCR is the gold standard for detecting various diseases such as HIV, HPV, and SARS-CoV-2. Beyond diagnostics, it has applications in gene research, detecting genetic mutations, and environmental studies. With its specificity and sensitivity, qPCR empowers researchers and medical professionals to make informed decisions.

In the medical and biotechnology fields, qPCR detects genetic mutations and infectious diseases like HPV, a leading cause of cervical cancer. In aquaculture, qPCR helps identify viruses that threaten shrimp health, such as IMNV and AHPND.

In forensics, qPCR is instrumental in analyzing DNA evidence from crime scenes to identify perpetrators. Meanwhile, environmental scientists use qPCR to monitor specific microorganisms in water or soil, ensuring ecosystem health.

Why Can PCR Results Vary?

Despite qPCR's advanced capabilities, discrepancies in results can still occur. For example, identical samples tested in different labs may yield different outcomes, raising questions like, “Why are the results inconsistent if the same qPCR technology is used?”

To understand this, it’s essential to recognize that qPCR is a highly technical process influenced by numerous factors. The process begins with DNA/RNA extraction, followed by amplification using specific primers and polymerase enzymes in a thermocycler—a machine that precisely controls temperatures during each cycle.

Though standardized, several factors can affect qPCR results, such as:
 

1. Sample Quality and Quantity

Contaminated or degraded DNA/RNA samples can hinder qPCR performance. For instance, DNA mixed with proteins or chemicals may disrupt amplification. High-quality DNA typically has an A260/280 ratio of around 1.8, while RNA has a ratio of approximately 2.0. Clean and intact samples are crucial for accurate and consistent results.
 

2. Primer Design and Quality

Primers guide qPCR to the specific DNA segment for amplification. Poorly designed primers—those that are too long, off-target, or form secondary structures like hairpins—can lead to errors. Careful primer design ensures the process targets the correct DNA segment, producing accurate results.
 

3. Protocol and Temperature Settings

qPCR relies on precise temperatures at each cycle stage. Deviations, even minor ones, can disrupt the process and compromise results. Adhering to correct protocols, including cycle duration and reagent concentrations, is vital for consistency.
 

4. Sterility and Contamination

qPCR’s sensitivity makes it prone to contamination from foreign DNA. Contaminants can come from non-sterile equipment, the laboratory environment, or cross-sample mixing. Maintaining sterility throughout the process is essential.
 

5. Reagent and Enzyme Quality

High-quality reagents, such as polymerase enzymes, buffers, and fluorescent dyes, are critical for efficient reactions. Degraded enzymes or damaged dyes can weaken signals, leading to inconsistent results. Using pure reagents ensures the success of qPCR.

Nusantics: Indonesia's Leading Precision Molecular Diagnostics Company

As a pioneer in precision molecular diagnostics in Indonesia, Nusantics offers state-of-the-art qPCR solutions. Here are some of our flagship products:
 

• PathoScan hrHPV qPCR Kit: Specialized reagents for detecting HPV, the virus causing cervical cancer. This product’s high accuracy supports early cervical cancer diagnosis.
• ShrimProtect: Real-time PCR reagents for detecting various shrimp diseases, providing fast and reliable results.
• CeKolam Services: An independent laboratory for diagnosing diseases in shrimp farming.

We utilize advanced qPCR technology and develop high-quality reagents to deliver consistent and reliable results. With a team of experienced laboratory professionals, Nusantics supports its partners in achieving optimal outcomes for cutting-edge diagnostics in human and animal health.

qPCR is a revolutionary technology with wide-ranging benefits across multiple fields. However, achieving consistent and accurate results requires meticulous execution and high-quality equipment and reagents. Nusantics is your trusted partner for molecular diagnostics, offering innovative solutions tailored to your needs. Make informed decisions with reliable data, powered by Nusantics.

Visit our website to explore more about our products and services in precision molecular diagnostics.

 

References:

1. Genome.gov. Retrieved January 21, 2025, from https://www.genome.gov/genetics-glossary/Polymerase-Chain-Reaction-PCR
2. Tahamtan, A., & Ardebili, A. (2020). Real-time RT-PCR in COVID-19 detection: Issues affecting the results. Expert Review of Molecular Diagnostics, 20(5), 453–454. https://doi.org/10.1080/14737159.2020.1757437
3. Lemke, T. L. (2005). Early diagnosis of myocardial infarction: Is there a future for non-enzymatic markers? Journal of Steroid Biochemistry & Molecular Biology, 96(3–4), 237–252. https://doi.org/10.1016/j.jsbmb.2005.09.016