In the realm of molecular biology, the quest for efficient, rapid, and sensitive techniques for detecting nucleic acids has been ongoing. One such groundbreaking method that has emerged in recent years is Loop-Mediated Isothermal Amplification (LAMP). This innovative technique has revolutionized nucleic acid detection by offering a robust, rapid, and cost-effective alternative to traditional PCR-based methods.

Principles of LAMP

Loop-Mediated Isothermal Amplification operates on a simple yet ingenious principle, allowing the amplification of DNA (and sometimes RNA) under isothermal conditions. Unlike conventional PCR, which necessitates thermal cycling, LAMP achieves amplification at a constant temperature, typically around 60-65°C. This is made possible through the use of a DNA polymerase enzyme with strand displacement activity, which facilitates continuous DNA synthesis without the need for thermal cycling.

The key player in this process is Bst (Bacillus stearothermophilus) DNA polymerase, a thermostable enzyme with robust strand displacement activity. Bst polymerase efficiently synthesizes DNA strands while displacing the existing strands, enabling the continuous amplification of target DNA sequences. This unique enzymatic property allows LAMP to proceed rapidly and efficiently under isothermal conditions.

The process begins with the design of four to six primers that target multiple regions of the DNA sequence of interest. These primers initiate the amplification process by binding to their specific target sequences on the DNA template. As the DNA polymerase extends the primers, it displaces the existing DNA strands, creating single-stranded regions for further primer binding and extension. This results in the formation of a unique stem-loop structure with multiple repeats of the target sequence. The reaction proceeds in a cyclical manner, with each cycle generating an exponentially increasing amount of DNA.

Applications and Advantages

The versatility and utility of Loop-Mediated Isothermal Amplification are reflected in its broad range of applications. From molecular diagnostics to pathogen detection and genetic analysis, LAMP has found use in various fields including medicine, veterinary science, agriculture, and environmental monitoring.

One of the most significant advantages of LAMP is its high specificity and sensitivity, which rivals that of traditional PCR methods. Additionally, LAMP offers rapid amplification under isothermal conditions, eliminating the need for expensive thermal cycling equipment and reducing assay time. Furthermore, LAMP assays can be performed using simple instrumentation, making them accessible even in resource-limited settings.

Detection Methods

The amplified DNA generated by LAMP can be detected using various methods depending on the specific application. Turbidity measurements, based on the accumulation of magnesium pyrophosphate during the reaction, offer a simple and cost-effective detection method. Colorimetric indicators, such as pH-sensitive dyes, provide a visual readout of amplification through color changes in the reaction tube. Fluorescence-based assays offer high sensitivity and quantitative analysis capabilities, making them ideal for research and diagnostic applications. Visual inspection of color changes in reaction tubes can also serve as a low-cost and straightforward detection method, particularly in field settings.

Case Study

The paper titled "Portable Real-Time Colorimetric LAMP-Device for Rapid Quantitative Detection of Nucleic Acids in Crude Samples," published in Scientific Reports, highlights a significant breakthrough in molecular diagnostics. This collaborative effort between the Institute of Molecular Biology and Biotechnology and BIOPIX DNA TECHNOLOGY PC marks a milestone in the development of a portable real-time colorimetric LAMP device. This innovative technology promises to revolutionize disease diagnostics, particularly in resource-limited settings.

The portable real-time colorimetric LAMP device represents a fusion of accuracy from lab-based quantitative analysis and the simplicity of point-of-care testing. Its design incorporates a plastic tube anchored vertically on a hot surface, with side walls exposed to a mini camera capable of capturing real-time color changes during LAMP amplification. This setup enables rapid analysis, quantification over a wide dynamic range, and compatibility with crude samples like saliva, tissue, and swabs.

Distinguishing itself from conventional diagnostic platforms, the device offers numerous competitive features. These include rapid analysis time, quantification of nucleic acids over nine log-units, compatibility with crude samples, low detection limit (< 5 copies/reaction), smartphone operation, and rapid prototyping through 3D printing. Additionally, users have the flexibility to select the dye of interest, such as Phenol red or Hydroxynaphthol blue (HNB), enhancing its versatility and practicality.

Central to the functionality of this device is the integration of SBS Genetech's Bst DNA/RNA Polymerase within the LAMP assay. Bst DNA/RNA Polymerase represents a fusion of Bst Polymerase and an exceptionally thermostable reverse transcriptase (tolerant up to 65°C), making it ideal for the isothermal amplification of both DNA and RNA templates. This unique composition enables the detection of low-sensitivity RNA molecules, rendering it highly recommended for isothermal amplification experiments utilizing RNA as a template.

The robust amplification capability of Bst DNA/RNA Polymerase, coupled with the real-time colorimetric detection method, ensures remarkable sensitivity, specificity, and tolerance to inhibitory substances. This combination empowers the device with the capability to cater to a wide range of diagnostic applications, promising accurate and reliable results even in challenging conditions.

Beyond Bst DNA/RNA Polymerase

At SBS Genetech, our pioneering spirit drives us to continually enhance our offerings in loop-mediated isothermal amplification (LAMP) technologies. Our Bst P DNA/RNA Polymerase stand as testament to our dedication to innovation and excellence.

Bst P DNA/RNA Polymerase stands as an enhanced iteration of Bst DNA/RNA Polymerase, meticulously refined through enzyme electronic re-structuring and evolution screening (utilizing in silico Design & in vitro Evolution). Specifically tailored for LAMP or RT-LAMP amplification of DNA or RNA, this upgraded version represents a notable leap forward in molecular biology. With its advanced design and optimized functionality, Bst P DNA/RNA Polymerase offers unparalleled performance and versatility, setting a new standard for nucleic acid amplification technologies.

Highlighted in the esteemed Science Journal on April 13, 2023, our glycerol-free Bst P DNA/RNA Polymerase underscores our commitment to pushing the boundaries of molecular biology research. This recognition reaffirms our position at the forefront of scientific advancement and underscores the transformative potential of our technologies.

Key features of the Bst P DNA/RNA Polymerase include:

• Integration of a hot start Aptamer, ensuring precise control over enzyme activity, even at low temperatures, and facilitating rapid activation for efficient amplification.
• Elevated reaction temperature to 70°C, minimizing primer dimer formation, enhancing amplification specificity, and ensuring efficient nucleic acid release from crude samples.
• Incorporation of Helicase, enabling Premium LAMP amplification (pLAMP) without the need for F3/B3 primers, while also assisting in strand unwinding to improve amplification uniformity.
• Complementing our enzyme portfolio is our customized lyophilized microbead service, offering tailored solutions to meet the unique requirements of your research projects. With customized primers and reaction volumes, we empower researchers to optimize experiments with precision and efficiency.

For more information on our Bst P DNA/RNA Polymerase and other groundbreaking products, visit the Science Journal article dated April 13, 2023, or contact us to discover how SBS Genetech can accelerate your research journey.

Related: 

• Bst DNA Polymerase Large Fragment
• Bst DNA Polymerase (comparable to 2.0)
• Bst Polymerase (comparable to 3.0)
• Bst Polymerase (glycerol-free)
• Bst DNA/RNA Polymerase (with superior reverse transcription activity)
• Bst DNA/RNA Polymerase (glycerol-free)
• Bst P DNA/RNA Polymerase (glycerol-free) (with superior reverse transcription activity & hot-start property)