LAMP Method: A Practical Solution for Detecting Peronospora destructor in Shallot Seed
Shallots are one of the key commodities relied upon by many farmers and the culinary industry. However, like other crops, shallots are vulnerable to various diseases that can reduce both the quality and quantity of the harvest. One of the most destructive diseases is downy mildew, caused by the pathogen Peronospora destructor. This disease can spread quickly through infected seeds, causing the plants to wither and become unproductive.
How can we prevent this disease from the outset? One of the latest solutions lies in early detection technology, such as the LAMP method (Loop-mediated Isothermal Amplification). LAMP enables rapid and accurate pathogen detection even before the seeds are planted. This way, farmers can avoid the spread of disease and ensure that shallot plants grow healthy and optimally.
In this article, we will explore how the LAMP method works, why early detection is essential, and its benefits for improved quality and yield in shallot production.
Utilization of the Loop-mediated Isothermal Amplification (LAMP) Method
The LAMP (Loop-mediated Isothermal Amplification) method can be effectively utilized to detect the pathogen Peronospora destructor, which causes downy mildew in shallot plants. This disease can significantly reduce shallot yields and often spreads through contaminated seeds.
LAMP works by amplifying specific DNA sequences of the pathogen, in this case, Peronospora destructor. This technique uses the enzyme Bst DNA polymerase and four primers that recognize six specific regions of the target DNA. Amplification is carried out at a constant temperature, around 63°C, requiring only an incubator or water bath instead of a thermocycler.
The LAMP amplification process has several main stages. The first stage is the formation of a dumb-bell structure, initiated by the separation and folding of DNA strands by the primers and polymerase enzyme. This structure forms the basis for subsequent amplification.
Then, DNA amplification proceeds cyclically. In this stage, the polymerase enzyme synthesizes new DNA from the dumb-bell structure. Amplification continues, exponentially creating more copies of the target sequence.
The resulting DNA undergoes repeated extension, forming more amplification products in a relatively short time, often around 40-60 minutes.
Using the LAMP method to detect Peronospora destructor in shallots is crucial because it can prevent destructive downy mildew, facilitate high-quality seed production, and reduce excessive pesticide use.
With LAMP, pathogen detection can be conducted early on, enabling more targeted and selective control measures. This approach not only minimizes excessive pesticide use but also helps maintain soil ecosystem balance and reduces harmful chemical residues in shallot products.
Advantages of the Loop-mediated Isothermal Amplification (LAMP) Method
The Loop-mediated Isothermal Amplification (LAMP) method has several advantages that make it effective for pathogen detection in seed crops. Here is a further explanation of LAMP's advantages compared to other methods like PCR:
Efficient Isothermal System
LAMP reactions occur at a constant temperature (usually around 60-65°C), meaning that it does not require a thermocycler like PCR does. In LAMP, a simple incubator or even a water bath is sufficient for the reaction, making this method more practical and suitable for field use or in laboratories with limited facilities. This efficiency makes LAMP highly suitable for applications in developing countries or in remote locations without access to fully equipped laboratories.
Faster Process Without Denaturation and Annealing Steps
The absence of denaturation and annealing steps, which are crucial in PCR, makes LAMP faster. PCR typically requires around 2-3 hours, whereas LAMP only requires about 1 hour. Positive results can often be observed within 40-45 minutes through visual analysis or agarose gel electrophoresis, and in as little as 11 minutes when using real-time turbidity detection.
Simple Visual Detection of Results
One of the main advantages of LAMP is its ability to visually detect positive results without the need for costly additional equipment. Positive results can be seen directly in the reaction tube through turbidity changes due to magnesium pyrophosphate precipitation or using intercalating dyes like SYBR Green I, which binds to double-stranded DNA and produces a green color. This allows for quick and simple detection without requiring additional equipment like electrophoresis.
High Sensitivity
LAMP is known for its high sensitivity, up to 10 times more sensitive than nested PCR. With this high sensitivity, LAMP can detect even very low levels of pathogen DNA or RNA, enabling early detection of infection or contamination in very small samples. This high sensitivity is particularly useful in the seed and agricultural industries, where infections in seeds are often at low levels but can spread rapidly after planting.
High Specificity Due to Specialized Primer Design
The LAMP reaction is supported by four primers that recognize six specific regions of the target DNA, significantly enhancing its specificity. These four primers ensure that only the target DNA is amplified, reducing the likelihood of false positives. This high specificity makes LAMP particularly well-suited for detecting specific pathogens in plants, such as identifying specific pathogens in various vegetable and horticultural crops.
Suitable for Different Types of Samples
In addition to DNA, the LAMP method can also be used to detect RNA with the help of reverse transcriptase enzymes, enabling the detection of pathogens with RNA genomes. This flexibility is very beneficial for detecting viruses and other pathogenic microorganisms that may infect plants.
Using the LAMP method, seed producers and farmers can detect Peronospora destructor at an early stage and prevent the spread of downy mildew in shallots, ultimately supporting healthier and more sustainable crop production.
How familiar are you with the LAMP method that can be used to test tomato seeds? For more interesting information, visit the IML Research blog regularly! And IML Research can conduct laboratory tests on tomato seeds using the LAMP method, make sure to test your tomato seeds at IML Research!
Author: Dherika
References
Lee, I.S., Kim, W, Gyeongpyo, J., & Kwangyeol, Y. (2024). Rapid Detection of a Downy Midew Pathogen, Peronospora destructor, in Infected Onion Tissues and Soils by Loop-Mediated Isothermal Amplification. Phytopathology: 1-7. https://doi.org/10.1094/PHYTO-11-23-0440-R.
Murwantoko. (2006). Metode Loop-mediated Isothermal Amplification (LAMP) dah Aplikasinya untuk Deteksi Penyakit Ikan. Jurnal Perikanan, 8(1): 1-8.
