Pathogen Detection Amplified

Advances in DNA amplification techniques have significantly contributed to the accurate detection of a wide range of pathogens. Among these techniques, loop-mediated isothermal amplification (LAMP) has emerged as a particularly powerful tool in pathogen detection. LAMP overcomes many of the limitations of traditional polymerase chain reaction methods, offering a simpler, faster, and more robust amplification method.

LAMP operates under isothermal conditions, meaning that it does not require complex thermal cycling protocols. Instead, it utilizes a set of four to six specially designed primers that recognize multiple target regions within the pathogen's DNA. These primers initiate strand displacement and amplification reactions, leading to the rapid and exponential production of DNA copies.

One of the significant advantages of LAMP is its high specificity. The use of multiple primers that target distinct regions of the pathogen's genome enhances the specificity of the amplification, reducing the likelihood of false-positive results. Furthermore, LAMP offers excellent sensitivity, enabling the detection of even low concentrations of pathogens. These properties have made it possible to accurately identify the presence of a wide range of pathogens, enabling timely and effective disease surveillance, outbreak management, and personalized treatment strategies.

LAMP techniques have not been utilized to their full potential, however, due to some limitations of the technology. Typical LAMP protocols require the use of specialized fluorescent dyes and other reagents, and as anyone that has ever set foot in a biology lab knows, these reagents can be extremely costly. Moreover, LAMP requires that some complex DNA purification protocols be followed, which limits who can perform such tests, and generally excludes point-of-care facilities from performing them.

A simple, low-cost LAMP sensing chip has recently been developed in a collaboration between researchers at Iowa State University and Texas A&M University that seeks to make the technology more widely accessible for diagnostic purposes in plants, animals, and humans. This new technique not only overcomes the limitations of existing approaches, but it also outperforms them — it has been demonstrated to detect disease pathogens with 10 times greater sensitivity.

The team designed a chip that contains a nanopore thin-film sensor housed within a LAMP reaction chamber. Primer sequences are immobilized on the surface of the sensor such that the DNA of the target pathogen will bind with it during the amplification process. When the reaction is complete, the LAMP products are washed out, but the pathogenic DNA remains attached to the immobilized primers. Through the use of a portable spectrometer with an optical fiber probe, the presence of DNA from the target pathogen can be measured.

As a validation of their sensor chip, the team prepared primers to detect the fungus Phytophthora infestans, which is known to cause the late blight disease that can ravage potato and tomato crops. Within 30 minutes, the sensor was able to detect the presence of the pathogenic DNA. It was observed that the chip could detect the target with incredible sensitivity — concentrations as low as 1 fg/μL could be detected.

Next up, the researchers intend to investigate how their LAMP chip might be able to distinguish between pathogens that have very similar sequences of DNA. They also have plans to enable quantification of detected DNA through the use of artificial intelligence algorithms and CRISPR gene-editing techniques. After such refinements are made, the team hopes to make their sensor available commercially for point-of-care applications.