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The recent pandemic caused worldwide health, economic, and social disruptions with its reverberations still felt today. A key takeaway from this event is the need for accurate and accessible tools for detecting and tracking infectious diseases. Timely identification is essential for early intervention, managing outbreaks, and preventing their spread. This article reviews several valuable tools employed in the detection and surveillance of infectious diseases.
Targeted Sequencing for Pathogen Detection
Targeted sequencing is a popular and highly efficient strategy for rapid pathogen identification. This approach encompasses a number of techniques that allow researchers to focus on sequences of interest. In the context of pathogen detection, targeted sequencing facilitates the in-depth analysis of specific segments within a pathogen’s genome. By focusing on these regions, scientists can quickly and accurately determine the identity of infectious agents. Two of the most common targeted sequencing techniques are hybridization capture and amplicon sequencing. Hybridization capture uses probes to isolate specific sequences, whereas amplicon-based sequencing amplifies selected areas with target-specific primers.
There are multiple advantages to employing targeted sequencing for infectious disease research. Targeted sequencing reduces costs, enables deep sequencing of specific regions, and accelerates the data analysis process. Additionally, its high degree of flexibility makes it ideal for a variety of applications, including research on low-frequency mutations and detecting low levels of pathogens.
A notable application of these targeted techniques was their involvement in monitoring the global spread of SARS-CoV-2. Patrick Finn, President and Chief Operating Officer at Twist Bioscience, highlighted how their target enrichment panels were instrumental in the accurate detection of the virus. Several of these panels are also designed to detect a broad spectrum of viral pathogens simultaneously, allowing scientists to identify the source of an infection and any co-infections.
The integration of synthetic controls can further enhance the capability of targeted sequencing in tracking pathogens. Finn elaborated on this, emphasizing that these controls add a crucial layer of accuracy and reliability to viral genome testing. Specifically, they serve as essential quality control measures across a broad spectrum of applications. This includes the verification and validation of diagnostic tests, not only for sequencing but also in RT-PCR assays.
Targeted sequencing is set to continue as a key tool in monitoring the spread of various infectious diseases. As highlighted by Finn, the adaptability of these panels is a significant advantage. They offer researchers the flexibility to focus on specific pathogens of interest. Moreover, the ease with which these panels can be modified or expanded is crucial for keeping pace with the constantly evolving landscape of infectious diseases.
Primer Monitoring Tools
Monitoring mutations in pathogens plays a critical role not only in evaluating the effectiveness of vaccines and drugs but also in ensuring that diagnostic assays are current and accurate. Changes within a pathogen's genome can significantly affect the detection capabilities of these tests, leading to the risk of false negatives. In response to emerging SARS-CoV-2 variants, scientists at New England Biolabs (NEB) developed an innovative primer monitoring tool.
This tool cross-references primer sequences against known SARS-CoV-2 variants from the NCBI GenBank database to evaluate potential impacts on the primers. In addition, researchers have the option to register for notifications. These alerts will inform them when a certain variant of the virus, which shares sequences with their specific primer set, reaches a specified threshold of concern.
Kaylinnette Pinet, a Development Scientist at NEB, highlighted the importance of their tool. Initially created to verify the functionality of their own primers, it has become invaluable for tracking sequencing data and assessing the impact of new variants on primers in various applications, including LAMP, qPCR, and sequencing. Recognizing its benefits, NEB made the tool publicly accessible to support the broader scientific community.
Researchers can also assist with the ongoing development of this tool by uploading sequences to public databases like NCBI, which are fundamental to its operation. “It will make our tools more powerful and more useful to everybody,” Pinet stressed. She also noted that while the primer monitor can predict the impact of mutations on primers, verifying these predictions in a wet lab is crucial for maintaining accuracy.
Bradley Langhorst, Group Leader at NEB, and a key developer of the tool, discussed plans for expanding their platform to track RSV and influenza by drawing from their experiences with SARS-CoV-2. A team member, Caiden Kumar, is currently adapting the tool to manage sequencing data from these viruses and fine-tuning it to accurately predict the effects of specific variants on primer sets.
Simplifying Molecular Diagnostics with LAMP
Colorimetric loop-mediated isothermal amplification (LAMP) assays proved to be another key resource during the pandemic. These isothermal amplification methods enable nucleic acid detection without the need for thermal cycling by relying on a strand-displacing polymerase for primer binding and initiation. Importantly, the amplification can be visualized by the eye through color changes from indicators sensitive to byproducts in the reaction. This allows for the rapid identification of a broad spectrum of RNA and DNA targets, and in many cases, specific nucleic acids from pathogens for detection.
“LAMP and the colorimetric LAMP came out of an effort to make simple accessible tools for molecular diagnostics,” stated Nathan Tanner, Associate Research Director at NEB. Tanner’s lab played a significant role in developing this technology, as they have been focused on broadening access to diagnostic tools for the detection of infectious diseases. Highlighting the simplicity of LAMP reaction chemistry, Tanner explained that it only requires incubation at 65°C and is even achievable in a hot cup of water. The results can be interpreted through a visual inspection, while some regulated diagnostics may require additional measuring of color changes.
Langhorst emphasized the ease and robustness of colorimetric LAMP as advantages over alternative techniques like qPCR. “We don't have to isolate RNA or DNA first,” he stated, underlining the elimination of steps like nucleic acid extractions. This simplification is crucial for applications in settings that lack lab equipment. It also shortens the workflow time and allows researchers to handle more difficult samples like saliva.
While the technology was instrumental during the pandemic, Tanner noted that it has a wide range of applications. Scientists have employed these assays to study filarial diseases, plant pathogens, and more. The World Mosquito Program also demonstrated colorimetric LAMP’s portability and accessibility by employing it to monitor mosquitos in the field1. Using small portable devices for incubation, researchers have shown that this technology can be used nearly anywhere, including the International Space Station2.
Tanner and Langhorst further detailed how NEB utilized this technology to protect its own employees. Amid the pandemic, the company's enzymes played a vital role in worldwide testing efforts. To prevent the spread of the virus among their staff and maintain uninterrupted production, they employed colorimetric LAMP. This technology was chosen for its speed and cost-effectiveness, enabling the company to continue its operations safely.
The most recent advancements to these colorimetric LAMP assays include a shift from pH change-based color indicators to metal sensing dyes. As explained by Tanner, this change enhances visual contrast and reduces sensitivity to sample conditions like pH and buffers. This new approach facilitates the development of fully buffered mixes, offering greater robustness against environmental conditions and compatibility with lyophilization for easier transportation and field diagnostics. It also eliminates the need for cold storage.
Looking ahead, Tanner foresees continued expansion in the uses of LAMP. “The next generation of these platforms will enable much more than COVID testing,” he stated. Current tests that employ this technology already have a flu A, flu B, and COVID combination assay, and they are available for at-home use. He anticipates the next versions could include RSV testing and more. “The technology is there now, and it will be exciting to see how it gets used,” he concluded.
References
- Gonçalves, D. D. S., Hooker, D. J., Dong, Y., Baran, N., Kyrylos, P., Iturbe-Ormaetxe, I., Simmons, C. P., & O'Neill, S. L. (2019). Detecting wMel Wolbachia in field-collected Aedes aegypti mosquitoes using loop-mediated isothermal amplification (LAMP). Parasites & vectors, 12(1), 404. https://doi.org/10.1186/s13071-019-3666-6
- Rubinfien, J., Atabay, K. D., Nichols, N. M., Tanner, N. A., Pezza, J. A., Gray, M. M., Wagner, B. M., Poppin, J. N., Aken, J. T., Gleason, E. J., Foley, K. D., Copeland, D. S., Kraves, S., & Alvarez Saavedra, E. (2020). Nucleic acid detection aboard the International Space Station by colorimetric loop-mediated isothermal amplification (LAMP). FASEB bioAdvances, 2(3), 160–165. https://doi.org/10.1096/fba.2019-00088
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