Combined with real time detection, LAMP (loop-mediated isothermal amplification) has been a popular isothermal amplification technology used to create COVID-19 molecular diagnostics. Among other reasons, this is likely because LAMP, specifically RT-LAMP (reverse transcription LAMP) in the case of SARS-CoV-2, is a robust method for specific detection of nucleic acids.
Though these methods are robust, it is still best to optimize the reaction as well as its components. This is especially true considering LAMP is well-recognized for producing spurious amplicons that can lead to false positive results1. While it is up to the assay developer to optimize the reaction, vendors must lead the way optimizing the components.
The strand displacing polymerase, e.g. Bst DNA Polymerase, which catalyzes the LAMP reaction, is an important component that has properties subject to direct improvement, e.g. via site-directed mutagenesis2. A quick and indirect way of “improving the polymerase” is by enhancing another equally-important component, the primers. The best way of doing this is by fine-tuning the purification methods used in the manufacturing process (Figure 1). Either Desalt or HPLC purification of LAMP oligonucleotides is common, and it is often possible to modify both methods to create optimized primers.
Custom LAMP Primers, a derivative of our iScale Oligos™, are intended to maximize the function of LAMP primers in assays to be used as life science research tools, molecular diagnostics, and laboratory developed tests.
Figure 1.High-level overview of the main steps used in the manufacturing process for Custom LAMP Primers (steps upstream from Synthesis and downstream from Purification have been omitted for simplicity).
For background on Custom LAMP Primer design as well as access to a complimentary design tool, please see the excellent resources available from New England Biolabs.
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In addition to being a robust method as described above, colorimetric RT-LAMP is rapidly growing in popularity as a molecular diagnostic, especially for SARS-CoV-2, because it allows for rapid detection with minimal instrumentation. Currently, several EUA (Emergency Use Authorization) tests use the primers in Table 1, which are described in the paper, Enhancing colorimetric loop-mediated isothermal amplification speed and sensitivity with guanidine chloride3. Direct testing by several end users has revealed that our Custom LAMP Primers have led to exceptional performance with the E1, N2, and As1e primer sets.
*Typical primer ratios means that FIP/BIP must be present at 8-10-fold higher than F3/B3, and LF/BF must be present at 2-fold higher for the LAMP chemistry to function properly.