The strand displacing polymerase used in LAMP reactions is an important component that is subject to direct improvement through site-directed mutagenesis. However, primer design is also critical to successful amplification.
GLAPD searches for candidate single primers in two groups, the target group and the background group. It then checks their commonality, specificity and tendency to bind to each other.
During LAMP amplification, forward and reverse internal primers target and duplicate specific analyte DNA sequences and generate loop structures. Then, outer and loop primers bind to these structures, initiating strand synthesis and displacement. The resulting displacement products are subsequently amplified by the Bst DNA polymerase. The process is carried out under isothermal conditions. The resulting products can be visualized using a colorimetric or fluorometric assay.
The RT-LAMP assay developed in this study can replace the gold standard RT-PCR test and is inexpensive and easy to perform. In addition, the assay can be conducted within 60 min. This test is also able to detect COVID-19. However, a high number of false-positive results due to misamplification is one limitation of the assay. To reduce this problem, the RT-LAMP assay was modified to include five primers instead of six. This modification significantly reduced the number of non-specific amplifications and was as effective as the gold standard RT-PCR. The new RT-LAMP assay was tested in serially diluted SARS-CoV-2 RNA samples and showed sensitivity of 89.5% and 92.2% in the colorimetric and fluorometric assays.
The physical distance between the two annealing sites in a LAMP signature is an important factor in optimizing the assay’s specificity. It also determines whether the resulting amplicon can be visualized by using fluorescent tags. In addition, the choice of linker sequences can have a significant impact on the predicted primer dimerization.
The multiple-sequence alignment (MSA) method is used to identify candidate primer regions for the loop, middle, and outer positions of the LAMP signature. The sequences are then analyzed to select the most promising combinations. The sequences with the lowest score are then down-selected based on their overlap and relative score.
A titration is then performed by running each primer set at different temperatures. Depending on the target sequence, seven reactions may be needed to find the optimal amplification temperature for that particular primer set. This process is time-consuming and labor-intensive, but it can be worthwhile in the long run. This step can also help you save money on reagents and improve the quality of your results.
When designing primers, it is important to consider the locations and characteristics of the oligonucleotides. These factors will determine the conditions under which oligos will bind to their target sequences in PCR and qPCR experiments. In particular, it is important to avoid inter-primer homology and the formation of hairpins. These structures may inhibit the binding of DNA polymerase, leading to false-positive results.
Another important aspect of primer design is GC content. This factor can significantly impact the melting temperature and annealing temperature of a primer. Ideally, a primer should have a GC content between 45 and 60%.
Aside from GC content, a primer’s length is also important in determining its melting temperature. A longer primer will have a lower melting temperature than a shorter one. The optimum length of a primer should be approximately 18 nucleotides. However, extending the length of a primer too far could cause it to form a secondary structure. This may reduce sensitivity and specificity of the resulting PCR.
Primer annealing temperature
The amplification mechanism of LAMP involves the insertion of large modular primers into weakened double-stranded DNA and subsequent displacement. The resulting dumbbell-shaped reaction intermediate is then subject to successive rounds of extension and strand displacement, which eventually leads to exponential DNA amplification.
Unlike PCR, which requires multiple temperatures to carry out the amplification process, LAMP only needs one temperature for all reactions. This enables the use of cheaper thermocyclers and eliminates the need for technical optimisation of cycling conditions.
In addition, LAMP is less complex to operate and requires only a few reagents, making it an excellent alternative to traditional methods of molecular detection. However, a positive control reaction is still required to ensure that the assay is functioning correctly. This can be performed by adding a known concentration of the target organism to the reaction mixture and monitoring its color change using a standard fluorometer. This can help the user identify whether the amplification is taking place or not.