Search Results for « LAMP »

This protocol describes how to perform colorimetric LAMP / RT-LAMP reactions with homemade buffers, together with home-brewed BstLF and MMLV enzymes. It is based on previous protocols of our lab (see LAMP/RT-LAMP Reaction protocol), but since two of the most common indicators used in colorimetric LAMP (phenol red and neutral red) require weakly buffered reactions, the buffer composition needed to be changed. This was done according to indications by Poole et al (2017). The colorimetric methods rely on the inherent proton production by DNA polymerases during the amplification process, which leads to a drop in the reaction pH that can be followed using these pH indicators. The results can be seen directly with the "naked eye", without the need for equipment or hands-on processing (Tanner et al., 2015).

Colorimetric Loop Amplification (LAMP) is a fast diagnostic tool which identifies presence of pathogenic RNA or DNA through a colour change. This method is not used widely because it is not as sensitive as real time PCR (lacks in sensitivity) and the negative control also becomes positive eventually (lacks in specificity). At Genomics WA, have developed a solution e-solution, which increases the sensitivity and specificity of colorimetric LAMP. Use of this solution keeps the negative control negative for a long time, thus enabling low copy numbers of pathogens to be detected. Our solution improves the sensitivity and specificity of colorimetric LAMP.

This protocol describes the preparation of reaction buffer for homemade LAMP / RT-LAMP reactions which involves the use of home-brewed BstLF and MMLV enzymes. It also contains the instructions to prepare the enzyme storage buffers. Home-brewed enzymes information: BstLF polymerase: expression and purification protocol. expression plasmid sequence. M-MLV reverse transcriptase: expression and purification protocol. expression plasmid sequence. Testing We test the buffers on SARS-CoV-2 LAMP or RT-LAMP reactions with synthetic dsDNA or ssRNA respectively. The general performance of Homemade buffer seems appropriate and no amplification was obtained for non-template controls (NTC). To properly formulate the buffer we tested different KCl concentrations which is known to be a critical factor in the buffer. For the home-brewed BstLF, we found that 50 mM KCl is the best concentration, displaying equal or better performance than the commercial buffer. No amplification was observed for 150 mM KCl formulation. [1] Zhang, Y. et al. Enhancing colorimetric loop-mediated isothermal amplification speed and sensitivity with guanidine chloride. Biotechniques69, (2020).

This protocol describes how to perform LAMP / RT-LAMP reactions with home-made reagents, which means home-made buffers and positive controls, together with home-brewed BstLF and MMLV enzymes. Home-brewed enzymes information: BstLF polymerase: expression and purification protocol. expression plasmid sequence. M-MLV reverse transcriptase: expression and purification protocol. expression plasmid sequence. Positive control information Sample preparation protocol here. Testing We test this protocol in LAMP or RT-LAMP reactions with SARS-CoV-2 synthetic dsDNA or ssRNA respectively. As shown in Figure 1, the general performance of reactions with homemade reagents seems appropriate compared with the commercial standard option. No amplification was observed in non-template controls (NTC). [1] Zhang, Y. et al. Enhancing colorimetric loop-mediated isothermal amplification speed and sensitivity with guanidine chloride. Biotechniques69, (2020).

This protocol collection specifies how to carry out LAMP or RT-LAMP (reverse transcription - Loop-mediated isothermal amplification) reactions with homemade reagents and home-brewed enzymes. It describes how to prepare all the solutions and buffers to perform the reactions. This collection was developed in the context of COVID-19 pandemic to provide access to RT-LAMP tools in accordance with the principles of ReClone community initiative. It is the result of information exchange and reagent sharing with a huge community of collaborators from ReClone, gLAMP and JOGL.

This protocol explains how to prepare dsDNA and ssRNA target positive control for LAMP and RT-LAMP reactions. Particularly it describes the pipeline for SARS-Cov-2 Nucleocapsid (N) gene positive control which is included in the ReClone collection. The general procedure involves amplifying the gene of interest with primers which add T7 promoter and T7 terminator to its sequence followed by T7 in vitro transcription to obtain the ssRNA. Sequence elaboration: Sequence OrfN_5 was designed and synthesized based on IDT SARS-CoV-2 positive control plasmid (CAT#10006625_2019).