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. 2021 Jul 20;11(7):2150. doi: 10.3390/ani11072150

Figure 1.

Figure 1

Comparison of polymerase chain reaction (PCR) insulated isothermal polymerase chain reaction (iiPCR) and loop-mediates isothermal amplification (LAMP) procedures. (a) PCR procedure is as follows; 1. sample is collected; 2. sample is purified; 3. contents for PCR are mixed including the purified sample, forward and reverse primers, and master mix buffer which includes Taq polymerase and dNTPs; 4. the reaction is ran on a thermocycler for ≥90 min cycling through three temperatures for the denaturation, annealing and extension stages; 5. PCR products are subjected to agarose gel electrophoresis for approximately 35 min at 100 amps to visualize results. (b) iiPCR follows a similar starting procedure to PCR where, 1. samples are collected, and 2. purified, 3. contents are mixed as such for PCR. However, reaction is conduced within capillary tubes with a copper ring at the base and lid, where the mixture is heated underneath to create a temperature gradient through convection; reactions last for around 1 h. This can be achieved through two options: 4.1. an automated portable machine, POCKIT™ (GeneReach USA, Lexington, MA, USA) where results are displayed in real time; alternatively, 4.2. an insulated box that requires the products to undergo (4.2.2) agarose gel electrophoresis for approximately 35 min at 100 amps to visualize results. (c) The LAMP procedure is as follows, 1. samples are collected and 2. mixed with 4–6 primers (F3, B3, forward inner primer and backward inner primer, and optional loop primers). LAMP can tolerate impurities in samples and therefore do not required to be purified. 3. The mixture is heated at a single temp temperature for typically ≤30 min. This can also be achieved by two options: 3.1. an automated machine, Genie III™, (OptiGene Horsham, Eng, UK), where results are displayed in real time; alternatively, 3.2. a heat source, such as a water bath, where products are visualized through (3.2.2) fluorescence for approximately 5 min to observe a color change. Created with BioRender.com.