This technique need not to have even high cost equipments (especially PCR or GelDoc or refrigerator). The specific LAMP primer set consist of F3(forward outer primer), B3 (backward outer primer), FIP (forward inner primer), and BIP (backward inner primer) These primers will be designed using the Primer Explorer program (http://www.loopamp.eiken.co.jp) to amplify the 18S ribosomal
RNA gene (http://www.ebi.ac.uk.com). The protocol could be optimized for each reaction; my experience for the study of Acanthamoeba spp rapid identification, LAMP was performed for 60 min at temp 65 C in 25 microL of a mixture containing 2 microL of extracted DNA, 40 pmol each of FIP and BIP, 5 pmol each of F3 and B3, 1.4 mM deoxynucleoside triphosphates, 0.8 M betaine and 1 microL of Bst DNA polymerase in buffer (20 mM Tris–HCl pH 8.8, 10 mM KCl, 10 mM (NH4)2SO4, 8 mM MgSO4, and 0.1% Tween 20). The reaction was terminated by heating (using water bath or heating block) for 2 min at temp 80 C. Turbidity of the LAMP reaction will be inspected visually in real time. The DNA products could also be visualized under UV light after addition of Loopamp fluorescent detection reagent (Eiken Chemical Co., Ltd.). LAMP products was confirmed by electrophoresis in 1.5% agarose gel and visualization by staining assay. The sensitivity and specificity were 100%. (see more at: Usa Lek-Uthai et al. Rapid identification of Acanthamoeba from contact lens case using loop-mediated isothermal amplification method. Experimental Parasitology Volume 121, Issue 4, April 2009, Pages 342-345). However, the study of genotyping and copy numbers for the Plasmodium spp specific genes (the species identification need not to have any DNA extraction method from fresh or dried blood spots) in different malaria endemic regions in Thailand since 2007 have been doing well (unpublished). Therefore, this technique might be useful for molecular public health in the developing country’s disease control program. LAMP took less time than qPCR, which required 2 h as compared to 1 h for the LAMP procedure. However, the cost of this technique is the cheapest (about 70 cents US per sample compared to 5–7 $US per sample using DNA amplification by realtime PCR.
Loop-Mediated Isothermal Amplification Method (LAMP): Low and Effective cost Novel Tool for Molecular Public Health