B-241 A Novel Approach to Develop Multiplex Loop Mediated Amplification (LAMP) Assays

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Abstract Background Loop mediated amplification (LAMP) technology is a sensitive, accurate and rapid method for point-of-care (POC) molecular diagnostic detection. One disadvantage is that current LAMP methods can detect only one target per reaction whereas in a standard real-time PCR assay, multiple targets (3–4) can be detected in a single reaction. This is because current methods of detection for amplification products in a LAMP assay are not sequence-specific. Common detection methods include turbidity, use of color-changing dye or intercalating dye in the reaction mix, gel electrophoresis, etc. These methods are not sequence-specific and, thus, unable to differentiate between targets if amplified in a single tube, with the exception of lateral flow devices or melt analysis, which add time, complexity, and cost. To overcome this challenge, we have developed a novel method which simplifies development of multiplex LAMP without use of any additional primers/probes/reagents or steps. Methods Triplex RT-LAMP assay was developed for rapid detection of foot-and-mouth disease virus (FMDV) at POC. For this, LAMP primers targeting two different regions of 3D pol gene of FMDV were designed. For use as internal control (IC), LAMP primers were also designed targeting a conserved region of the bovine 18S rRNA gene. For multiplexing, best performing primer sets for each target were labelled with a unique fluorophore at 5′ end and a quencher (BHQ-1) at 3′ end. FMDV-1 primers were labeled with Cy5 fluorophore, FMDV-2 primers were labelled with Texas Red (TxR) fluorophore, and 18S rRNA with FAM fluorophore. Triplex RT-LAMP assay was performed using a specially formulated Multiplex Isothermal Master Mix but without any intercalating dye added to the mix. All 3 primer mixes were added to the Master Mix and reactions were carried out in a real-time thermocycler at 72°C for 30 min, with data collected every 30 seconds. Reaction kinetics were monitored as an increase in fluorescence associated with the accumulation of double-stranded DNA in respective channels (FAM for 18S rRNA, Cy5 for FMDV-1, and TxR for FMDV-2). Assay sensitivity was determined by testing 10-fold serial dilution of respective template, individually as well as mixed together. Results Results obtained showed feasibility of triplex RT-LAMP assay for simultaneous detection of 3 targets in a single reaction. Sensitivity and specificity of triplex RT-LAMP was comparable to control reactions (RT-LAMP assay with intercalating dye) for each target. No cross reactivity or non-specific amplification was observed with any of the primer designs. Conclusions This newly developed method allows detection of up to 3 targets in a single reaction without affecting the assay performance. This method is much easier to develop and optimize than previously reported methods as it uses standard LAMP primers without need of any additional primers/probes.

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