Development of a Novel Triplex Reverse Transcription Loop Mediated Amplification (RT-LAMP) Assay

Yogesh Chander,1Kurt Throckmorton,1Alyssa Hassinger,1Phil Brumm,1Sunil Mor,2Victor Ngu Ngwa,3and David Mead1

1Varizymes, Middleton, WI 53562; 2South Dakota State University, Brookings, SD 57007; 3University of Ngaoundere, Cameroon

Introduction:

Conventional LAMP methods can detect only one target per reaction, as opposed to 3-4 in a standard real-time PCR assay. These methods include measuring turbidity, using color-changing or intercalating dyes, and gel electrophoresis, among others. These are not sequence-specific and are thus unable to differentiate between targets if amplified in a single tube, with the exception of lateral flow devices, which add time, complexity, and cost.

Varizymes’ TORCH™ technology enables real-time detection of multiple targets (up to 3) in a single LAMP reaction. This technology works with both DNA and RNA targets, uses fluorophore/quencher-labeled primers, and, unlike other multiplexing methods, requires no additional primers, probes, or steps relative to conventional LAMP. Examples of detection of 2 targets (duplex LAMP) and 3 targets (triplex LAMP) in single LAMP reactions are shown below.

Duplex LAMP: Virulent Aeromonas hydrophila (vAh) is a causative agent of motile Aeromonas septicemia (MAS) in channel catfish. A duplex LAMP assay using TORCH technology was developed for rapid detection and differentiation of vAh from non-virulent A. hydrophila (non-vAh). For this, vAh-specific primers were labeled with Cy5 fluorophore and universal Ah primers were labelled with FAM fluorophore. Duplex LAMP was performed using a specially formulated Multiplex Isothermal Master Mix without any intercalating dye added. Figure 1 shows amplification with both primer sets in a single well.

Figure 1. Duplex LAMP for detection of virulent Aeromonas hydrophila (vAh) using vAh-specific primers (Cy5-labeled, purple) and universal Ah primers (FAM-labeled, blue). Both primer sets were combined in a LAMP reaction (without any intercalating dye) carried out in a real-time thermocycler at 66°C with data collected every 30 sec.

Triplex RT-LAMP: Foot-and-mouth disease (FMD) is a highly contagious RNA viral disease of cloven-hoofed animals, caused by foot-and-mouth disease virus (FMDV). To develop a triplex LAMP assay for rapid detection of FMDV at point-of-care, LAMP primers targeting two regions of the FMDV genome were designed and labeled with different fluorophores, Cy5 (FMDV-1) and Texas Red (FMDV-2). In addition, LAMP primers for bovine 18S rRNA (internal control) were designed and labeled with FAM fluorophore. Triplex RT-LAMP was performed using a specially formulated Multiplex Isothermal Master Mix without any intercalating dye added. The results shown in Figure 2 demonstrate simultaneous detection of all 3 targets in a single well with sensitivity and specificity comparable to control single-target reactions with intercalating dye (not shown).

Figure 2. Development of Triplex RT-LAMP using VariSafe™ RNA controls (Catalog # 1106) including target sequences of FMDV-1 (Cy5, purple), FMDV-2 (Texas Red, red), and 18S rRNA (FAM, blue). All 3 primer sets were combined in an RT-LAMP reaction (without any intercalating dye) carried out in a real-time thermocycler at 66°C with data collected every 30 sec. 10-fold serial dilutions of the targets are indicated with -1, -2, and -3.

CONCLUSIONS

Varizymes’ TORCH technology is a newly developed method for multiplex LAMP and RT-LAMP assay design.  Using this technology, we have performed single-tube detection of 2 and 3 targets with time-to-results and sensitivity comparable to conventional monoplex LAMP.  Since TORCH technology uses the same primers as a conventional LAMP assay without additional detection primers or reagents, assay development and optimization are much easier than other multiplex LAMP approaches.

Please contact us at info@varizymes.com for custom VariSafe™ RNA or Multiplex LAMP assay development questions.

REFERENCES

Zhang Y, Tanner NA. Development of multiplexed reverse-transcription loop-mediated isothermal amplification for detection of SARS-CoV-2 and influenza viral RNA. Biotechniques. 2021;70(3):167-174.

ACKNOWLEDGEMENTS This work was supported by a grant from USDA-NAHLN to Dr. Sunil Mor (PI), Dr. Yogesh Chander (co-PI), and Dr. Victor N Ngwa.

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Yogesh Chander, PhD

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Alyssa Hassinger

Research Scientist

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