Carboxamide and N-alkylcarboxamide additives can greatly reduce non specific amplification in Loop-Mediated Isothermal Amplification for Foot-and-Mouth disease Virus (FMDV) using Bst 3.0 polymerase

Abstract

Foot-and-Mouth disease Virus (FMDV) is a highly infectious RNA virus that causes severe economic losses in cloven-hoofed animals. Early detection is needed to control epidemics, and loop-mediated isothermal amplification (LAMP) can be performed using inexpensive and commonly available equipment with a short processing time, but existing assays for FMDV still require an additional reverse transcriptase enzyme to convert RNA to cDNA prior to amplification. We sought to develop a novel RT-LAMP assay for FMDV with carboxamide and N-alkylcarboxamide additives to reduce non-specific amplification in combination with an improved commercially available polymerase (Bst 3.0) with efficient reverse transcriptase activity. SYBR Green I dye was used for sensitive visual detection of amplification products from our LAMP assay within 15 min without the need for a colorimeter. In the presence of a carefully titrated mixture of carboxamide and N-alkylcarboxamide additives, longer reactions of up to 1 h were also possible on both RNA and cDNA without the appearance of non-specific amplification products, thereby increasing the potential robustness of the assay by allowing a greater window of time in which to detect weak positives.

Keywords: Amide additives; Bst 3.0; Foot-and-Mouth disease Virus (FMDV); Loop-Mediated Isothermal Amplification (LAMP); Reverse transcriptase.