Development of reverse transcription loop-mediated isothermal amplification combine with lateral flow device as a detection assay for swine influenza virus

Abstract

Swine influenza causes high morbidity and low mortality in pigs. The disease spreads rapidly in pig farms causing the delay of pig weight to reach market. Moreover, pigs play important role as “mixing vessel” of influenza viruses. Pigs can support infection and replication of influenza viruses from avian, human and swine origins. These lead to major concerns of the important of swine influenza which are not only the economic impacts but also the human health. There is the need of accurate and rapid diagnosis for swine influenza virus (SIV) to minimize further spreading of the viruses in pig population and to reduce the risk of multiple infections of influenza viruses. The objective of this study was to develop a Reverse Transcription Loop-Mediated Isothermal Amplification combine with Lateral Flow Device (RT-LAMP-LFD) assay for the detection of SIVs and to evaluate the performance, sensitivity and specificity of the assay. This study was conducted in two phases: the development of RT-LAMP-LFD assay for SIVs detection and the evaluation of the performance, sensitivity and specificity of the assay. The result showed that RT-LAMP-LFD assay was developed with six newly designed primers and with optimum condition at 63oC and 30 minutes. The detection limit of the assay was 1.14 pg/µl, which is 10-fold higher than real time RT-PCR assay, RT-LAMP-LFD assay is specific method for the detection of SIV, there were no cross reaction with other important viruses of pigs (PRRSV, PCV2, Pseudorabies virus and PEDV). From our result the newly developed RT-LAMP-LFD assay has good performance with 100% sensitivity, 100% specificity and perfect percentage of agreement (Kappa = 1) compared to reference assays (viral isolation and/or real time RT-PCR). In conclusion, this RT-LAMP–LFD assay has a potential for patent technology as a rapid diagnostic test for SIV detection and could be developed for low-cost or in house kits. The RT-LAMP-LFD assay could be implemented and applied at first point care of SIV outbreaks to help control and prevent the spread of disease in pig farms.