Simple Summary
Due to recombination or mutation among different porcine epidemic diarrhea virus (PEDV) strains, existing vaccines may exhibit reduced protective efficacy, resulting in significant economic losses. Consequently, developing a method to identify different genotypes of PEDV is of paramount importance. In this study, we established a loop-mediated isothermal amplification (LAMP) method for the differential diagnosis of PEDV GI and GII genotypes. Two sets of primers, PEDV-LM and PEDV-LS, were designed based on the M and S genes of PEDV, respectively. PEDV-LM exhibited specificity for all PEDV strains, while PEDV-LS specifically targeted the PEDV GI genotype. In the experiments, both sets of LAMP primers had detection limits of 1 × 102 copies, which is 100 times more sensitive than RT-PCR. This assay demonstrated specific amplification of PEDV and PEDV GI genotypes without cross-amplification with other viruses. Additionally, the assay results can be visualized by adding nucleic acid dye. When tested on clinical samples, our novel method performed comparably to RT-qPCR. Therefore, this method can be used for the detection of different PEDV genotypes.
Abstract
Porcine epidemic diarrhea virus (PEDV), a primary pathogen causing diarrhea in pigs, particularly in piglets, has a mortality rate of up to 100%. Field PEDV strains circulating in pig production can be phylogenetically divided into two genotypes, GI and GII. Differential diagnosis of clinical strains with different genotypes is helpful for understanding disease epidemiology, vaccine selection, and prevention and control measures. The loop-mediated isothermal amplification method (LAMP), a novel nucleic acid amplification technique, has been utilized to detect a variety of pathogens in practice. In this study, we developed a distinguished RT-LAMP method to identify genotypes GI and GII strains of PEDV. Two pairs of primers, PEDV-LM and PEDV-LS, were designed based on the membrane and spike genes of PEDV, respectively. PEDV-LM primers exhibited specificity for all PEDV strains, while PEDV-LS primers specifically targeted the PEDV genotype GI. The diagnostic sensitivity of both primers was 1 × 102 copies/reaction, which is 100 times more sensitive than RT-PCR. The RT-LAMP reaction process was completed at 65 °C for 40 min just in a water bath or metal bath. A cross-reactivity assay confirmed that this method is specific for PEDV GI and GII, with no cross-amplification observed with other swine-origin viruses such as PDCoV, PoRV, PRV, and PRRSV. Therefore, this refined LAMP technique offers a rapid, sensitive, and reliable method with which to detect and differentiate between PEDV GI and GII, making it a superior tool for the large-scale clinical surveillance of PEDV infections.