Reverse transcription-PCR assays for the differentiation of various US porcine epidemic diarrhea virus strains

Abstract

Concurrently, several porcine epidemic diarrhea virus (PEDV) variants are circulating in US swine farms, including the original US and the spike insertion-deletion (S-INDEL) strains. In this study, reverse transcription (RT)-PCR assays for the detection and differentiation of different US PEDV variants were developed based on the differences in the S1 domain of the spike (S) gene. This assay successfully differentiated three PEDV strains: PC22A (the original US virulent), Iowa106 (S-INDEL), and PC177 (S-197DEL) that was derived from cell culture adaptation and has a 197 amino acid-deletion in the S1 domain. The assays did not amplify the porcine deltacoronavirus OH-FD22 strain or transmissible gastroenteritis virus Miller strain. It is the first report on the development of RT-PCR assays allowing the detection and differentiation of all major types of US PEDV variants.

Keywords: Porcine epidemic diarrhea virus; Reverse transcription-PCR; Spike gene.

Fig. 1

Location of external and internal primers and their combinations to generate five different sizes of product.

Fig. 2

Specificity of reverse transcription-PCR assays. PCR products were analyzed by electrophoresis on a 1.5% agarose gel stained with EZ-VISION^® THREE 6× loading buffer. (a) PCR products of primary amplification with primer pair F1/UR and using cDNA as templates; (b) PCR products of secondary amplification with primer pair F2/UR using 10⁻² diluted first round PCR products as template; (c) PCR products of secondary amplification with primer pair F3/UR and using 10⁻² diluted first round PCR products as templates. M: 100 bp ladder DNA marker (PHENIX Research Products, NC, USA); Lane 1: PEDV strain PC177; Lane 2: PEDV strain PC22A; Lane 3: PEDV strain Iowa106; Lane 4: a mixture of PC177, PC22A and Iowa106 with 1:1:1 ratio; Line 5: a mixture of PC177, PC22A and Iowa106 with 10:1:1 ratio; Line 6: a mixture of PC177, PC22A and Iowa106 with 1:10:1 ratio; Line 7: a mixture of PC177, PC22A and Iowa106 with 1:1:10 ratio; Line 8: porcine transmissible gastroenteritis virus (TGEV); Line 9: porcine deltacoronavirus (PDCoV).

Fig. 3

Sensitivity of the primary PCR assays with the individual primer set. The sensitivity of each PCR assay was determined using 10-fold serial dilutions (from original cell culture supernatants to 1:10⁵ in RNase-free water) of each strain. PCR products were analyzed by electrophoresis on a 1.5% agarose gel stained with EZ-VISION^® THREE 6× loading buffer. M: 100 bp ladder DNA marker (PHENIX Research Products, NC, USA); Lines 1–6: 10⁰–10⁻⁵ diluted virus stocks. (a) PCR products of PC177 strain with primer pair F1/UR; (b) PCR products of PC22A strain with primer pair F2/UR; (c) PCR products of Iowa106 strain with primer pair F3/UR.

Fig. 4

Nucleotide alignment of representative PEDV strains in the F2 and F3 primer regions. (a) Sequence and location of primer F2; and (b) Sequence and location of primer F3.