Complete Genomic Sequence Characteristics and Establishment of qPCR Detection Technique of Sweet Potato Virus E in China

Sweet potato virus E (SPVE) is a novel virus infecting Ipomoea batatas. The objectives of this study are to determine the complete genomic sequence of SPVE Xuzhou isolate (SPVE-XZ) in China, analyze the genomic sequence characteristics of SPVE-XZ, and develop the quantitative PCR (qPCR) assay for the specific detection of SPVE. This study will provide theoretical basis and technical support for the detection, monitoring and controlling SPVE in China.

The complete genomic sequence of SPVE-XZ was obtained by using small RNA deep sequencing, combined with RT-PCR and RACE technologies. The sequence alignment and phylogenetic relationship analysis of SPVE-XZ were performed by using software MegAlign and MEGA11. The primers of qPCR for rapid detection of SPVE were designed, and the qPCR detection method for SPVE was established by optimizing the annealing temperature and primer concentration. The specificity and sensitivity of the qPCR were determined. The developed qPCR technology was used to detect sweet potato samples collected from Jiangsu Province and Shandong Province.

The complete genomic sequence of SPVE-XZ was 10 919 nt, excluding the 3′-terminal poly (A) tail, containing the typical open reading frame of 10 560 nt in length and encoding a putative large polyprotein of 3 519 amino acids. The 5′UTR and 3′UTR of SPVE-XZ were 129 and 230 nt, respectively. PISPO and PIPO were produced with the frameshift in P1 and P3, respectively. Genomic sequence analysis showed that the genomic nucleotide sequence identity between SPVE-XZ and Korean SPVE-GS isolate was 98.6%, and the amino acid sequence identity of polyprotein between them was 98.5%. Phylogenetic trees were constructed based on the coat protein gene sequences and polyprotein amino acid sequence using the neighbor-joining method. The result showed that SPVE-XZ was clustered with SPVE-GS in these phylogenetic analyses. The established qPCR method could detect SPVE specifically, however sweet potato virus 2 (SPV2), sweet potato virus C (SPVC), sweet potato virus G (SPVG), sweet potato feathery mottle virus (SPFMV), sweet potato chlorotic stunt virus (SPCSV), sweet potato latent virus (SPLV), sweet potato chlorotic fleck virus (SPCFV), cucumber mosaic virus (CMV) could not be detected in this qPCR system. The lowest sensitivity of the qPCR was 3.12×10² copies/μL and it was 100 times that of conventional RT-PCR. SPVE was detected in 1 sample of 6 samples collected from Shandong Province and 13 samples of 52 samples collected from Jiangsu Province by using the established qPCR detection method.

The complete genome sequence of SPVE-XZ is 10 919 nt. The genome structure of SPVE-XZ is consistent with SPVE-GS reported in Korean. The established qPCR detection system for SPVE is specific and highly sensitive, which can be used for the rapid detection of SPVE.