我国甘薯E病毒全基因组序列特征及荧光定量检测技术的建立

doi:10.3864/j.issn.0578-1752.2023.20.007

Abstract

Abstract:

【Objective】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. 【Method】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. 【Result】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 3.12×10²copies/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. 【Conclusion】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.

Key words: Ipomoea batatas, sweet potato virus E (SPVE), complete genomic sequence, phylogenetic analysis, qPCR detection

TANG Wei, ZHANG ChengLing, YANG DongJing, MA JuKui, CHEN JingWei, GAO FangYuan, XIE YiPing, SUN HouJun. Complete Genomic Sequence Characteristics and Establishment of qPCR Detection Technique of Sweet Potato Virus E in China[J].Scientia Agricultura Sinica, 2023, 56(20): 4010-4020.

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References 40

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引物名称 Primer name	序列 Sequence (5′-3′)	片段大小 Length (bp)	退火温度 TM (℃)	备注 Note
SPVE-F1	CAATACAACACAACAAAAATCAAAG	1708	58	基因组扩增Genome amplification
SPVE-R1	GAAATCCTCCCACTCTCCATA	1708	58	基因组扩增Genome amplification
SPVE-F2	ATTCAAAAGGTGGAATCTGA	1803	55	基因组扩增Genome amplification
SPVE-R2	CACATAGCAATACCCATCTTTC	1803	56	基因组扩增Genome amplification
SPVE-F3	TTTGCCAATTACTTCGATATT	2745	54	基因组扩增Genome amplification
SPVE-R3	CCCTGCATCAATTAAGAGG	2745	50	基因组扩增Genome amplification
SPVE-F4	AACTGCTGACAATATTCTCGTATAC	2776	52	基因组扩增Genome amplification
SPVE-R4	AAGGTCAAATAACATGCATTTC	2776	50	基因组扩增Genome amplification
SPVE-F5	TAGTGAAAAATGGGTCTTAGATAGA	2529	50	基因组扩增Genome amplification
SPVE-R5	GACAACATAAGTAGATTTTAAAGGC	2529	55	基因组扩增Genome amplification
SPVE-F6	CAATTCGACAACACCCGCT	~400	56	3′RACE
M4T	GTTTTCCCAGTCACGACTTTTTTTTTTTTTTTT	~400		3′RACE
SPVE-InR	ATGGAGTTGTGCGAGCATAAC	~400	55	5′RACE
SPVE-OutR	GTCTCTTCAGCCTCTTCCC		55	5′RACE
Q_T	CCAGTGAGCAGAGTGACGAGGA CTCGAGCTCAAGCTTTTTTTTTTTTTTTTT			5′RACE
Q_O	CCAGTGAGCAGAGTGACG			5′RACE

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

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