Biotechnology Bulletin ›› 2024, Vol. 40 ›› Issue (6): 126-133.doi: 10.13560/j.cnki.biotech.bull.1985.2024-0129

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Establishment and Application of Real-time PCR for Sugarcane Striate Virus

WANG Chao-min1(), HE Mei-dan1, WANG Wen-zhi2, YUAN Qian-hua1, ZHANG Shu-zhen2(), SHEN Lin-bo2()   

  1. 1. College of Tropical Agriculture and Forestry, Hainan University, Haikou 570223
    2. Institute of Tropical Bioscience and Biotechnology of Chinese Academy of Tropical Agricultural Sciences, Haikou 571101
  • Received:2024-02-01 Online:2024-06-26 Published:2024-05-06
  • Contact: ZHANG Shu-zhen, SHEN Lin-bo E-mail:3492787588@qq.com;zhangshuzhen@itbb.org.cn;shenlinbo@itbb.org.cn

Abstract:

【Objective】 It is to establish a rapid, sensitive and specific SYBR Green I quantitative PCR method for the detection of sugarcane striate virus(SStrV).【Method】 A specific amplification primer was designed from the conserved sequence of the SStrV genome sequence, the recombinant plasmid pMD19-T-SStrV-qN containing SStrV gene was constructed as a positive plasmid standard. Using it as template, the SStrV fluorescence quantitative PCR assay was established. And the sensitivity, specificity, stability, and subsequently of this method were tested, then the SStrV loads in different tissue sites of sugarcane were detected.【Result】 The recombinant plasmid containing SStrV genome sequence was diluted into a standard at a 10-fold ratio, and they were used as a template for real-time PCR, the standard curve y = -3.337 x + 38.197 was obtained, and the correlation coefficient r2 = 0.999 was obtained, indicating that the Cq value was linearly related to the logarithm of the copy number of the standard concentration. With this established real-time PCR, the least detection limit was 13 copies of the recombinant plasmid/μL, which was 100 times more sensitive than ordinary PCR. The method specifically detected SStrV with high specificity, and the coefficient of variation within and between groups was n 0.13%-0.94%, indicating that the method was of good repeatability. The load of SStrV was significantly different among different tissue sites of the sugarcane, and the load of SStrV was the highest in +4 leaves, which was significantly different from other tissue sites.【Conclusion】 The SYBR Green I quantitative PCR method is established to provide an efficient quantitative detection method for the diagnosis of SStrV, and it is determined that +4 leaves are the best sampling site for the detection of SStrV in sugarcane.

Key words: sugarcane striate virus, real-time PCR, viral load, detection