Acta Entomologica Sinica ›› 2020, Vol. 63 ›› Issue (2): 159-165.doi: 10.16380/j.kcxb.2020.02.005

• RESEARCH PAPERS • Previous Articles     Next Articles

Development and application of a TaqMan real-time fluorescent quantitative PCR method for rapid detection of Tomato chlorosis virus

WANG Ji-Cheng, LI Jie, DING Tian-Bo, CHU Dong*   

  1.  (Key Laboratory of Integrated Crop Pest Management of Shandong Province, College of Plant Health and Medicine, Qingdao Agricultural University, Qingdao, Shandong 260109, China)
  • Online:2020-02-20 Published:2020-02-25

Abstract: 【Aim】 The aim of this study is to develop a TaqMan real-time fluorescent quantitative PCR (TaqMan RT-qPCR) method to rapidly detect the tomato chlorosis virus (ToCV) in a single whitefly (Bemisia tabaci) vector. 【Methods】 A pair of specific primers and one TaqMan probe were designed based on the conserved sequence of ToCV coat protein, and then TaqMan RT-qPCR was developed for viral detection. The sensitivity and specificity of TaqMan RT-qPCR were compared to those of the conventional PCR. Finally, this method was applied to rapidly detect ToCV in a single adult of B. tabaci. 【Results】 The cycle threshold (Ct) on the standard curve of TaqMan RT-qPCR of ToCV showed a linear relationship with the template concentration, and the amplification efficiency was 98%. The minimum concentration of this virus detection method was 8.3×10 copies/μL, and the sensitivity was 1 000 times as high as that of the conventional PCR. This method had no cross-reactivity with two important tomato viruses in the field, i.e., tomato yellow leaf curl virus (TYLCV) and tomato spotted wilt virus (TSWV). Detection of ToCV in a single adult of B. tabaci showed that the viruliferous rate of ToCV in B. tabaci in greenhouses was 100% and that in the field was 30%. 【Conclusion】 The TaqMan RT-qPCR method developed in this study can detect ToCV in a single whitefly (B. tabaci) rapidly and efficiently, providing technical support for the prevention and control of this virus disease.

Key words: Tomato chlorosis virusBemisia tabaci, coat protein, viruliferous rate, amplification efficiency, TaqMan RT-qPCR