Abstract:

【Objective】 The objective of this study is to develop a real-time fluorescent quantitative PCR (RT-qPCR) method to detect the Tomato chlorosis virus (ToCV) and its concentration in plant and the vector Bemesia tabaci.【Method】 Firstly, the special primers ToCVqF/R were designed using Primer 3.0 for RT-qPCR based on the highly conserved region of the minor coat protein (CPm). The primers specificity was evaluated by Primer-BLAST. Secondly, the fragment was amplified using the primers ToCVqF/R by PCR within the ToCV-infected, Tomato yellow leaf curl virus (TYLCV)-infected, and Tomato spotted wilt virus (TSWV)-infected tomato samples, and cloned into pMD^TM 18-T Vector. The positive clone was selected and multiplied in LB liquid medium containing ampicillin. Furthermore, the recombinant plasmid was extracted from the clone and served as the ToCV standard. Then, the standard curve was generated for quantitative analysis using the ten-fold dilution of the recombinant plasmid. In addition, the detection sensitivity of RT-qPCR was also evaluated and compared to that of reverse transcription-polymerase chain reaction (RT-PCR). Finally, this method was used to detect and quantify ToCV in eight tomato samples with suspected ToCV infection and single B. tabaci after acquiring ToCV for 24 h, according to the amplification curves, melting curves and Ct values.【Result】 The purpose strips could be successfully amplified with the primers ToCVqF/R only in the ToCV-infected tomato sample and positive control, which indicated that the primers was highly specific. Additionally, the sequencing result of the fragment was in according with the target gene CPm. The ToCV standard curve showed that the Ct values gradually increased as the concentration of recombinant plasmid decreased. Among the concentration range of 2.7×10³-2.7×10⁹ copies/μL, the standard curve showed a good linear relationship between Ct values and plasmid concentration. The correlation coefficient of the standard curve was 0.9911, and the amplification efficiency was 100%. The Straight-line equation is y=-3.32×lgx+40.06 (y and x represent the Ct value and plasmid concentration, respectively). The sensitivity of RT-qPCR was 100 times higher than regular RT-PCR. The minimum detectable concentration of ToCV plasmid standard in RT-qPCR assay is 2.7×10³ copies/μL, while that in RT-PCR assay is 2.7×10⁵ copies/μL. This method could successfully detect the ToCV within tomato samples, and the result was consistent with RT-PCR assay. Six of eight tomato samples were infected by ToCV, the viral titers of which were 2.48×10⁵, 2.21×10⁵, 7.97×10⁴, 3.74×10⁷, 3.37×10⁷ and 2.78×10⁶ copies/μL, respectively. After 24 h feeding on ToCV infected tomato plants, 100% of the B. tabaci acquired this virus successfully, and the virus concentration in single whitefly ranged from 6.46×10² to 2.55×10⁴ copies/μL.【Conclusion】 The RT-qPCR method is applicable for accurate and sensitive detection of ToCV both in plants and vector insects, which will provide a technical support for the monitoring and early warning of this virus.

Key words: Tomato chlorosis virus (ToCV), real-time fluorescent quantitative PCR (RT-qPCR), Bemisia tabaci, detection