A rapid detection tool for VT isolates of Citrus tristeza virus by immunocapture- reverse transcriptase loop mediated isothermal amplification assay

Authors: SELVARAJ, VIJAYANANDRAJ - Foreign Agricultural Service (FAS, USDA); MAHESHWARI, YOGITA - Foreign Agricultural Service (FAS, USDA); HAJERI, SUBHAS - Central California Tristeza Eradication Agency; Yokomi, Raymond - Ray

Submitted to: PLOS ONE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/27/2019
Publication Date: 9/5/2019
Citation: Selvaraj, V., Maheshwari, Y., Hajeri, S., Yokomi, R.K. 2019. A rapid detection tool for VT isolates of Citrus tristeza virus by immunocapture- reverse transcriptase loop mediated isothermal amplification assay. PLoS One. 14(9):e0222170. https://doi.org/10.1371/journal.pone.0222170.
DOI: https://doi.org/10.1371/journal.pone.0222170

Interpretive Summary: Citrus tristeza virus (CTV) has different strains that range from symptomless to severe stem pitting (SP) independent from rootstock use that significantly reduces tree vigor, fruit production and quality. The VT genotype strains in California has been shown to cause SP. Therefore, research was conducted here to develop a rapid and simple method to detect VT strains using a reverse transcription loop-mediated isothermal amplification (RT-LAMP). This assay was sensitive down to a level of 11.3 copies of CTV RNA within 10-11 minutes and was specific to VT strains. To facilitate on-site detection, an immunocapture step was incorporated where CTV antibodies are pre-coated in PCR tubes and crude citrus extract added and incubated. This step allows virions to attach to the walls of the PCR tubes and eliminates the need for nucleic acid extraction and purification. This is followed by RT-LAMP. The IC-RT-LAMP was compared with real time RT-PCR (RT-qPCR) with 40 CTV-infected field citrus trees infected with T30, RB, VT, and mixtures of these isolates. The IC-RT-LAMP was 100% in agreement with the RT-qPCR results. Therefore, this simplified test suggests that it can be used as an on-site tool to rapidly identify VT strains of CTV.

Technical Abstract: Severe strains of Citrus tristeza virus (CTV) cause quick decline and stem pitting in citrus which lead to huge economic losses. A reverse-transcriptase loop-mediated amplification (RT-LAMP) assay was developed in this study to detect VT strains that are typically associated with CTV virulence. The sensitivity of the RT-LAMP was determined by ten-fold serial dilutions of CA-VT-AT39 RNA in comparison to one-step RT-digital droplet (dd) PCR. RT-LAMP assay detected up to 0.002 ng of RNA with an amplification time of 10 minutes 35 seconds that was equivalent to 11.3 copies as determined by RT-ddPCR. The RT-LAMP assay detected CA-VT-AT39 and did not cross-react with other CTV genotypes tested (T36, T30, RB, S1 and T68). The RT-LAMP assay was improved by capturing the CTV virions from citrus crude leaf sap using CTV-IgG (IC-RT-LAMP) without nucleic acid extraction steps to facilitate rapid on-site detection. IC-RT-LAMP assay was optimized with two-fold dilutions of CTV-IgG ranging from 1:500 to 1:16,000. The IC-RT-LAMP assay detected the CA-VT-AT39 virions in all the dilutions. The minimum amplification time of 6 minutes 45 seconds was obtained with 1:500 and 1:1000 of CTV-IgG concentrations. The sensitivity limit of detection of IC-RT-LAMP assay with infected crude leaf sap of CA-VT-AT39 was up to 1:320 with a maximum amplification time of 9 minutes 8 seconds. The IC-RT-LAMP assay was validated for VT genotype by comparing to IC-RT-qPCR using the CTV infected 40 field samples. A100% agreement was observed between tests regardless of single or mixed infections of CTV genotypes. Therefore, the IC-RT-LAMP assay can serve as useful tool in the management of virulent strains of CTV.