Rapid Detection and Molecular Characterization of Citrus Tristeza Virus (CTV) in Citrus Plants

Abstract

Citrus tristeza virus (CTV) is responsible for worldwide destruction of citrus orchards leading to huge economic losses in citrus growing countries. World-wide cultivation and huge production demands makes citrus an important fruit industry for Pakistan enabling it to generate huge foreign revenues each year. However, CTV spread mainly carried out by infected root stocks and aphid species is causing devastating effects on citrus orchards. Eradication upon early detection and use of virus-free rootstocks is the only solution to this problem, due to which CTV detection has gained importance. In this study, development of an isothermal amplification method was coupled with integration of genetic analysis, for rapid and accurate screening of CTV. Emergence of recombinant strains can cause destruction and are threat to diagnostic procedures. High divergence within isolated CTV strains was present showing identity of 93% to 100% at nucleotide level and 95% to 100% at protein level. Moreover, phylogenetic analysis of CTV isolates showed relatedness with worldwide genotypes T3 and T30+VT genotypes. The T30+VT was not previously the major genotype which indicates variability of CTV in our region. This variability can be due to mixed infection giving rise to severely infecting strains having capability to escape from detection procedures. In-case of RT-PCR detection, RNA isolation steps are of foremost importance along-with established positive and negative controls. Conventional (CTAB and TENS-PCI) and kit based methods indicated A260/280 and A260/230 > 1.8 showing high quality isolated RNA quality with reproducible amplification. Furthermore, CTV detection at TAS-ELISA, RT-PCR and RT-nested PCR differed drastically in-terms of detection sensitivity and specificity. TAS-ELISA proved to be least sensitive as compared to nucleic acid testing procedures. The real time assay was 10-folds more sensitive than RT-PCR, whereas RT-nested PCR (RT-nPCR) provided additional advantage by providing large amplicons that can be used in sequencing reactions required for molecular characterization of CTV. However, RT-PCR and RT-nPCR also have certain limitations of being highly expensive, laborious and require highly trained professionals. Therefore, in the final phase of this study Reverse-Transcriptase-Recombinase Polymerase Amplification (RT-RPA) assay which is an isothermal amplification technology was developed. Sequenced data alongwith other submitted sequences available was curated for primer designing. RT-RPA operations at single temperature make it instrument free and user-friendly approach. The developed RT-RPA assay was successfully implemented for CTV detection coupled with a crude extract isolation procedure to further facilitate steps from sample preparation till target detection. The reaction time of RT-RPA is 20 minutes, it’s highly specific with a detection limit of 0.2 ng/ μL. Such features make RT-RPA rapid, simple, specific, sensitive and user-friendly tool for CTV detection. Beside this, it is a cost-effective detection tool. Application of RT-RPA to detect CTV rapidly could be used a promising candidate for adapting into field test formats or certification laboratories for rapid selection of CTV-free planting material for further propagation and distribution to smallholder farmers.