Detection of Cucumber mosaic Virus (CMV) from Parsley by One-step RT-LAMP

Background and Aim: Cucumber mosaic virus (CMV) is a linear, single stranded positive sense RNA genome virus which belongs to the genus cucumovirus and family Bromoviridae. CMV has the widest host range among plant viruses. The CMV genome is composed of three positive single-stranded RNAs (RNA۱, RNA۲ and RNA۳) and two subgenomic RNAs (RNA۴ and RNA۴A). In order to investigate the incidence of virus infecting Parsley, ۸۰ samples with yellowing and deformation symptoms were collected from different locations of Arak during the spring of ۲۰۱۸.Methods: A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay has been used to diagnose Cucumber mosaic Virus (CMV) in this study. A RT-LAMP assay amplified DNA with four oligonucleotide primers which recognize six distinct regions on the target DNA in conjunction with two loop primers to accelerate the reaction and a heat-resistant DNA polymerase with strand displacement activity . LAMP specific primers corresponding to the conserved coat protein gene were designed using Primer ExplorerV۴. Total RNA was extracted using RNA column extraction kit (Denazist Asia, Iran). The reaction was performed in a ۲۵ µl mixture containing a final concentration of ۰.۰۲ mM RNA, ۰.۰۴ mM dNTP, ۰.۰۴ mM DDT, ۰.۰۲ mM RNasin, ۰.۰۶ mM of primer FIP, ۰.۰۶ mM of primer BIP, ۰.۰۴ mM of primer LB, ۰.۰۴ mM of primer LF, ۰.۰۲ mM of primer F۳, ۰.۰۲ mM of primer B۳, ۰.۰۸ mM Betaine, ۰.۰۴ mM MgSO۴, ۰.۱ mM Bst (۱۰x) buffer, ۰.۱ mM RNase-free water, ۰.۲ mM of M-Mulv RT and ۰.۰۴ mM Bst DNA polymerase. Reaction mixtures were incubated at ۶۳◦C for ۱ h and terminated in an ۸۰◦C water bath for ۲ min.Results: Visual inspection of the LAMP amplification products was observed in ۲۵ tubes by turbidity due to the formation of precipitate of magnesium pyrophosphate. Five microliters of product were electrophoresed on a ۱% agarose gel stained with ethidium bromide. On the other hand, Vigna unguiculata which was inoculated with the extract of the collected samples, showed necrotic local lesion.Conclusion: Some advantages of RT-LAMP method are reducing assay time, amplicon confirmation by hybridization and use of simpler equipment (i.e., no need for a thermocycler).

Keywords:

RT-LAMP , Turbidity , Cucumber mosaic Virus , hybridization