ردیابی ویروس موزاییک کرفس (CeMV) در گیاه گشنیز با استفاده از واکنش One-step RT-LAMP

Coriander (Coriandrum sativum L.) belongs to Apiaceae family, is an annual, medicinal and aromatic plant. Area under coriander cultivation has increased over recent years, due to planning and implementing appropriate crop patterns. This study was aimed to the identification of coriander viral diseases in Arak greenhouses. A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay is a novel method, which has been used to diagnose Celery Mosaic Virus (CeMV) 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 .During spring of 2016, a total number of 44 coriander plants with symptoms including mottle, mosaic, yellowing and leaf curly top were collected. LAMP specific primers corresponding to the conserved coat protein gene were designed using Primer ExplorerV5 software (specific for LAMP) respectively. Total RNA was extracted using RNA column extraction kit (Denazist Asia, Iran). The reaction was performed in a 25 µl mixture containing a final concentration of 0.02 mM RNA, 0.04 mM dNTP, 0.04 mM DDT, 0.02 mM RNasin, 0.06 mM of primer FIP, 0.06 mM of primer BIP, 0.04 mM of primer LB, 0.04 mM of primer LF, 0.02 mM of primer F3, 0.02 mM of primer B3, 0.08 mM Betaine, 0.04 mM MgSO4, 0.1 mM Bst (10x) buffer, 0.1 mM RNase-free water, 0.2 mM of M-Mulv RT and 0.04 mM Bst DNA polymerase. Reaction mixtures were incubated at 63◦C for 1 h and terminated in an 80◦C water bath for 2 min. Visual inspection of the LAMP amplification products was observed in 8 tubes by turbidity due to the formation of precipitate of magnesium pyrophosphate. Five microliters of product were electrophoresed on a 1% agarose gel stained with ethidium bromide. On the other hand, Chenopodium amaranticolor and C. quinoa which were inoculated with the extract of the collected samples, showed mottle and chlorotic local lesion. Some advantages of turbidity of magnesium pyrophosphate in RT-LAMP method are rapid detection, high sensitivity, ease of work and cost-effective.