Suprimescript rt pcr premix 2

To design the mRT-PCR primer sets of CTV, CYVCV, CVEV, and CLBV, more than 10 isolates reported from different geographical regions were retrieved from the NCBI database and aligned using MUSCLE method. A primer binding site was selected as conserved regions. The primer sets for mRT-PCR of CTV were designed on p61 region; CYVCV were designed on RNA-dependent RNA polymerase (RdRp) region; CVEV were designed on coat protein (CP) region and CLBV were designed on CP region. The amplified product sizes of each primer set were designed as different lengths, and melting temperature of all primer sets were adjusted to 57℃ for efficiency of this method. The sequence of primer sets for mRT-PCR developed in this study was detailed in Table 4. A mRT-PCR system was established by designing primer sets to identify the four viruses. One-step RT-PCR was performed using SuPrimeScript RT-PCR Premix 2× (Genetbio), and four viruses were simultaneously detected using the CTV 13169F/13907R, CYVCV 102F/698R, CVEV 4831F/5303R, and CLBV 7120F/7505R primer sets designed in this study.

Total RNA was extracted from leaf samples of citrus using easy-spin Total RNA Extraction kit (iNtRON Biotechnology, Seongnam, Korea). RNA quality was assessed using the Tapestation RNA Screen Tape (Agilent, Santa Clara, CA, USA). Subsequently, cDNA libraries were prepared with TruSeq Stranded Total RNA and a Ribo-Zero Plant kit (Illumina, San Diego, CA, USA), removing plant-derived ribosomal RNA to boost the viral nucleic acid in NGS reads. Sequencing was performed on an Illumina NovaSeq 6000 system following the manufacturer’s instructions at Macrogen Inc. (Seoul, Korea). For NGS analysis, total RNA was extracted from kumquat, Persian lime, Eureka lemon, and calamansi in Gongju, and mixed to prepare the cDNA library. In Taean, total RNA was extracted from natsudaidai, satsuma mandarin, ‘Kanpei’, ‘Shiranui’, and lemon. The reverse transcription polymerase chain reaction (RT-PCR) experiment was performed using total RNA isolated from each citrus host. Gene-specific primer sets (Table 2) were used for the four viruses (CTV, CYVCV, CVEV, and CLBV) identified from the NGS results. One-step RT-PCR using SuPrimeScript RT-PCR Premix 2× (Genetbio, Daejeon, Korea) was conducted to diagnose viral disease. After purifying the product using an Expin Combo GP kit (GeneAll, Seoul, Korea), nucleotide sequences were analyzed by Sanger sequencing (BIONICS, Daejeon, Korea).