Judul: Primer Design for Detection Dickeya spp. and Pectobacterium spp. in Potato with Multiplex PCR
Abstrak: Potato (Solanum tuberosum L.) is an important horticultural crop both on industrial need and household consumption. The high demand of potatoes is fulfilled by importation. The importation of potatoes carried out by the Indonesian government has a potential threat such as pathogens from outside that do not yet exist in Indonesia or OPTK A1. There are several OPTK A1 potatoes, Dickeya dadantii, Dickeya solani, Dickeya zeae, Pectobacterium atrosepticum and Pectobacterium wasabiae. This research aims to design multiplex PCR primers associated with OPTK A1 in Dickeya spp. and Pectobacterium spp.. The research was carried out by conducting scientific research in stages including 16S rRNA homology analysis, pelI and maeA gene homology analysis, pelI and maeA gene polymorphism analysis, primers design, and secondary primer structure analysis. Five pairs of primary candidates match to the criteria. Five pairs candidate primers were successfully designed for detection of Dickeya spp. and Pectobacterium spp. respectively: WD2F 5'-agccagttccgagtgtaagc-3' and WD2R 5'-ggtttctgcttgtcgctgtc-3' (D. dadantii); JD1F 5'-tgaacgcatcgccgtact-3' and JD1R 5'-gccaacgatggtgagggtt-3' (D. zeae); FA2F 5'-cgtcattagccgcccaaac-3' and FA2R 5'-gcagttgccgctttcacag-3' (D. solani); SA2F 5'-ggtgccctgaatgtcatcg-3' and SA2R 5'-cgtctgaacgatccgctgta-3' (P. atrosepticum); GI1F 5'-cgggttagacagtggcatca-3' and GI1R 5'-cgtcgcacagatgaaatccg-3' (P. wasabiae). Five primers candidate have primer lengths from 18 to 21 Mer, Tm values from 59.57 to 60.39 ˚C, % GC from 55% to 57% and do not have any potential secondary structure. Furthermore, each primer candidate generates 108, 445, 500, 213, and 316 bp amplicons. These candidate primers have the potential to be used in multiplex PCR detection and need further in vitro testing to prove the effectiveness., Kentang (Solanum tuberosum L.) merupakan tanaman hortikultura yang dibutuhkan baik dalam skala industri maupun skala rumah tangga, tetapi pemenuhan kebutuhan kentang nasional pemerintah Indonesia masih melakukan importasi. Importasi kentang yang dilakukan oleh pemerintah Indonesia memiliki potensi ancaman masuknya patogen dari luar yang belum ada di Indonesia atau OPTK (Organisme Penganggu Tanaman Karantina) A1 . OPTK A1 kentang diantaranya yaitu, Dickeya dadantii, Dickeya solani, Dickeya zeae, Pectobacterium atrosepticum dan Pectobacterium wasabiae. Penelitian ini bertujuan mendapatkan rancangan primer untuk mendeteksi Dickeya spp. dan Pectobacterium spp. pada kentang dengan multiplex PCR. Penelitian melalui beberapa tahapan yaitu analisis homologi 16S rRNA, analisis homologi gen pelI dan maeA, analisis polimorfisme sekuen gen pelI dan maeA, perancangan primer, dan analisis struktur sekunder. Lima pasang primer didapatkan sesuai dengan kriteria untuk digunakan dalam deteksi menggunakan teknik multiplex PCR. Lima pasang primer yang berhasil dirancang untuk deteksi Dickeya spp. dan Pectobacterium spp. diantaranya yaitu WD2F 5'-agccagttccgagtgtaagc-3' and WD2R 5'-ggtttctgcttgtcgctgtc-3' (D. dadantii); JD1F 5'-tgaacgcatcgccgtact-3' and JD1R 5'-gccaacgatggtgagggtt-3' (D. zeae); FA2F 5'-cgtcattagccgcccaaac-3' and FA2R 5'-gcagttgccgctttcacag-3' (D. solani); SA2F 5'-ggtgccctgaatgtcatcg-3' and SA2R 5'-cgtctgaacgatccgctgta-3' (P. atrosepticum); GI1F 5'-cgggttagacagtggcatca-3' and GI1R 5'-cgtcgcacagatgaaatccg-3' (P. wasabiae). Lima kandidat primer memiliki panjang primer 18 hingga 21 mer, nilai Tm berkisar 59,57-60,39 ˚C, %GC berkisar 55%-57%, dan tidak ditemukan nilai energi bebas gibs lebih dari -9 kcal/mol. Masing-masing kandidat primer menghasilkan amplikon sebesar 108, 445, 500, 213, dan 316 bp. Lima kandidat primer merupakan primer spesifik. Kandidat primer tersebut memiliki potensi untuk digunakan dalam deteksi dengan multiplex PCR dan perlu pengujian lebih lanjut secara in vitro untuk membuktikan keefektifannya.
Keyword: multiplex pcr, optk a1, potato, primer design