* Corresponding Author
Optimasi Metode PCR untuk Deteksi Pectobacterium carotovorum, Penyebab Penyakit Busuk Lunak Anggrek
https://doi.org/10.22146/jpti.9813 Tri Joko(1*), Nanda Kusumandari(2), Sedyo Hartono(3)
(1) Fakultas Pertanian Universitas Gadjah Mada
(2) Fakultas Pertanian Universitas Gadjah Mada
(3) Fakultas Pertanian Universitas Gadjah Mada
(*) Corresponding Author
Abstract
Soft rot is one of the most important diseases of orchids caused by Pectobacterium carotovorum. The conventional methods for the detection of pathogen is tedious and time consuming. In recent years, numerous molecular diagnostic approaches for the detection of P. carotovorum have been developed, including various PCR-based assays. Optimization of PCR technique to DNA amplification is essential for time and material efficiency, which will make detection to be rapid and more appropriate. The purposes of this study were to decide concentration of DNA and primer, and also the concentration of bacterial pure cultures and primer to amplify 16S rRNA gene fragment. Optimization of PCR was done by using various concentration of DNA, pure cultures of bacteria, and primer to amplify the 16S rRNA gene sequence. The results showed that the most optimum concentration to amplify 16S rRNA gene sequence at DNA and primer concentration were 63,4 ng/µl and 10 pmol, while pure cultures and primer concentrations were at 8×109 CF U/ml and 10 pmol respectively.
Penyakit busuk lunak yang disebabkan oleh Pectobacterium carotovorum merupakan salah satu penyakit penting pada tanaman anggrek. Deteksi patogen secara cepat dan akurat dapat dilakukan secara molekular menggunakan teknik Polymerase chain reaction (PCR). Optimasi metode PCR perlu dilakukan untuk mengefisienkan waktu dan penggunaan bahan sehingga proses deteksi dapat dilakukan dengan cepat dan tepat. Penelitian ini bertujuan untuk menentukan konsentrasi DNA dengan primer maupun konsentrasi kultur murni bakteri dengan primer yang paling tepat untuk mendapatkan fragmen gen 16S rRNA. Optimasi PCR dilakukan menggunakan beberapa variasi pengenceran pada DNA, kultur murni bakteri, dan primer untuk mengamplifikasi gen 16S rRNA. Hasil penelitian menunjukkan bahwa konsentrasi yang paling optimal untuk mengamplifikasi gen 16S rRNA yaitu DNA dan primer masing-masing sebesar 63,4 ng/µl dan 10 pmol, sedangkan konsentrasi kultur murni dan primer sebesar 8×109 CFU/ml dan 10 pmol.
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Agrios, G.N. 2005. Plant Pathology, 5th Edition. Academic Press. New York. 922 p.
Ali, B.A., T.H. Huang, H.H. Salem, & Q.D. Xie. 2006. Influence of Thermal Cycler Day-today Reproducibility of Random Amplified Polymorphic DNA Fingerprints. Biotechnology 5: 324–329.
Ausubel F.M, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, & K. Struhl (eds.). 1990. Current Protocols in Molecular Biology. New York: Greene Publishing Associates and Wiley-Inter Science.
Case, R.J., Y. Boucher, I. Dahllöf, C. Holmström, W.F. Doolittle, & S. Kjelleberg. 2007. Use of 16S rRNA and rpoB Genes as Molecular Markers for Microbial Ecology Studies. Applied and Environmental Microbiology 73: 278–288.
Clarridge, J.E. 2004. Impact of 16S rRNA Gene Sequence Analysis for Identification of Bacterial on Clinical Microbiology and Infectious Diseases. Clinical Microbiology Review 17: 840–862.
Erlich, H.A. 1989. Polymerase Chain Reaction. Journal of Clinical Immunology 9: 437−447.
Greisen, K., A. Loeffelholz, Purohit, & D. Leong. 1993. PCR Primers and Probes for the 16s rRNA Gene of Most Species of Pathogenic Bacteria, Including Bacteria Found in Cerebrospinal Fluid. Journal of Clinical Microbiology 32: 335–51.
Harini, S.S., M. Leelombik, M.N.S. Kameshwari, & N. Sathyanarayana. 2008. Optimization of DNA Isolation and PCR-RAPD Methods for Molecular Analysis of Urginea indica Kunth. International Journal of Integrative Biology 2: 138–144.
Heuer, H., K. Hartung, G. Wieland, I. Kramer, & K. Samalla. 1999. Polynucleotide Probes That Target a Hypervariable Region of 16S rRNA Genes to Identify Bacterial Isolate Corresponding to Bands of Community Fingerprint. Applied and Environmental Microbiology 65: 1045–1049.
Joko, T., D. Kiswanti, S. Subandiyah, & Hanudin. 2011. Occurence of Bacterial Soft Rot of Phalaenopsis Orchids in Yogyakarta and West Java, Indonesia, p. 255–265. In Y. Koentjoro (ed.), Proceeding of Internasional Seminar on “Natural Resources, Climate Change, and Food Security in Developing Countries”. 27−28 June 2011. Surabaya, Indonesia.
Joko, T., H. Hirata, & S. Tsuyumu. 2007a. Sugar Transporter (MfsX) of Major Facilitator Super family is Required for Flagella-mediated Pathogenesis in Dickeya dadantii 3937. 2007. Journal of General Plant Pathology 73: 266−273.
Joko, T., H. Hirata, & S. Tsuyumu. 2007b. The Sugar Transporter (MfsX) is Required Also for Fitness to Plant Environments in Dickeya dadantii 3937. Journal of General Plant Pathology 73: 274−280.
Marchesi, J.R., T. Sato, A.J. Weightman, T.A. Martin, J.C. Fry, S.J. Hiom, & W.G. Wade. 1998. Design and Evaluation of Useful Bacterium Specific PCR Primer that Amplify Genes Coding for Bacterial 16S-rRNA. Applied and Environmental Microbiology 64: 795–799.
Padmalatha, K. & M.N.V. Prasad. 2006. Optimization of DNA Isolation and PCR Protocol for RAPD Analysis of Selected Medicinal and Aromatic Plants of Conservation Concern from Peninsular India. African Journal Biotechnology 5: 230–234.
Rychlik, W., W.J. Spencer, & R.E. Rhoads. 1990. Optimization of the Annealing Temperature for DNA Amplification In vitro. Nucleic Acids Research 18: 6409–6412.
Sambrook, J., E.F. Fritsch, & T. Maniatis. 1989. Molecular Cloning: A Laboratory Manual. 2nd edition. Cold Spring Harbor Laboratory Press. New York.
Schaad N.W., J.B. Jones, & W. Chun. 2001. Laboratory Guide for Identification of Plant Pathogenic Bacteria. APS Press USA. 373 p.
Yuwono, T. 2006. Teori dan Aplikasi Polymerase Chain Reaction. Andi Offset. Yogyakarta. 237 p.
DOI: https://doi.org/10.22146/jpti.9813
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