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.


Keywords


detection, PCR optimization, Pectobacterium carotovorum, soft rot disease; deteksi, optimasi PCR, penyakit busuk lunak

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DOI: https://doi.org/10.22146/jpti.9813

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