Deteksi Vibrio harveyi dengan Metode Amplifikasi DNA pada Gen toxR
DOI:
Abstract Views:
730 times
PDF Downloads:
561 times
Abstract
Abstrak -Infeksi hewan akuakultur oleh Vibrio harveyi dapat menyebabkan kematian serta kerugian ekonomi. Kemampuan untuk mendeteksi bakteri tersebut secara dini dapat mencegah penyebarannya dalam akuakultur. Penelitian ini bertujuan untuk mengembangkan metode untuk mendeteksi Vibrio harveyi melalui amplifikasi gen toxR. Amplifikasi DNA dilakukan dengan dua metode, yakni amplifikasi isotermal termediasi loop (loop-mediated isothermal amplification, LAMP) dan reaksi berantai polimerase (PCR). Amplifikasi menggunakan metode LAMP menunjukan perlu dilakukan optimasi protokol maupun desain primer untuk mencegah perolehan hasil false positive. Amplifikasi menggunakan metode PCR menghasilkan produk berukuran 229 pasang basa yang spesifik pada Vibrio harveyi dengan batas deteksi hingga 0,526 ng.µL-1 (setara 2,09 × 106 CFU.mL-1).
Kata kunci: Akuakultur, LAMP, PCR, toxR, Vibrio harveyi
Abstract -Vibrio harveyi infection in aquacultures may cause death and economical loss. Rapid detection of this bacteria may prevent its dispersal in aquacultures. The goal of this research was to develop method in detection of Vibrio harveyi via amplification of toxR gene. DNA amplification was carried out with two methods, loop-mediated isothermal amplification (LAMP) and polymerase chain reaction (PCR). Amplification with LAMP suggest optimization of either protocol or primer design was needed to prevents false positive results. Amplification with PCR yields 229 bp-length product specific to Vibrio harveyi with detection limit up to 0.526 ng.µL-1 (equals to 2.09 × 106 CFU.mL-1).
Keywords: Aquaculture, LAMP, PCR, toxR, Vibrio harveyi
Downloads
References
Austin, B. (2010). Vibrios as casual agents of zoonoses. Veterinary Microbiology, 140(3-4), 310-317.
Austin, B., & Zhang, X. H. (2006). Vibrio harveyi: a significant pathogen of marine vertebrates and invertebrates. Letters in Applied Microbiology, 43(2), 119- 124.
Cao, Y. T., Wu, Z. H., Jian, J. C., & Lu, Y. S. (2010). Evaluation of a loop-mediated isothermal amplification method for the rapid detection of Vibrio harveyi in cultured marine shellfish. Letters in Applied Microbiology, 51(1), 24-29.
Chen, W. P., & Kuo, T. T. (1993). A simple and rapid method for the preparation of gram-negative bacterial genomic DNA. Nucleic Acid Research, 21(9), 2260.
Conejero, M. J., & Hedreyda, C. T. (2003). Isolation of partial toxR gene of Vibrio harveyi and design of toxR-targeted PCR primers for species detection. Journal of Applied Microbiology, 95(3), 602-611.
Dhama, K., Karthik, K., Chakraborty, S., Tiwari, R., Kapoor, S., Kumar, A., & Thomas, P. (2014). Loop-mediated Isothermal Amplification of DNA (LAMP): A New Diagnostic Tool Lights the World of Diagnosis of Animal and Human Pathogens: A Review. Pakistan Journal of Biological Sciences, 17(2), 151-166.
Direktorat Jenderal Perikanan Budidaya. (n.d.). Udang Vaname dan Udang Windu Masih Andalan Ekspor Indonesia. Retrieved Juni 15, 2018, from Direktorat Jenderal Perikanan Budidaya: http://www.djpb.kkp.go.id/arsip/c/246/Udang- Vannamei-dan-Udang-Windu-Masih-AndalanEkspor-Indonesia/?category_i d=13
Felix, F., Nugroho, T. T., Silalahi, S., & Octavia, Y. (2011). Skrining Bakteri Vibrio sp. Asli Indonesia Sebagai Penyebab Penyakit Udang Berbasis Teknik 16S Ribosomal DNA. Jurnal Ilmu dan Teknologi Kelautan Tropis, 3(2), 85-99.
Fernandez-Soto, P., Mvoulouga, P. O., Akue, J. P., Aban, J. L., Santiago, B. V., Sanchez, M. C., & Muro, A. (2014). Development of a Highly Sensitive Loop-Mediated Isothermal Amplification (LAMP) Method for the Detection of Loa loa. PLOS One, 9(4), e94664.
Gandelman, O., Jackson, R., Kiddle, G., & Tisi, L. (2011). Loop-Mediated Amplification Accelerated by Stem Primers. International Journal of Molecular Sciences, 12(12), 9108-9124.
Garno, Y. S. (2004). Pengembangan Budidaya Udang dan Potensi Pencemarannya pada Perairan Pesisir. Jurnal Teknologi Lingkungan, 5(3), 187-192.
Hsieh, K., Mage, P. L., Csordas, A. T., Eisenstein, M., & Soh, H. T. (2014). Simultaneous elimination of carryover contamination and detection of DNA with uracil-DNA-glycosylase-supplemented loop-mediated isothermal amplification (UDG-LAMP). Chemical Communications, 50(28), 3747-3749.
Hundenborn, J., Thurig, S., Kommerell, M., Haag, H., & Nolte, O. (2013). Severe Wound Infection with Photobacterium damselae ssp. damselae and Vibrio harveyi, following a Laceration Injury in Marine Environment: A Case Report and Review of the Literature. Case Reports in Medicine, 2013, 1-7.
Liu, P. C., Lee, K. K., & Chen, S. N. (1996). Pathogenicity of different isolates of Vibrio harveyi in tiger prawn, Penaeus monodon. Letters in Applied Microbiology, 22(6), 413-416.
Meagher, R. J., Priye, A., Light, Y. K., Huang, C., & Wang, E. (2018). Impact of primer dimers and self-amplifying hairpins on reverse transcription loop- mediated isothermal amplification detection of viral RNA. Analyst, 143(8), 1924-1933.
Nagamine, K., Hase, T., & Notomi, T. (2002). Accelerated reaction by loop- mediated isothermal amplification using loop primers. Molecular and Cellular Probes, 16(3), 223-229.
Notomi, T., Okayama, H., Masubuchi, H., Yonekawa, T., Watanabe, K., Amino, N., & Hase, T. (2000). Loop-mediated isothermal amplification of DNA. Nucleic Acids Research, 28(12), e63.
Oakey, H. J., Levy, N., Bourne, D. G., Cullen, B., & Thomas, A. (2003). The use of PCR to aid in the rapid identification of Vibrio harveyi isolates. Journal of Applied Microbiology, 95(6), 1293-1303.
Pang, L., Zhang, X. H., Zhong, Y., Chen, J., Li, Y., & Austin, B. (2006). Identification of Vibrio harveyi using PCR amplification of the toxR gene. Letters in Applied Microbiology, 43(3), 249-255.
Pascual, J., Macian, M. C., Arahal, D. R., Garay, E., & Pujalte, M. J. (2010). Multilocus sequence analysis of central clade of the genus Vibrio by using the 16S rRNA, recA, pyrH, rpoD, gyrB, rctB and toxR genes. International Journal of Systematic and Evolutionary Microbiology, 60, 154-165.
Robertson, P. A., Calderon, J., Carrera, L., Stark, J. R., Zherdmant, M., & Austin, B. (1998). Experimental Vibrio harveyi infections in Penaeus vannamei larvae. Diseases of Aquatic Organisms, 32, 151-155.
Saulnier, D., Haffner, P., Goarant, C., Levy, P., & Ansquer, D. (2000). Experimental infection models for shrimp vibriosis studies: a review. Aquaculture, 191(1-3), 133-144.
Wang, D.-G., Brewster, J. D., Paul, M., & Tomasula, P. M. (2015). Two Methods for Increased Specificity and Sensitivity in Loop-Mediated Isothermal Amplification. Molecules, 20(4), 6048-6059.
This work is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License.
- Articles published in Keluwih: Jurnal Sains dan Teknologi are licensed under a Creative Commons Attribution-ShareAlike 4.0 International license. You are free to copy, transform, or redistribute articles for any lawful purpose in any medium, provided you give appropriate credit to the original author(s) and the journal, link to the license, indicate if changes were made, and redistribute any derivative work under the same license.
- Copyright on articles is retained by the respective author(s), without restrictions. A non-exclusive license is granted to Keluwih: Jurnal Sains dan Teknologi to publish the article and identify itself as its original publisher, along with the commercial right to include the article in a hardcopy issue for sale to libraries and individuals.
- By publishing in Keluwih: Jurnal Sains dan Teknologi, authors grant any third party the right to use their article to the extent provided by the Creative Commons Attribution-ShareAlike 4.0 International license.
