Metode Direct PCR yang Cepat dan Hemat Biaya untuk DNA Barcoding Anggrek Obat Dendrobium crumenatum Sw.
Abstract
This study aims to develop a rapid and cost-effective Direct PCR method for amplifying the rbcL gene without requiring DNA isolation, particularly for Dendrobium crumenatum Sw., an orchid species known for its traditional medicinal uses. The method involved soaking fresh leaves of D. crumenatum in TE buffer, followed by incubation at 55ºC. The incubation product was Directly used as a PCR template. The results showed a clear DNA band of 600 bp, corresponding to the target rbcL gene. BLAST analysis confirmed that the DNA sequence shared 84-85% identity with other species in GenBank. In conclusion, the Direct PCR method proved to be effective for DNA barcoding applications, offering advantages in speed and cost-efficiency over conventional DNA isolation methods.
Keywords: Medicinal Orchids, Dendrobium crumenatum Sw., Direct PCR, DNA Barcoding
References
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Ben-Amar, A., & Mliki, A. (2021). Timely gene detection assay and reliable screening of genetically engineered plants using an improved Direct PCR-based technology. Transgenic Research, 30(3), 263–274. https://doi.org/10.1007/s11248-021-00250-1
Cakova, V., Bonte, F., & Lobstein, A. (2017). Dendrobium: Sources of active ingredients to treat age-related pathologies. Aging and Disease, 8(6), 827-849. https://doi.org/10.14336/AD.2017.0214
CBOL Plant Working Group, Hollingsworth, P. M., Forrest, L. L., Spouge, J. L., Hajibabaei, M., Ratnasingham, S., van der Bank, M., Chase, M. W., Cowan, R. S., Erickson, D. L., Fazekas, A. J., Graham, S. W., James, K. E., Kim, K.-J., Kress, W. J., Schneider, H., van AlphenStahl, J., Barrett, S. C. H., van den Berg, C., & Little, D. P. (2009). A DNA barcode for land plants. Proceedings of the National Academy of Sciences, 106(31), 12794–12797. https://doi.org/10.1073/pnas.0905845106
Choudhary, P., Das, S., Chakdar, H., Singh, A., Goswami, S. K., & Saxena, A. K. (2019). Rapid high throughput template preparation (rHTTP) method: A novel cost-effective method of Direct PCR for a wide range of plants. BMC Biotechnology, 19(1), Article 69. https://doi.org/10.1186/s12896-019-0560-4
de Vere, N., Rich, T. C. G., Trinder, S. A., & Long, C. (2015). DNA barcoding for plants. In J. Batley (Ed.), Plant Genotyping: Methods and Protocols, 101–118. Springer. https://doi.org/10.1007/978-1-4939-1966-6_8
Doyle, J. (1991). DNA protocols for plants. In G. M. Hewitt, A. W. B. Johnston, & J. P. W. Young (Eds.), Molecular Techniques in Taxonomy, 283–293. Springer. https://doi.org/10.1007/978-3-642-83962-7_18
Fassler, J., & Cooper, P. (2011). BLAST glossary. In BLAST® Help [Internet]. National Center for Biotechnology Information (US). https://www.ncbi.nlm.nih.gov/books/NBK62051/
Fitri, N. E., Su’udi, M., & Ikrimah, S. W. (2024). DNA barcode characteristic of Dendrobium crumenatum based on ITS2. Life Science and Biotechnology, 2(1), Article 1. https://doi.org/10.19184/lsb.v2i1.48632
Friar, E. A. (2005). Isolation of DNA from plants with large amounts of secondary metabolites. In Methods in Enzymology, 395, 1–12. https://doi.org/10.1016/S0076-6879(05)95001-5
Gong, L., Xu, J., Guo, M., Zhao, J., Xin, X., Zhang, C., Ni, X., Hu, Y., & An, F. (2024). Octahydroindolizine alkaloid Homocrepidine A from Dendrobium crepidatum attenuate P. acnes-induced inflammatory in vitro and in vivo. Journal of Ethnopharmacology, 333, 118455. https://doi.org/10.1016/j.jep.2024.118455
He, L., Su, Q., Bai, L., Li, M., Liu, J., Liu, X., Zhang, C., Jiang, Z., He, J., Shi, J., Huang, S., & Guo, L. (2020). Recent research progress on natural small molecule bibenzyls and its derivatives in Dendrobium species. European Journal of Medicinal Chemistry, 204, 112530. https://doi.org/10.1016/j.ejmech.2020.112530
Hwang, H., Bae, S.-C., Lee, S., Lee, Y.-H., & Chang, A. (2013). A rapid and simple genotyping method for various plants by direct-PCR. Plant Breeding and Biotechnology, 1(3), 290–297. https://doi.org/10.9787/PBB.2013.1.3.290
Kang, Y., Deng, Z., Zang, R., & Long, W. (2017). DNA barcoding analysis and phylogenetic relationships of tree species in tropical cloud forests. Scientific Reports, 7(1), Article 12564. https://doi.org/10.1038/s41598-017-13057-0
Kongkatitham, V., Dehlinger, A., Wang, M., Poldorn, P., Weidinger, C., Letizia, M., Chaotham, C., Otto, C., Ruprecht, K., Paul, F., Rungrotmongkol, T., Likhitwitayawuid, K., Böttcher, C., & Sritularak, B. (2023). Immunomodulatory effects of new phenanthrene derivatives from Dendrobium crumenatum. Journal of Natural Products, 86(5), Article 5. https://doi.org/10.1021/acs.jnatprod.3c00107
Kumar, D., & Samantaray, S. D. (2021). Identification of nutritionally important protein in Amaranthus genes. Journal of Pharmacognosy and Phytochemistry, 10(4), 238–248. https://www.phytojournal.com/archives/2021.v10.i4.14157/identification-of-nutritionally-important-protein-in-amaranthus-genes
Li, Y., Zhao, H., Yan, X., Li, M., Chen, P., & Zhang, S. (2017). A universal method for direct PCR amplification of plant tissues. Analytical Methods, 9(11), 1800–1805. https://doi.org/10.1039/C6AY03156K
Maulidya, N. N., Rohimah, S., Ramadany, Z., Ratnasari, T., & Su’udi, M. (2020). Assessment of the DNA barcodes characteristic of Phalaenopsis deliciosa based on matK, rbcL, and ITS. Biogenesis: Jurnal Ilmiah Biologi, 8(2), 138-144. https://doi.org/10.24252/bio.v8i2.13278
Miura, M., Tanigawa, C., Fujii, Y., & Kaneko, S. (2013). Comparison of six commercially available DNA polymerases for direct PCR. Revista do Instituto de Medicina, 55(6), 401–406. https://doi.org/10.1590/S0036-46652013000600005
Newmaster, S. G., Fazekas, A. J., & Ragupathy, S. (2006). DNA barcoding in land plants: Evaluation of rbcL in a multigene tiered approach. Canadian Journal of Botany, 84(3), 335–341. https://doi.org/10.1139/b06-047
Nie, G., Zhang, Y., Zhou, Z., Xu, J., Wang, H., Chen, D., & Wang, K. (2021). Dynamic evaluation of the protective effect of Dendrobium officinale polysaccharide on acute alcoholic liver injury mice in vitro and in vivo by NIR fluorescence imaging. Analytical and Bioanalytical Chemistry, 413(23), 5715–5724. https://doi.org/10.1007/s00216-021-03546-7
Omonhinmin, C., & Onuselogu, C. (2022). rbcL gene in global molecular data repository. Data in Brief, 42, 108090. https://doi.org/10.1016/j.dib.2022.108090
Pere, K., Mburu, K., Muge, E. K., Wagacha, J. M., & Nyaboga, E. N. (2023). Molecular discrimination and phylogenetic relationships of Physalis species based on ITS2 and rbcL DNA barcode sequence. Crops, 3(4), Article 4. https://doi.org/10.3390/crops3040027
Perwitasari, D., Rohimah, S., Ratnasari, T., Sugiharto, B., & Su’udi, M. (2020). DNA barcoding of medicinal orchid Dendrobium discolor Lindl. Tanimbar using rbcL and ITS genes. Buletin Penelitian Tanaman Rempah Dan Obat, 31, 8–20. https://doi.org/10.21082/bullittro.v31n1.2020.8-20
Raskoti, B. B., & Ale, R. (2021). DNA barcoding of medicinal orchids in Asia. Scientific Reports, 11(1), 23651. https://doi.org/10.1038/s41598-021-03025-0
Rohimah, S., Ratnasari, T., & Su’udi, M. (2021). DNA barcoding of Thrixspermum longipilosum based on internal transcribed spacer 2 (ITS2) region. IOP Conference Series: Earth and Environmental Science, 743(1), 012092. https://doi.org/10.1088/1755-1315/743/1/012092
Sandrasagaran, U. M., Subramaniam, S., & Murugaiyah, V. (2014). New perspective of Dendrobium crumenatum orchid for antimicrobial activity against selected pathogenic bacteria. Pakistani Journal of Botany, 46(2), 719–724. https://www.pakbs.org/pjbot/PDFs/46(2)/45.pdf
Scobeyeva, V. A., Omelchenko, D. O., Dyakov, L. M., Konovalov, A. S., Speranskaya, A. S., & Krinitsina, A. A. (2018). Comparison of some plant DNA extraction methods. Russian Journal of Genetics, 54(5), 576–586. https://doi.org/10.1134/S1022795418050095
Song, C., Ma, J., Li, G., Pan, H., Zhu, Y., Jin, Q., Cai, Y., & Han, B. (2022). Natural composition and biosynthetic pathways of alkaloids in medicinal Dendrobium species. Frontiers in Plant Science, 13, 850949. https://doi.org/10.3389/fpls.2022.850949
Su’udi, M., Budyartini, D. W., & Ramadany, Z. (2022). DNA barcoding Dendrobium linearifolium Teijsm. & Binn. berdasarkan penanda molekuler ITS2. Al-Kauniyah: Jurnal Biologi, 15(1), Article 1. https://doi.org/10.15408/kauniyah.v15i1.16710
Su’udi, M., Ulum, F. B., Ardiyansah, M., & Fitri, N. E. (2024). Evaluasi lokus potensial matK dan ITS2 untuk DNA barcoding Bulbophyllum lobbii Lindl. Al-Kauniyah: Jurnal Biologi, 17(2), Article 2. https://doi.org/10.15408/kauniyah.v17i2.33897
Teoh, E. S. (2019). Orchids as aphrodisiac, medicine or food. Springer. https://doi.org/10.1007/978-981-13-0843-2
Ye, M., Liu, J., Deng, G., Cai, X., Zhang, X., Yao, L., Wu, J., He, X., Peng, D., & Yu, N. (2022). Protective effects of Dendrobium huoshanense polysaccharide on D-gal induced PC12 cells and aging mice: In vitro and in vivo studies. Journal of Food Biochemistry, 46(12), e14496. https://doi.org/10.1111/jfbc.14496
Zhang, Y., Zhang, Q., Zhou, J., & Zou, Q. (2022). A survey on the algorithm and development of multiple sequence alignment. Briefings in Bioinformatics, 23(3), bbac069. https://doi.org/10.1093/bib/bbac069
Antil, S., Abraham, J. S., Sripoorna, S., Maurya, S., Dagar, J., Makhija, S., Bhagat, P., Gupta, R., Sood, U., Lal, R., & Toteja, R. (2023). DNA barcoding, an effective tool for species identification: A review. Molecular Biology Reports, 50(1), 761–775. https://doi.org/10.1007/s11033-022-08015-7
Bateman, R. (2001). Evolution and classification of European orchids: Insights from molecular and morphological characters. Journal Europäischer Orchideen, 33, 33–119. dikutip dari https://www.researchgate.net/publication/287854443_Evolution_and_classification_of_European_orchids_Insights_from_molecular_and_morphological_characters
Ben-Amar, A., & Mliki, A. (2021). Timely gene detection assay and reliable screening of genetically engineered plants using an improved Direct PCR-based technology. Transgenic Research, 30(3), 263–274. https://doi.org/10.1007/s11248-021-00250-1
Cakova, V., Bonte, F., & Lobstein, A. (2017). Dendrobium: Sources of active ingredients to treat age-related pathologies. Aging and Disease, 8(6), 827-849. https://doi.org/10.14336/AD.2017.0214
CBOL Plant Working Group, Hollingsworth, P. M., Forrest, L. L., Spouge, J. L., Hajibabaei, M., Ratnasingham, S., van der Bank, M., Chase, M. W., Cowan, R. S., Erickson, D. L., Fazekas, A. J., Graham, S. W., James, K. E., Kim, K.-J., Kress, W. J., Schneider, H., van AlphenStahl, J., Barrett, S. C. H., van den Berg, C., & Little, D. P. (2009). A DNA barcode for land plants. Proceedings of the National Academy of Sciences, 106(31), 12794–12797. https://doi.org/10.1073/pnas.0905845106
Choudhary, P., Das, S., Chakdar, H., Singh, A., Goswami, S. K., & Saxena, A. K. (2019). Rapid high throughput template preparation (rHTTP) method: A novel cost-effective method of Direct PCR for a wide range of plants. BMC Biotechnology, 19(1), Article 69. https://doi.org/10.1186/s12896-019-0560-4
de Vere, N., Rich, T. C. G., Trinder, S. A., & Long, C. (2015). DNA barcoding for plants. In J. Batley (Ed.), Plant Genotyping: Methods and Protocols, 101–118. Springer. https://doi.org/10.1007/978-1-4939-1966-6_8
Doyle, J. (1991). DNA protocols for plants. In G. M. Hewitt, A. W. B. Johnston, & J. P. W. Young (Eds.), Molecular Techniques in Taxonomy, 283–293. Springer. https://doi.org/10.1007/978-3-642-83962-7_18
Fassler, J., & Cooper, P. (2011). BLAST glossary. In BLAST® Help [Internet]. National Center for Biotechnology Information (US). https://www.ncbi.nlm.nih.gov/books/NBK62051/
Fitri, N. E., Su’udi, M., & Ikrimah, S. W. (2024). DNA barcode characteristic of Dendrobium crumenatum based on ITS2. Life Science and Biotechnology, 2(1), Article 1. https://doi.org/10.19184/lsb.v2i1.48632
Friar, E. A. (2005). Isolation of DNA from plants with large amounts of secondary metabolites. In Methods in Enzymology, 395, 1–12. https://doi.org/10.1016/S0076-6879(05)95001-5
Gong, L., Xu, J., Guo, M., Zhao, J., Xin, X., Zhang, C., Ni, X., Hu, Y., & An, F. (2024). Octahydroindolizine alkaloid Homocrepidine A from Dendrobium crepidatum attenuate P. acnes-induced inflammatory in vitro and in vivo. Journal of Ethnopharmacology, 333, 118455. https://doi.org/10.1016/j.jep.2024.118455
He, L., Su, Q., Bai, L., Li, M., Liu, J., Liu, X., Zhang, C., Jiang, Z., He, J., Shi, J., Huang, S., & Guo, L. (2020). Recent research progress on natural small molecule bibenzyls and its derivatives in Dendrobium species. European Journal of Medicinal Chemistry, 204, 112530. https://doi.org/10.1016/j.ejmech.2020.112530
Hwang, H., Bae, S.-C., Lee, S., Lee, Y.-H., & Chang, A. (2013). A rapid and simple genotyping method for various plants by direct-PCR. Plant Breeding and Biotechnology, 1(3), 290–297. https://doi.org/10.9787/PBB.2013.1.3.290
Kang, Y., Deng, Z., Zang, R., & Long, W. (2017). DNA barcoding analysis and phylogenetic relationships of tree species in tropical cloud forests. Scientific Reports, 7(1), Article 12564. https://doi.org/10.1038/s41598-017-13057-0
Kongkatitham, V., Dehlinger, A., Wang, M., Poldorn, P., Weidinger, C., Letizia, M., Chaotham, C., Otto, C., Ruprecht, K., Paul, F., Rungrotmongkol, T., Likhitwitayawuid, K., Böttcher, C., & Sritularak, B. (2023). Immunomodulatory effects of new phenanthrene derivatives from Dendrobium crumenatum. Journal of Natural Products, 86(5), Article 5. https://doi.org/10.1021/acs.jnatprod.3c00107
Kumar, D., & Samantaray, S. D. (2021). Identification of nutritionally important protein in Amaranthus genes. Journal of Pharmacognosy and Phytochemistry, 10(4), 238–248. https://www.phytojournal.com/archives/2021.v10.i4.14157/identification-of-nutritionally-important-protein-in-amaranthus-genes
Li, Y., Zhao, H., Yan, X., Li, M., Chen, P., & Zhang, S. (2017). A universal method for direct PCR amplification of plant tissues. Analytical Methods, 9(11), 1800–1805. https://doi.org/10.1039/C6AY03156K
Maulidya, N. N., Rohimah, S., Ramadany, Z., Ratnasari, T., & Su’udi, M. (2020). Assessment of the DNA barcodes characteristic of Phalaenopsis deliciosa based on matK, rbcL, and ITS. Biogenesis: Jurnal Ilmiah Biologi, 8(2), 138-144. https://doi.org/10.24252/bio.v8i2.13278
Miura, M., Tanigawa, C., Fujii, Y., & Kaneko, S. (2013). Comparison of six commercially available DNA polymerases for direct PCR. Revista do Instituto de Medicina, 55(6), 401–406. https://doi.org/10.1590/S0036-46652013000600005
Newmaster, S. G., Fazekas, A. J., & Ragupathy, S. (2006). DNA barcoding in land plants: Evaluation of rbcL in a multigene tiered approach. Canadian Journal of Botany, 84(3), 335–341. https://doi.org/10.1139/b06-047
Nie, G., Zhang, Y., Zhou, Z., Xu, J., Wang, H., Chen, D., & Wang, K. (2021). Dynamic evaluation of the protective effect of Dendrobium officinale polysaccharide on acute alcoholic liver injury mice in vitro and in vivo by NIR fluorescence imaging. Analytical and Bioanalytical Chemistry, 413(23), 5715–5724. https://doi.org/10.1007/s00216-021-03546-7
Omonhinmin, C., & Onuselogu, C. (2022). rbcL gene in global molecular data repository. Data in Brief, 42, 108090. https://doi.org/10.1016/j.dib.2022.108090
Pere, K., Mburu, K., Muge, E. K., Wagacha, J. M., & Nyaboga, E. N. (2023). Molecular discrimination and phylogenetic relationships of Physalis species based on ITS2 and rbcL DNA barcode sequence. Crops, 3(4), Article 4. https://doi.org/10.3390/crops3040027
Perwitasari, D., Rohimah, S., Ratnasari, T., Sugiharto, B., & Su’udi, M. (2020). DNA barcoding of medicinal orchid Dendrobium discolor Lindl. Tanimbar using rbcL and ITS genes. Buletin Penelitian Tanaman Rempah Dan Obat, 31, 8–20. https://doi.org/10.21082/bullittro.v31n1.2020.8-20
Raskoti, B. B., & Ale, R. (2021). DNA barcoding of medicinal orchids in Asia. Scientific Reports, 11(1), 23651. https://doi.org/10.1038/s41598-021-03025-0
Rohimah, S., Ratnasari, T., & Su’udi, M. (2021). DNA barcoding of Thrixspermum longipilosum based on internal transcribed spacer 2 (ITS2) region. IOP Conference Series: Earth and Environmental Science, 743(1), 012092. https://doi.org/10.1088/1755-1315/743/1/012092
Sandrasagaran, U. M., Subramaniam, S., & Murugaiyah, V. (2014). New perspective of Dendrobium crumenatum orchid for antimicrobial activity against selected pathogenic bacteria. Pakistani Journal of Botany, 46(2), 719–724. https://www.pakbs.org/pjbot/PDFs/46(2)/45.pdf
Scobeyeva, V. A., Omelchenko, D. O., Dyakov, L. M., Konovalov, A. S., Speranskaya, A. S., & Krinitsina, A. A. (2018). Comparison of some plant DNA extraction methods. Russian Journal of Genetics, 54(5), 576–586. https://doi.org/10.1134/S1022795418050095
Song, C., Ma, J., Li, G., Pan, H., Zhu, Y., Jin, Q., Cai, Y., & Han, B. (2022). Natural composition and biosynthetic pathways of alkaloids in medicinal Dendrobium species. Frontiers in Plant Science, 13, 850949. https://doi.org/10.3389/fpls.2022.850949
Su’udi, M., Budyartini, D. W., & Ramadany, Z. (2022). DNA barcoding Dendrobium linearifolium Teijsm. & Binn. berdasarkan penanda molekuler ITS2. Al-Kauniyah: Jurnal Biologi, 15(1), Article 1. https://doi.org/10.15408/kauniyah.v15i1.16710
Su’udi, M., Ulum, F. B., Ardiyansah, M., & Fitri, N. E. (2024). Evaluasi lokus potensial matK dan ITS2 untuk DNA barcoding Bulbophyllum lobbii Lindl. Al-Kauniyah: Jurnal Biologi, 17(2), Article 2. https://doi.org/10.15408/kauniyah.v17i2.33897
Teoh, E. S. (2019). Orchids as aphrodisiac, medicine or food. Springer. https://doi.org/10.1007/978-981-13-0843-2
Ye, M., Liu, J., Deng, G., Cai, X., Zhang, X., Yao, L., Wu, J., He, X., Peng, D., & Yu, N. (2022). Protective effects of Dendrobium huoshanense polysaccharide on D-gal induced PC12 cells and aging mice: In vitro and in vivo studies. Journal of Food Biochemistry, 46(12), e14496. https://doi.org/10.1111/jfbc.14496
Zhang, Y., Zhang, Q., Zhou, J., & Zou, Q. (2022). A survey on the algorithm and development of multiple sequence alignment. Briefings in Bioinformatics, 23(3), bbac069. https://doi.org/10.1093/bib/bbac069
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2024-12-31
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