Authors : Obayiuwana, Amarachukwu Chigozie; Nwose, Adaobi Maureen

Volume/Issue : Volume 10 - 2025, Issue 6 - June

Google Scholar : https://tinyurl.com/24y3cjex

DOI : https://doi.org/10.38124/ijisrt/25jun1254

Note : A published paper may take 4-5 working days from the publication date to appear in PlumX Metrics, Semantic Scholar, and ResearchGate.

Abstract : The PCR-based restriction fragment length polymorphisms (PCR-based RFLP) is a method applied in molecular biology which leverage the differences in cells’ homologous DNA molecules from different locations on sites of action of restriction enzyme. RFLP acts as a molecular marker and is specific to a single clone or combination of restriction enzyme. This method involves the PCR amplification of 16S rRNA genes, a highly conserved region with variable regions for bacterial species differentiation. The PCR method uses two primers to amplify the 16S rRNA genes isolated from total bacterial community DNA. The amplicons from the PCR are subjected to restriction enzymes digest. The resulting restriction fragments are resolved according to their size using gel electrophoresis. The patterns are visualized and used to classify the bacterial community into diverse groups. Representative amplicons of each group are sequenced to determine their identity. In this study, PCR-based RFLP analysis was employed to study the effect of different antibiotics at varying concentrations on bacterial community in various bioreactors. Following the standard procedures, the method was able to classify and give the identity of 254 selected bacterial isolates from sludge samples of the various reactors into 7 groups, which includes Pseudomonas gessardii group, Staphylococcus saprophyticus group, Acinetobacter sp. group, Bacillus sp. group, Myroides marinus group, and Enterobacter sp. From their different patterns and subsequent sequencing. Generally, our result shows that PCR-RFLP is a veritable tool for studying and identifying the diversity of bacterial communities and can be employed in studying the recombination rate providing genetic distance between loci.

Keywords : Bioreactors, Bacteria Diversity, Genes, PCR-RFLP, 16S rRNA

References :

1. D. Cheng, H. H. Ngo, W. Guo, Y. Liu, S. W. Chang, D. D. Nguyen, L. D. Nghiem, J. Zhou, and B. Ni, “Anaerobic membrane bioreactors for antibiotic wastewater treatment: Performance and membrane fouling issues”, Bioresource Technology. 267:714-724, 2018.
2. W. Liu, L. Terence, M. H. Cheng, and J. F. Larry,  “Characterization of Microbial Diversity by Determining Terminal Restriction Fragment Length Polymorphisms of Genes Encoding 16S rRNA”, Applied and Environmental Microbiology. 63 (11): 4516–4522, 1997.
3. R. Chaudhary, and G. K. Maurya,  “Restriction Fragment Length Polymorphism”, In: Vonk, J., Shackelford, T. (eds) Encyclopedia of Animal Cognition and Behavior, Springer, Cham, 2019.
4. J. Zhang, Z. Zhao, C. Zhu, E.Wang, B. Brunel, S. Li, Q. Zheng, Z. Feng and H. Zhang, “Diverse Peanut Bradyrhizobial Communities in Chinese Soils: Insights from Eastern, Central, and Northern Henan Province”, Microbial Ecology, 88:65, 2025
5. S. K. Sardar, S. Kobayashi, K. Das, Y. Saito-Nakano, S. Dutta, T. Nozaki and S. Ganguly, “Development of a simple PCR-RFLP technique for detection and differentiation of E. histolytica, E. dispar and E. moshkovskii”, Parasitology Research. 122 (1):139-144, 2023
6. Y. Hao, Z. Pei, and S. M. Brown, “Methods in Microbiology. Bioinformatic Applications for the Analysis of Microbial Communities 16S rRNA-Based Approach and Taxonomic Diversity”, The Human Microbiome. 44: 1-18, 2017.
7. A. J. Martinez-Murcia, S. G. Acinas, and F. Rodriguez-Valera, “Evaluation of prokaryotic diversity by restrictase digestion of 16S rDNA directly amplified from hypersaline environments”, FEMS Microbiology Ecology 17:247-256, 1995.
8. A. A. Massol-Deya, D. A. Odelson, R. F. Hickey, J. M. Tiedje, “Bacterial community fingerprinting of amplified 16S and 16–23S ribosomal DNA gene sequences and restriction endonuclease analysis” (ARDRA). In: Akkermans, A. D. L., Van Elsas, J. D., De Bruijn, F. J. (eds). Molecular Microbial Ecology Manual. Springer, Dordrecht, 1995.
9. J. Vonk,  and T. K. Shackelford, “Encyclopedia of Animal Cognition and Behavior”, Springer Nature Switzerland AG, (eds.), 2019.
10. A. C. Obayiuwana, A. Ogunjobi, M. Yang,  and M. Ibekwe, “Characterization of bacterial communities and their antibiotic resistance profiles in wastewaters obtained from pharmaceutical facilities in Lagos and Ogun States, Nigeria”, International Journal of Environmental Research and Public Health. 15:1365-1378, 2018.
11. S.Urrea-Valencia, , D. R. Melo, P. Gonçalves, C. W. Galvão, and R. M. Etto, “Molecular Techniques to Study Microbial Wastewater Communities-Review”. Environmental Sciences. 64: e21200193, 2021.