Validation of Genetic Markers for AmpC, AAC, and RNR3 Genes in Potentially Present Pathogens Organisms in Fecal Sludge
DOI:
https://doi.org/10.33394/bioscientist.v11i2.9623Keywords:
Pathogen, PCR, Web-Based Bioinformatics Programs, Oligonucleotide, Molecular.Abstract
The issue of sanitation and water pollution due to fecal waste has become a serious concern in Indonesia, particularly after it was revealed that approximately 70% of the community's drinking water sources are contaminated with human feces. Determining safe waste management requirements necessitates indicators confirming the contamination-free nature of the waste management methods. Pathogen testing indicators for fecal waste are highly diverse. The current standard testing is the culture-based method for microorganisms and microscopy observation for helminths. However, molecular detection tools for these pathogens can give higher sensitivity and specificity in a relatively shorter time of technical processing. In this research, we designed primers for molecular-based diagnostics on three pathogens exhibiting extreme survival capabilities under various environmental conditions. The species-coupled genes are AmpC for Escherichia coli, AAC for Mycobacterium tuberculosis, and RNR3 for Ascaris lumbricoides. The design of molecular-based biomarkers and their specificity tests were conducted using web-based bioinformatics programs to prevent unintended dimers. Ultimately, primers were validated by conventional gradient PCR on available positive controls and a sample from processed fecal sludge. The PCR validation test showed that the designed primers were validated to amplify the intended target with the right size without unspecific bands on the positive controls. Meanwhile, the AAC gene for E. coli was detected in the sample from the fecal sludge.References
Babben, S., Perovic, D., Koch, M., & Ordon, F. (2015). An Efficient Approach for the Development of Locus Specific Primers in Bread Wheat (Triticum aestivum L.) and Its Application to Re-Sequencing of Genes Involved in Frost Tolerance. Plos One, 10(11), e0142746. https://doi.org/10.1371/journal.pone.0142746
Bakhtiarizadeh, M. R., Najaf-Panah, M. J., Mousapour, H., & Salami, S. A. (2016). Versatility of Different Melting Temperature (Tm) Calculator Software for Robust PCR and Real-Time PCR Oligonucleotide Design: A Practical Guide. Gene Reports, 2, 1-3. https://doi.org/10.1016/j.genrep.2015.11.001
Barbosa, V. B., de Oliveira Martins, E., & Weber, G. (2019). Nearest-Neighbour Parameters Optimized for Melting Temperature Prediction of DNA/RNA Hybrids at High and Low Salt Concentrations. Biophysical Chemistry, 251, 106189. https://doi.org/10.1016/j.bpc.2019.106189
Benito, M., Menacho, C., Chueca, P., Ormad, M. P., & Goñi, P. (2020). Seeking the Reuse of Effluents and Sludge from Conventional Wastewater Treatment Plants: Analysis of the Presence of Intestinal Protozoa and Nematode Eggs. Journal of Environmental Management, 261, 110268. https://doi.org/10.1016/j.jenvman.2020.110268
Chahal, C., van den Akker, B., Young, F., Franco, C., Blackbeard, J., & Monis, P. (2016). Pathogen and Particle Associations in Wastewater. Advances in Applied Microbiology, 97, 63-119. https://doi.org/10.1016/bs.aambs.2016.08.001
Coleman, J. P., & Smith, C. J. (2014). Microbial Resistance☆. In Reference Module in Biomedical Sciences. Elsevier. https://doi.org/10.1016/B978-0-12-801238-3.05148-5
Das, K., Chowdhury, P., & Ganguly, S. (2015). Internal Transcribed Spacer 1 (ITS1) Based Sequence Typing Reveals Phylogenetically Distinct Ascaris Population. Computational and Structural Biotechnology Journal, 13, 478-483. https://doi.org/10.1016/j.csbj.2015.08.006
Davi, M. J. P., Jeronimo, S. M. B., Lima, J. P. M. S., & Lanza, D. C. F. (2021). Design and In Silico Validation of Polymerase Chain Reaction Primers to Detect Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Scientific Reports, 11(1), Article 1. https://doi.org/10.1038/s41598-021-91817-9
Fadila, R. H. (2021). Pemanfaatan Lumpur Tinja sebagai Pupuk Kompos pada Instalasi Pengolahan Lumpur Tinja (IPLT) Pulo Gebang. Thesis. Universitas Sahid Jakarta.
Fitriana, S., Dian, M. A. P., & Setiawan, A. (2017). Pemanfaatan Tinja Menjadi Pupuk Cair Organik di Kelurahan Tambakrejo. E-Dimas, 8(1), 96-103. https://doi.org/10.26877/e-dimas.v8i1.1378
Gupta, G., Tak, V., & Mathur, P. (2014). Detection of AmpC β Lactamases in Gram-Negative Bacteria. Journal of Laboratory Physicians, 6(1), 1-6. https://doi.org/10.4103/0974-2727.129082
Hata, H., Kitajima, T., & Suyama, A. (2018). Influence of Thermodynamically Unfavorable Secondary Structures on DNA Hybridization Kinetics. Nucleic Acids Research, 46(2), 782. https://doi.org/10.1093/nar/gkx1171
Hendling, M., & Barišić, I. (2019). In-Silico Design of DNA Oligonucleotides: Challenges and Approaches. Computational and Structural Biotechnology Journal, 17, 1056-1065. https://doi.org/10.1016/j.csbj.2019.07.008
Irianto, J., Zahra., Hananta, M., Anwar, A., Yunianto, A., Azhar, K., Lestary, H., Cahyorini., Laelasari, E., Marina, R., Sari, P., Sugiharti., Widjiastuti, B., Setiadi, T. R. S., Agita, S., Rachmat, B., Lasut, D., Hermawan, A., Puspita, T., & Widyastuti, A. (2021). Studi Kualitas Air Minum Rumah Tangga Tahun 2020. Laporan Akhir. Pusat Penelitian dan Pengembangan.
Joshi, R. S., Jamdhade, M. D., Sonawane, M. S., & Giri, A. P. (2013). Resistome Analysis of Mycobacterium tuberculosis: Identification of Aminoglycoside 2’-Nacetyltransferase (AAC) as Co-Target for Drug Desigining. Bioinformation, 9(4), 174-181. https://doi.org/10.6026/97320630009174
Koh, C. S., & Sarin, L. P. (2018). Transfer RNA Modification and Infection – Implications for Pathogenicity and Host Responses. Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms, 1861(4), 419-432. https://doi.org/10.1016/j.bbagrm.2018.01.015
Kon, H., Lurie-Weinberger, M., Cohen, A., Metsamber, L., Keren-Paz, A., Schwartz, D., Carmeli, Y., & Schechner, V. (2023). Occurrence, Typing, and Resistance Genes of ESBL/AmpC-Producing Enterobacterales in Fresh Vegetables Purchased in Central Israel. Antibiotics (Basel, Switzerland), 12(10), 1528. https://doi.org/10.3390/antibiotics12101528
Krzyzanowski, F., Zappelini, L., Martone-Rocha, S., Dropa, M., Matté, M. H., Nacache, F., & Razzolini, M. T. P. (2014). Quantification and Characterization of Salmonella spp. Isolates in Sewage Sludge with Potential Usage in Agriculture. BMC Microbiology, 14(1), 263. https://doi.org/10.1186/s12866-014-0263-x
Lubis, M. F. (2019). Pembuatan Briket Berbahan Dasar Slack di PDAM Tirtanadi Instalasi Pengolahan Lumpur Tinja Cemara. Thesis. Universitas Sumatera Utara.
Medeiros, P. H. Q. S., Lima, A. Â. M., Guedes, M. M., Havt, A., Bona, M. D., Rey, L. C., Soares, A. M., Guerrant, R. L., Weigl, B. H., & Lima, I. F. N. (2018). Molecular Characterization of Virulence and Antimicrobial Resistance Profile of Shigella Species Isolated from Children with Moderate to Severe Diarrhea in Northeastern Brazil. Diagnostic Microbiology and Infectious Disease, 90(3), 198-205. https://doi.org/10.1016/j.diagmicrobio.2017.11.002
Miranda, P., & Weber, G. (2021). Thermodynamic Evaluation of the Impact of DNA Mismatches in PCR-Type SARS-CoV-2 Primers and Probes. Molecular and Cellular Probes, 56, 101707. https://doi.org/10.1016/j.mcp.2021.101707
Nurfadillah, A., Wahdaniar., Musyahadah, U. S. A., Amir, N. I., Irma, A., Miladiarsi., & Adri, T. A. (2023). Prevalence of CTX-M Gene as an ESBL Marker in Clinical Klebsiella pneumoniae Isolates. Journal of Health Sciences and Medical Development, 2(01), 20-26. https://doi.org/10.56741/hesmed.v2i01.164
Park, M., Won, J., Choi, B. Y., & Lee, C. J. (2020). Optimization of Primer Sets and Detection Protocols for SARS-CoV-2 of Coronavirus Disease 2019 (Covid-19) Using PCR and Real-Time PCR. Experimental & Molecular Medicine, 52(6), Article 6. https://doi.org/10.1038/s12276-020-0452-7
Rychlik, W., Spencer, W. J., & Rhoads, R. E. (1990). Optimization of the Annealing Temperature for DNA Amplification In Vitro. Nucleic Acids Research, 18(21), 6409-6412. https://doi.org/10.1093/nar/18.21.6409
SantaLucia, J., & Hicks, D. (2004). The Thermodynamics of DNA Structural Motifs. Annual Review of Biophysics and Biomolecular Structure, 33(1), 415-440. https://doi.org/10.1146/annurev.biophys.32.110601.141800
Sanz-GarcÃa, F., Anoz-Carbonell, E., Pérez-Herrán, E., MartÃn, C., LucÃa, A., Rodrigues, L., & AÃnsa, J. A. (2019). Mycobacterial Aminoglycoside Acetyltransferases: A Little of Drug Resistance, and a Lot of Other Roles. Frontiers in Microbiology, 10, 46. https://doi.org/10.3389/fmicb.2019.00046
Sari, N. I. P., Mertaniasih, N. M., Soedarsono., & Maruyama, F. (2019). Application of Serial Tests for Mycobacterium tuberculosis Detection to Active Lung Tuberculosis Cases in Indonesia. BMC Research Notes, 12(1), 313. https://doi.org/10.1186/s13104-019-4350-9
Sinthawaty, N. (2021). Retrieved November 10, 2023, from BPK Perwakilan Provinsi Lampung. Interactwebsite: https://lampung.bpk.go.id/pemerintah-pusat-bangun-instalasi-pengolahan-lumpur-tinja-iplt-di-bakung/
Ulaganeethi, R., Rajkumari, N., Saya, G. K., & Dorairajan, G. (2023). Retrieved November 10, 2023, from NCBI Nucleotide Database. Interactwebsite: http://www.ncbi.nlm.nih.gov/nuccore/OQ778747.1
Untergasser, A., Cutcutache, I., Koressaar, T., Ye, J., Faircloth, B. C., Remm, M., & Rozen, S. G. (2012). Primer3-New Capabilities and Interfaces. Nucleic Acids Research, 40(15), e115. https://doi.org/10.1093/nar/gks596
Wang, Z., Moffitt, A. B., Andrews, P., Wigler, M., & Levy, D. (2022). Accurate Measurement of Microsatellite Length by Disrupting its Tandem Repeat Structure. Nucleic Acids Research, 50(20), e116. https://doi.org/10.1093/nar/gkac723
Ye, J., Coulouris, G., Zaretskaya, I., Cutcutache, I., Rozen, S., & Madden, T. L. (2012). Primer-BLAST: A Tool to Design Target-Specific Primers for Polymerase Chain Reaction. BMC Bioinformatics, 13, 134. https://doi.org/10.1186/1471-2105-13-134
Yesudas, J. P., Blinov, N., Dew, S. K., & Kovalenko, A. (2015). Calculation of Binding Free Energy of Short Double Stranded Oligonucleotides Using MM/3D-RISM-KH Approach. Journal of Molecular Liquids, 201, 68-76. https://doi.org/10.1016/j.molliq.2014.11.017
