Optimization of PCR Primers for Detection of Extended-Spectrum Beta-Lactamase Targeting CTX-M and TEM Genes
DOI:
https://doi.org/10.5614/3bio.2025.7.1.5Keywords:
Polymer Chain Reaction, primer optimization, in silico analysis, CTX-M , TEM genesAbstract
Extended-Spectrum Beta Lactamase (ESBL) is an enzyme that inhibit the activity of third-generation cephalosporin antibiotics. CTX-M and TEM are the genes encoding the ESBLs. The horizontal spread of these gene from one bacteria to another leads to increased bacterial resistance. Molecular-based bacterial identification methods such as Polymer Chain Reaction are methods that are currently used because they provide faster and more specific results. Therefore, in this study, a method for identifying bacteria that produce ESBL was created by targeting CTX-M and TEM genes. In this study, two pairs of primers were designed using in silico method, then the characteristics of the primers were analyzed. The primer annealing temperature was optimized using 55and 60oC temperatures. The results of the in silico analysis showed that both pairs of primers met the ideal characteristics of a primer, as the Tm, %GC content, and secondary product values fulfilled the required criteria. Meanwhile, the results of the primer annealing temperature optimization indicated that the optimal temperature for the PCR method using both primers was 60C.
References
] Aboumarzouk OM. Extended spectrum beta-lactamase urinary tract infections. Urology Ann. 2014 [cited 2024 Des 13];6(2):114-5. doi:10.4103/0974-7796.130641.
] Liakopoulos A, Mevius D, Ceccarelli D. A review of SHV extended-spectrum beta-lactamase: neglected yet ubiquitous. Front Microbiol. 2016 [cited 2024 Des 25];7:1374. doi:10.3389/fmicb.2016.01374.
] Alby K, Miller MB. Mechanism and Detection of Antimicrobial Resistance. In: Carroll KC, Pfaller MA, Landry ML, et al., editors. Manual of Clinical Microbiology. 12th ed. Washington, DC: ASM Press; 2019. p. 1253-77.
] Joyanes P, Conejo MC, Martez-Martez L, Perea EJ. Evaluation of the VITEK 2 system for the identification and susceptibility testing of three species of nonfermenting gram-negative rods frequently isolated from clinical samples. J Clin Microbiol. 2001 Sep;39(9):3247?53. doi:10.1128/JCM.39.9.3247-3253.2001.
] Severin JA, Mertaniasih NM, Kuntaman K, Lestari ES, Purwanta M, Lemmens-Den Toom N, Duerink DO, Hadi U, van Belkum A, Verbrugh HA, Goessens WH; Study Group ?Antimicrobial Resistance in Indonesia: Prevalence and Prevention? (AMRIN). Molecular isolates from Surabaya, Indonesia. J Antimicrob Chemother. 2010 Mar;65(3):465?9. doi: 10.1093/jac/ dkp471. PMID: 20053690.
] Ghafourian S, Sadeghifard N, Soheili S, Sekawi
Z. Extended spectrum beta-lactamases: definition, classification and epidemiology. Curr Issues Mol Biol. 2015 [cited 2024 Nov 1];17:11-22. doi:10.21775/
cimb.017.011.
] Rohit A, Deekshit VK, Balaraj M, Shetty VA, Abraham
G. CTX-M type extended-spectrum ?-lactamase in Escherichia coli isolated from extra-intestinal infections in a tertiary care hospital in south India. Indian J Med Res. 2019 [cited 2024 Nov 1];149:281-4. doi:10.4103/
ijmr.IJMR_191_17.
] Tamma PD, Aitken SL, Bonomo RA, Mathers AJ, van Duin D, Clancy CJ. Treatment of Extended-Spectrum ?-lactamase Producing Enterobacterales (ESBL-E), Carbapenem-Resistant Enterobacterales (CRE), and Pseudomonas aeruginosa with Difficult-to-Treat Resistance (DTR-P. aeruginosa). Clin Infect Dis. 2022 [cited 2024 Nov 5];75:187-212. doi:10.1093/cid/ciac268.
] Nachimuthu R, Kannan VR, Bozdogan B, Krishnakumar V, Pandian K, Manohar P. CTX-M-type ESBL-mediated resistance to third-generation cephalosporins and conjugative transfer of resistance in Gram-negative bacteria isolated from hospitals in Tamil Nadu, India. Access Microbiol. 2021 [cited 2025 Jan 25];3:000142. doi:10.1099/acmi.0.000142.
] Basu C, editor. PCR Primer Design. 2nd ed. New York: Humana Press; 2015 [cited 2024 Nov5]. doi:10.1007/978- 1-4939-2365-6.
] Takahashi M, Morikawa K, Akao T. Short-length Homologous Region exhaustive Search algorithm (SHRS): A primer design algorithm for differentiating bacteria at the species, subspecies, or strain level based on a whole genome sequence. J Microbiol Methods. 2022 [cited 2024 Nov 5];203:106605. doi:10.1016/j. mimet.2022.106605.
] Nadipineni C. Guide for Designing Species Specific Primer. J Plant Physiol Pathol. 2021 [cited 2023 Jan 25];9(7):123-34. doi:10.4172/2329-955X.1000167.
] Khaira A, Achyar A, Zulyusri, Atifah Y, Putri DH, Violita. Primer Design and Optimization of Annealing Temperature for Analysis of Glutathione Reductase Gene Expression in Rice (Oryza sativa L.). J Biol Sci Technol Manag.2023 [cited 2024 Nov 5];5(1):142-8. doi:10.5614/3bio.2023.5.1.3.
] Clark DP, Pazdernik NJ, McGehee MR. Molecular Biology. 3rd ed. London: Academic Cell; 2019 [cited 2024 November 13]. doi:10.1016/C2017-0-01404-5.
] Chakraborty AK, Nandi S. A Method of Universal Primer Design for the Detection of Diverged CTX-M Beta- Lactamases in Multi-Drug Resistant Superbugs. STM J. 2019; [cited 2024 Nov 13]9(2):1-6.
] Bevan ER, McNally A, Thomas CM, Piddock LJ, Hawkey PM. Acquisition and Loss of CTX-M-Producing and Non-producing Escherichia coli in the Fecal Microbiome of Travelers to South Asia. mSphere. 2018; [cited 2024 Nov 13]. 3(6):9-18.doi:10.1128/mSphere.00695-18.
] Husna A, Rahman M, Badruzzaman AT, Sikder MH, Islam MR, Rahman T, et al. Extended-Spectrum ?-Lactamases (ESBL): Challenges and Opportunities. Biomedicines. 2023; [cited 2024 Nov 13]; 11(10):2937.. doi:10.3390/
biomedicines11102937.
] Nomura K, Onda K, Murase H, Hashiya F, Ono Y, Terai G, et al. Development of PCR Primers Enabling the Design of Flexible Sticky Ends for Efficient Concatenation of Long DNA Fragments. RSC Chem Biol. 2024; [cited 2025 Jan 27];5:360-71.. doi:10.1039/D3CB00277A.
] Yu K, Karwowska S, Sharma A, Liesenfeld O, Scudder SA. Polymerase Chain Reaction. In: Jorgensen JT, editor. Companion and Complementary Diagnostics: From Biomarker Discovery to Clinical Implementation. London: Academic Press; 2019. [cited 2022 Jan 27]; p.
-102.. doi:10.1016/B978-0-12-813539-3.00005-9.
] Purwakasih DB, Achyar A. Primer Design and in Silico PCR for Detection of Shigella sp. on Refilled Water Samples. Serambi Biol. 2021 [cited 2025 Jan 27];6(1):1-
] Bajpai, T., Pandey, M., Varma, M., & Bhatambare, G.S. Prevalence of TEM, SHV, and CTX-M Beta-lactamase Genes in the Urinary Isolates of a Tertiary Care Hospital. Avicenna J Med. 2017 [cited 2025 Jan 25]; 7(1):12?16. DOI: 10.4103/2231-0770.197508.
] Mann, T., Humbert, R., Dorschner, M., Stamatoyannopoulos, J., & Noble, W.S. A thermodynamic approach to PCR primer design. Nucleic Acids Res. 2009 [cited 2025 Jan 25]; 37(13):e95. DOI: 10.1093/nar/
gkp366.
] Mlynarcik, P., Roderova, M., & Kolar, M. Primer Evaluation for PCR and Its Application for Detection of Carbapenemases in Enterobacteriaceae. Jundishapur J Microbiol. 2016 [cited 2025 3 Jan 25]; 9(1):e29314. DOI: 10.5812/jjm.29314.
Downloads
Published
Issue
Section
License
Copyright (c) 2025 Dwi Elfira Kurniati, Catur Riani, Renny Hardiyanti, Dian Rachmawati
This work is licensed under a Creative Commons Attribution 4.0 International License.
Authors retain copyright and grant the journal right of first publication with the work simultaneously licensed under a Creative Commons Attribution (CC-BY) 4.0 License that allows others to share the work with an acknowledgment of the work’s authorship and initial publication in this journal.
