Molecular Detection of Staphylococcus Aureus: A Performance Comparison of Colony PCR and Conventional PCR Targeting the icaB Gene

Authors

• Farah Badri Abed, Higher Institute of Forensic Sciences, Al-Nahrain University, Jadriya, Baghdad, IraqFollow
• Shahrazad Hazim Muhi, Higher Institute of Forensic Sciences, Al-Nahrain University, Jadriya, Baghdad, IraqFollow
• Sahar Mustafa Ibrahim, Higher Institute of Forensic Sciences, Al-Nahrain University, Jadriya, Baghdad, IraqFollow
• Riyam Alaa Mohammed, ASCO Learning Center, Baghdad, IraqFollow

Article Type

Article

Abstract

Background: Staphylococcus aureus is an important nursing disease that requires a prompt and effective diagnosis to implement a strategy to curb its transmission and spread. Although the gold standard is the conventional PCR using column-purified genomic DNA, Colony PCR with crude cell lysates saves a lot of time and cost of the test and its performance features have not been well established.
Objectives: This paper will compare Colony PCR (boiling method) and Conventional PCR (ABIOpuretm kit extraction) to amplify icaB gene (141 bp), which is a major determinant of biofilm formation in S. aureus and then assess their application with respect to downstream application.
Methods: Clinical and reference S. aureus isolates (n = 9) underwent parallel DNA preparation: direct colony boiling in TE buffer versus column purification. PCR amplification employed a standardized thermal profile (95 °C denaturation, 57 °C annealing, 72 °C extension; 30 cycles). Amplicons were resolved on 1.5% agarose gels stained with ethidium bromide. DNA concentration was measured by fluorometry, and amplification quality was assessed by band intensity, smear background, and reproducibility.
Results: the two techniques demonstrated 100 and 100 percent efficiency in the detection of the target icaB and 141 bp bands were clear in all the isolates. Colony PCR had a processing time that was cut by half but had unstable band intensities and low-molecular-weight smears that showed the presence of residual proteins and fragmented DNA. The traditional PCR generated clean and consistent high-intensity bands, with little background. Crude lysates did not have proper quantification and purification of their DNA, which restricted their use in the quantitative PCR and Next-Generation Sequencing.
Conclusions: Colony PCR offers a fast and inexpensive method of qualitative screening of S. aureus genes, but its inconsistent quality of template and carryover of the inhibitor limits its application in quantitative and genomic studies. There are still assays that require tight quantification and high-fidelity sequencing and column-purified DNA is vital. The performance difference as well as the applicability of Colony PCR can be improved by optimizing the rapid cleanup protocols in the future.

Keywords

Staphylococcus aureus, Colony PCR, Conventional PCR, icaB gene, DNA extraction

Recommended Citation

Abed, Farah Badri; Muhi, Shahrazad Hazim; Ibrahim, Sahar Mustafa; and Mohammed, Riyam Alaa (2026) "Molecular Detection of Staphylococcus Aureus: A Performance Comparison of Colony PCR and Conventional PCR Targeting the icaB Gene," Muthanna Medical Journal: Vol. 13: Iss. 3, Article 12.
Available at: https://muthmj.mu.edu.iq/journal/vol13/iss3/12

Creative Commons License

This work is licensed under a Creative Commons Attribution 4.0 International License.