Preliminary evaluation of a SYBR Green-based screening approach for selected fungal targets in simulated cerebrospinal fluid samples

Elif Ayça Şahin; Ayşe Kalkancı; Emrah Çeltikçi; Mustafa Çağlar Şahin; Işıl Fidan; Kayhan Çağlar

Preliminary evaluation of a SYBR Green-based screening approach for selected fungal targets in simulated cerebrospinal fluid samples

Abstract

Aims: Most commercial multiplex polymerase chain reaction (PCR) panels primarily detect bacterial and viral pathogens and usually include only Cryptococcus among fungal targets. However, other fungi such as Candida and Aspergillus are increasingly recognized as important causes of central nervous system (CNS) infections, especially in immunocompromised patients. This study aimed to preliminarily develop a multiplex real-time PCR approach for the detection of selected fungal targets in cerebrospinal fluid (CSF)-based samples. The assay was initially designed as a SYBR Green-based screening method; however, because overlapping melting temperatures limited reliable target discrimination, a three-target probe-based confirmation step was subsequently incorporated for Candida spp., Aspergillus spp., and Fusarium spp. Methods: Target-specific primers were used for Candida spp., Aspergillus spp., Fusarium spp., Mucor spp., Rhizopus spp., and Trichosporon spp. in the SYBR Green-based screening step. In addition, probe-based primer/probe sets were evaluated for Candida spp., Aspergillus spp., and Fusarium spp. to improve target-specific discrimination. The assay was evaluated using CSF-based simulated samples and tested against selected bacterial backgrounds. Results: The SYBR Green-based screening step produced amplification signals for the selected fungal targets, but melting curve analysis alone was insufficient for reliable discrimination because of overlapping Tm values. In the probe-based format, Candida spp., Aspergillus spp., and Fusarium spp. generated target-specific signals in their assigned fluorescence channels, with no off-target amplification in the tested bacterial background. These findings support the added value of probe-based confirmation while highlighting the preliminary nature of the assay. Conclusion: This preliminary study suggests that an in-house multiplex real-time PCR approach may provide a feasible framework for detecting selected fungal targets in CSF-based simulated samples. While SYBR Green-based amplification enabled initial screening, overlapping melting temperatures limited reliable target discrimination. Probe-based testing in a limited subset suggested a potential role in reducing interpretive ambiguity. Further validation with clinically positive CSF samples is required before clinical implementation.

Keywords

Supporting Institution

This study was funded by the Scientific Research Projects Unit of Gazi University.

Project Number

TSG-2022-7508

Ethical Statement

This study was approved by the Gazi University Clinical Research Ethics Committee (No/Date:71/24.01.2022).

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Details

Primary Language

English

Subjects

Medical Infection Agents, Medical Mycology

Journal Section

Research Article

Authors

Elif Ayça Şahin ^*
0000-0002-2215-2756
Türkiye

Ayşe Kalkancı
0000-0003-0961-7325
Türkiye

Emrah Çeltikçi
0000-0001-5733-7542
Türkiye

Mustafa Çağlar Şahin
0000-0002-5141-8154
Türkiye

Işıl Fidan
0000-0002-2552-7971
Türkiye

Kayhan Çağlar
0000-0001-7257-6453
Türkiye

Publication Date

July 20, 2026

Submission Date

April 26, 2026

Acceptance Date

June 29, 2026

Published in Issue

Year 2026 Volume: 9 Number: 4

IZ

https://izlik.org/JA87EC32PT

Cite

RIS / Bibtex

APA

Şahin, E. A., Kalkancı, A., Çeltikçi, E., Şahin, M. Ç., Fidan, I., & Çağlar, K. (2026). Preliminary evaluation of a SYBR Green-based screening approach for selected fungal targets in simulated cerebrospinal fluid samples. Journal of Health Sciences and Medicine, 9(4), 1104-1111. https://izlik.org/JA87EC32PT

AMA

1.Şahin EA, Kalkancı A, Çeltikçi E, Şahin MÇ, Fidan I, Çağlar K. Preliminary evaluation of a SYBR Green-based screening approach for selected fungal targets in simulated cerebrospinal fluid samples. J Health Sci Med / JHSM. 2026;9(4):1104-1111. https://izlik.org/JA87EC32PT

Chicago

Şahin, Elif Ayça, Ayşe Kalkancı, Emrah Çeltikçi, Mustafa Çağlar Şahin, Işıl Fidan, and Kayhan Çağlar. 2026. “Preliminary Evaluation of a SYBR Green-Based Screening Approach for Selected Fungal Targets in Simulated Cerebrospinal Fluid Samples”. Journal of Health Sciences and Medicine 9 (4): 1104-11. https://izlik.org/JA87EC32PT.

EndNote

Şahin EA, Kalkancı A, Çeltikçi E, Şahin MÇ, Fidan I, Çağlar K (July 1, 2026) Preliminary evaluation of a SYBR Green-based screening approach for selected fungal targets in simulated cerebrospinal fluid samples. Journal of Health Sciences and Medicine 9 4 1104–1111.

IEEE

[1]E. A. Şahin, A. Kalkancı, E. Çeltikçi, M. Ç. Şahin, I. Fidan, and K. Çağlar, “Preliminary evaluation of a SYBR Green-based screening approach for selected fungal targets in simulated cerebrospinal fluid samples”, J Health Sci Med / JHSM, vol. 9, no. 4, pp. 1104–1111, July 2026, [Online]. Available: https://izlik.org/JA87EC32PT

ISNAD

Şahin, Elif Ayça - Kalkancı, Ayşe - Çeltikçi, Emrah - Şahin, Mustafa Çağlar - Fidan, Işıl - Çağlar, Kayhan. “Preliminary Evaluation of a SYBR Green-Based Screening Approach for Selected Fungal Targets in Simulated Cerebrospinal Fluid Samples”. Journal of Health Sciences and Medicine 9/4 (July 1, 2026): 1104-1111. https://izlik.org/JA87EC32PT.

JAMA

1.Şahin EA, Kalkancı A, Çeltikçi E, Şahin MÇ, Fidan I, Çağlar K. Preliminary evaluation of a SYBR Green-based screening approach for selected fungal targets in simulated cerebrospinal fluid samples. J Health Sci Med / JHSM. 2026;9:1104–1111.

MLA

Şahin, Elif Ayça, et al. “Preliminary Evaluation of a SYBR Green-Based Screening Approach for Selected Fungal Targets in Simulated Cerebrospinal Fluid Samples”. Journal of Health Sciences and Medicine, vol. 9, no. 4, July 2026, pp. 1104-11, https://izlik.org/JA87EC32PT.

Vancouver

1.Elif Ayça Şahin, Ayşe Kalkancı, Emrah Çeltikçi, Mustafa Çağlar Şahin, Işıl Fidan, Kayhan Çağlar. Preliminary evaluation of a SYBR Green-based screening approach for selected fungal targets in simulated cerebrospinal fluid samples. J Health Sci Med / JHSM [Internet]. 2026 Jul. 1;9(4):1104-11. Available from: https://izlik.org/JA87EC32PT