Patojen Prototheca Türlerinin Tespitinde Konvansiyonel PCR Yönteminin Duyarlılığının Değerlendirilmesi

Abstract

Bu çalışmada, iki patojen Prototheca suşunun konvansiyonel PCR ve yeniden PCR (re-PCR) yöntemleriyle moleküler olarak tespit edilebilirlik sınırı (Limit of Detection, LOD) değerlendirilmiştir. P. ciferrii (SAG 2063) ve P. bovis (LZ-5) suşlarına ait DNA örnekleri, 1:2 oranında seri dilüsyonlara tabi tutulmuş ve PCR yöntemiyle değerlendirilmiştir. Konvansiyonel PCR ile P. ciferrii suşunda yaklaşık 9.1𝚡10-4 ng/µL, P. bovis suşunda ise yaklaşık 5.34𝚡10-6 ng/µL DNA konsantrasyonuna kadar amplifikasyon gözlenmiştir. Re-PCR uygulaması sayesinde her iki suşta da daha düşük DNA düzeylerinde bant elde edilmiştir. Bu veriler, Prototheca türlerinin çok düşük konsantrasyonlarda bile moleküler düzeyde saptanması için yeterli hassasiyete sahip olduğunu ve re-PCR uygulamasının tanısal duyarlılığı artırabileceğini göstermektedir.

Keywords

• Mikroalg
• Tanısal eşik
• Nadir patojenler
• Çevresel tespit

Evaluating the Sensitivity of Conventional PCR for Detecting Pathogenic Prototheca Species

Abstract

This study aimed to evaluate the molecular detection limit (Limit of Detection, LOD) of two pathogenic Prototheca strains using conventional PCR and re-amplification PCR (re-PCR) methods. Genomic DNA samples from Prototheca ciferrii (SAG 2063) and Prototheca bovis (LZ-5) were subjected to 1:2 serial dilutions and evaluated by PCR. In conventional PCR, amplification was observed up to approximately 9.1𝚡10-4 ng/µL for P. ciferrii and 5.34𝚡10-6 ng/µL for P. bovis. Re-PCR allowed the detection of amplification products at even lower DNA concentrations in both strains. These findings demonstrate that Prototheca species can be detected at very low DNA concentrations using molecular techniques and that re-PCR may enhance diagnostic sensitivity.

Keywords

• Microalgae
• Diagnostic threshold
• Rare pathogens
• Environmental detection

References

1. [1] Libisch, B., Picot, C., Ceballos-Garzon, A., Moravkova, M., Klimesová, M., Telkes, G., Chuang, S.-T., & Le Pape, P. 2022. Prototheca infections and ecology from a One Health perspective. Microorganisms, 10(5), 938.
2. [2] Vasco-Julio, D., Huilca-Ibarra, M., Ledesma, Y., Echeverria, G., Guerrero-Freire, S., Jagielski, T., Bastidas-Caldes, C., & de Waard, J. H. 2023. The development of a multiplex PCR assay for fast and cost-effective identification of the five most significant pathogenic Prototheca species. Pathogens, 12(8), 1018.
3. [3] Solymosi, K. 2012. Plastid structure, diversification and interconversions I: Algae. Current Chemical Biology, 6, 167–186.
4. [4] Maciszewski, K., Wilga, G., Jagielski, T., Bakuła, Z., Gawor, J., Gromadka, R., & Karnkowska, A. 2024. Reduced plastid genomes of colorless facultative pathogens Prototheca (Chlorophyta) are retained for membrane transport genes. BMC Biology, 22, 294.
5. [5] Atkinson, A. W., Gunning, B. E. S., & John, P. C. L. 1972. Sporopollenin in the cell wall of Chlorella and other algae: Ultrastructure, chemistry, and incorporation of ¹⁴C-acetate, studied in synchronous cultures. Planta, 107, 1–32.
6. [6] Pore, R. S., Barnett, E. A., Barnes, W. C., & Walker, J. D. 1983. Prototheca ecology. Mycopathologia, 81, 49–62.
7. [7] Jagielski, T., Iskra, M., Bakuła, Z., Rudna, J., Roeske, K., Nowakowska, J., Bielecki, J., & Krukowski, H. 2022. Occurrence of Prototheca microalgae in aquatic ecosystems with a description of three new species, Prototheca fontanea, Prototheca lentecrescens, and Prototheca vistulensis. Applied and Environmental Microbiology, 88(11), e01092–22.
8. [8] Leimann, B. C. Q., Monteiro, P. C. F., Lazéra, M., Ulloa Candanoza, E. R., & Wanke, B. 2004. Protothecosis. Medical Mycology, 42(2), 95–106.

9. [9] Lass-Flörl, C., & Mayr, A. 2007. Human protothecosis. Clinical Microbiology Reviews, 20(4), 230–242.
10. [10] Hillesheim, P. B., & Bahrami, S. 2011. Cutaneous protothecosis. Archives of Pathology & Laboratory Medicine, 135(7), 941–944.
11. [11] Cullen, G. D., Yetmar, Z. A., Fida, M., & Abu Saleh, O. M. 2023. Prototheca infection: A descriptive study. Open Forum Infectious Diseases, 10(6), ofad294.
12. [12] Huilca-Ibarra, M. P., Vasco-Julio, D., Ledesma, Y., Guerrero-Freire, S., Zurita, J., Castillejo, P., Barceló Blasco, F., Yanez, L., Changoluisa, D., Echeverría, G., Bastidas-Caldes, C., & de Waard, J. H. 2022. High prevalence of Prototheca bovis infection in dairy cattle with chronic mastitis in Ecuador. Veterinary Sciences, 9(12), 659.
13. [13] Jagielski, T., Dyląg, M., Roesler, U., & Murugaiyan, J. 2017. Isolation of infectious microalga Prototheca wickerhamii from a carp (Cyprinus carpio): A first confirmed case report of protothecosis in a fish. Journal of Fish Diseases, 40(10), 1417–1421.
14. [14] Stockinger, B. G., & Doster, A. R. 2017. Disseminated protothecosis in a Ruwenzori long-haired fruit bat (Rousettus lanosus). Journal of Zoo and Wildlife Medicine, 48(4), 1260–1263.
15. [15] Camboim, E. K. A., Garino, F. J., Dantas, A. F. M., Simões, S. V. D., Melo, M. A., Azevedo, E. O., Mota, R. A., & Riet-Correa, F. 2011. Protothecosis by Prototheca wickerhamii in goats. Mycoses, 54(3), e196–e200.
16. [16] Schöniger, S., Roschanski, N., Rösler, U., Vidovic, A., Nowak, M., Dietz, O., Wittenbrink, M. M., & Schoon, H.-A. 2016. Prototheca species and Pithomyces chartarum as causative agents of rhinitis and/or sinusitis in horses. Journal of Comparative Pathology, 155(2–3), 121–125.
17. [17] Macedo, J. T. S. A., Riet-Correa, F., Dantas, A. F. M., & Simões, S. V. D. 2008. Cutaneous and nasal protothecosis in a goat. Veterinary Pathology, 45(3), 352–354.
18. [18] Jagielski, T., Bakuła, Z., Gawor, J., Maciszewski, K., Kusber, W.-H., Dyląg, M., Nowakowska, J., Gromadka, R., & Karnkowska, A. 2019. The genus Prototheca (Trebouxiophyceae, Chlorophyta) revisited: Implications from molecular taxonomic studies. Algal Research, 43, 101639.
19. [19] Hunter, M. E., Dorazio, R. M., Butterfield, J. S. S., Meigs-Friend, G., Nico, L. G., & Ferrante, J. A. 2017. Detection limits of quantitative and digital PCR assays and their influence in presence–absence surveys of environmental DNA. Molecular Ecology Resources, 2,221-229.
20. [20] Chen, J., Hu, X., Li, G., Wan, P., Shao, Z., Jin, E., Liu, X., Yang, Q., Long, A., & Qian, Y. 2024. Investigation of Prototheca bovis infection and its correlation with dairy herd improvement data from a dairy farm in Central China. Veterinary Sciences, 11(1), 37.
21. [21] Capra, E., Cremonesi, P., Cortimiglia, C., Bignoli, G., Ricchi, M., Moroni, P., Pesce, A., Luini, M., & Castiglioni, B. 2014. Simultaneous identification by multiplex PCR of major Prototheca spp. isolated from bovine and buffalo intramammary infection and bulk tank. Letters in Applied Microbiology, 59(6), 642–647.
22. [22] Göksal, A. E. 2025. Isolation and Characterization of Prototheca sp. from Environmental Samples, Ege University, Graduate School of Natural and Applied Science, MSc. Thesis, 115p, Izmir.
23. [23] Mendonca, A., Santos, H., Franco-Duarte, R., & Sampaio, P. 2022. Fungal infections diagnosis–past, present and future. Research in Microbiology, 173(3), 103915.
24. [24] Sancha Dominguez, L., Cotos Suárez, A., Sánchez Ledesma, M., & Muñoz Bellido, J. L. 2024. Present and future applications of digital PCR in infectious diseases diagnosis. Diagnostics, 14(9), 931.
25. [25] Alanio, A., & Bretagne, S. 2014. Difficulties with molecular diagnostic tests for mould and yeast infections: where do we stand? Clinical Microbiology and Infection, 20, 36–41.
26. [26] Bashashati, M., & Sabouri, F. 2022. Development of two novel SYBR green-based quantitative PCR assays for detection and quantification of Mycoplasma gallisepticum and Mycoplasma synoviae. Turkish Journal of Veterinary & Animal Sciences, 46(2), Article 7.
27. [27] Alborzi, A., Larki, S., & Zeinali, A. 2020. Evaluation of larval culture and conventional PCR methods for the detection of Strongylus vulgaris in equines of Iran. Turkish Journal of Veterinary & Animal Sciences, 44(4), Article 8.
28. [28] Susever, S., & Yeğenoğlu, Y. 2011. Evaluation of the significance of molecular methods in the diagnosis of invasive fungal infections: Comparison with conventional methods. Mikrobiyoloji Bülteni, 45(2), 325–335.
29. [29] Al-Farha, A. A. B., Hemmatzadeh, F., Tearle, R., Jozani, R., Hoare, A., & Petrovskı, K. 2020. Comparison of culture and PCR for detection of field isolates of bovine milk mollicutes. Kafkas Üniversitesi Veteriner Fakültesi Dergisi, 26(3), 337–342.