Evcil Hayvanlardan İzole Edilen Mantarların Teşhisinde Kullanılan Konvansiyonel ve ITS Dizi Analizi Metotlarının Karşılaştırılması

Bu çalışmanın amacı, evcil hayvanlardan izole edilen mantar türlerinin identifikasyonunda altın standart olan konvansiyonel kültür yöntemi ve Internal Ara Bölgeler (ITS) dizi analizi yönteminin karşılaştırılmasıdır. Çalışmada, mantar enfeksiyonlarından şüphelenilen toplam 35 hayvan (15 kedi, 11 köpek, 4 at ve 5 tavuk) incelendi. 35 örnek içinde 20 tanesinin mantar kültürleri pozitif bulundu. Pozitif örnekler arasında 8 (%40) tanesi konvansiyonel yöntemlerle ağırlıklı olarak dermatofit türleri olarak bulundu. İzolatların ITS bölgeleri (ITS1, ITS2 ve ITS bölgesinin tamamı) amplifiye edildi, sekanslandı ve elde edilen sonuçlar kültür yöntemiyle karşılaştırıldı. Hem konvansiyonel hem de dizi analizi yöntemiyle elde edilen identifikasyon sonuçları 18 (%90) örnekte birbiri ile uyumlu bulundu. Bu sonuçlar doğrultusunda, ITS bölgesinin tamamının dizi analizi, mantarların hızlı teşhisi için kullanılabileceğini ortaya koydu. ITS gen bölgesinin tamamına ait dizi analizi sonuçlarının fenotipik identifikasyon sonuçları ile uyumlu olması, ITS gen bölgesi dizi analizinin konvansiyonel yöntemlerin doğrulanmasında bir araç olarak kullanılabileceğini ortaya koymuştur.

Comparison of Internal Transcribed Spacer Region Sequencing and Conventional Methods Used in the Identification of Fungi Isolated from Domestic Animals

The aim of this study was to compare gold standard conventional culture method and internal transcribed spacer (ITS) sequence-basedanalysis in the identification of fungi isolated from domestic animals. A total of 35 animals (15 cats, 11 dogs, 4 horses and 5 chickens)suspected for fungal infection were examined. Of the 35 samples, 20 were found to be positive for fungal culture. Among positive samples8 (40%) were predominantly found to be dermatophyte species by conventional methods. The ITS regions (ITS1, ITS2 and complete ITS) offungal isolates were also amplified, sequenced and the results were compared with conventional culture method. The identification resultsof 18 (90%) fungal species were found to be compatible with both conventional culture and sequencing methods. Comparison of the resultsdemonstrated that, complete ITS regions gene sequencing could be used for the identification of medically important fungi rapidly. Sincethe results of complete ITS regions gene sequencing were found to be compatible with the results of phenotypic identification, it can beconcluded that ITS regions gene sequencing of fungal isolates can be also used as a confirmative tool of conventional culture methods.

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  • 1. Xu J: Fungal DNA barcoding. Genome, 59, 913-932, 2016. DOI: 10.1139/ gen-2016-0046
  • 2. Irinyi L, Serena C, Garcia-Hermoso D, Arabatzis M, Desnos-Ollivier M, Vu D, Cardinali G, Arthur I, Normand AC, Giraldo A, da Cunha KC, Sandoval-Denis M, Hendrickx M, Nishikaku AS, Melo ASD, Merseguel KB, Khan A, Parente Rocha JA, Sampaio P, Briones MRD, Ferreira RCE, Muniz MD, Castanon-Olivares LR, Estrada-Barcenas D, Cassagne C, Mary C, Duan SY, Kong FR, Sun AY, Zeng XY, Zhao ZT, Gantois N, Botterel F, Robbertse B, Schoch C, Gams W, Ellis D, Halliday C, Chen S, Sorrell TC, Piarroux R, Colombo AL, Pais C, de Hoog S, Zancope- Oliveira RM, Taylor ML, Toriello C, Soares CMD, Delhaes L, Stubbe D, Dromer F, Ranque S, Guarro J, Cano-Lira JF, Robert V, Velegraki A, Meyer W: International Society of Human and Animal Mycology (ISHAM)- ITS reference DNA barcoding database-the quality controlled standard tool for routine identification of human and animal pathogenic fungi. Med Mycol, 53 (4): 313-337, 2015. DOI: 10.1093/mmy/myv008
  • 3. Irınyi L, Lackner M, De Hoog GS, Meyer W: DNA barcoding of fungi causing infections in humans and animals. Fungal Biol, 120 (2): 125-136, 2016. DOI: 10.1016/j.funbio.2015.04.007
  • 4. Kiraz N, Oz Y, Aslan H, Erturan Z, Ener B, Akdagli SA, Muslumanoglu H, Cetinkaya Z: Is the extraction by Whatman FTA filter matrix technology and sequencing of large ribosomal subunit D1-D2 region sufficient for identification of clinical fungi? Mycoses, 58 (10): 588-597, 2015. DOI: 10.1111/myc.12365
  • 5. Winn WC JR, Allen SD, Janda WM, Koneman E, Procop G, Schreckenberger P, Woods G: Laboratory approach to the diagnosis of fungal infections. In, Koneman’s Color Atlas and Textbook of Diagnostic Microbiology. 6th ed., 1156-1166, Lippincott Williams, Philadelphia, 2006.
  • 6. Winn WC Jr AS, Janda WM, Koneman E, Procop G, Schreckenberger P, Woods G: Hyaline molds and hyalohyphomycosis. In, Koneman’s Color Atlas and Textbook of Diagnostic Microbiology. 6th ed., 1172-1187, Lippincott Williams, Philadelphia, 2006.
  • 7. Liu D, Coloe S, Baird R, Pedersen J: Rapid mini-preparation of fungal DNA for PCR. J Clin Microbiol, 38(1): 471, 2000.
  • 8. Kumar M, Shukla PK: Use of PCR targeting of internal transcribed spacer regions and single-stranded conformation polymorphism analysis of sequence variation in different regions of rRNA genes in fungi for rapid diagnosis of mycotic keratitis. J Clin Microbiol, 43 (2): 662-668, 2005. DOI: 10.1128/Jcm.43.2.662-668.2005
  • 9. CLSI: Interpretive Criteria for Identification of Bacteria and Fungi by DNA Target Sequencing; Approved Guideline. In, Institute CaLS, (Ed): CLSI document MM18-A. Wayne, PA ABD 2008.
  • 10. Raja HA, Miller AN, Pearce CJ, Oberlies NH: Fungal identification using molecular tools: A primer for the natural products research community. J Nad Prod, 80, 756-770, 2017. DOI: 10.1021/acs.jnatprod.6b01085
  • 11. Pryce TM, Palladino S, Kay ID, Coombs GW: Rapid identification of fungi by sequencing the ITS1 and ITS2 regions using an automated capillary electrophoresis system. Med Mycol, 41 (5): 369-381, 2003. DOI: 10.1080/13693780310001600435
  • 12. Sulaiman IM, Jacobs E, Simpson S, Kerdahi K: Molecular identification of isolated fungi from unopened containers of greek yogurt by DNA sequencing of internal transcribed spacer region. Pathogens, 3 (3): 499- 509, 2014. DOI: 10.3390/pathogens3030499
  • 13. Wagner K, Springer B, Pires VP, Keller PM: Molecular detection of fungal pathogens in clinical specimens by 18S rDNA high-throughput screening in comparison to ITS PCR and culture. Sci Rep, 8:6964, 2018. DOI: 10.1038/s41598-018-25129-w
  • 14. Ninet B, Jan I, Bontems O, Lechenne B, Jousson O, Panizzon R, Lew D, Monod M: Identification of dermatophyte species by 28S ribosomal DNA sequencing with a commercial kit. J Clin Microbiol, 41 (2): 826-830, 2003. DOI: 10.1128/Jcm.41.2.826-830.2003
  • 15. Bialek R, Ibricevic A, Fothergill A, Begerow D: Small subunit ribosomal DNA sequence shows Paracoccidioides brasiliensis closely related to Blastomyces dermatitidis. J Clin Microbiol, 38 (9): 3190-3193, 2000.
  • 16. Bretagne S, Costa JM, Marmoratkhuong A, Poron F, Cordonnier C, Vidaud M, Fleuryfeith J: Detection of aspergillus species DNA in bronchoalveolar lavage samples by competitive PCR. J Clin Microbiol, 33 (5): 1164-1168, 1995.
  • 17. Einsele H, Hebart H, Roller G, Loffler J, Rothenhofer I, Muller CA, Bowden RA, vanBurik JA, Engelhard D, Kanz L, Schumacher U: Detection and identification of fungal pathogens in blood by using molecular probes. J Clin Microbiol, 35 (6): 1353-1360, 1997.
  • 18. Henry T, Iwen PC, Hinrichs SH: Identification of Aspergillus species using internal transcribed spacer regions 1 and 2. J Clin Microbiol, 38 (4): 1510-1515, 2000.
  • 19. Chou HH, Wu WS: Phylogenetic analysis of internal transcribed spacer regions of the genus Alternaria, and the significance of filament-beaked conidia. Mycol Res, 106, 164-169, 2002. DOI: 10.1017/S0953756201005317
  • 20. Kusaba M, Tsuge T: Phylogeny of Alternaria fungi known to produce host-specific toxins on the basis of variation in internal transcribed spacers of ribosomal DNA. Curr Genet, 28 (5): 491-498, 1995. DOI: 10.1007/ Bf00310821
  • 21. Vujanovic V, Labrecque M: Potentially pathogenic and biocontrol Ascomycota associated with green wall structures of basket willow (Salix viminalis L.) revealed by phenotypic characters and ITS phylogeny. Biocontrol, 53 (2): 413-426, 2008. DOI: 10.1007/s10526-007-9092-2
  • 22. Spicker AR: Dermatophytosis. http://www.cfsph.iastate.edu/ DiseaseInfo/factsheets.php; 2013. Accessed: 05.12.2018
  • 23. Kwiatkowski NP, Babiker WM, Merz WG, Carroll KC, Zhang SX: Evaluation of nucleic acid sequencing of the D1/D2 region of the large subunit of the 28S rDNA and the internal transcribed spacer region using SmartGenes IDNS Software for identification of filamentous fungi in a clinical laboratory. J M
Kafkas Üniversitesi Veteriner Fakültesi Dergisi-Cover
  • ISSN: 1300-6045
  • Yayın Aralığı: Yılda 6 Sayı
  • Başlangıç: 1995
  • Yayıncı: Kafkas Üniv. Veteriner Fak.
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