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Saf kültür olarak stoklanmış bazı mikrofungusların ITS, β-tubulin ve Aktin gen dizilerine göre moleküler tanısı

Year 2018, Volume: 9 Issue: 1, 1 - 17, 25.04.2018

Abstract

Bu çalışmada, Trakya Üniversitesi Fen Fakültesi Biyoloji Bölümü Mikrobiyoloji laboratuvarında saf kültür olarak stoklanmış bununla beraber; cins veya tür düzeyinde morfolojik olarak teşhis edilemeyen bazı mikrofunguslar moleküler olarak tanımlanmıştır. Çalışmada kullanılan mikrofunguslar daha önce tür düzeyinde morfolojik olarak tanımlanamamış izolatlardan oluşmuştur. Örnekler, MEA besiyerine ekilip 25ºC’de 7 gün inkübe edilmiştir. Besiyerinden alınan örnekler, analiz yapılana kadar -20ºC’de saklanmıştır. DNA izolasyonu için funguslara özel, kimyasal (SDS ve CTAB), biyokimyasal (proteinazK vb.) ve fiziksel (0.1 mm çaplı boncuklar) parçalama yöntemlerini bir arada kullanan DNA izolasyon kitleri kullanılmıştır. Çeşitlilik çalışmaları, PCR tabanlı fungal çeşitlilik çalışma kitleri ile yapılmıştır. Tüm izolatlar için ITS gen dizisi kullanılmış, cinse bağlı olarak β-tubulin ve Actin gen dizilimlerini hedeflenmiştir. PCR ile çoğaltılan DNA dizilimleri “Sanger Sequencing Yöntemi” ile dizilenmiştir. Elde edilen dizilerin hangi organizmalara ait olduğu NCBI ve EBI gibi uluslararası nükleik asit data bankalarında mevcut dizilimlerle, elde edilen filotiplerin dizilimleri karşılaştırılarak belirlenmiştir. Toplam 61 mikrofungus örneği, cins düzeyinde 3 gruba (A,B,C) ayrılmış olup, gruplara göre ilgili gen bölgeleri moleküler teşhis için analiz edilmiştir. 3 grup için hem ilgili gen bölgeleri hem de ITS bölgeleri baz alınarak yapılan moleküler analizler sonucunda, 6 örneğin tür teşhisi yapılamamış, 56 tür moleküler düzeyde teşhis edilmiştir.

References

  • Abacı Ö, Haliki A. Fungal Tanıda Kullanılan Polimeraz Zincir Reaksiyonu, Orlab On-Line Mikrobiyoloji Dergisi, İzmir,2005.
  • Bridge PD, Arora DK. Interpretation of PCR methods for Species Definition, P:357, 63-84, Bridge PD (ed), CAB1 Publishing, Wallingford, 1998.
  • Edel V. Polymerase Chain Reaction İn Mycology: An Overview, P:357, Bridge PD, CAB1 Publishing, Wallingford, 1998.
  • Henry T, Iwen PC,Hinrichs SH. Identification of Aspergillus species using internal transcribed spacer regions 1 and 2. Journal of Clinical Microbiology. 38: 1510-1515, 2000.
  • Levetin E, Horner WE, Scott JA. Taxonomy of allergenic fungi. Journal of Allergy Clin Allergy Prac. 4 (3): 375-385, 2016.
  • Manian S, Sreenivasaprasad S, Mills PR. DNA extraction method for PCR in mycorrhizal fungi, 307-310. Letters in Applied Microbiology. 33 (4): 307-310, 2001.
  • Samson RA, Houbraken J, Thrane U, Frisvad JC, Andersen, B. Food and Indoor Fungi, CBS-KNAW Fungal Diversity Centre, Utrecht, The Netherlands, 2010.
  • Takamatsu S, PCR Applications in Fungal Phylogeny, P:357, 125-152, Bridge PD (ed), CABI Publishing, Wallingford, 1998.
  • Wessner DR, Dupont C, Charles TC. Microbiology. John Wiley & Sons. 867 pp + Ekler. ABD, 2013.

Molecular Based Identification of Some Stocked Microfungi as pure culture according to ITS, Beta-tubulin and Actin Genes

Year 2018, Volume: 9 Issue: 1, 1 - 17, 25.04.2018

Abstract

Some microfungi deposited in laboratuary as pure culture and unidentified morphologically as genus or species level and these fungi were identified by molecular methods in study. The used microfungi were comprised unidentifed species and can not be identify as colonially and morphologically previously. We used ITS gene for all isolates, also according to the genus we used beta-tubulin and actin genes targeting PCR based fungal biodiversity working kits. DNA sequences were sequenced by Sanger Method, and obtained sequences were analysed as bioinformatics and completed filogenetically analysis. Then samples inoculated to MEA at 25°C for 7 days. All samples obtained from MEA media were preserved at -20°C until analysis. For DNA isolation were used the spesific kits, using together digestion method such as chemical (SDS and CTAB), biochemical (proteinaseK etc.) and physical (beat with 0.1 mm diameter) were used for DNA isolation. DNA’s maintained in silica colones. In the last stage of isolation, nucleic acides were dissolved in the water DNase/Pyrogen free. After that, sequence series analysised on spectrophotometer and so determined, purity of DNA. Studies of diversity is made with PCR based fungal biodiversity working kits. After the PCR application, DNA series sequenced using by “Sanger-Sequencing Protocol”. Sequence series found that belong to which organisms on NCBI and EBI. After that, philotypes were compared with similar organisms. A total of 61 Microfungi samples , the genus level 3 groups (A, B, C) is divided , according to group related gene regions were analyzed for molecular diagnostics. Both gene regions related to 3 groups based on the results of molecular analyzes of ITS , 6 species diagnosis has not been made , for example , 56 species have been identified at the molecular level .

References

  • Abacı Ö, Haliki A. Fungal Tanıda Kullanılan Polimeraz Zincir Reaksiyonu, Orlab On-Line Mikrobiyoloji Dergisi, İzmir,2005.
  • Bridge PD, Arora DK. Interpretation of PCR methods for Species Definition, P:357, 63-84, Bridge PD (ed), CAB1 Publishing, Wallingford, 1998.
  • Edel V. Polymerase Chain Reaction İn Mycology: An Overview, P:357, Bridge PD, CAB1 Publishing, Wallingford, 1998.
  • Henry T, Iwen PC,Hinrichs SH. Identification of Aspergillus species using internal transcribed spacer regions 1 and 2. Journal of Clinical Microbiology. 38: 1510-1515, 2000.
  • Levetin E, Horner WE, Scott JA. Taxonomy of allergenic fungi. Journal of Allergy Clin Allergy Prac. 4 (3): 375-385, 2016.
  • Manian S, Sreenivasaprasad S, Mills PR. DNA extraction method for PCR in mycorrhizal fungi, 307-310. Letters in Applied Microbiology. 33 (4): 307-310, 2001.
  • Samson RA, Houbraken J, Thrane U, Frisvad JC, Andersen, B. Food and Indoor Fungi, CBS-KNAW Fungal Diversity Centre, Utrecht, The Netherlands, 2010.
  • Takamatsu S, PCR Applications in Fungal Phylogeny, P:357, 125-152, Bridge PD (ed), CABI Publishing, Wallingford, 1998.
  • Wessner DR, Dupont C, Charles TC. Microbiology. John Wiley & Sons. 867 pp + Ekler. ABD, 2013.
There are 9 citations in total.

Details

Primary Language Turkish
Journal Section MYCOLOGY
Authors

Ahmet Asan 0000-0002-4132-3848

Elcin Tuney This is me

Burhan Sen This is me

Publication Date April 25, 2018
Published in Issue Year 2018 Volume: 9 Issue: 1

Cite

APA Asan, A., Tuney, E., & Sen, B. (2018). Saf kültür olarak stoklanmış bazı mikrofungusların ITS, β-tubulin ve Aktin gen dizilerine göre moleküler tanısı. Mantar Dergisi, 9(1), 1-17. https://doi.org/10.30708/mantar.295146

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