Rapid, Accurate and Low-Cost Innovative Approach for Molecular Identification of Aspergillus species

Öz

Some of Aspergillus species are the most common food spoilage fungi contaminating economically important agricultural products. They also produce carcinogenic, teratogenic and mutagenic mycotoxins that threaten human health. For all these reasons, precise and accurate identification of Aspergillus species is of great importance. In this study, the Mycelium Tissue- Polymerase Chain Reaction (MT-PCR) based molecular technique was developed and therefore the genomic DNA isolation step which used as a template in the Polymerase Chain Reaction was eliminated. The mycelium tissue PCR-based molecular technique has some advantages. For example, it does not require expensive and specialized equipment, not require expensive chemicals such as proteinase K, RNAse, other enzymes and not use toxic chemicals such as fenol/kloroform during the process of DNA isolation. The classic genomic DNA extraction procedure based on mycelium from liquid cultivations is also relatively time-consuming. For molecular identification of Aspergillus isolates were amplified two different gene regions (β-tubulin: benA, Calmodulin: CaM) by using tissue samples in this study. Then, all PCR products were purified and were sent to base sequence analysis. Sequence analysis results were examined using bioinformatic tools and the isolates were identified to species level as Aspergillus tubingensis R. Mosseray.

Anahtar Kelimeler

• Aspergillus
• Calmodulin Gene
• Βeta-Tubulin Gene
• Polymerase Chain Reaction

Aspergillus Türlerinin Moleküler Tanımlanması için Hızlı, Doğru ve Düşük Maliyetli Yenilikçi Yaklaşım

Öz

Aspergillus türlerinin bazıları ekonomik açıdan önemli tarımsal ürünleri kontamine ederek en yaygın gıda bozulmalarına sebep olan mantarlardır. Bu türler ayrıca insan sağlığını tehdit eden karsinojenik, teratojenik ve mutajenik etkilere sahip mikotoksinleri üretmektedirler. Tüm bu nedenlerden dolayı Aspergillus türlerinin kesin ve doğru tanımlamaları büyük önem taşımaktadır. Bu çalışmada Misel Doku-Polimeraz Zincir Reaksiyon (MD-PZR) temelli moleküler teknik geliştirilerek Polimeraz Zincir Reaksiyonunda kalıp olarak kullanılan genomik DNA basamağı ortadan kaldırılmıştır. Bu misel doku-PZR temelli moleküler tekniğinin pahalı ve özel donanım gerektirmemesi, proteinaz K, RNAaz veya başka enzimler gibi pahalı kimyasallara ihtiyaç duyulmaması, genomik DNA izolasyon işlemleri sırasında fenol/kloroform gibi toksik kimyasalların kullanılmaması gibi avantajları bulunmaktadır. Ayrıca sıvı kültür ile misel üretimine dayalı klasik DNA ekstraksiyon protokolleri zaman alıcıdır. Bu çalışmada Aspergillus izolatlarının moleküler tanımlanması için misel doku örnekleri kullanılarak iki farklı gen bölgesi (β-tubulin: benA, Calmodulin: CaM) çoğaltılmıştır. Daha sonra tüm PZR ürünleri saflaştırılıp ve dizi analizine gönderilmiştir. Sekans analiz sonuçları biyoinformatik araçlar kullanılarak incelenmiş ve izolatlar tür düzeyinde Aspergillus tubingensis (Divane Asper) R. Mosseray olarak tanımlanmışlardır.

Anahtar Kelimeler

• Aspergillus
• Kalmodulin Geni
• Beta-Tubulin Geni
• Polimeraz Zincir Reaksiyonu

Destekleyen Kurum

Ege Üniversitesi, Mühendislik Fakültesi, Biyomühendislik Bölümü ve TÜBİTAK 2209-A Üniversite Öğrencileri Yurt İçi Araştırma Projeleri Destek Programı tarafından desteklenmiştir.

Teşekkür

Çalışmalarımız esnasında laboratuvar imkânlarını bizden esirgemeyen Prof. Dr. Elif Esin TUNA’ya, Arş. Gör. Dr. Arzu YILDIRIM’a çok teşekkür ederiz. Bu çalışma TÜBİTAK 2209-A Üniversite Öğrencileri Yurt İçi Araştırma Projeleri Destek Programı tarafından desteklenmiştir. Finansal desteğinden ötürü TÜBİTAK’a teşekkür ederiz.

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