Türkiye'de yoncada önemli thrips türlerinin hızlı tespiti için yeni bir kapiler jel elektroforez tabanlı fragment analiz yöntemi

Abstract

Thripsler, birçok endüstriyel üründe önemli verim kayıplarına neden olur. Bu zararlılar Türkiye'deki karantina organizmaları arasında yer aldığından, etmenlerin hızlı tespiti yeni alanlara yayılmalarını önlemek için önemlidir. Mitokondriyal sitokrom oksidaz I (COI) barkodlama geninin analizleri; moleküler yöntemlerden biri olarak thrips teşhislerinde yaygın olarak kullanılmaktadır. Ancak COI geninin fragman uzunluğu çok kısa olduğundan, PCR sonrası agaroz jel üzerinde fragman boyutlarını ayırt etmek çok zordur. Bu çalışmada, daha önce farklı araştırmacılar tarafından kullanılan primer çiftleri kullanılarak Thrips tabaci Lideman, Frankliniella occidentalis (Pergande) ve Frankliniella intonsa (Trybom) türleri için Kapiler Jel Elektroforez (CGE) sistemi entegre edilerek yeni bir tanımlama yöntemi geliştirilmiştir. Analiz, özellikle kısa parça uzunluklu COI geninde birbirine yakın parça uzunluklarının ayrılmasında hata payını en aza indirerek, elde edilmiş güçlü sinyaller üretir. Bu sebeple, jel elektroforezi adımı ortadan kaldırılarak, tehlikeli kimyasallara maruz kalmadan güvenilir tespitler elde edilmiştir. Yeni yöntem, tespit süresini kısaltmış ve düşük DNA konsantrasyonuna sahip tek bir thripsin saptanmasındaki işlem hatalarını da en aza indirmiştir. Kapiler jel elektroforezi tabanlı fragman analizi ile toplam 83 thrips bireyi (52 F. intonsa, 31 F. occidentalis) tespit edilebilmiştir. Yeni yöntem, üç farklı thrips türünün tespiti için benzersiz, spesifik ve hızlı olarak değerlendirilmektedir. Ayrıca yakın gelecekte kısa fragman boyutlarına sahip farklı thrips türlerinin tanımlanmasında da kullanılabileceği düşünülmektedir.

Keywords

• Frankliniella intonsa
• Frankliniella occidentalis
• Mitokondriyal sitokrom oksidaz I
• Multipleks polimeraz zincir reaksiyonu
• Thysanoptera

A novel capillary gel electrophoresis based fragment analysis method for the rapid detection of important thrips species on alfalfa in Turkey

Abstract

Thrips cause significant yield reduction in several industrial crops. Since these pests are also included in the quarantine organisms of Turkey, the rapid detection of agents is important to prevent their spread to new areas. Mitochondrial cytochrome oxidase I (COI) barcoding gene assay; one of the molecular methods is widely used in thrips identification. However, as the COI gene has a very short fragment length, it is very difficult to distinguish fragment sizes on agarose gel after PCR. In this study, a new identification method was developed by integrating the Capillary Gel Electrophoresis (CGE) system for Thrips tabaci Lideman, Frankliniella occidentalis (Pergande) and Frankliniella intonsa (Trybom) species, using primer pairs previously used by different researchers. The assay produces strong signals obtained by minimizing the margin of error in the separation of fragment lengths close to each other, especially in the short fragment length COI gene. Therefore, by eliminating the gel electrophoresis step, reliable detections could be obtained without exposure to hazardous chemicals. The novel method shortened the detection time and minimized the process mistakes on the detection of a single thrips with a low DNA concentration. Total 83 thrips individual (52 F. intonsa, 31 F. occidentalis) were able to be detected with this capillary gel electrophoresis based fragment analysis. The novel method is evaluated as unique, specific and quick for the detection of three different thrips species. It is also thought to be able to utilize for identification of different thrips species with short fragment sizes in the foreseeable future.

Keywords

• Frankliniella intonsa
• Frankliniella occidentalis
• mitochondrial cytochrome oxidase I
• multiplex polymerase chain reaction
• Thysanoptera

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