Diagnosis of Peach latent mosaic viroid (PLMVd) and Hop stunt viroid (HSVd) by RT-PCR using different extraction protocols

Abstract

RT-PCR method was performed using four different nucleic acid extraction methods to identify viroids; peach latent mosaic viroid (PLMVd) and hop stunt viroid (HSVd) which causes serious damage to stone fruits. Leaf samples were collected from fruit orchards showing viroid-like symptoms in İzmir province. Silica capture, citric buffer, lithium chloride, and ames buffer methods were used to extract total nucleic acids. The four extraction methods were compared using samples collected during the vegetation period from naturally infected trees (plum, apricot, and peach). They were evaluated with RT-PCR tests. In 64 stone fruit tree samples, only the silica capture method gave reliable and accurate results in RT-PCR molecular tests for the detection of PLMVd and HSVd. The other wielded nucleic acid extraction methods were found to be ineffective for the isolation of the viroid RNAs.

Keywords

• TNA extraction
• RT-PCR
• viroid
• stone fruit
• nucleic acid

Peach latent mosaic viroid (PLMVd) ve Hop stunt viroid (HSVd)’in farklı ekstraksiyon metotları kullanılarak RT-PCR ile teşhisi

Abstract

Sert çekirdekli meyve ağaçlarında ekonomik zarara neden olan peach latent mosaic viroid (PLMVd) ve hop stunt viroid (HSVd)’leri RT-PCR ile tanılamak için 4 farklı nükleik asit ekstraksiyon yöntemi kullanılmıştır. Yaprak örnekleri İzmir ilinde farklı meyve bahçelerinden viroid benzeri belirti gösteren ağaçlardan alınmıştır. Silica capture, lithium chloride, citric buffer ve ames buffer yöntemleri kullanılarak total nükleik asit ekstraksiyonu gerçekleştirilmiştir. Dört farklı ekstraksiyon yöntemi erik, kayısı ve şeftali ağaçlarından farklı dönemlerde toplanan örnekler kullanılarak birbiri ile karşılaştırılmıştır. Altmış dört farklı sert çekirdekli bitki örneği RT-PCR test yöntemi ile değerlendirilmiş ve silica capture ekstraksiyon yöntemi, kullanılan yöntemler arasında en güvenilir sonucu vermiştir. Diğer 3 ekstraksiyon yöntemi ile yapılan ekstraksiyonlarda istenilen sonuç alınamamıştır.

Keywords

• TNA ekstraksiyonu
• RT-PCR
• viroid
• sert çekirdekli meyve
• nükleik asit

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