Ceren ÖZKUL

Dışkı Örneklerinde Real-Time PCR Analizleri için Bakteriyel DNA Izolasyon Yöntemlerinin Karşılaştırılması

Moleküler mikrobiyoloji çalışmalarında en önemli basamak yüksek kalitede yeterli DNA eldesidir. Bu çalışmada iki hazır ticari izolasyon kiti (QIAamp DNA Stool Mini Kit, Qiagen and PSP Spin Stool DNA Plus Kit, Inivtek) ve modifiye fenol-kloroform ekstraksiyon yöntemi Real-Time qPCR analizleri için belirli bakterilerin dışkıdan izolasyonundaki etkinleri bakımından karşılaştırılmıştır. DNA miktarı Invitek kitinde en yüksek oranda elde edilmiştir. Dışkıya ve PCR karışımına bilinen miktarda hücre ve DNA eklenme deneyleri ve qPCR sonuçları fenol-kloroform ekstraksiyon yönteminin etkinliğinin düşük olduğunu göstermiştir. Hazır ticari dışkıdan izolasyon kitleri DNA eldesi ve saflığı açsıından qPCR deneylerinde kullanılmak üzere daha doğru sonuçlar vermektedir.

Comparison of Bacterial DNA Extraction Methods From Stool Samples for Quantitative Real-Time PCR Analysis

The most crucial step in molecular microbiology studies is choosing an appropriate procedure to obtain sufficient, highquality DNA. Two commercially available kits (QIAamp DNA Stool Mini Kit, Qiagen and PSP Spin Stool DNA Plus Kit, Invitek)and a modified in-house phenol-chloroform extraction method was compared for their efficiency in isolation of certainbacteria from human stool samples for Real-Time qPCR. DNA yield was significantly higher in Invitek when compared to other methods. Spiking experiments and qPCR results revealed that efficiency was in phenol-chloroform extraction was lowerwhen compared to other two commercial kits. Commercial kits have better results in terms of DNA recovery and purity, andhave more accurate results for qPCR experiments.

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