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2003, Cilt 33, Sayı 1, Sayfa(lar) 066-070
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Comparison of Two Different Extraction Methods in Isolation of DNA from Fungi and Interpretation of Sequence Analysis
Nilgün Işık
İstanbul Üniversitesi İstanbul Tıp Fakültesi, Mikrobiyoloji ve Klinik Mikrobiyoloji Anabilim Dalı, İstanbul
Keywords: Fungi, extraction techniques

In immunsuppressed patients, particularly in leukemia, lymphoma, and transplantation patient groups, fungal infections are known to cause high mortality and morbidity rates. Classical diagnosis tests such as hemoculture and serology are not sufficiently specific and sensitive in early diagnosis of fungal infections. For this reason currently molecular biological methods that are more specific and sensitive are in used. In this study, blood samples with EDTA from 120 immunsuppressed patients were studied; DNA was isolated from the blood samples by using DNAzol method and commercial DNA extraction kit (Qiagen) samples on which PCR had done were monitorized on %2 agarose gel. Positive samples were noted to give 500 bp single bands, which is specific to fungal products on agarose gel. Qiagen purification kits were used for analyses of the samples. Amounts of PCR products were measured and mixed with %96 and %70 percent alcohol. After that DNA filtered from columns were purified. PCR products were labeled by using Qiagen Dye Ex kit. Sequence analyses were performed by ABI Prism377 (Perkin Elmer) device. Sequence analyses were evaluated by using blast programme and the samples evaluated, as positive samples were observed to posses gene sequences specific to fungus species. When the results of two different extraction techniques, applied to 120 blood samples, were compared, it is observed that Qiagen extraction techniques showed better results in DNA isolation. 20 positive samples were found by Qiagen techniques and only 5 were found by DNAzol techniques. This shows us, in choosing the extraction techniques used for diagnosis of infection agents, besides cost effectiveness the techniques, methods which are more specific and sensitive, should be chosen. Following extraction (Qiagen) and PCR, 20 positive samples were found 35% of the positivities were found to be A.niger, 60% A.fumigatus and 5% mixed infections (A.niger/A.fumigatus). Recently, particularly in studies, in determination of whether PCR products are really the target strain or not, it is stressed that sequence analysis has to be done and verified. In our study, primarily two of the extraction techniques used in DNA isolation for diagnosis of fungal infections, were compared. Qiagen techniques were used in extractions, which are expensive but have better PCR results than DNAzol techniques. The products specific to 18S rRNA gene sequences in PCR results were observed to give single band evaluated by sequence analysis, positive samples were observed to posses gene sequences from A.fumigatus and A.niger species.

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Ana Sayfa | Dergi Hakkında | Yayın Kurulu | Telif Hakkı Devir Formu | Arşiv | Yayın Arama | Yazarlara Bilgi | Etik Politikalar | İletişim