Last data update: May 13, 2024. (Total: 46773 publications since 2009)
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Query Trace: Munoz-Cadavid C[original query] |
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Improving molecular detection of fungal DNA in formalin-fixed paraffin-embedded (FFPE) tissues: comparison of five tissue DNA extraction methods using panfungal PCR
Munoz-Cadavid C , Rudd S , Zaki S , Patel M , Moser S , Brandt M , Gomez B . J Clin Microbiol 2010 48 (6) 2147-2153 DNA extraction from formalin-fixed paraffin-embedded (FFPE) tissues is difficult and requires special protocols in order to extract small amounts of DNA suitable for amplification. Most described methods report an amplification success rate between 60 and 80%, therefore there is a need to improve molecular detection and identification of fungi in FFPE tissue. Eighty-one archived FFPE tissues with a positive Gomori methenamine silver stain (GMS) were evaluated using five different commercial DNA extraction kits with some modifications. Three different panfungal PCR assays were used to detect fungal DNA and two housekeeping genes were used to assess the presence of amplifiable DNA and detect PCR inhibitors. The sensitivities of the five extraction protocols were compared and the quality of DNA detection (calculated for each kit as the number of housekeeping gene PCR positive / total samples) was 60-91% among the five protocols. The efficiency of the three different panfungals used (calculated as number of panfungal PCR positive / number of housekeeping gene PCR positive) was 58-93%. The panfungal PCR using ITS 3 and 4 primers yielded a product in most FFPE tissues. Two of the five DNA extraction kits (TaKaRa and QIAGEN) showed similar and promising results. However, one method (TaKaRa) could extract fungal DNA from 69 of the 74 FFPE tissues from which a housekeeping gene could be amplified, and was also cost effective, with a non-laborious protocol. Factors such as sensitivity, cost, and labor will help guide the selection of the most appropriate method for the needs of each laboratory. |
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