tailieunhanh - Candida infections detection and epidemiology - part 3

Một số phương pháp nhanh chóng có được mô tả cho chuẩn bị mẫu máu cho PCR của Candida spp. Loại bỏ các hồng cầu bằng cách ly tâm và sử dụng của bề (plasma) 19 kết quả trong một mất hơn 50% của đầu vào ban đầu của C. albicans. | Chapter 2 C. albicans cells followed by RNA isolation NASBA amplification and enzymatic beadbased detection reproducibly a sensitivity of 1-10 cfu was obtained. Preparation of clinical samples. Blood samples were stored at -70 C until further processing. Short term storage of the blood samples had no negative effect on the sensitivity of the NASBA amplification. On the contrary lysis of blood cells was more complete and less dependent on blood composition than without storage at -70 C. Several rapid methods have been described for blood sample preparation for PCR of Candida spp. Removal of erythrocytes by centrifugation and the use of the supernatant plasma 19 resulted in a loss of more than 50 of the initial input of C. albicans. A rapid method described by Fujita12 which included lysis of erythrocytes and leukocytes in TE buffer with proteinase K and Tween 20 detergent followed by pelleting the yeast cells was not suitable for the reproducible processing of 1 ml blood because of the low numbers of yeast cells present in our samples. The first steps of the method described by Buchmann6 included lysis of blood cells by detergents breakdown of human DNA by DNase pelleting of the yeast cells followed by a labour intensive DNA extraction from spheroplasts. The method described by Van Deventer32 which was based on the Buchmann method but included glass beads for the isolation of Candida DNA was not directly suitable for processing more than 200 pl blood. We optimized this method by preceding the sample preparation with freeze thawing the sample optimizing the lysis of blood and yeast cells and the use of a RNeasy column to bind fungal nucleic acids in the presence of guanidine thiocyanate4. This resulted in a rapid sample preparation method. Using this method we were able to process 1 ml of blood with a sensitivity of 1 cfu per ml of whole blood in a reproducible manner Fig. 2 . The data obtained by agarose gel electrophoresis were confirmed by an enzymatic bead assay