1. Storage of the samples (Area 1)
5 ml of Ethylenediaminetetraacetic acid (EDTA)- anticoagulated blood samples are shipped on dry ice to the laboratory. Within one hour after arrival at the hospital`s receiving desk samples will be stored in a -86°C freezer (Forma Scientific, Marietta, USA) until preparation of the samples. Freezers are locked and secured by a temperature alarm system giving a noise signal at a temperature above -70°C.
2. Registration of the samples (Area 2)
Shipment and receiving data (including any problems during transportation process) will be recorded for each individual parcel/sample.
Samples are registered by the PILZDATA program based on Novell/Windows. Access to the data is possible for 24 h a day for the responsible persons of this study and the laboratory technician. All samples are registered also in a registration book as a backup including sample number, date of arrival, date of preparation and remarks.
3. DNA extraction of patient samples (Area 3)
- Lysis of the erythrocytes with the hypotonic Red cell lysis buffer
|Tris pH 7,6 (Merck)||10 mM|
|Magnesium chloride (Merck)||5 mM|
|Sodium chloride (Merck)||10 mM|
Store at 4°C, expiration date 3 months.
40 ml Red cell lysis buffer are added to the 5 ml blood sample and incubated on a horizontal shaker for 10 min, then centrifuged for 10 min at 1500 g. Then the supernatant is decanted and the pellet resuspended. This process has to be repeated once.
- Lysis of the leukocytes with White cell lysis buffer containing Proteinase K
|Tris pH 7,6 (Merck)||10 mM|
|EDTA pH 8 (Merck)||10 mM|
|Sodium chloride (Merck)||50 mM|
|Proteinase K (Boehringer Mannheim)||200 µg/ml|
Store at -20°C, expiration date 3 months
1 ml White cell lysis buffer is added to the pellet. The sample is incubated for 45 min at 65°C and then centrifuged for 10 min at 2000 g. The supernatant has to be decanted and the pellet resuspended.
- Recombinant lyticase buffer for generating sphaeroblasts
|Tris pH 7,5 (Merck)||50 mM|
|EDTA (Merck)||1 mM|
|ß-mercaptoethanol (Sigma)||0.2 %|
|Recombinant lyticase (Sigma)||1 U / 100 µl|
Store at -20°C, expiration date 3 months
500 µl lyticase buffer (5 U) is added to the pellet. The sample is incubated for 45 min at 37°C and then centrifuged for 10 min at 2500 g. The supernatant has to be decanted and the pellet resuspended.
- QIAmp DNA Mini Kit (Qiagen No. 51306) for sphaeroblast lysis and protein precipitation
180 µl ATL-buffer (Kit) and 20 µl Proteinase K (Kit) are added to the sample and incubated for 20 min at 55°C. Then, 200 µl AL-buffer (Kit) is added and incubated for 10 min at 70°C (Lysis and precipitation).
After that, 210 µl 100% Ethanol (Merck) is added to the sample.
The whole volume has to be pipetted into the spin column and centrifuged for 1 min at 5000 g.
500 µl AW1-buffer (Kit) is pipetted into the column, centrifuged for 1 min at 5000 g. This has to be repeated once with AW2-buffer (Washing steps).
50µl AE-buffer (Kit) is pipetted into the column, centrifuged for 1 min at 5000 g (first elution), then 50 µl AE-buffer is pipetted again into the column and centrifuged for 1 min at 9000 g into the same Eppendorf cup (second elution).
The extracted DNA can be stored at -80°C in the freezer (Area 1).
4. DNA extraction of positive controls (Area 4)
DSM-Strains (German Collection of Microorganisms) of the medically important fungal species of Aspergillus (A. fumigatus, A. flavus, A. niger, A. terreus, A. versicolor) and Candida (C. albicans, C. tropicalis, C. glabrata, C. krusei, C. parapsilosis) are permanently grown at 30°C in the laboratories of the Department of Medical Microbiology on Sabouraud-Glucose-Agar (Becton Dickinson, Heidelberg, No. 4354083). For Candida spp. fungal cultures are inoculated into sterile NaCl-Solution. For Aspergillus spp. plates are covered with 10 ml sterile NaCl-Solution. The optical densities of the solutions are measured in a spectral photometer against BaCl2 and H2SO4 (Mc Farland-Index 0.5 = 106 fungal cells). Serial dilutions are performed from 105-100 including dilutions at 10, 5 and 1 CFU/ml. Blood samples (5 ml) from healthy volunteers are spiked with the fungal suspensions (500 µl). DNA is extracted immediately in Area 4 using the protocol above. DNA is transfer into Area 1 for storage.
5. Storage of the extracted DNA (Area 1)
All extracted DNA is stored in a -86°C freezer (Forma Scientific, Marietta, USA) until preparation of the samples. Freezers are locked and secured by a temperature alarm system giving a noise signal at a temperature above -70°C. For storage purpose, numbered boxes with a letter/number file system are used.
6. Photometric quantitation of extracted DNA (Area 3)
DNA is quantitated in a spectral photometer (Dr. Lange, Berlin). All samples are measured at 260 nm (DNA), 270 nm (RNA), 280 nm (proteins) and 320 nm. Results are recorded on prints and saved in a book together with the patient numbers, respectively. All samples are diluted to a final concentration of 100 ng/µl genomic DNA. 100 ng/µl DNA are run in the PCR assay.
7. PCR (Area 5)
Before entering Area 5 people must wear one-way visitor gowns. Everybody preparing a PCR must wear sterile latex surgical gloves, face masks, single-use gowns and hair net. Instead of H2O ice we use thermo-isolation blocks.
All reagents necessary for PCR are stored in a separate freezer. New lot numbers (Taq-polymerase, nucleotides) are tested for their sensitivity, respectively.
Temperature profile of the PCR:
|1 x||94°C||4 min|
|34 x||94°C||30 s|
|34 x||62°C||1 min|
|34 x||72°C||2 min|
|1 x||72°C||5 min|
Final PCR volume: 50 µl (10 µl sample + 40 µl PCR reaction)
Primer sequences (made by Roth):
Binding in a conserved region of the 18ssu rRNA gene of fungi
5`- ATT GGA GGG CAA GTC TGG TG
5`- CCG ATC CCT AGT CGG CAT AG
2.Exon of the DR-B gene, human
5`- ACT GGA ACA GCC ACA AGG AC
5`- CCG CTG CAC TGT GAA GCT CT
PCR reaction mix: PCR ELISA (Dig labeling, Nr. 1636120, Boehringer Mannheim)
|Reagents||Final concentration||Final Volume|
|PCR buffer, 10 x||1 x||5 µl|
|MgCl2||1 mM||2 µl|
|Dig labeling mix||200 µM||5 µl|
|Primers||25 pM each||1 µl, 1 µl|
|Taq-Polymerase||2,5 U||0.3 µl|
PCR is performed in a GeneAmp 9600 from Perkin Elmer.
8. Agarose gel electrophoresis (Area 6)
Sample are either stored at -20°C overnight or prepared immediately in Area 6 for gel electrophoresis:
10 µl PCR product are mixed with 2.5 µl 5 x buffer.
The 100 bp marker from Life Technologies is used as a control.
5 x buffer:
|Tris (Merck)||50 mM|
|EDTA (Merck)||50 mM|
|Bromphenol blue (Sigma)||0.1%|
We are running a 2% agarose gel (Agarose Type II from Sigma) in 1 x TAE-buffer. The 10 x TAE-buffer is commercially available from Life Technologies, Karlsruhe.
The GelStar DNA staining (FMC Bioproducts) is used in a 1:10000 dilution before pipetting the gel.
The electrophoresis is run in a Horizon 11.14 chamber (Life Technologies) at 90 V for 2 h.
DNA is visualized by UV-light (Fluolink, Biometra) and photographed by a Polaroid camera. Photos are stored in a book.
9. PCR-ELISA (Area 6)
All samples are hybridized in the PCR-ELISA assay with the A. fumigatus and the Candida spp. oligo.
Oligonucleotides: Biotin-labeled at 3` (made by Roth)
Candida spp: 5`- GGA CCA TCG TAA TGA TTA ATA GGG ACG
A. fumigatus: 5`- CAT GGC CTT CAC TGG CTG TGG GGG GAA CCA
Dig detection, Nr. 1636111, Boehringer Mannheim
2 x 20 µl of the PCR product of all samples are pipetted into two Eppendorf cups, respectively.
10 µl of the control DNA (delivered by Boehringer Mannheim) in the dilutions 1:1, 1:10, 1:100 is pipetted into Eppendorf cups.
Then 20 µl denaturation solution (Kit) is added to each well, incubation for 10 min at room temperature.
Preparation of the hybridization solution:
oligos are labeled at 3`with biotin.
for the Candida spp. oligonucleotide:
add 30% DMSO (Sigma) to hybridization solution
Add 200 µl hybridization solution (Kit) to each Eppendorf cup, pipette 200 µl hybridization mixture into the ELISA wells, incubation for 3 h at 40°C on a shaker.
Decant the hybridization mixtures, add 250 µl washing solution (Kit), wash 3 times at 40°C.
Dilute the anti-Dig-POD (Kit) 1:100 in conjugate buffer (at least 1 hour before), add 200 µl to each well, incubation for 30 min at 40°C on a shaker.
Decant the conjugate solution, add 250 µl washing solution (Kit), wash 6 times at 40°C.
Prepare the substrate solution (Kit), 1 ABTS /5 ml substrate buffer (at least 30 min before).
Add 200 µl substrate solution (Kit) to each well, incubation for 30 min at 40°C on a shaker.
Read the adsorbance at 405 nm (reference filter 492 nm) in a SLT Rainbow reader.