organism detected in blood culture: Negative organism detected using array: Negative
Growth protocol
Blood samples were drawn into aerobic and anaerobic blood culture bottles (BacT/Alert®, bioMérieux, France) and were incubated in the blood culturing equipment BacT/ALERT 3 D (bioMérieux) for up to 5 or 6 days, at which time they were reported as negative when no sign of micro-organism growth was detected. If during the cultivation period possible growth was observed by the blood culturing instrument, it was identified and reported according to CLSI guidelines.
Extracted molecule
genomic DNA
Extraction protocol
The DNA was extracted using an automated nucleic acid extraction instrument Nuclisens® easyMAG™ (bioMérieux, France) according to the manufacturer’s protocol (Generic 1.0.6). The elution volume was 55 µl.
Label
biotin
Label protocol
The PCR reaction mixture contained 1 uM of gBF primer mixture (Metabion, Germany), 1 uM of biotin-labeled gBR primer mixture (Metabion, Germany), 0.165 µM of SaurF primer (Metabion, Germany), 0.165 µM of biotin-labeled SaurR primer (Metabion, Germany), 0.25 µM of mecAF primer (Metabion, Germany), 0.25 µM biotin-labeled mecAR primer (Metabion, Germany), 1x Hot Start Taq® PCR buffer (Qiagen, Germany), in which the final concentration MgCl2was 2.0 mM, 300 uM of each of dNTP (Finnzymes, Finland), 1.5 g/l BSA (EuroClone, Italy), 0.125 U/ul Hot Start Taq® DNA polymerase (Qiagen, Germany), 1.5 ul of isolated DNA, and water to bring the total volume to 15 ul. In the blood culture dataset, 1.5 ul of PCR control template was added in the reaction and the equivalent amount of water was reduced. A negative control, i.e., sterile water was included in each test series. All the reverse primers were biotinylated at their respective 5′-end. The PCR was performed using a Mastercycler® epgradient S thermal cycler (Eppendorf, Germany). The following PCR program was used: a denaturation step at 95°C for 15 minutes, 36 cycles of 10 seconds at 96°C, 35 seconds at 52°C, 10 seconds at 72°C, 5 cycles of 5 seconds at 96°C, 30 seconds at 65°C, 5 cycles of 5 seconds at 96°C and finally 30 seconds at 68°C. After the PCR, the success of the amplification of double-stranded DNA and single-stranded DNA was ascertained by gel electrophoresis using a 2% agarose gel containing SYBR® Green II (Invitrogen, USA) or using Agilent BioAnalyzer (Agilent Technologies, USA).
Hybridization protocol
All incubation steps except that of the last precipitation reaction were performed under continuous agitation of 550 rpm at 25°C. Briefly, a first a prewash with 500 µl of water from 30 to 55°C for 5 to 10 minutes was done. Hybridization at 55°C for 10 minutes, of 1 ul of the biotinylated target and 99 ul hybridization buffer (250 mM Na2HPO4, 4.5% SDS, 1mM EDTA, 1xSSC) took place on a microarray. When hybridization control oligonucleotides were included, they were added to the hybridization buffer. After hybridization, the microarray was washed in 500 µl of 0.2xSCC at 20°C for 5 minutes. Incubation with 100 µl of blocking buffer (2% milk powder, 6xSSPE, 0.005% Triton-X100) was performed for 5 minutes at 30°C. Then 100 µl of 1:5000 dilution of streptavidin-conjugated horseradish peroxidase in PBS was applied for 10 minutes at 30°C followed by a similar washing step as described above. Finally, 100 µl of 3, 3′, 5, 5′-tetramethylbenzidine (TMB) analog (Seramun Grün; Seramun Diagnostica, Germany) was added for the precipitation reaction at 25°C for 10 minutes.
Scan protocol
Microarray images were generated by ATR-01 Reader (Clondiag).
Description
no additional information
Data processing
The array images were analyzed with the Prove-it™ Advisor software (Mobidiag, Finland, www.mobidiag.com). The software performed image analyses and result reporting, including the identification of the bacterial targets and the evaluation of the control probes. This took place automatically without user involvement in adjusting any of the parameters. The target identifications were made by software using multiple parameters such as signals from the probe oligonucleotides on the array. These were interpreted using built-in rules and parameters specific for each assay type. All the probes for a specific bacterial target were required to be positive for that target to be classified as positively identified, except for the CNS probes of which only 2 of 4 specific oligonucleotides were required to be positive. If both CNS and S. epidermidis probes in the analyses were positive, only S. epidermidis was reported due to its identification by species-specific probes. The original array layout contained spots, which were not included in the final probe panel. The software reports signal intensities as percentages between 0-100%. The scanner gives 8-bit images. Median signal intensity reported in the data table (these are the raw data). The processed data are composed of ORGANISM NAME or NEGATIVE answers depending if the pathogen is detected in the sample or not. The processed data are linked as a supplementary file on the Series GSE17221 record.
