Streptococcus pneumoniae detection in blood
a streptococcus pneumoniae and probe technology, applied in the field of primers and probes for the detection of streptococcus pneumoniae, can solve the problems of invasiveness and inability to perform routinely, affecting the diagnosis of pneumococcal pneumonia, and difficult to establish definitive diagnosis, so as to reduce the chance of false positive results
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example 1
[0096]Blood Contamination Using a S. pneumoniae Culture in Stationary Phase and Contaminated Blood Using a S. pneumoniae Culture in Exponential Phase
[0097]S. pneumonia serotype 1 (code 070799) was cultured in exponential and in stationary phase. Culture technique of S. pneumonia is known for the person skilled in the art. From each culture, 10−1, 10−2, 10′13, 10−4, 10−5 and 10−6 dilutions were prepared in Todd Hewitt Broth medium.
[0098]Whole blood samples were collected from voluntary humans into collection tubes containing EDTAK2 anticoagulant. Aliquots were generated from the collected samples such as each aliquot contained 2 ml of blood. The aliquots were then contaminated by the different dilutions of S. pneumonia cultured in exponential and in stationary phase. The contaminated samples were then frozen at −80° C.
example 2
Total RNA Extraction and Purification
[0099]Contaminated blood samples were defrosted by placing them in a mixture of ice and water for a period of 90 minutes. Afterwards, an equal volume of heated acidic phenol was added to each sample. The mixture of the sample and the acidic phenol was then mixed about 5 min at 65° C. The mixture was then centrifuges 20 min at 14.000 g at 4° C. 500 μl of the supernatant were then collected. RNA was purified from the obtained supernatant using the RNeasy midi kit of Qiagen according to the manufacturer's instructions. The purity of the isolated RNA was then monitored using spectrophotometric measurements.
example 3
[0100]cDNA Production
[0101]10 μl of the purified DNA are mixed with 1 μl of the random primers. Secondary structures are then denaturated in a thermocycler for 5 min at 70° C. Afterwards, 14 μl of mastermix is added and retro transcription is performed for 10 min at 25° C. followed by a cycle of 60 min at 50° C. The Go script reverse Transcriptase® of Promega was used for retro-transcription of the purified RNA.
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