Thursday, September 8, 2011

Direct molecular detection of bacterial DNA in blood.

Today, pathogen-specific and broad-range PCR are employed for direct detection of bacterial DNA in blood. These assays have high sensitivity and allow fast identification because lack of the need of blood culture. The main pathogen-specific PCR disadvantageis that it is effective only when a specific infection is suspected, and in that way is not good in general diagnosis of febrile patients (Peters at al., 2004)
Nevertheless, multiplex PCR assays are available for these cases. Primers for multiplex detection can be included in a single reaction tube what allows to determine several bacteria in a sample (Singh et al., 2006)
PCR with universal primers that is often called broad-range PCR assay has been developed. The principle of that procedure is detection 16S rRNA bacteria gene. This gene is present in all bacteria and encodes the same product in all bacteria, whereas numbers of copies of these genes can be various (Barken et al., 2007)
The 16S rRNA method allows to detect any bacterial DNA present in blood, including not only living bacteria, but dead and even degraded bacteria. This means that broad-range PCR has high sensitivity, and can be used for detection only of real bacteremia. With PCR using the laboratory exists contamination of blood samples.
In any way this problem can be solved by correct blood collection, accurate DNA extraction and careful control of reagents (Peters at al., 2004)
The products of PCR can be identified by capillary sequencing or pyrosequencing.
Sequencing is an effective technique for analysis. This automatic technique does not allow identification of multiple pathogens.
Pyrosequencing is another method that has recently emerged as a rapid real time technique for sequencing of short DNA fragments. It is useful in classification, recognition, sub-typing and SNP-typing of bacteria (Barken et al., 2007)
In summary, all pluses and minuses of molecular detection techniques of bacteria in blood are presented in (table 9).
Beyond all questions every detection method has advantages and disadvantages, but in future present rates of molecular diagnostics development can allow to create optimal method for detection of all bacterial infections in blood.
Furthermore all molecular methods used for detection and identification of bacteria in blood or blood cultures are limited in identification of increasing resistance to antibiotics.

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