Summary of the technology
The method of the invention is a solution that allows the simultaneous isolation of microbial DNA from the blood. In this method the isolation is carried out by compilation of enzymatic, mechanical and thermal lysis. This approach enables to obtain DNA from all types of organisms, irrespective of the structure of the cells.The obtained precipitate is subjected to further preparations, using a commercial DNA isolation kit according to the protocol provided by the manufacturer's procedure. The procedure results in obtaining the DNA ready for further stages of the analysis, e.g. performing a PCR reaction to detect fungi.
The most important and most difficult problem in the treatment of bloodstream infections, determining the effectiveness of therapy and, consequently, the cost and time of hospitalization, is the effective diagnosis of factors responsible for the systemic inflammatory response in the course of sepsis. Determination of etiologic factors allows for selection of appropriate antibiotic therapy. The material subjected to diagnostic testing is blood taken from the patient showing clinical signs of sepsis. Currently, the "gold standard" diagnostic method is testing for microbial growth after inoculation in culture media specific to selected pathogen groups. This method is relatively simple and inexpensive, but also time-consuming – results can be available as late as in 5 days. Moreover, identification of pathogens with this method often fails due to low sensitivity; microbial growth can be detected in only about 15-20% of the cultures.
Description of the technology
The offer is a new method to isolate microbial DNA from blood samples using combination of mechanical, thermal and enzymatic lysis. The method has been optimized for detection of blood infections caused by pathogenic bacteria and fungi so that it can be utilised to rapid isolation of high-quality material for molecular diagnostic PCR. Microbiological diagnosis of blood is one of the most problematic diagnostic challenges. The presence of bacteria (bacteremia) or fungi (fungemia) in the blood very often results in sepsis – systemic inflammation caused by the infection. Sepsis is one of the most pressing problems of modern medicine. Mortality caused by sepsis of bacterial or fungal etiology is high: in the U.S. more than 200 000 people die each year due to sepsis, and there are further 150 000 fatal cases in the European Union. Moreover, even in the developed countries sepsis occur in 2-4 newborns per 1000 and it is the main cause of newborn death.
Detection of microbiological agents in blood can be improved using molecular detection methods based on polymerase chain reaction (PCR). Sensitivity of those methods is much higher than in the case of the culture methods, while the diagnostic results are not affected by prior use of antibiotics. This allows for immediate introduction of the treatment, even before blood sampling for analysis. Unfortunately, the methods of molecular biology also have limitations. The difficulty is to isolate DNA template of adequate quality and high concentration. The cells of bacteria and fungi show different susceptibility to lysis, which is a prerequisite for obtaining DNA from them. In the case of bacteria, the cell wall of the species from the Gram-positive group is thicker and resistant to degradation, which makes it necessary to use special enzymatic lysis. The fungal cell wall, on the other hand, has completely different chemical composition than the bacterial wall, so standard procedures used with bacteria fail. In addition, the cell walls of yeast-like fungi and mould fungi are structurally different, which greatly complicates the process of DNA isolation. Molecular diagnostics is also impaired by the heme present in the blood, which is a potent inhibitor of DNA polymerases used in the PCR method. Most of the blood processing procedures available does not allow for the complete elimination of the PCR inhibition effect, which in turn may lead to a false negative diagnostic result. There are ways to reduce the inhibitory effect of heme on the reaction, such as using bovine serum albumin, glycerol or dextran, but they reduce PCR sensitivity and limit its diagnostic usefulness. The method of the invention is a solution that allows the simultaneous isolation of microbial DNA from the blood. In this method the isolation is carried out by compilation of enzymatic, mechanical and thermal lysis. This approach enables
Main advantages of its use
- Combination of enzymatic, mechanical and thermal lysis
- Increased sensitivity of micro-organism detection in the blood (detection from 100 CFU/ml of blood)
- Minimising heme concentration in the sample, so that the PCR reaction is not inhibited
- No need to use special chemical protectors DNA polymerase
- Reduced costs.
- Simplified DNA isolation method
- The subject of invention is a new method to isolate microbial DNA from blood samples to obtain a high-quality material for molecular diagnostic PCR. Thus it finds an application in the biomedical field. Users of this technology may include biotechnological companies conducting research covering DNA issues (eg. for biomedical applications).