Pre-analytical Sample Treatment and DNA Extraction Protocols for the Detection of Bacterial Pathogens from Whole Blood

Abstract

Molecular diagnostics is an increasing popular approach for the direct detection and identification of pathogenic bacteria in clinical samples. Conventional culture techniques are time-consuming and therefore causing a delay in the diagnosis of the patient. Alternative techniques based on nucleic acid amplification offer a shorter turn-around-time and the ability to identify fastidious and non-cultivable organisms. However, molecular detection of bacteria in blood, by for example PCR, RT-PCR, or sequencing of the 16S rDNA genes is often complicated by the presence of PCR-inhibitory compounds. Here we describe several different methods for the extraction of bacterial DNA from whole blood samples. The methods differ regarding costs, hands-on time as well as regarding sensitivity. In combination with a model PCR the detection limits that can be reached using the different methods range from 1,000 to 50 cfu/ml.

Development of a qualitative real-time PCR for microbiological quality control testing in mammalian cell culture production

“…Only for the study of Hansen et al . (), which used the MolYsis™ pre‐analytical sample treatment in combination with the MagNA Pure LC DNA Isolation Kit III for species‐specific detection of methicillin‐resistant S. aureus , sensitivities could be considered comparable. With respect to the detection of fungal organisms, Khot and Fredricks () compiled a comprehensive review of 23 PCR‐based assays for detection of fungal pathogens, of which only eight were using a broad‐range primer (Khot and Fredricks ), and only two studies were using real‐time PCR technology.…”

“…Though studies discussed above apparently report 'superior' sensitivities, they neither include the removal of mammalian cells, nor address the detection of fungal organisms and they sometimes needed concentration of sample prior to extraction or did not report a real NEC. Only for the study of Hansen et al (2013), which used the MolYsis TM pre-analytical sample treatment in combination with the MagNA Pure LC DNA Isolation Kit III for species-specific detection of methicillin-resistant S. aureus, sensitivities could be considered comparable. With respect to the detection of fungal organisms, Khot and Fredricks (2009) compiled a comprehensive review of 23 PCR-based assays for detection of fungal pathogens, of which only eight were using a broad-range primer (Khot and Fredricks 2009), and only two studies were using real-time PCR technology.…”

Extraction of human genomic DNA from whole blood using a magnetic microsphere method

“…For magnetic microspheres to be marketable, they need to have a uniform particle size, a strong magnetic response, good stability, and a surface rich in active functional groups. [1][2][3][4][5][6] How to improve the binding efficiency and specificity of biological macromolecules on the surfaces of magnetic microspheres and expand their scope of application has been the focus of intense research interest.…”

“…Blood is an ideal source of human genomic DNA. [1][2][3] Being able to prepare human genomic DNA from whole blood with high purity and in sufficient quantities from fresh trace blood is important both in basic science research and in the clinical setting. However, extracting genomic DNA by traditional methods is a timeconsuming process, and phenol and chloroform are toxic reagents that endanger health.…”

“…However, extracting genomic DNA by traditional methods is a timeconsuming process, and phenol and chloroform are toxic reagents that endanger health. [2][3][4][5][6] Further, traditional methods, such as phenol extraction, isopropanol precipitation, the formamide lysate method, nonorganic solvent extraction, and glass particle adsorption, have been found to be ineffective for extracting genomic DNA from trace, dried, and frozen blood. Therefore, it is necessary to find a more convenient and efficient method for obtaining human genomic DNA.…”