Maxim Brevnov

A sample extraction method for faster, more sensitive PCR-based detection of pathogens in blood culture

Three mechanistically different sample extraction methodologies, namely, silica spin columns, phenol-chloroform, and an automated magnetic capture of polymer-complexed DNA (via an Automate Express instrument), were compared for their abilities to purify nucleic acids from blood culture fluids for use in TaqMan assays for detection of Staphylococcus aureus. The extracts from silica columns required 100- to 1000-fold dilutions to sufficiently reduce the powerful PCR inhibitory effects of the anticoagulant sodium polyanetholsulfonate, a common additive in blood culture media. In contrast, samples extracted by either phenol-chloroform or the Automate Express instrument required little or no dilution, respectively, allowing for an approximate 100-fold improvement in assay sensitivity. Analysis of 60 blood culture bottles indicated that these latter two methodologies could be used to detect lower numbers of pathogens and that a growing S. aureus culture could be detected 2 hours earlier than when using silica columns. Of the three tested methodologies, the Automate Express instrument had the shortest time to result, requiring only approximately 80 minutes to process 12 samples. These findings highlight the importance of considering the mechanism when selecting a DNA extraction methodology, given that certain PCR inhibitors act in a similar fashion to DNA in certain chemical environments, resulting in copurification, whereas other methodologies use different chemistries that have advantages during the DNA purification of certain types of samples.

Automated Extraction of DNA from Forensic Sample Types Using the PrepFiler Automated Forensic DNA Extraction Kit

The HID EVOlution—Extraction System (Tecan Group Ltd., Mannedorf, Switzerland) was developed to automate DNA extraction from biological samples using the PrepFiler Automated Forensic DNA Extraction Kit (Applied Biosystems, Foster City, CA). The system consists of a Tecan Freedom EVO 150 robot (Tecan Group Ltd., Mannedorf, Switzerland), a graphical user interface designed for use with Freedom EVOware software v 2.1 SPI (Tecan Group Ltd., Mannedorf, Switerland) as well as instrument hardware and plastic to support the PrepFiler reagents and protocol. The DNA quality and quantity obtained were comparable to that observed with the corresponding manual extraction protocol. Purified DNA was free of inhibitors and ready for downstream applications, such as real-time quantitative PCR and PCR for short tandem repeat (STR) analysis. The DNA quantity and quality obtained were consistent as demonstrated by the quantification and STR results. Our studies indicate that the HID EVOlution—Extraction System can easily be adopted in forensic laboratories to alleviate some of the bottlenecks of sample preparation in forensic laboratories.

AutoMate Express™ Forensic DNA Extraction System for the Extraction of Genomic DNA from Biological Samples*

Abstract:  The AutoMate Express™ Forensic DNA Extraction System was developed for automatic isolation of DNA from a variety of forensic biological samples. The performance of the system was investigated using a wide range of biological samples. Depending on the sample type, either PrepFiler™ lysis buffer or PrepFiler BTA™ lysis buffer was used to lyse the samples. After lysis and removal of the substrate using LySep™ column, the lysate in the sample tubes were loaded onto AutoMate Express™ instrument and DNA was extracted using one of the two instrument extraction protocols. Our study showed that DNA was recovered from as little as 0.025 μL of blood. DNA extracted from casework‐type samples was free of detectable PCR inhibitors and the short tandem repeat profiles were complete, conclusive, and devoid of any PCR artifacts. The system also showed consistent performance from day‐to‐day operation.