To accelerate differentiation between Staphylococcus aureus and Coagulase Negative Staphylococci (CNS), this study aimed to compare six different DNA extraction methods from 2 commonly used blood culture materials, i.e. BACTEC and Bact/ALERT. Furthermore, we analyzed the effect of reduced blood culture times for detection of Staphylococci directly from blood culture material. A real-time PCR duplex assay was used to compare 6 different DNA isolation protocols on two different blood culture systems. Negative blood culture material was spiked with MRSA. Bacterial DNA was isolated with: automated extractor EasyMAG (3 protocols), automated extractor MagNA Pure LC (LC Microbiology Kit MGrade), a manual kit MolYsis Plus, and a combination between MolYsis Plus and the EasyMAG. The most optimal isolation method was used to evaluate reduced bacterial culture times. Bacterial DNA isolation with the MolYsis Plus kit in combination with the specific B protocol on the EasyMAG resulted in the most sensitive detection of S.aureus, with a detection limit of 10 CFU/ml, in Bact/ALERT material, whereas using BACTEC resulted in a detection limit of 100 CFU/ml. An initial S.aureus load of 1 CFU/ml blood can be detected after 5 hours of culture in Bact/ALERT3D by combining the sensitive isolation method and the tuf LightCycler assay.
Het doel van deze studie was het testen van een dertigtal familieleden op Charcot-Marie-Tooth type 1A met behulp van een real time kwantitatieve polymerase kettingreactie. Duplicatie van het gen werd bij 50 % van de familieleden gevonden, overeenkomend met Mendeliaanse overerving.
This report presents the highlights of the 7th European Meeting on Molecular Diagnostics held in Scheveningen, The Hague, The Netherlands, 12-14 October 2011. The areas covered included molecular diagnostics applications in medical microbiology, virology, pathology, hemato-oncology,clinical genetics and forensics. Novel real-time amplification approaches, novel diagnostic applications and new technologies, such as next-generation sequencing, PCR lectrospray-ionization TOF mass spectrometry and techniques based on the detection of proteins or other molecules, were discussed. Furthermore, diagnostic companies presented their future visions for molecular diagnostics in human healthcare.
New innovative methods to determine the DNA sequences of different bacterial species are rising. In the field of microbiology, these methods are very important since it is now possible to determine all the genetic characteristics of the bacterium in one step! This enables to define e.g. the species family, drug resistance or relatedness to other bacteria in outbreak evaluations which is necessary to efficiently treat the bacteria or target potential outbreaks. For many years, PCR-based methods have been the technique of choice to determine DNA sequences (including next-generation sequencing techniques). Recently, a new technique has been introduced to the market that is based on single molecule real-time sequencing (SMRT) with the possibility to determine the DNA sequence of a bacterium. This SMRT MinION sequencing technique is housed on an USB stick and is known for its user-friendliness and huge data output. However, before such a new technique can be implemented and presented in laboratories and used for educational purposes, methods should be harmonized and evaluated to proof its applicability. Harmonisation of the methodology regarding new laboratory techniques is very important to be able to compare results generated by different laboratories. A single consistent protocol, applied in each lab, is essential to obtain the best results in interlaboratory comparisons. During this KIEM-hbo project, we – i.e. Avans UAS, Maastricht University Medical Center and the company IS-diagnostics – will determine the DNA sequence of bacterial species and mixes thereof with a harmonized protocol for an interlaboratory comparison. We will compare this technique to the IS-PRO, an existing technology. Finally a workshop will be organized for medical technicians and other SMRT sequencing users to evaluate the protocols. This will, generate an up-to-date and harmonized sequencing protocol which can be expanded to future research and diagnostics in the different areas.
