SepsiTest™-UMD Kit

SepsiTest-UMD detects and identifies bacteria and fungi in primary sterile material without culture. The kit combines sample pre-treatment, DNA isolation, universal rDNA PCR and sequencing-based identification.

Principle

The MolYsis pre-treatment lyses the human cells and degrades the released human DNA, so the enriched microbial DNA reaches the PCR with a much lower host background; that suppresses false results from non-specific primer binding. Universal primers then amplify the V3/V4 region of the bacterial 16S rRNA gene and the V8/V9 region of the fungal 18S rRNA gene. The amplicons are sequenced with the supplied primers SeqGP16, SeqGN16 and SeqYeast18, and the sequences identified by BLAST.

Sensitivity

The minimum titres reported are 10 cfu/ml for Candida albicans and Candida glabrata, 20 cfu/ml for Staphylococcus aureus, Staphylococcus epidermidis and Enterococcus faecalis, 40 cfu/ml for Escherichia coli, 50 cfu/ml for Klebsiella pneumoniae and Moraxella catarrhalis, 75 cfu/ml for Streptococcus pneumoniae and 80 cfu/ml for Pseudomonas aeruginosa.

Sample material and instruments

Body fluids including ascites, bronchoalveolar lavage, blood, blood culture, cerebrospinal fluid, plasma, pleural fluid, pus and synovial fluid; swabs from mouth, nasopharynx, wounds and bone; and tissue such as abscess material, biopsies, heart valves and prostheses. One millilitre is used for fluids and swabs, and tissue pieces of at most 0.5 by 0.5 by 0.5 cm. The end-point PCR protocol is validated on the Eppendorf Mastercycler, and real-time protocols on the Roche LightCycler 96, 480 and Nano, the Bio-Rad Opticon and CFX96, the Applied Biosystems 7500 Fast, the Stratagene Mx3000P and Mx3005P, the Qiagen Rotor-Gene and the peqSTAR 96Q.

Controls and limitations

The kit contains a Control DNA as internal extraction control, which monitors extraction, DNA quality and the absence of PCR inhibitors, plus positive PCR controls at two concentrations. Frozen and thawed specimens are not suitable, and the kit is not to be used for safety level 3 or 4 pathogens. The manufacturer states that sequencing results must be assessed by a clinician, to exclude contamination and organisms without clinical relevance. The device is CE-marked under Directive 98/79/EC and continues to be placed on the market under the transitional provisions of Regulation (EU) 2017/746.

• Lysis Buffer: A solution containing chaotropic salts and detergents that disrupt the red blood cells and release the microbial DNA into solution.
• Magnetic Beads: Beads coated with specific oligonucleotide probes that capture and isolate microbial DNA from the sample.
• Wash Buffer: A solution used to wash the magnetic beads and remove any remaining contaminants.
• Elution Buffer: A solution used to release the captured microbial DNA from the magnetic beads.
• Control DNA: A synthetic DNA that is added to the sample as an internal control to ensure that the assay is working correctly.
• PCR Master Mix: A solution containing all of the reagents necessary for PCR amplification of the captured microbial DNA.
• DNA Size Marker: A DNA ladder used to determine the size of the amplified PCR products.
• PCR Primers: Specific primers that amplify conserved regions of the microbial DNA for detection.
• PCR Enzyme: A heat-stable DNA polymerase enzyme used to catalyze the PCR reaction.
• DNase/RNase-free Water: Water that has been treated to remove contaminating DNases and RNases, which can degrade the extracted DNA.

The kit also includes instructions for use, as well as a protocol for the detection of microbial DNA in blood samples using real-time PCR.