FAQ - Pathogen DNA Isolation MolYsis™

  • Why has human DNA to be removed before PCR assaying of bacteria and fungi? The human genome contains unspecific binding sites for e.g. pan-bacterial primers. This may give rise to false-positive signals in PCR assays. Moreover, unspecific binding limits the accessibility of primers to the specific sites on the 16S rRNA gene. As a result, the sensitivity of detection is reduced. Last, the removal of human DNA allows the easy processing of larger volumes (up to 10 ml) by a quick mini spin column protocol which in turn increases the sensitivity of detection of bacteria. Altogether, degradation of human/animal DNA reduces the constraints otherwise connected with universal 16S rDNA PCR using whole blood and other primary body fluids.
  • How is human DNA removed during blood processing?Samples are treated with a buffer lysing the human/animal cells, while bacteria and fungi are unaffected. The released human DNA is enzymatically degraded and bacterial and fungi cells are sedimented at the end. The protocol is completed within 45 min (6 parallels). Thereafter, any procedure for DNA extraction can be performed to isolate the microbial DNA, including manual and automatic protocols.
  • Do I have to change my validated DNA purification system? Not necessarily. Because of its modular nature, MolYsis™ is adaptable to any DNA purification system. In particular, MolYsis™ Basic, MolYsis™ Basic5 and MolYsis™ Basic10 kits are units that are designed to treat 0.2 ml (pediatric), 1 ml and 5-10 ml liquid samples, respectively, before mini isolation of microbial DNA is performed by other kits, including Qiagen, Roche, Promega, Biomerieux and Epicentre. Make sure that ingredients of DNA isolation kits of other manufacturers are DNA-free (reagents, buffers, spin columns, plastics). If you are uncertain, ask your supplier. Automated DNA isolation products can also be combined with MolYsis™ Basic, MolYsis™ Basic5 and MolYsis™ Basic10 (see below).
  • Can MolYsis™ be used with automated DNA purification systems? MolYsis™ Basic (kit for removal of human DNA) has been successfully tested in conjunction with liquid handling DNA extraction systems, including Qiagen QiaCube®, Roche Magnapure®, Chemagen Chemagic® MSM I, Biomerieux easyMag®, FujiFilm QuickGene® 810 and PSS Magtration®. Make sure that the reagents used are DNA-free. Ask your supplier.
  • What kind of consumables are to be used? Sterile pipette tips with filters are obligate to avoid carryover contamination during DNA preparation. Also, sterile 1.5 and 2.0 ml polypropylene tubes, as long as not supplied with MolYsis™ kits, should be used. Many manufacturers of plastic consumables guarantee absence of human DNA. Only few suppliers test for the absence of bacterial DNA. We have made good experience using Biosphere® pipette tips and plastic vials (Sarstedt, Germany).
  • What kind of samples can be processed with MolYsis™?
    • Samples evaluated for kits of the MolYsis™ Basic and MolYsis™ Complete series include:
      Human origin: Whole blood, synovial fluid, pleural fluid, cerebrospinal fluid, ascites fluid, pus, broncho-alveolar lavage, nasal wash fluid, urine
      Animal origin: Whole blood from mouse, rat, and monkey, chinese hamster ovary cell culture, monkey renal cell culture
    • Samples evaluated for MolYsis™ Plus:
      Blood culture
  • What is the sample volume that can be processed?
    • MolYsis™ Basic is for the treatment of 0.2 ml pediatric samples.
    • MolYsis™ Basic5 can be used for 1 and 5 ml samples.
    • MolYsis™ Basic10 can be used for 5-10 ml samples.
    • MolYsis™ Complete5, the complete system of human DNA removal and microbial DNA isolation, is available for 1 ml and 5 ml samples.
    • MolYsis™ Complete10 allows the processing of 5-10ml samples.

 

  • What is the sensitivity of detection of bacteria and fungi in blood? Spiking experiments showed that bacteria and fungi can be detected at low titres in whole blood. For instance, with 5 ml whole blood using MolYsis™ Complete5 and an universal 16S PCR assay (Mastermix 16S Complete) or an universal 18S assay the detection limits are (100%; 6 parallel determinations): Staphylococcus aureus, 12 cfu/ml; Escherichia coli, 24 cfu/ml; Candida albicans, 5 cfu/ml. 

 

  • Can blood cultures be used for isolation of microbial DNA? MolYsis™ Plus is a kit specially developed for the isolation of microbial DNA from blood cultures. The procedure includes removal of PCR inhibitors and human DNA and the isolation of microbial DNA from 0.2 ml blood culture. In a study it was shown that bacterial growth in blood cultures can be detected by PCR significantly earlier when using MolYsis™ Plus than the microbial detection system (Gebert et al. 2008; see references).You do not find your question posted? Direct your question directly to our team of experts This email address is being protected from spambots. You need JavaScript enabled to view it.!