FAQ - DNA-free PCR Reagents

  • Why is universal 16S rDNA PCR a promissing option of bacteria detection?
    For two reasons universal 16S rDNA PCR is a favourable way of bacteria detection in samples. First, the use of highly conserved regions in the 16S rRNA gene allows the amplification of sequences from any bacterial strain. With Mastermix 16S Primer and Mastermix 16S Complete, assays are supplied that use primers binding to the conserved regions of >345 bacterial species, including Gram-positive and Gram-negative pathogens. Second, because of the multicopy status of the 16S rRNA gene and other reasons, the sensitivity of detection of bacterial DNA has been found to be very high (see below).
  • What is the sensitivity of detection?
    The analytical sensitivity of the assay (Mastermix 16S Complete) is ≤200 fg DNA (P. aeruginosa). Expressed as viable counts per assay (25 µl), 1 (S. epidermidis), 4.5 (S. pneumoniae), 7.5 (E. coli), 11 (K. pneumoniae) and 23 (P. aeruginosa) cfu can be detected.
  • What reagents can bear contaminations by bacterial DNA?
    Principally all. In particular, to our experience, the Taq DNA polymerase and primers are mostly prone to DNA contamination due to the manufacturing processes. Molzym has invested great efforts to develop a purification process for the elimination of bacterial DNA from PCR reagents – guaranteed.
  • How can bacteria be identified when detected by 16S rDNA PCR?
    The direct way is sequence analysis. Amplicons are purified in a simple procedure, using a commercial PCR purification kit (e.g. QiaQuick, Qiagen, Germany). The purified amplicon is then included in a sequencing reaction.
  • Can MolTaq 16S and Mastermix 16S products be used for Real-Time PCR?
    Yes. All DNA-free PCR products of Molzym are fully compatible and optimised for Real-Time PCR, using fluorescent dye or probe detection systems.
  • Does Molzym manufacture customized mastermixes?
    Yes. For further information and inquiries on customised manufactured mastermixes, please click here.
FAQ - Sequencing primers – SeqGP and SeqGN
  • For what application do I need the primers?
    The primers are used in conjunction with Mastermix16S Complete and Mastermix 16S Primer. Both primers bind to conserved sites within the 16S rRNA gene region amplified in the assays. Primer SeqGP is group-specific including, among others, Gram-positive Staphylococcus, Streptococcus. Enterococcus, Listeria, Micrococcus, Bacillus and Lactococcus species. Primer SeqGN includes Gram-negative Pseudomonas, Acinetobacter, Serratia, Enterobacter, Escherichia, Proteus, Klebsiella, Stenotrophomonas, and Legionella species. Using both sequencing primers, mixed infections by bacteria in the two different groups can be analysed.
  • Do the primers differentiate between Gram-positive and Gram-negative bacteria?
    The sequencing primers comprise large Gram-specific groups, but there are some exceptions. As related to clinical applications, the most important organisms covered by primer SeqGP are the Firmicutes (among others, Bacillaceae, Staphylococcaceae, Streptococcaceae, Enterococcaceae) and Actinobacteria (among others, Micrococcaceae). Exceptions comprise few Gamma-Proteobacteria like Edwardsiella spp. Primer SeqGN includes the Gamma Proteobacteria (among others, Pseudomonadaceae, Moraxellaceae, Enterobacteriaceae, Xanthomonadaceae and Sphingomonadaceae. Few Gram-positives are identified, including Clostridium, Corynebacterium, Mycobacterium and Propionibacterium species.
  • What to do with overlapping sequences?
    Overlapping sequences are the result of mixed strains in the extract that could not be resolved by the sequencing primers SeqGP and SeqGN. Typical examples are mixtures of Gram-positive or Gram-negative or strains. In this case we recommend using a special tool for resolving mixed sequences, Isentio RipSeq (www.isentio.com).
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