This kit is designed to make a reliable risk assessment for cylindrospermopsin in swimming waters. The Cylindrospermopsin Cyanotox qPCR detection kit is based on the fast, sensitive, and proven primers/probe qPCR technique. The used qPCR primers and probe recognizes the CyrA gene. CyrA is a gene from the biochemical pathway for the production of cylindrospermopsin and is only singular present in this pathway. This makes it a good candidate to determine the copy number of microcystin biochemical pathways in water samples.
Toxin information
Cylindrospermopsin (abbreviated to CYN, or CYL) is a cyanotoxin produced by a variety of freshwater cyanobacteria. CYN is a polycyclic uracil derivative containing guanidino and sulfate groups. It is also zwitterionic, making it highly water soluble. CYN is toxic to liver and kidney tissue and is thought to inhibit protein synthesis and to covalently modify DNA and/or RNA. It is not known whether cylindrospermopsin is a carcinogen, but it appears to have no tumour initiating activity in mice.
CYN was first discovered after an outbreak of a mystery disease on Palm Island, Queensland, Australia. The outbreak was traced back to a bloom of Cylindrospermopsis raciborskii in the local drinking water supply, and the toxin was subsequently identified. Analysis of the toxin led to a proposed chemical structure in 1992, which was revised after synthesis was achieved in 2000. Several analogues of CYN, both toxic and non-toxic, have been isolated or synthesised.
C. raciborskii has been observed mainly in tropical areas, however has also recently been discovered in temperate regions of Australia, North, South America, New Zealand and Europe. However, CYN-producing strain of C. raciborskii has not been identified in Europe, several other cyanobacteria species occurring across the continent are able to synthesize it.
Primer design
The primers and probe are specially designed to be used with eDNA samples and have the following properties:
- Highest possible sensitivity (<10 DNA copies per reaction).
- Strong fluorescence signal with low background noise. Isolated environmental samples contain residues of naturally occurring auto fluoresce substances that will interfere with the measurements. A strong fluorescence signal from the analyses is required for these kind of samples.
- 100% specificity. Isolated DNA from environmental samples contains billions of DNA fragments from bacteria, protozoa, plants, animals, etc. Not only species from the same order must be taken in account during primer/probe design, but all known DNA sequences must be checked for nonspecific binding of the primers and probe. This is validated by experimental and bioinformatical studies.
The kit is developed and optimized to be used on eDNA isolates purified using the eDNA isolation kit (#SYL002) from Sylphium molecular ecology.
Kit contents
The kit contains materials for an 3-fold analyses on 54 samples including all necessary controls.
- Positive control (CyrA gene)
- 2x Sylphium qPCR mix (100 reactions) without primers and probes
- 2x Primer/probe mix (100 reactions) for detection of CyrA gene (FAM dye)
- 1x Taq DNA polymerase (200 reactions)
- Protocol and primer/probe validation report
Manual
