This kit is designed to make a reliable risk assessment for anatoxin in swimming waters. The Anatoxin Cyanotox qPCR detection kit is based on the fast, sensitive, and proven primers/probe qPCR technique. The used qPCR primers and probe recognizes the AnaF gene. AnaF is a gene from the biochemical pathway for the production of anatoxin and homo-anatoxin and is only singular present in this pathway. This makes it a good candidate to determine the copy number of anatoxin biochemical pathways in water samples.
Anatoxin, also known as Very Fast Death Factor (VFDF), is a secondary, bicyclic amine alkaloid and cyanotoxin with acute neurotoxicity. It was first discovered in the early 1960s in Canada, and was isolated in 1972. The toxin is produced by seven different genera of cyanobacteria and has been reported in North America, South America, Central America, Europe, Africa, Asia, and Oceania. Symptoms of anatoxin exposure include loss of coordination, muscular fasciculations, convulsions and death by respiratory paralysis. Its mode of action is through the nicotinic acetylcholine receptor (nAchR) where it mimics the binding of the receptor’s natural ligand, acetylcholine. As such, anatoxin-a has been used for medicinal purposes to investigate diseases characterized by low acetylcholine levels. Due to its high toxicity and potential presence in drinking water, anatoxin poses a threat to animals, including humans. (source: wikipedia)
The primers and probe are specially designed to be used with eDNA samples and have the following properties:
- Highest possible sensitivity (<100 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.
Other eDNA isolation methods/kits can be used as well. Please contact us to get more information how to use the obtained isolates from other methods/kits to get reliable results with the kit.
The kit contains materials for an 3-fold analyses on 54 samples including all necessary controls.
- Positive control (AnaF gene)
- 2x Sylphium qPCR mix (100 reactions) without primers and probes
- 2x Primer/probe mix (100 reactions) for detection of AnaF gene (FAM dye)
- 1x Taq DNA polymerase (200 reactions)
- Protocol and primer/probe validation report