Not all cyanobacterial strains produce toxins. However, the toxin-producing strains cannot be distinguished from the nontoxin-producing strains by traditional light microscopy, commonlyused to monitor water bodies. An alternative for the differentiation of potentially toxic strains from nontoxic strains is to use molecular methods to detect the presence of toxin biosynthetic genes. Such methods are already available and could be used for the detection and identification of potential microcystin and nodularin producers present in environmental samples (Attogene catalog number NA2024).
Screening for the toxin itself, can be very costly. In turn, real time PCR for the detection of a gene region responsible for assembling in cyanobacterial strains and environmental samples can be a key indicator for the prescense of cyanobacteria capable of expressing the aetokthonotoxin toxin. Attogen has thus, designed primer pairs and probes targeting a the conserved gene region in order to enable the amplification and detection of several producer genera using real time PCR. Screening for the toxin genes can save significant costs and act as a triage for samples needing to be analyzed for the toxin itself.
Cyanobacterial neurotoxin aetokthonotoxin (AETX), a peculiar pentabrominated biindole alkaloid implicated in fatal Vacuolar Myelinopathy. This neurodegenerative disease was first recorded in 1994 during an outbreak of bald-eagle poisonings at De Gray Lake in Arkansas, USA. AETX was experimentally confirmed to be produced by the true branching heterocytous cyanobacterium Aetokthonos hydrillicola. The production of AETX is dependent on bromide (Br−) availability, and likely linked to its hyper-accumulation by the host plan. Thus regular monitoring of A. hydrillicola (accompanied by assessment of Br− and AETX levels) is highly advisable to predict the possible threat of further VM outbreaks.
The cyanobacterial AetA gene which encodes the unique FAD-dependent halogenase involved in the pathway for AETX synthesis has been adapted to develop a -aetokthonotoxin specific quantitative PCR (qPCR) assay.
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Real time qPCR kit for AetA gene
For screening aetokthonotoxin gene cluster
Use in combination with Attogene Algae DNA isolation kit
N-(Propargyl-PEG2)-PEG3-N-Boc is a branched PEG linker with an alkyne group and a t-butyl ester. The alkyne group reacts with azide-bearing compound in coppper catalyzed azide-alkyne Click Chemistry reaction. The Boc group can be deprotected under acidic conditions. The amino group is reactive with carboxylic acids, activated NHS esters, and carbonyls. Reagent grade, for research purpose.
Document
N-(Propargyl-PEG2)-PEG3-N-Boc is a branched PEG linker with an alkyne group and a t-butyl ester. The alkyne group reacts with azide-bearing compound in coppper catalyzed azide-alkyne Click Chemistry reaction. The Boc group can be deprotected under acidic conditions. The amino group is reactive with carboxylic acids, activated NHS esters, and carbonyls. Reagent grade, for research purpose.
The 16S V3-V4 Library Preparation Kit for Illumina consists of the reagents and components required for library preparation of the 16S V3-V4 amplicon libraries to be used for next-generation sequencing on Illumina platforms. All molecular reagents including primers, enzyme mixes, indexes, and buffers are provided. Instructions for PCR clean up with the AMPure XP Magnetic Beads (supplied by customer) are also included for rapid purification of nucleic acid products generated at two steps of the workflow. The library prep workflow could be used for purified DNA inputs from different sources including stool, soil, water, saliva, plant, urine, skin swab, vaginal swab, cheek swab, nasal swab, plasma/serum, tongue swab, gum swab, and others.
The 16S V3-V4 Library Preparation Kit for Illumina has a streamlined procedure that reduces the handling time such that the library prep procedure can be completed in approximately 4 hours (see diagram below). Input DNA is first subjected to targeted PCR to amplify the V3-V4 region of the DNA encoding 16S rRNA. The post-PCR reaction is then cleaned up using AMPure XP beads. Dual index primers are then added using a limited-cycle PCR. The indexed amplicons flanked by 5′ and 3′ barcoded adaptors are then cleaned using AMPure XP beads. The libraries are then ready for quantification, pooling and sequencing.
Details
Minimum amount of starting material:
2.5 µL of DNA (5 ng/µL)
Time to complete library preparation:
4 hours
Storage Conditions and Product Stability Norgen’s 16S V3-V4 Library Prep Kit for Illumina is shipped as one kit box (for the 24 prep kit) or two sub-component kits (for the 96 prep kit). All kits should be stored at -20°C upon arrival.
All kit components should remain stable for at least 1 year when stored at the specified storage conditions.
