Detection of zebra mussel larvae (Dreissena polymorpha)
Information (04-06-09)
Our laboratory offers detection zebra mussel larvae by molecular techniques on samples supplied by the customer. The molecular detection, unlike detection by light microscopy, reduces the risk of false negatives, amplifying the DNA of the larvae present in the sample, and is a faster method, especially when they have to process a large number of samples. The detection limit of the test locates a single larva in the sample supplied, as a result of filtering a known volume of water as a larva is a multicellular organism with many copies of target DNA. In this case, it is not possible to determine the number of larvae present as do the number of CFUs (colony -forming units of bacteria or fungi) or viruses detected, since in these cases there is only one copy of the genome in each UFC or in each virion. Our system includes the design of an internal control test, made with a genomic sequence of an amplicon of greater length having at the ends specific sequences of zebra mussels, and we have cloned into a plasmid vector to replicate in Escherichia coli. This plasmid is introduced into the sample to begin DNA extraction and be amplified simultaneously for conducting the test duplex PCR, where appropriate, detect the presence of inhibitors of the PCR test, and to avoid false negatives proof.
Our method has been designed from the known genomic sequences zebra mussels, and using DNA samples extracted from this species have found it quite specific in the same and has no cross-reactions with other bivalve species.
Sample Collection
In principle, filtering water, water mass target, we leave it in the hands of the customer, in the case of dams, can count on the support of staff and materials attached thereto (boat, nursery, ...) . This on the one hand, avoids the risks that a national company moves from one reservoir to another with their teams, although they must be cleaned and disinfected in accordance with appropriate protocols, it does pose a possible outbreak scattering mussel invasion. On the other hand, enlist the help of attached to water bodies personnel often reduce test times, because it avoids the permit application process for each state / federation / reservoir.
The protocol filtering is variable depending on various aspects: In general, recommended to be filtered at least 10 to 100 liters, through a sieve or mesh plankton 50 micron mesh. If a body of water large (eg reservoir) and have boat, ideally recommend taking at least two samples (surface and integrated throughout the water column), at least two points of mass water (eg, central reservoir area and near the dam), to ensure a certain representative data. If you do not have boat could be pumped a known volume of water from the shore, one or more points of the body of water.
An important aspect to take into account the client, is to avoid contamination with sampling equipment, because the method detects DNA, and it can be deposited in the sampling equipment and cause contamination between samples. For this reason, the material used for sampling should be disposable single use. An alternative possibility is treatment with sodium hypochlorite -lejía- sampling material, to destroy this DNA had been left from a previous sampling.
In any case, to avoid contamination of samples, our laboratory has designed a simple for water filtration equipment through a system with a disposable mesh, and would prepare and supply it to the customer, if we request, which would avoid having to resort to using plankton sleeves high cost.
The most important is to have a filtering a body of water the largest possible volume, as long as the conditions of the body of water permitted (depending on the concentration of particulate matter), for example 100 to 200 liters, does not increase just the effort of collecting the samples and allows, as an added precaution, to preserve a portion of the sample for microscopic analysis, if one wanted to contrast a negative result obtained by molecular techniques.
Conservation and transport Samples
Our laboratory could provide customers everything necessary for the preservation and transportation of samples taken by them: bottles to deposit water samples of small volumes and cold accumulators and container expanded polystyrene, to ensure the arrival of refrigerated samples to our laboratory. However, these recommendations and design team sampling and remission are general, and are customized according to customer needs and can even supply mesh filter for single use and avoid the risk of contamination between samples.
If requested tests provide additional information.
Response time
Regarding the response time, if negative samples, can have the results in 3 or 4 days, and if positive and require sequencing to confirm that this sequence of zebra mussels, about 10 days (these are timeouts on the normal working conditions of our laboratory).
Cost of testing
Information by email to: ivami@ivami.com