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Crayfish disease diagnostics

Towards a Nordic standard

image of Crayfish disease diagnostics

Crayfish utilization is a long-standing Scandinavian tradition, which unfortunately has suffered a major drawback with the introduction of crayfish plague disease about a hundred years ago. In spite of intensive research many problems in management and diagnosis of this disease are not yet solved. To evaluate ongoing research and diagnostic methodology in different countries, representatives of diagnostic laboratories involved in crayfish disease diagnostics were invited to a workshop in Kuopio to discuss the problems with crayfish diseases. The workshop was held at the Kuopio unit of the Finnish Food Safety Authority Evira. Participants from Finland, Sweden, Norway, Estonia and Latvia were present, as well as the OIE expert from the reference laboratory for crayfish plague. In the programme of the first day the state of crayfish stocks, crayfish diseases and diagnostic methods used in each country were presented and discussed. During the second day diagnostic methods were discussed in detail, as well as research projects concerning crayfish diseases. The workshop has been made possible by way of a grant from the Nordic Council of Ministers.

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A TaqMan real-time PCR assay for specific and quantitative detection of Aphanomyces astaci

Many limitations are connected to the diagnosis of crayfish plague based on classic approaches. Isolation of A. astaci in pure culture is highly specific if successful, but poses a considerable risk for false negatives. Direct microscopy commonly reveals a developed infection, but poses a risk for false positives (observation of oomycetes other than A. astaci) and false negatives (no observations despite presence of the agent). Provided the required specificity and caution, molecular detection represents a powerful tool for rapid and sensitive detection of A. astaci from a broad range of materials, including early infections, degraded remnants and historical samples. However, the reliability of the results depends heavily on the personnel handling of the samples during processing and analysis. Even though a molecular method has proved specific to A. astaci, positive results are reliable only if all steps during sample processing and preparation, DNA-isolation, PCR setup and post-PCR work have not introduced cross contaminations. In order to control for false positives during these steps, we always include environmental controls (open tube(s) with milliQ water following all working processes), DNA isolation controls (notemplate tube(s) included in all steps of the DNA extraction) and negative PCR-controls. During sample preparation, we divide the samples (cuticle, eyes, telson, muscle etc) in two equal series of sub-samples annotated A and B. All controls are tested together with the A subsamples. If positives are detected in any of the controls, the results will not be trusted or reported. In such cases, or if conclusions are difficult to draw based on the A-results alone, the B sub-samples (including new sets of controls) will processed and analyzed separately.

English

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