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Sampling protocol

Specific requirements for avoiding contamination

The largest problem associated with specimen collection is contamination. PCR is a very sensitive test that functions on the replication of nucleic acid. It is therefore imperative that all procedures are quality controlled and the equipment and surfaces upon which specimens are collected are clean. In addition to the following protocols we have put together an instructional video describing how to collect different types of samples for DNA analysis.

Recommendations:

  1. When handling animals, use a fresh (previously unused) pair of gloves for each sample
  2. Use a fresh scalpel/tooth pick/swab for each sample
  3. Use only recommended consumables (e.g. swabs and DS water)
  4. Use a fresh dissection board (cutting surface; e.g. a Petri dish) for each sample
  5. Use a fresh storage container for each sample
  6. Use freshly prepared storage media (e.g. >96 % ethanol, Longmire buffer or DS water)

Blood samples

This sample type is primarily for disease screening and genotyping from birds. We prefer blood samples to be collected into a lysis buffer and stored in screw cap tubes. For most of our routine screening 10-15 ul of blood is sufficient and is collected into the lysis buffer at a ratio of 1 part blood: 2 parts lysis buffer. We can supply the buffer and collection tubes on request.

Tissue

Our preferred source of material from trapped animals is from ear tissue. For small mammals such as rats, stoats, cats and mice we require a piece of ear approximately 4-5mm square. This can then be stored dry, frozen, or immersed in 95% ethanol.

Hair samples

Hair samples can be a good source of quality DNA and are easily transported and stored. If samples are being physically removed from an animal, make sure at least 10 coarse hairs are pulled that retain their follicles. If hair samples are being collected via rubber bands from hair collection tubes, then cut the entire band from the tube. Samples are then stored in manilla envelopes along with a piece of filter paper to ensure the sample remains dry. These can then be posted directly to us.

Faecal samples

These are not ideal to work with but are generally good for identifying species using mtDNA. For individual identifications there are more problems with obtaining sufficient DNA to provide a reliable genotype. However, these issues can be overcome if errors are taken into account and by carrying out sufficient replicates. We can discuss possibilities for this type of sample with you if there is no other alternative.

Saliva swabs

These can be taken from carcasses or from eggshell fragments. Swabbing the sample should occur as soon as possible and prior to freezing as DNA is degraded by time and the freeze-thaw process.

  1. Prior to swabbing, prepare a swab or cotton bud by cutting it in half. Only handle the cut end of the cotton bud.
  2. Put on surgical gloves and with the prepared dry cotton bud swab the surface of the sample, paying particular attention to the vicinity of any puncture wounds. Swab around the edge and within any wounds. If wounds are not apparent swab the entire surface of the sample.

d

  1. Air dry cotton bud swabs individually for approximately 24 hours. Ensure cotton bud swabs cannot touch anything which may have had a predator species in contact with it.
  2. Once cotton bud swab has been air dried for 24 hours, place cotton bud swab in a manila envelope along with a piece of filter paper.  The filter paper will absorb moisture still present. Seal envelope.

d

  1. Label manila envelope with details of where the sample was found (GPS coordinates if possible); suspected predator; or any other identification number; estimated length of time the animal/bird was dead; and the name of the person who collected and swabbed it.
  2. As soon as possible send manila envelope containing cotton bud swab to Ecogene.

Chytrid samples

The following methodology has been provided by the CSIRO Australian Animal Health Laboratory (AAHL).

Swabs

It is recommended to use Medical Wire & Equipment Co. (UK) MW100-100 sourced from bioMérieux in Australia. Alternative swabs have not been validated.

Medical Wire & Equipment Co. (UK) MW100-100 swabs

  1. Swab comprehensively (e.g. repeat 2-3 times) on the underside of feet, legs and drink patch. Place the swab back into the container (does not require drying) and store. (e.g. containers can be placed in ‘backpack’ for the duration of a field trip)
    Note: Trials have shown that such samples can be stored at room temperature (23°C) for at least one month without loss of sensitivity.
  2. Upon return to the laboratory, samples should be stored at 4°C, or colder, to inhibit growth of other organisms; this is a precautionary measure
  3. Submit sample to laboratory

Tissue Samples

  1. Collect samples using either a fresh scalpel or tooth pick (depending upon the nature of the sample). REFER to details above about SPECIFIC REQUIREMENTS on how to maintain the integrity of the sample(s).
  2. Place the sample(s) into a clean (unused) collection container(s) and either
    1. place at 4°C or colder or
    2. add 70% ethanol and store at room temperature or 4°C until required
  3. Submit sample to laboratory