Sample preparations for a detection of Mycobacterium in fish, feed and environmental samples by PCR-RCBH
Suppalak PunttinaowaratAquatic Animal Health Research Institute, Bangkok 10900 Thailand
The movement of live aquatic animals has recently become of international concern since it is a potential route for introducing new pathogens into the aquatic environment, which in turn, may have detrimental effects on indigenous species. Since mycobacteriosis, caused by Mycobacterium spp., has now been reported in a wide range of both food fish and ornamental fish, some countries require certification stating that imported animals are Mycobacterium-free. Detection of Mycobacterium is time consuming because of their slow growth. A number of different methods have been used to identify Mycobacterium based on biochemistry, mycolic acid profiles, antibody-based and DNA-based methods., AAHRI is the only recognised authority in Thailand to provide health certification for live fish, which are being exported world-wide. The fish are certified as Mycobacterium-free, based on a polymerase chain reaction-reverse cross blot hybridization (PCR-RCBH) method previously described by Puttinaowarat et al (2002).
Sample preparation is a crucial step in the PCR procedure. Each type of sample (fish tissue, food, sediment, etc.) differs in the amount of organic material that is present, and this can interfere with the isolation of DNA as well as the DNA amplification. Therefore, a selection of methods used to prepare different types of samples from fish farms for use in PCR are described below. There is no one ideal method for DNA extraction, however the methods outlined are based on the author’s own personnel experience, which may be useful to others doing similar types of work.
One advantage of the PCR is that extracted DNA does not have to be purified to the same extent as for other procedures. However, some samples may contain inhibitors of the PCR and therefore some precautions may need to be taken during sample preparation to prevent inhibition.
Bacteria from pure cultures can be prepared for PBR in two different ways depending on the purity of DNA required.
(a) Bacterial lysatesA small amount of bacteria (around 1 mg; 108 bacteria) is suspended in a vial containing 0.5 ml of TE buffer (10 mMTris-HCL, 1 mM EFTA, pH 8.0) and 0.5 ml of 150-212 mm glass beads. The bacterial suspension is incubated at 75oC for 150 min to kill bacteria and then vortexed at a maximum speed for 1 min. The supernatant is diluted 1/100 with TE buffer and used as a template in the PCR.
It should be noted that this extraction procedure is not suitable for bacteria cultured on Ogawa medium isince the bacteria contain a high amount of organic material from the culture medium, and bacteria should instead be grown on Modified Saulton's Agar.
(b) DNA isolationBacteria previously incubated at 75oC for 15 min are then centrifuged at 3000 xg for 30 min. The pellets are washed twice with TE buffer and resuspended in 5 mL of TE buffer. Lysozyme is added at a final concentration of 1mg mL-1 and the bacteria are incubated for 90 min at 37oC. Proteinase K and sodium dodecyl sulfate (SDS) are then added at a final concentration of 0.1 mg mL-1 and 1% (w/v) respectively, and the bacteria are incubated for a further 1 h at 60oC with continuous shaking. The mixture is added to Tris-saturated phenol, pH 8.0 (Sigma) at a ratio of 1:1 (v:v) and mixed on a rotator shaker for 10 min, then centrifuged at 3000 xg for 5 min. The upper aqueous phase is transferred to a fresh tube and the procedure repeated with chloroform only. DNA, present in the final solution, is precipitated by adding cold ethanol (-20oC) and NaCl at a final concentration of 70% and 0.2 M respectively, and incubating at –20oC for at least 1 h. The tubes are centrifuged at 3000 xg at –20oC for 30 min. The supernatant is discarded and 70% cold ethanal is gently added to the pellets taking care not to disturb them. The tubes are again centrifuged at 3000 xg at –20oC for 5 min and the supernatant is removed. The pellets are dried in a desiccator before re-dissolving them in 3 mL of TE buffer with an overnight incubation at 4oC.
Fish samplesInfected tissue samples commonly sampled are spleen, kidney and liver. Any fat surrounding the tissue should be trimmed off. The samples are placed on an eppendorf and grinded with a small pestle. The samples are then treated with proteinase K solution (0.1 ma mL-1 proteinase K in 20 mM Tris-HCL pH 8.3, 0.5% Triton X-100, 1 mM EDTA) at 60oC overnight and treated as described by Boom et al (1990)
Blood samplesBlood (1 ml) is collected from a caudal vein of the animal. To prevent clotting, 30 l of 0.2M EDTA is added to the sample. It should be noted that the use of heparin is not recommended, as it will inhibit the PCR. However, EDTA is also a PCR inhibitor, so excess amounts should be avoided. The sample is then centrifuged at 8,000 xg for 3 min and the middle layer (white blood cell) is collected and treated with 1 ml lysis buffer (0.1M Tris, 0.32M sucrose, 1% Triton x-100, pH 7.5). The mixture is centrifuged at 9,000 xg for 5 min and the supernatant discarded. The following solutions are then added to the pellet: 50 l of STE buffer (0.5M Tris, 0.01M EDTA, 1M NaCl, pH 7.5), 25 l of 10% SDS, 20 l of 5 mg ml-1 proteinese K and 550 l of water and then incubate at 37oC for 12 h. The sample is treated with phenol and chloroform twice and 0.1 volume of 3M Na-acetate and 0.6 volume of iso-propanol is added to the pellet. The mixture is centrifuged at 8,000 xg for 4 min. The supernatant is removed and the pellet is washed with cold 70% ethanol and allowed to dry. The pellet of DNA is then diluted with 100 l of TE buffer (10 mM Tris-HCl, 1mM EDTA, pH 8.0).
FeedLive feed including artemia, rotifer and water flea are washed twice with sterile water and then incubated with proteinase K overnight at 60oC. The digested sample is subsequently treated as described by Boom et al (1990).
Sediment The sediment on the bottom of fish tank is collected and treated twice with phenol and chloroform and the DNA solution is then precipitate in ethanol as described above.
Water Fifty ml of water sample is collected and centrifuged at 4,000 xg for 20 min. The supernatant is removed and the pellet is treated as described by Boom et al (1990).
PCR-RCBH is a powerful method to detect and identify Mycobacterium spp. The limit detection of the method is equivalent to 20 mycobacteria (Puttinaowarat et al, 2002). The detection of mycobacteria in environmental samples is important as it can indicate possible routes of infection. However, it should be noted that the presence of bacteria in the environment does not necessarily mean that the fish are infected. Infection may be the result of a number of different factors including the health status of the fish, the environmental condition of the water in which the fish are held, as well as the number of the bacteria present in the environment. Although mycobacteriosis is not currently a disease notifiable to the Office International des Epizooties (OIE), methods of disease-screening should be discussed internationally with a view to developing a standardised method to detect Mycobacterium in fish and environmental samples which is both sensitive and reproducible.
Boom, R., Sol, C. J. A., Salimans, M. M. M., Jansen, C.L., Wertheim van Dillen, P. M. E., & van der Noordaa, J. (1990). Rapid and simple method for purification of mucleic acids. Journal of Clinical Microbiology 28(3), 495-503. Puttinaowarat, S., Thompson, K.D., Kolk, A. & Adams, A. (2002) Identification of Mycobacterium spp. isolated from snakehead (Channa striata) and Siamese fighting fish (Betta splendens) using polymerase chain reaction-reverse cross blot gybridisation (PCR-RCBH). Journal of Fish Diseases 25:235-243.