1. Unless otherwise stated procedures described molecular biology grade distilled water should be used in all the procedures described.

2. Since the HAV genome is RNA and RNases are ubiquitous and highly resistant to heat inactivation, the plastic material (and whenever possible all solutions) should be auto-claved at 121°C for 45 min.

3. Preparation of these reagents is not necessary when RNA extraction kits such as the QIAamp Viral RNA Mini Kit (QIAGEN, Hilden Germany) are used. The advantages in using this kit are that it is less time-consuming and that the RNA recovery is similar or even better with certain kinds of samples such as shellfish. However, the price is comparatively expensive.

4. All ethanol solutions are prepared from 96° absolute ethanol.

5. The reason to autoclave (121°C for 20 min) most of the Southern blot reagents is not to prevent RNA degradation but rather to avoid bacterial contamination that could interfere with the hybridization reaction as well as immunological detection.

6. If the prehybridization solution is not used immediately, it should be kept on ice to avoid renaturation of the salmon DNA.

7. Since the initial pH of this buffer is around 1.5, the addition of NaOH beads is recommended to increase the pH abruptly to 7.0; at this point, adjust the pH with 1 N NaOH.

8. This step refers to a method for the removal of some potential inhibitors of the ulterior molecular techniques (14). At the same time it allows the viral particles present in the sample to be concentrated, since the initial volume of the sample is 50 mL and the final volume is 500 ^L, which corresponds to a 100X concentration.

9. Owing to the crucial problem of cross-contamination in the PCR-based diagnostic procedures, physical separation between each step should be kept at maximum. The nucleic acid extraction should be performed in a separate and specific room and in a vertical laminar flow hood (P2 level) since HAV is a human pathogen.

10. It is important to autoclave the microcentrifuge tubes with their lids open to avoid potential cross-contamination. For the same reason, use different pipets to add and remove reagents from the tubes, and change the filter tips at each step.

11. Completely resuspend the silica pellet from the stock.

12. Completely resuspend the silica pellet after each wash.

13. Manipulate the guanidinium isothiocyanate wastes very carefully, as well as all the reagents and materials in contact with it, and dispose of them in a container whose one-third part is 10 N NaOH.

14. Carefully eliminate all the silica particles. If the first supernatant has silica contamination, repeat the centrifugation step.

15. If further processing is delayed, keep the nucleic acid-containing supernatant frozen at -70°C.

16. Because of the crucial problem of cross-contamination in the PCR-based diagnostic procedures, physical separation between each step should be kept at maximum. The RT and PCR reaction mix should be prepared in a different room separately from the nucleic acid extraction room. The thermocycler should be located in a third separate facility, and, last but not least, the amplimer-containing tubes should be open and the gels run in a fourth separate room.

17. The DNA bands may also be stained with the traditional ethidium bromide. However, since environmental samples usually present low viral loads, the GelStar stain is suggested to increase the sensitivity (around 0.5-1 log higher).

18. The gel analyzer could be replaced by a UV transilluminator coupled to a Polaroid camera.

19. Avoid touching the nylon membrane with the fingers even when using gloves.

20. Use a tube to roll the air bubbles out gently.

21. The membrane can be used immediately for prehybridization or can be stored dry at 4°C for future use.

22. Longer blocking times may also be performed.

23. To reduce the background, it is preferable to incubate the membrane in Petri dishes rather than plastic bags. It is not useful to increase the incubation time since the background increases correspondingly.

24. To reduce the background, it is important to remove the CSPD reagent completely by changing the plastic bag container.

25. Since it is a photographic development, typical dark room conditions should be observed.

26. Longer exposition times may be used to increase the intensity of the bands. The same membrane may be used for several photographic expositions at different times.

27. Since the genotyping is based on a DNA sequence, it is important to use a DNA polymerase with proofreading activity such as the Pwo polymerase included in the Expand enzyme.

28. Avoid excising agarose by cutting the band as accurately as possible.

29. This mix contains all the reagents necessary for the sequencing reaction with the exception of the specific primer and template and should be maintained on ice during preparation of the complete sequencing mix.

30. Since HAV is not very variable, only one strand is sequenced. However, if the quality of the sequence is not good (double sequence, low intensity of peaks, and so on), the second strand is also analyzed by using the VP1-3285 primer. When mutations not described in any database are detected, the second strand is also analyzed to confirm it.

31. The volume necessary to set up a reaction with 3-10 ng of DNA. The remaining amount to a final volume of 10 ^L is added as distilled water.

32. It is preferable to continue with the DNA precipitation on the same day.


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