3. Add 1 U Taq DNA polymerase to a final volume of 50 mL.

4. Overlay all reaction mixtures with 100 mL of sterile mineral oil.

5. As thermal cycling conditions for amplification, use the following parameters: 97°C for 1 min, 46°C for 2 min, 72°C for 1 min (15 cycles), final extension at 72°C for 7 min (1 cycle).

6. Identification of the P-type isolates requires adding sufficient VP4 template to 5 ^L 10X PCR buffer (100 mM Tris-HCl, pH 9.0, 500 mM KCl), 8 ^L of stock dNTP mix, 1.5 mM MgCl2, 2.5 U Taq DNA polymerase, 20 pmol of each P-typing primer, and 20 pmol of the VP4 common primer, Con2 (Tables 1 and 3).

7. The cycling parameters for the P-typing reaction are as follows; 97°C for 1 min, 50°C for 2 min, 72°C for 1 min (20 cycles), with a final extension at 72°C for 5 min (1 cycle).

8. Visualize all PCR products by agarose gel (1.5%) electrophoresis as previously described in Subheading 3.3.1.

4. Notes

1. The RNA isolation procedure described was used to isolate RNA directly from fecal matter. The method can also be applied to tissue culture samples, but the quality and quantity of RNA may not be as good. Alternative methods are also available.

2. Contamination with exogenous ribonucleases can result in the degradation of RNA; therefore proper pretreatment of reagents and experimental apparatus is a prerequisite. To reduce ribonuclease contamination of laboratory equipment, all surfaces and apparatus can be thoroughly wiped with RNase AWAY (Molecular Bioproducts, San Diego, CA).

Glassware and nonsterile plasticware can be immersed in a solution of 0.2% (v/v) DEPC and then autoclaved at 121°C for 30 min. Gloves should be worn at all times during procedures.

3. Caution: care should be taken when handling phenol/chloroform. As phenol is an acid, it can cause skin burns. This reagent should only be handled in a safety cabinet with extraction ventilation.

4. The presence of DNA in RNA samples could potentially interfere with subsequent analysis of the rotaviral genome and therefore it should be removed. DNA is degraded by treatment with a specific endonuclease enzyme, DNase 1. This enzyme digests DNA but does not affect the integrity of RNA.

5. Prepare sufficient gel volumes according to the PAGE apparatus used.

6. Isobutanol is added to prevent oxygen from diffusing into the gel, as this would inhibit polymerization.

7. The loading buffer described in Subheading 2.2.1. can be substituted for another appropriate loading buffer if preferred. The volume of sample that is loaded into the gel will depend on the size of the gel wells and thus can be adjusted accordingly.

8. Using excess RNA template can be detrimental to the RT-PCR assay, as it appears to inhibit the reaction. In general, small volumes of dsRNA template (1-5 ^L) often produce better results.

9. Stronger binding forces exist between RNA-RNA compared with DNA-DNA and DNA-RNA; thus more stringent denaturing conditions are required to prevent strand reannealing. Prolonged denaturation (5 min) and the addition of nucleic acid destabilizers, such as DMSO, in the RT step are often recommended (34).

10. It is not essential but recommended to include RNasin in the assay to inactivate any contaminating ribonucleases that may be present in the reaction.

11. The thermocycler used in this study was a Pharmacia ATAQ controller (Pharmacia, Uppsala, Sweden).

12. The amount of cDNA template used in the serotyping PCR assays can be identified by performing initial template titrations. In general, 1-2 ^L of cDNA template is sufficient for amplification. However, if the RT-PCR products are very weak, use more template.


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