PCR Methods

3.3.1. Reverse Transcriptase (RT)-Mediated PCR

1. Denature 1-5 ^L of the dsRNA template (see Note 8) by heating to 97°C in the presence of 3.5 ^L DMSO for 5 min (see Note 9). Place the reaction tubes immediately on ice.

2. To amplify full-length gene segment 9 (1062 bp) encoding VP7, assemble a PCR reaction cocktail containing the following: 5 ^L 10X PCR buffer, 8 ^L of the working dNTP mix (final concentration of each dATP, dCTP, dGTP, dTTP was 200 ^M), 1.5 mM MgCl2, 10 pmol of each primer (Table 1), 2.5 U Taq DNA polymerase, 4 U AMV-RT, 0.5 ^L RNasin, and 1 mM DTT (which is added to the denatured template; see Note 10). The final reaction volume is 50 ^L. Include an appropriate negative control containing all of the above except template dsRNA.

3. Centrifuge the tubes for 10 s and overlay the mixture with 100 ^L of sterile mineral oil to prevent evaporation during thermal cycling.

5. Then heat all reaction tubes to 70°C to inactivate the AMV-RT enzyme and cool on ice for 1 min. This step results in the production of a single-stranded complementary DNA (cDNA) copy of one of the double-stranded RNA molecules.

6. Amplify the cDNA products by PCR using the following cycling parameters: 94°C for 1 min, 48°C for 2 min, 72°C for 1 min (30 cycles) and finally 72°C for 7 min (1 cycle) to complete extension (see Note 11).

7. To amplify partial length gene segment 4 (867 bp), the same reaction conditions are required, with the substitution of 40 pmol of each VP4 forward and VP4 reverse primer (Table 1) to target gene segment 4 specifically.

8. Perform reverse transcription of the dsRNA template as previously described (step 4 above). The reaction parameters for the VP4 primer pair are as follows; 94°C for 1 min, 53°C for 2 min, 72°C for 1 min (30 cycles), with a final extension at 72°C for 7 min (1 cycle).

9. Resolve RT-PCR products in a 1.5% (w/v) agarose gel, containing ethidium bromide (0.1 mg/mL) and visualize by UV illumination.

3.3.2. Purification of PCR Products

1. Purify RT-PCR products using a PCR purification kit according to the manufacturer's instructions (QIAGEN).

The RT-PCR products are subsequently used in seminested PCR assays to identify the G- and P-type of each isolate. In addition, the VP7 serotype could also be identified by direct DNA sequencing of the RT-PCR fragments followed by nucleotide comparison with sequenced strains in the databases using BLAST search tools (www.ncbi.nih.nlm.gov/BLAST).

3.3.3. Seminested PCR

1. Analyze PCR products from the RT-PCR reactions by seminested PCR to identify G-and/or P-types.

2. Identify the G-type of each isolate by adding sufficient template (see Note 12) to 5 ^L of 10X PCR buffer (100 mMTris-HCl, pH 9.0, 500 mM KCl), 8 ^L of stock dNTP mix, 1 mM MgCl2, 10 pmol of each G-serotype primer, and 10 pmol of the common reverse primer, RVG9 (Tables 1 and 2 and Fig. 3).

Table 2

Characteristics of G-Typing Primers

Table 2

Characteristics of G-Typing Primers

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