Reverse transcription-PCR (RT-PCR) was performed to detect five groups of enteric viruses: panenteroviruses (i.e., poliovirus, echoviruses, and coxsackieviruses), rotavirus, HAV, and NLV genogroups 1 and 2. We used a single-tube, large-volume RT-PCR format that has been previously described by Abbaszadegan et al. (2
). Reactions were not multiplexed. In brief, 50 μl of chromatography column eluate, 50 μl of nuclease-free water, and 4 μl (2 μg) of random hexamers (Promega, Madison, Wis.) were mixed, heated for 4 min at 99°C, placed on ice, and then supplemented with 186 μl of RT reaction mixture. The mixture components and their final concentrations were as follows: 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 3 mM MgCl2
, 10 mM dithiothreitol, 70 μM concentrations of each deoxynucleoside triphosphate (Applied Biosystems, Foster City, Calif.), 200 U of RNasin (Promega), and 500 U of SuperScript II reverse transcriptase (Life Technologies, Rockville, Md.). Reaction tubes were inserted into a thermal cycler (RoboCycler; Stratagene, La Jolla, Calif.), and the following thermal profile was run: 25°C for 15 min, 42°C for 60 min, and 99°C for 5 min and then 4°C until PCR amplification. After the RT reaction, an 8.6-μl PCR cocktail was added containing 10 U of Taq
DNA polymerase (Applied Biosystems) and 0.4 μM concentrations of each primer (Integrated DNA Technologies, Coralville, Iowa). Primer pairs are listed in Table 1
. Amplification conditions for enteroviruses, rotavirus, and HAV included an initial denaturation step for 4 min at 96°C, followed by 35 cycles of denaturation (94°C for 75 s), annealing (55°C for 75 s), and extension (72°C for 75 s). The amplification conditions for NLVs G1 and G2 were similar to the other virus groups, except that there were 40 cycles and the annealing and extension temperatures were 50 and 60°C, respectively. All amplifications ended with a final extension period of 72°C for 7 min. Reaction products were electrophoresed by using a 1.6% agarose gel containing ethidium bromide, and an amplicon of the size expected for the virus group tested (Table 1
) was detected by UV light illumination (Gel-Doc System; Bio-Rad Laboratories, Hercules, Calif.).
Note that a separate RT reaction with 50 μl of chromatography column eluate was run for each of the five virus groups tested. Given the range in final concentrated sample volumes obtained in the present study (11 to 36 ml), the extraction volume of 500 μl, and the column eluate volume of 750 μl, each RT-PCR assay analyzed 0.1 to 0.3% of the original sample volume.
RT-PCR controls included a negative control of the beef extract eluent, a negative control of the RT and PCR cocktails, and a positive control of each virus tested, seeded into beef extract and carried through the same RNA extraction and RT-PCR steps as the field samples. RT-PCRs were batched by using one positive control per batch to minimize the possibility of amplicon contamination.