Identification of co-infection by rotavirus and parvovirus in dogs with gastroenteritis in Mexico

Ortega, Ariadna Flores; Martínez-Castañeda, José Simón; Bautista-Gómez, Linda G.; Muñoz, Raúl Fajardo; Hernández, Israel Quijano

In the case of rotavirus, two-step RT-PCR was performed for each of the samples, using the ImProm-II TM Reverse Transcription System kit (Promega, USA). In the first step, cDNA synthesis was accomplished. 3 L of RNA of each purification plus 2 L (0.5 g/reaction) of primer dT was used in each reaction. All reactions were incubated at 70 • C for 15 min and subsequently at 4 • C for 5 min. Next, 1 L of ImProm-II TM Reverse Transcriptase (1 u/L) was added, as well as 1 L dNTP All reactions were performed under the following conditions: 1 cycle at 94 • C 5 min, followed by 35 cycles at 94 • C 1 min, 61 • C 1 min, 72 • C for 2 min and a final extension at 72 • C 5 min. Amplicons were loaded in a 2.0% agarose gel (stained with 0.5 mg/mL of ethidium bromide) and visualized by means of a UV Bio-Imaging Systems Mini Bis Pro transilluminator (Fig. 1) .