Determination of Rotavirus, Sapovirus and Norovirus Co-Infection among Children Suffering from Gastroenteritis Referred to Ahvaz Abuzar Hospital, Southern Iran

cute gastroenteritis is one of the most common disease in infants and children both in developing and developed countries. Almost 80% of acute gastroenteritis is due to viruses (1). Between various kinds of diarrheal viruses, rotavirus is the most important cause of severe gastroenteritis in infants and children in the world (2). Norovirus and Sapovirus however are also considered to be significant global cause of gastroenteritis (3, 4). Transmission of these viruses occurs through fecal-oral route and might be other unknown modes of transmission (5, 6). The aim of this study was to determine co-infection of Rotavirus, Norovirus and Sapovirus in fecal specimens of children suffering from gastroenteritis referred to Ahvaz Abuzar Hospital. One hundred eighty fecal specimens were collected from children up to 5 years old suffering from acute gastroenteritis. For determination of viral co-infection we used two methods. First we used an ELISA kit for detection of rotaviruses and then the positive and negative samples were tested by the RTPCR method to detection of Noroviruses and Sapoviruses RNA. After suspension of about 5 grams of samples in ELISA buffer, ELISA test was done according to manufacture instructions. All tests carried out in duplicate. Viral RNA was extracted by Fermentas extraction kit (Lithuania) according to manufacturer’s instruction and eluted in 50μl of RNase-free sterile water. Reverse transcription was carried out in final volume 20 μl: 4μl 5x RT buffer , 1μl dNTPs (10 mM), 1μl Random hexamer (0.2 u/μl), 0.5μl RNase inhibitor (40 u/μl), 0.5μl RT enzyme (200 u/μl), 0.5μl MgCl2 (50mM), 6.5μl DEPC (RNase free water) and 6μl extracted RNA. The tubes were incubated at 42 ̊C for 1 hour. Then, the cDNA was stored at -20 ̊C until subsequent use as template in PCR reaction. 5μl of these cDNA was used as template for PCR by using specific primers. The rimer of PCR reaction for detection of sa o irus was as follows: R 0 : 5 -T AT TC T ACA CAA AAC CC, R: 5 T TAN AT CAR TCA TCA CC; The amplicon size of this primer was 320 bp. PCR condition was as follow: 35 cycles of amplification (5min in 94 0 C , 1min in 94 ̊C , 55 seconds in 51 ̊C and 50 seconds in 72 ̊C) and final extension at 6 min in 72 ̊C(7).The primer of PCR reaction for detection of Norovirus was as follow: N 5 : 5’-CTT GTT GGT TTG AGG CCA TAT ’, N 6R: 5’-ATA AAA GTT GGC ATG AAC A’. These primers amplify 470 bp. PCR A