Real-Time Reverse Transcription-PCR for Detection of Rotavirus and Adenovirus as Causative Agents of Acute Viral Gastroenteritis in Children

Abstract

Viral pathogens are the most common cause of gastroenteritis in developed countries. Human rotavirus and adenovirus infections are major causes of acute outbreaks and sporadic cases of gastroenteritis, occurring primarily among children less than 2 years of age. Patient hospitalization is often required, with enormous infection control implications. This work describes the development of real-time PCR assays for the detection of group F adenovirus, rotavirus A, and rotavirus C from stool specimens. Two hundred twenty stool samples from pediatric patients exhibiting symptoms of diarrhea and/or vomiting were examined. PCR results were compared with those of virus detection by electron microscopy and latex agglutination antigen detection. The incorporation of an internal-control RNA that was spiked into individual stool extracts functioned as an internal validation for the reporting of PCR-negative results. Rotavirus C was not detected by real-time PCR in the patient stool samples examined. Real-time reverse transcription-PCR resulted in 175% and 111% increases in the rates of detection of adenovirus F and rotavirus A, respectively, compared with latex agglutination testing. Molecular detection increased the number of stool specimens in which causative agents of gastroenteritis were identified by 155% compared to electron microscopy. Genotyping of a proportion of the rotavirus and adenovirus strains identified only genotype G1 rotavirus and both adenovirus genotypes 40 and 41 in circulation within the patient cohort examined. The results highlight the significance of rapid molecular methods for the routine screening of stool samples in hospital laboratories to provide rapid definitive diagnoses.

Genotypic Distribution of Rotavirus in Phnom Penh, Cambodia: An Association of G9 with More Severe Diseases

“…Probes and primers used for rotavirus detection were combinations of probes and primers designed for this study or modified from published literature ( Table 1). 11 Real-time RT PCR assays were conducted in a 15 μL reaction volume containing 1-μL nucleic acid template, TaqMan EZ buffer (Applied Biosystems, Foster City, CA), 0.3 mM of dATP, dCTP, dGTP each, 0.6 mM dUTP, 3.0 mM manganese acetate, 0.3 μM of forward and reverse primer each, 50 nM of TaqMan probe (Table 1), 1.5 U rTth DNA polymerase, and 0.15 U uracyl N-glycosylase (UNG). Reaction mixtures were incubated in an ABI7900 Sequence Detection System (Applied Biosystems).…”

“…A 20 μL reaction contained 1× PCR buffer, 0.8 mM dNTP mixture, 2.5 mM MgCl 2 , 0.5 U Taq DNA polymerase (Applied Biosystems), 3 μL of RT mixture, and 200 nM of each genotyping primer; P-typing: Con-3d and P [4]d, P [6] d, P [8], P [8]m, P [8]G1, P [9], P [10], or P [11]; for G-typing: 9Con1 and 9T1-1d, 9T1-2, 9T1-3Pd, 9T-4, or 9T-9B; for G12-typing: G12Fd and G12Rd (Table 1) was mixed and subjected to 40 cycles of amplification consisting of denaturation for 30 seconds at 95°C, annealing for 2 minutes at 50°C and extension for 1 minute at 72°C. The resulting PCR products were analyzed on a 1.5% agarose gel along with 100-base pair DNA ladder (Invitrogen), and visualized by ethidium bromide staining.…”

Circulation of 3 Lineages of a Novel Saffold Cardiovirus in Humans

“…Co-infection in the Brazil cohort was assessed by using recently published methods of real-time RT-PCR for norovirus, rotavirus, enterovirus, parechovirus, adenovirus, and astrovirus (18)(19)(20)(21)(22)(23). For the Germany cohorts, testing was done with the IDEIA rotavirus, adenovirus, and astrovirus antigen enzyme immunoassays (DakoCytomation, Ely, UK) and nested RT-PCRs as described before (23).…”

Detection and Genotyping of Viral Gastroenteritis in Hospitalized Children Below Five Years Old in Cairo, Egypt

“…Nearly 3.9% of reported deaths are due to rotavirus infection, whereas enteric adenovirus is the second leading cause of acute diarrhea after rotavirus (1,2). Both viruses, as extremely etiological agents for diarrhea, constitute a major health problem in developing countries (3).…”