Gastroenteritis is a clinical illness of humans and other animals characterized by vomiting and diarrhea, caused by a variety of pathogens including viruses. An increasing number of viral species have been associated with gastroenteritis or have been found in stool samples as new molecular tools are developed. In this work, a DNA microarray capable in theory of parallel detection of more than 100 viral species was developed and tested. Initial validation was done with 10 different virus species and an additional 5 species were validated using clinical samples. Detection limits of 1x103 virus particles of human adenovirus C (HAdV), human astrovirus (HAstV) and group A rotavirus (RV-A) were established. Furthermore, when exogenous RNA was added, the limit for RV-A detection decreased in one log. In a small group of clinical samples from children with gastroenteritis (n = 76), the microarray detected at least one viral species in 92% of the samples. Single infection was identified in 63 samples (83%) and more than one virus was identified in 7 samples (9%). The most abundant virus species were RV-A (58%), followed by anellovirus (15.8%), HAstV (6.6%), HAdV (5.3%), Norwalk virus (6.6%), human enterovirus (HEV; 9.2%), human parechovirus (1.3%), Sapporo virus (1.3%) and human bocavirus (1.3%). To further test the specificity and sensitivity of the microarray, the results were verified by RT-PCR detection of 5 gastrointestinal viruses. The RT- PCR assay detected a virus in 59 samples (78%). The microarray showed good performance for detection of RV-A, HAstV and calicivirus, while the sensitivity for HAdV and HEV was low. Furthermore some discrepancies in detection of mixed infections were observed, and were addressed by RT-qPCR of the viruses involved. It was observed that differences in the amount of genetic material favored the detection of the most abundant virus. The microarray described in this work should help to understand the etiology of gastroenteritis in humans and animals.
DNA Microarray for Detection of Gastrointestinal Viruses
SOTO DEL RIO, MARIA DE LOS DOLORES;
2015-01-01
Abstract
Gastroenteritis is a clinical illness of humans and other animals characterized by vomiting and diarrhea, caused by a variety of pathogens including viruses. An increasing number of viral species have been associated with gastroenteritis or have been found in stool samples as new molecular tools are developed. In this work, a DNA microarray capable in theory of parallel detection of more than 100 viral species was developed and tested. Initial validation was done with 10 different virus species and an additional 5 species were validated using clinical samples. Detection limits of 1x103 virus particles of human adenovirus C (HAdV), human astrovirus (HAstV) and group A rotavirus (RV-A) were established. Furthermore, when exogenous RNA was added, the limit for RV-A detection decreased in one log. In a small group of clinical samples from children with gastroenteritis (n = 76), the microarray detected at least one viral species in 92% of the samples. Single infection was identified in 63 samples (83%) and more than one virus was identified in 7 samples (9%). The most abundant virus species were RV-A (58%), followed by anellovirus (15.8%), HAstV (6.6%), HAdV (5.3%), Norwalk virus (6.6%), human enterovirus (HEV; 9.2%), human parechovirus (1.3%), Sapporo virus (1.3%) and human bocavirus (1.3%). To further test the specificity and sensitivity of the microarray, the results were verified by RT-PCR detection of 5 gastrointestinal viruses. The RT- PCR assay detected a virus in 59 samples (78%). The microarray showed good performance for detection of RV-A, HAstV and calicivirus, while the sensitivity for HAdV and HEV was low. Furthermore some discrepancies in detection of mixed infections were observed, and were addressed by RT-qPCR of the viruses involved. It was observed that differences in the amount of genetic material favored the detection of the most abundant virus. The microarray described in this work should help to understand the etiology of gastroenteritis in humans and animals.
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