Sensitive detection of multiple rotavirus genotypes with a single reverse transcription-real-time quantitative PCR assay.
Rotaviruses are one of the major causes of diarrhea in infants and children under 5 years old, especially affecting developing countries. In natural disasters, fecal matter and potable waters can mix, allowing low, yet infective, concentrations of rotavirus to be present in water supplies, constituting a risk for the population. Any of the most commonly detected rotavirus genotypes could originate an outbreak. The development of a fast and sensitive method that could detect the broadest possible range of rotavirus genotypes would help with efficient diagnosis and prevention. We have designed a reverse transcription (RT)-real-time quantitative PCR approach targeted to the rotaviral VP2 gene, based on a multiple-sequence alignment of different human rotaviral strains. To overcome the high nucleotide sequence diversity, multiple forward and reverse primers were used, in addition to a degenerate probe. The performance of the assay was tested on isolates representing the most prevalent human genotypes: G1P[8], G2P[4], G3P[8], G4P[8], G9P[8], and G12P[8]. The developed method improved classical rotavirus detection by enzyme-linked immunosorbent assay and nested RT-PCR by 5 and at least 1 order of magnitude, respectively. A survey of 159 stool samples indicated that the method can efficiently detect a broad range of rotavirus strains, including different G-P genotype combinations of human, porcine, and bovine origin. No cross-reactivity was observed with other enteric viruses, such as astrovirus, sapovirus, and norovirus.
Department of Biotechnology and Systems Biology, National Institute of Biology, Vecna pot 111, 1000, Ljubljana, Slovenia. ion.gutierrez@nib.si
Ion
I
Gutiérrez-Aguirre
Andrej
A
Steyer
Jana
J
Boben
Kristina
K
Gruden
Mateja
M
Poljsak-Prijatelj
Maja
M
Ravnikar
D001483:Base Sequence; D036022:Capsid Proteins / Q000235:genetics; D002675:Child, Preschool; D005243:Feces / Q000821:virology; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012401:Rotavirus / Q000145:classification* / Q000302:isolation & purification*; D012400:Rotavirus Infections / Q000175:diagnosis* / Q000821:virology*; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D014773:Virology / Q000379:methods*
D036022:Capsid Proteins; D015345:Oligonucleotide Probes; C442545:VP2 protein, Rotavirus