A novel RT-multiplex PCR for detection of Aichi virus, human parechovirus, enteroviruses, and human bocavirus among infants and children with acute gastroenteritis

A novel reverse transcription-multiplex polymerase chain reaction assay was developed to detect Aichi virus, human parechovirus, enteroviruses, and human bocavirus. A mixture of four pairs of published specific primers, 6261 and 6779, ev22(+) and ev22(−), F1 and R1, 188F and 542R, was used to amplify the viral genomes and specifically generate four different amplicon sizes of 519, 270, 440, and 354 bp for Aichi virus, human parechovirus, enteroviruses, and human bocavirus, respectively. A total of 247 fecal specimens previously screened for rotavirus, adenovirus, norovirus, sapovirus, and astrovirus-negative, collected from infants and children with acute gastroenteritis in Japan from July 2007 to June 2008, were tested further for the presence of the four viruses, Aichi virus, human parechovirus, enteroviruses, and human bocavirus, by RT-multiplex PCR. The total detection rate of these viruses was 26.7% (66 out of 247 samples). Of these, HPeV, EVs, and HBoV were identified in 20, 41, and 5 specimens. No Aichi virus was found among these subjects. The sensitivity and specificity of RT-multiplex PCR were assessed and demonstrated a strong validation against RT-monoplex PCR. This is the first report of detecting these types of viruses in fecal samples from infants and children with acute gastroenteritis by RT-multiplex PCR.