Abstract
Enterovirus 71 (EV71) immunomagnetic enrichment technique and routine detection methods were combined to detect swab environmental specimens to elucidate the role of environmental specimens in the spread of EV71. Immunomagnetic beads with specific enrichment of EV71 virus were prepared, then the beads were used to absorb the EV71 virus from environmental samples. Obtained immunomagnetic bead-virus complexes were detected by RT-PCR, RT-qPCR and cell culture. Isolated virus were subjected to VP1 full-length amplification and homology analysis was performed. A total of 4 µg of EV71 monoclonal antibody was mixed with 50 µl magnetic beads, and the highest coating efficiency was reached after incubating at room temperature for 2 h. Satisfactory enrichment effect was achieved by adding 50 µl immunomagnetic beads to 1.5 ml sample and shaking at room temperature for 2 h. The method of EV71 enrichment has high sensitivity and specificity. A total of 346 specimens after enrichment by immunomagnetic beads, the positive rates of RT-qPCR, RT-PCR and cell culture were 20.52, 5.78, and 9.25%, respectively, which were also significantly higher than those before enrichment (15.90, 3.47 and 4.05%; P<0.05). After enrichment with immunomagnetic beads, isolation rate of EV71 virus from case specimens and home environment specimens increased from 27.45 to 43.14% and from 0 to 5.29%, respectively. In home environment-positive specimens, positive rate of toys and stationery was high (52.00 and 24.00%, respectively). In kindergarten environmental samples, the positive rate of RT-qPCR was 6.12%, and EV71 virus was not isolated. Sequence analysis showed that the nucleotide homology of case isolates and home environment isolates was 98.0-100%.