Risk factors for severe bronchiolitis caused by respiratory virus infections among Mexican children in an emergency department

Robledo-Aceves, Mireya; Moreno-Peregrina, María de Jesús; Velarde-Rivera, Fernando; Ascencio-Esparza, Elba; Preciado-Figueroa, Francisco M.; Caniza, Miguela A.; Escobedo-Melendez, Griselda

Briefly, the viral genomes were manually obtained from 200 mL of samples with an extraction kit included in the assay, then eluted in 20 mL of elution buffer. RT-PCR was carried out in 2 different ready-to-use amplification tubes with specific primers for the respiratory viruses listed below. One multiplex-PCR tube for amplification of coronavirus, metapneumovirus (subtypes A and B), parainfluenza virus 1, 2, 3, and 4 (subtypes A and B), and RSV type A; and another multiplex-PCR tube for amplification of adenovirus; bocavirus; enterovirus, influenza virus C, metapneumovirus, and RSV type B. These tubes have a mixture of retrotranscriptase and DNA polymerase enzymes for amplification both RNA and DNA viruses. During the amplification process, the amplified products were labeled with biotin. These biotin markers act as reference system for the automatic alignment of the array grid, and serve as control of the reagents performance. A specific 120-to 330-base pair fragment of the viral genomes was amplified. In addition, an internal control (200 mL of solution dilution) was used to ensure the proper performance of the process of amplification.