Computational protocol: Human Bocavirus Infections in Hospitalized Children and Adults

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Protocol publication

[…] All pediatric (from case-patients and controls) and adult (case-patients only) NPA specimens were previously analyzed by using a multiplex real-time PCR assay for influenza A and B viruses, human respiratory syncytial virus (hRSV), and human metapneumovirus (hMPV) (,). For symptomatic children, viral cultures and antigen detection assays were performed upon request by the treating physician. Remaining specimens were frozen at –80°C until subsequent HBoV PCR studies.Nucleic acids were extracted from 200 μL of NPA by using the QIAamp viral RNA Mini Kit (QIAGEN, Inc., Mississauga, Ontario, Canada). A duplex HBoV PCR (TaqMan assay) was used to amplify conserved regions of NP-1 and NS-1 genes as described (), except that the NS-1 forward primer was replaced with primer 5′-TAG TTG TTT GGT GGG ARG A-3′. Probes were labeled with 6-carboxyfluorescein (FAM) or tetrachloro-6-carboxyfluorescein (TET) at the 5′ end and with a quencher at the 3′ end. Amplicons were 81 bp (NP-1) and 74 bp (NS-1), respectively. Duplex amplification was conducted by using 1 µmol/L NS-1 forward primer and 0.4 µmol/L NS-1 reverse primer and the 2 NP-1 primers. Taqman probes were used at concentrations of 0.1 mmol/L for the NP-1 gene and 0.2 mmol/L for the NS-1 gene (). The amplification master mixture consisted of 2.5 mmol/L MgCl2, 3.33 mg/mL bovine serum albumin, 0.2 mmol/L of each of the 4 deoxynucleotide triphosphates (Amersham Biosciences, Uppsala, Sweden), 10 mmol/L Tris-HCl, 50 mmol/L KCl, 0.625 U Promega Taq DNA polymerase (Fisher Scientific, Markham, Ontario, Canada) combined with TaqStart antibody (BD Biosciences Clontech, Palo Alto, CA, USA), and 3 μL DNA in a final volume of 25 μL. PCR amplification (180 s at 94°C and 45 cycles for 10 s at 95°C, 30 s at 58°C, and 30 s at 72°C) was performed in a Smart Cycler thermal cycler (Cepheid, Sunnyvale, CA, USA). A PCR extension step of 5 min at 72°C was performed at the end of the cycling protocol. An HBoV infection was defined by a positive PCR result for NP-1 and NS-1. The duplex assay had a sensitivity of 10 genome copies for NP-1 and NS-1 gene targets on the basis of quantification analysis of positive control plasmids.Half of the HBoV-positive samples were randomly selected for phylogenetic analysis, which consisted of amplifying and sequencing a 842-bp region of the VP1/VP2 genes as described (). The VP1/VP2 nucleotide sequences from this study, as well as prototype sequence type (ST)1 and ST2 (), were entered into a multiple alignment generated by ClustalW software version 1.83 (www.molecularevolution.org/software/clustalw) and corrected through final visual inspection with the SeqLab application (Wisconsin package version 10.3; Accelrys, San Diego, CA, USA). Phylogenetic analyses were conducted with the MEGA version 3.1 software () by using the distance method and the neighbor-joining algorithm with Kimura-2 parameters. Topologic accuracy of the tree was evaluated by using 1,000 bootstrap replicates. […]

Pipeline specifications

Software tools Clustal W, MEGA
Application Phylogenetics
Organisms Human bocavirus, Homo sapiens
Diseases Bronchitis, Pneumonia, Respiratory Tract Infections