Computational protocol: Incidence of human rabies and characterization of rabies virus nucleoprotein gene in dogs in Fujian Province, Southeast China, 2002–2012

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Protocol publication

[…] A total of eight rabid dogs were sacrificed, and 81 seeming healthy (asymptomatic) dogs were euthanized. Canine brain tissues were collected and stored at −70 °C for the subsequent experiments. Total RNA was extracted from canine brain specimens using a Trizol reagent (Invitrogen; Carlsbad, CA, USA) according to the manufacturer’s instructions. N gene was amplified from the brain specimens using a nested reverse transcription polymerase chain reaction (RT-PCR) assay [] with the primers described in Table [], while primers N1/N2 and N3/N4 were used for detection of virus nucleic acid, and primers N1/N5 and N6/N2 were used for the amplification of full-length N gene (Table ). PCR products were purified with the PCR product extraction kit (Omega Bio-Tek, Inc., Norcross, GA, USA), and cloned into the pMD18-T vector with a TA cloning kit (TaKaRa; Dalian, China) following the manufacturers’ instructions. Transformed clones were identified by restriction enzyme digestion and subjected to DNA sequencing. To increase the accuracy, we selected three clones of each fragment for sequencing. The sequence of the three clones that exhibited 100% homology was identified as the target sequence. Sequences were then aligned and compared with the N gene sequences of the reference strains obtained from GenBank using the software Bioedit version 7.0.1 (DNASTAR, Inc.; Madison, WI, USA) and MEGA version 4.0. […]

Pipeline specifications

Software tools BioEdit, MEGA
Application Phylogenetics
Organisms Homo sapiens, Canis lupus familiaris, Rabies lyssavirus
Diseases Rabies, Virus Diseases