Computational protocol: Complete Genomic Characterization of Porcine Reproductive and Respiratory Syndrome Virus Strain HB-XL

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Protocol publication

[…] PRRSV strain HB-XL, used in this study, was originally isolated from swine cultivated in Hebei, China. Total nucleic acid was extracted from the cell culture supernatant of a single passage using the Viral RNA Kit (Tiangen, Beijing, China), used for reverse transcription and PCR amplification with PrimeSTARHS polymerase(Takala). Twelve pairs of specific primers were designed in to obtain the full-length sequence of HB-XL. The PCR was done under the following conditions in a thermal cycler: 1 cycle of 3 min at 98 °C; 33 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, and elongation at 72 °C for 1 min 37 s; and 1 cycle of 10 min at 72 °C. The amplified products were analyzed by electrophoresis in a 1% agarose gel. The 12 fragments of the complete genome of HB-XL were termed seq1–seq12 (0.8 kb–1.5 kb), respectively, according to their sizes (). PCR products were excised and the gel purified using the PureLink Quick Gel Extraction Kit (Tiangen, Beijing, China). The products were subcloned into pEASY-Blunt cloning vectors (Trangen, Beijing, China) and subsequently subjected to Sanger Sequencing reactions (Invitrogen, Beijing, China). Genomic analyses were conducted using the ClustalW program in the DNAStar. Phylogenetic trees were constructed using the distance-based neighbor-joining method in the MEGA 5. […]

Pipeline specifications

Software tools Clustal W, MEGA
Application Phylogenetics
Organisms Sus scrofa, Porcine reproductive and respiratory syndrome virus
Diseases Respiratory Insufficiency, Sprains and Strains, Virus Diseases, Porcine Reproductive and Respiratory Syndrome
Chemicals Amino Acids