![]() Thus, global surveillance and intensive studies to understand the behavior and pathogenicity of these viruses are critical for understanding and controlling the spread of infections. These viruses have caused pandemics resulting in the death of millions of people worldwide and still pose threats and challenges to public health. The influenza A viruses (IAVs) which belong to the Orthomyxoviridae family, consist of eight segments (ie, PB2, PB1, PA, HA, NP, NA, M, and NS) 1. In conclusion, the novel cloning method for influenza A virus will contribute to a significant reduction in the time required for genetic studies of emerging influenza viruses. Our results demonstrate that this method-one-Pot cloning for influenza A virus-was efficient in terms of required time and cloning rate. We successfully cloned all genes from broad subtypes of influenza A viruses (H1-H12, N1-N9) and rescued by the RG system. The specifically designed viral gene and vector primers used for CPEC-PCR have improved cloning efficiency ranging from 63.6 to 100% based on the results of gene-specific colony PCR which was additionally confirmed by enzyme digestion. In this study, we improved influenza genome cloning into the pHW2000 vector for an RG system by incorporating a sequence-independent circular polymerase extension cloning (CPEC) approach which requires only 2 steps (reverse transcription and one-pot CPEC-PCR) and takes about 4 hours before the transformation. enzyme digestion and ligation) and exhibits low cloning efficiency compared to the sequence-independent cloning method. ![]() However, the conventional sequence-dependent method for cloning influenza genome segments is time-consuming and requires multiple processes (eg. The reverse genetics (RG) system of influenza A viruses is well established.
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