Nucleotide amplification and sequencing of the GC-rich region between matrix and fusion protein genes of peste des petits ruminants virus.

Peste des petits ruminants virus (PPRV) causes a highly devastating disease of sheep and goats, that threatens the conservation of small wild ruminants. The development of PPRV vaccines, diagnostics and therapeutics, greatly depends on in-depth genomic data. Yet, high guanine-cytosine (GC) content between matrix (M) and fusion (F) genes of PPRV poses difficulty for both primer design and nucleotide amplification. In turn, this has led into absence or low nucleotide sequence coverage in this region. This poses a risk of missing important part of the genome that could help to infer viral evolution. Here, an overlapping long-read primer-based amplification strategy was developed to amplify the GC-rich fragments between M-F gene junction using nexus gradient polymerase chain reaction (PCR). The resulting amplicons were sequenced by dideoxynucleotide cycle sequencing and compared with other PPRV nucleotide sequences available at GenBank. Our findings indicate clear PCR amplification products with expected size of the GC-rich fragments on agarose gel electrophoresis. The sequencing results of these fragments indicate 99.5 % nucleotide identity with PPRV strain KY628761. An extremely difficult PCR target of 67.4 % GC contents was successfully amplified and sequenced using this long-read primer approach. The long-read primer set may be used in tiling multiplex PCR for complete genome sequencing of PPRV.

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