Computational protocol: Measles Virus Strain Diversity, Nigeria and Democratic Republic of the Congo

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[…] Specific cDNA of MV nucleoprotein was synthesized by reverse transcription by using SuperscriptIII Reverse Transcriptase (Invitrogen, Merelbeke, Belgium) and random hexamers (Invitrogen). MV cDNA was amplified by nested PCR by using primers MN5 (nt 1113–1134, 5′-GCCATGGGAGTAGGAGTGGAAC-3′ []) and MN6 (nt 1773–1754, 5′-CTGGCGGCTGTGTGGACCTG-3′ []) for the first round and primers Nf1a (nt 1199–1224, 5′-CGGGCAAGAGATGGTAAGGAGGTCAG-3′) and Nr7a (nt 1725–1703, 5′-AGGGTAGGCGGATGTTGTTCTGG-3′) for the second round. Both PCRs were performed in a total volume of 25 μL that contained 1.8 mmol/L MgCl2, 1× PCR buffer, 0.2 mmol/L dNTPs, 0.5 U Platinum Taq (Invitrogen), and 0.8 μmol/L forward and reverse primer (Eurogentec, Seraing, Belgium). One microliter of cDNA or 5 μL of first-round product (diluted 50× in water) was added as template. Cycling conditions were initial denaturation at 94°C for 2 min; 35 (first round) or 30 (second round) cycles of amplification at 94°C for 30 s, 55°C (first round) or 58°C second round) for 1 min, and 72°C for 1 min; and a final extension at 72°C for 5 min.Nested PCR products were purified by using the Jetquick PCR product Purification Spin Kit (Genomed, Lohne, Germany). Twenty-five cycles of cycle sequencing (2-min elongation) were performed by using a BigDye Terminator version 3.1 Cycle Sequencing kit (Applied Biosystems, Nieuwerkerk, the Netherlands) with Nf1a or Nr7a primers (0.5 μmol/L) and 10 ng of purified PCR product. Cycle sequencing products were analyzed on an ABI 3130 Genetic Analyzer (Applied Biosystems). Sequences were aligned by using ClustalW (), and phylogenetic trees were constructed by using the neighbor-joining method (Kimura 2-parameter) and MEGA4 software (). All new sequences were submitted to GenBank under accession nos. FN985102–FN985162. […]

Pipeline specifications

Software tools Clustal W, MEGA
Application Phylogenetics
Organisms Measles morbillivirus