Exosap cleanup procedure

Some PCR products obtained by targeting the CQ11, ace-2 and cytb were randomly chosen and sequenced to confirm the PCR results and to determine whether nucleotide polymorphisms were informative to distinguish between Cx. pipiens forms. PCR products were purified using the ExoSAP cleanup procedure (Amersham Biosciences, Piscataway, NJ, USA). Cycle sequencing was performed using BigDye Terminator v.3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) and analyzed using a capillary automated sequencer 3500 Genetic Analyzer (Ruo. Hitachi, Foster City, CA, USA). Sequences were aligned using BioEdit 7.1.9 [31 ] and identified by comparison with sequences deposited in the GenBank database.

Specific TOSV detection was carried out by sequencing PCR products as described previously [24 (link)]. The PCR products were purified using the ExoSAP cleanup procedure (Amersham Biosciences, France). All nucleotide sequences were obtained using the Big Dye Terminator v.3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) and the 3130 automated sequencer (Applied Biosystems, Foster City, CA, USA). Sequences were edited and aligned using DNA Baser Sequence Aligner v3.5.4 software (Heracle BioSoft SRL, www.DnaBaser.com) to obtain optimal sequence alignment files. A BLAST analysis at the NCBI database was made to retrieve sets of homologues exhibiting high scores to the L gene of TOSV.
To build phylogenetic tree, the maximum parsimony [26 (link)] method calculating bootstrap confidence values of 100 bootstrapping trials, using the MEGA 5.2 software, was used.

The specificity of the duplex PCR was confirmed by sequencing PCR amplicons of A. marginale and A. phagocytophilum using primers M4-OvMar-F/M4-Mar-R for msp4 gene and Msp2-3 F/Msp2-3R for msp2, respectively. Thirteen randomly chosen positive PCR products were purified using the ExoSAP cleanup procedure (Amersham Biosciences, Piscataway, NJ, USA). All nucleotide sequences were obtained using the Big Dye Terminator v.3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) and the 3130 automated sequencer (Applied Biosystems). The sequences were edited and aligned using DNA Baser Sequence Aligner v3.5.4 software (Heracle BioSoft SRL, www.DnaBaser.com) to obtain optimal sequence alignment files. A BLAST analysis was made in the NCBI database to retrieve sets of homologues exhibiting high scores with the partial msp2 and msp4 gene of A. phagocytophilum and A. marginale, respectively.