Wolbachia Detection Using Nested PCR

Prescreening for Wolbachia presence was first performed by nested PCR amplification of the Wolbachia-specific ftsZ (cell division) gene using the primer pair ftsZF3/R3 (5′-GCAAATACYGATGCTCARGC-3′ and 5′-ATCAATRCCAGTTGCAAGAA-3′), followed by ftsZF4/R4 (5′-CTAAGGGDCTTGGTGCTGGT-3′ and 5′-ACYTCTTCRCGCACTCTATT-3′). Four other genes were also amplified (dnaA, coxA, fbpA, and gatB), as described by Lefoulon et al. (24 (link)) (Table S2 in the supplemental material). For all PCRs, negative controls (no DNA) were also performed to rule out contamination artifacts. The PCR products were Sanger sequenced after purification using the NEB Monarch PCR purification kit. The sequences were analyzed by comparison with available sequences extracted from Wolbachia complete or draft genome sequences and the addition of sequences from Wolbachia from Zootermopsis angusticollis and Zootermopsis nevadensis (Table S3 in the supplemental material).