Molecular Detection of Rickettsia and Chlamydia

A 539 base pair amplicon for 17kDa and a 650 base pair amplicon for ompA gene was amplified as previously described [33 (link)] using Platinum taq (Thermo Fisher Scientific). Cycling conditions for ompA amplification were 95°C for 2 min and 45 cycles of 95°C for 30 sec, 42°C for 35 sec, and 60°C for 2 min. Cycling conditions for 17kDa amplification were 95°C for 2 min and 45 cycles of 95°C for 30 sec, 57°C for 60 sec, and 72°C for 2 min. PCR products were resolved on a 2% agarose gel and purified using the QIAquick PCR Purification Kit (Qiagen). Samples were sequenced on the SeqStudio (Thermo Fisher Scientific) using the BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific) according to the manufacturer’s recommendations. Forward and reverse reads were aligned using CLC Genomics Workbench (Qiagen) and a consensus sequence for each gene was generated for BLAST analysis. Sequences of 17kDa and ompA genes and references from GenBank were imported and aligned in Geneious Prime 2022.11.0.14.1. The sequences were MAFFT aligned and exported to MEGA 10.2.6 [34 (link)] where maximum likelihood trees were created at 1,000 bootstrap iterations.