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BACKGROUND: It has been recognized that infectious agents, such as different bacteria and viruses, may play a role in the developing of rheumatoid arthritis (RA). Recently, the mycoplasma species has been implicated in the pathogenesis of RA.

Aim: The aim of this study was to design a multiplex PCR for rapid and simultaneous detection of Mycoplasma pneumoniae, Mycoplasma hominis, and Mycoplasma arthritidis in the synovial fluid of patients with rheumatoid arthritis (RA).

METHODS: A total of 131 synovial fluid (SF) samples from patients with RA were assayed. Mycoplasma pneumoniae (ATCC: 29342), M. hominis (native strain), and the synthetic complete genome of M. arthritidis mitogen (MAM) superantigen were used as controls. All SF samples were subjected to DNA extraction separately and multiplex PCR was performed. The PCR products were confirmed by sequencing

RESULTS: The designed multiplex PCR was able to detect M. pneumoniae, M. hominis, and M. arthritidis in the SF of patients with RA with a frequency of 30 (22.9%), 23 (17.5%) and 13 (9.9%), respectively.

CONCLUSION: In this study, the overall detection of the Mycoplasma species in RA patients was 53.4%; thus, we recommend the application of multiplex PCR assays when searching for a specific anti mycoplasma treatment for RA patients.