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Comment
. 2017 Aug 18;85(9):e00326-17.
doi: 10.1128/IAI.00326-17. Print 2017 Sep.

Reply to Tantibhedhyangkul et al., 'Suspected Mycoplasma Contamination in the Study "Toll-Like Receptor 2 Recognizes Orientia tsutsugamushi and Increases Susceptibility to Murine Experimental Scrub Typhus"'

Mohammad Gharaibeh  1 , Monica Hagedorn  1 , Stefanie Lilla  1 , Matthias Hauptmann  1 , Holger Heine  2 , Bernhard Fleischer  1   3 , Christian Keller  4
Affiliations
Comment

Reply to Tantibhedhyangkul et al., 'Suspected Mycoplasma Contamination in the Study "Toll-Like Receptor 2 Recognizes Orientia tsutsugamushi and Increases Susceptibility to Murine Experimental Scrub Typhus"'

Mohammad Gharaibeh et al. Infect Immun. .
No abstract available

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Figures

FIG 1
FIG 1
Decontamination of O. tsutsugamushi Karp from Mycoplasma sp. by mouse passage before the study of TLR2 involvement by Gharaibeh et al. O. tsutsugamushi Karp was propagated in L929 mouse fibroblasts (ZK2009). To decontaminate the strain from Mycoplasma sp., it was inoculated i.p. into a BALB/c mouse, reisolated from the spleen in L929 cell culture, and continuously propagated (ZK2013). DNA was prepared from reference samples ZK2009 and ZK2013 that had been stored at −20°C or Mycoplasma pneumoniae as a positive control (EZ1; Qiagen, Hilden, Germany) and eluted in 60 μl of AVE buffer (Qiagen). (A) A mycoplasma-specific PCR was run with the GPO-3/MGSO primer pair (MGSO, 5′-TGCACCATCTGTCACTCTGTTAACCTC-3′; GPO-3, 5′-GGGAGCAAACAGGATTAGATACCCT-3′) (2). The reaction mixture contained, in a total reaction volume of 25 μl, 5 μl of template, 400 nM GPO-3, 200 nM MGSO (both from MWG-Biotech, Ebersberg, Germany), 0.2 mM deoxynucleoside triphosphates, and 0.5 U of HotStarTaq DNA polymerase (Qiagen, Hilden, Germany). The cycling protocol was 94°C for 15 min; 35 cycles of 94°C for 20 s, 58°C for 30 s, and 72°C for 30 s; and 72°C for 7 min. (B) To detect O. tsutsugamushi-specific DNA, a PCR targeting a 409-bp fraction of the 56-kDa protein-encoding gene with the primer pair OtsuF/OtsuR was used (OtsuF, 5′-AATTGCTAGTGCAATGTCTG-3′; OtsuR, 5′-GGCATTATAGTAGGCTGAG-3′) (3). The reaction mixture contained, in a total reaction volume of 25 μl, 2 μl of template, 400 nM OtsuF, 400 nM OtsuR (both from TIB MOLBIOL, Berlin, Germany), 0.2 mM deoxynucleoside triphosphates, and 0.5 U of HotStarTaq DNA polymerase (Qiagen, Hilden, Germany). The cycling protocol was 94°C for 15 min; 40 cycles of 94°C for 20 s, 58°C for 30 s, and 72°C for 30 s; and 72°C for 7 min. Amplification products were visualized on a 2.2% agarose gel (Lonza, Basel, Switzerland). The values to the left of the marker lanes are molecular sizes in kilodaltons.
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References

    1. Gharaibeh M, Hagedorn M, Lilla S, Hauptmann M, Heine H, Fleischer B, Keller C. 2016. Toll-like receptor 2 recognizes Orientia tsutsugamushi and increases susceptibility to murine experimental scrub typhus. Infect Immun 84:3379–3387. doi:10.1128/IAI.00185-16. - DOI - PMC - PubMed
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    1. Ogawa M, Uchiyama T, Satoh M, Ando S. 2013. Decontamination of mycoplasma-contaminated Orientia tsutsugamushi strains by repeating passages through cell cultures with antibiotics. BMC Microbiol 13:32. doi:10.1186/1471-2180-13-32. - DOI - PMC - PubMed
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