S.B. Schwartz, K.A. Thurman, S.L. Mitchell, B.J. Wolff, J.M. Winchell

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Presentation transcript:

Genotyping of Mycoplasma pneumoniae isolates using real-time PCR and high- resolution melt analysis S.B. Schwartz, K.A. Thurman, S.L. Mitchell, B.J. Wolff, J.M. Winchell Clinical Microbiology and Infection Volume 15, Issue 8, Pages 756-762 (August 2009) DOI: 10.1111/j.1469-0691.2009.02814.x Copyright © 2009 European Society of Clinical Infectious Diseases Terms and Conditions

FIG. 1. Real-time PCR high-resolution melt (HRM) assay. (a) The HRM profile for type 1 and type 2 isolates in the normalized graph mode; and (b) the difference graph illustrates deviations between types 1 and 2. The type 2 isolates have been selected as the standard, displaying the deviations of type 1 isolates from the type 2 isolates. Clinical Microbiology and Infection 2009 15, 756-762DOI: (10.1111/j.1469-0691.2009.02814.x) Copyright © 2009 European Society of Clinical Infectious Diseases Terms and Conditions

FIG. 2. Sequence alignment. Alignment of the variable regions derived from the c.1900-bp product of Mycoplasma pneumoniae clinical isolates 2p (type 1) and 1136 (type 2) and the prototypical type 2 reference strain FH. The type 1 reference strain M129 sequence [24] is identical to 2p. The top row illustrates the consensus sequence. The accession numbers for the sequences are FJ215694, FJ215693, and FJ215695 for strains 1136, FH, and 2p, respectively. Clinical Microbiology and Infection 2009 15, 756-762DOI: (10.1111/j.1469-0691.2009.02814.x) Copyright © 2009 European Society of Clinical Infectious Diseases Terms and Conditions

FIG. 3. Restriction fragment length polymorphism analysis of HaeIII-digested PCR fragments. M129 and FH were used as the reference strains for type 1 and type 2, respectively. Characteristic banding patterns for each type are shown. A broad-range molecular weight marker is shown in lane 13 for both a and b. (a) Representative gel illustrating HaeIII-digested type-specific banding patterns from fragment 1 (generated by primers ADH1/ADH2) for ten isolates along with reference strains. Type 1 isolates (lane 2 – M129; lanes 4, 6, 7, 8) and type 2 isolates (lane 1 – FH; lanes 3, 5, 9, 10, 11, 12) are shown; and (b) representative gel illustrating HaeIII-digested type-specific banding patterns from fragment 2 (generated by primers ADH3/ADH4) for ten isolates along with reference strains. Type 1 isolates (lane 2 – M129; lanes 4, 5, 6, 7, 10, 11, 12) and type 2 isolates (lane 1 – FH; lanes 3, 8, 9) are shown. Clinical Microbiology and Infection 2009 15, 756-762DOI: (10.1111/j.1469-0691.2009.02814.x) Copyright © 2009 European Society of Clinical Infectious Diseases Terms and Conditions