Computational protocol: Culture-Independent Detection and Genotyping of Mycoplasma pneumoniae in Clinical Specimens from Beijing, China

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Protocol publication

[…] By comparing the genome sequences of 20 M. pneumoniae clinical isolates with those of the reference strains, we observed remarkable differences in large fragment insertions or deletions, mainly between genes mpn 459 (corresponding to the type 1 strain, M129) and mpna 5864 (corresponding to the type 2 strain, 309). The type 1 strains had an approximately 3-kb insertion encoding three genes, mpn 457–459, whereas the type 2 strains had an approximately 5-kb insertion encoding five genes, mpna 5861–5865. Therefore, the primers and probes, designed with Primer Express software (version 3.0; Life Technologies-Applied Biosystems, Grand Island, NY, USA), were based on the conserved sequences of the 3kb and 5kb insertion fragments within the type 1 and type 2 strain, respectively. These primers and probes are listed in . Each PCR mixture was prepared in a total volume of 25 μl and contained the following per reaction: 12.5 μl Platinum Quantitative PCR SuperMix-UDG (Life Technologies-Invitrogen), 1.5 μl MgCl2 (50 mM), 0.5 μM final concentration of each primer, 0.2 μM final concentration of each probe, 1.25 U Platinum Taq DNA polymerase (5 U/μl; Life Technologies-Invitrogen), 1 μl PCR nucleotide mix (10 mM), 5 μl nucleic acid extracted from each specimen, and nuclease-free water to achieve a 25 μl final volume. Real-time PCR for each target was performed in the CFX96 Real-time PCR Detection System (Bio-Rad, Hercules, CA, USA) under the following conditions: predenaturation at 95°C for 2 min, followed by 45 cycles at 95°C for 15 s and 56°C for 15 s. The data were analyzed with the CFX Manager Software (version 2.1; Bio-Rad). […]

Pipeline specifications

Software tools Primer Express, CFX Manager
Application qPCR
Organisms Mycoplasma pneumoniae, Homo sapiens
Diseases Pneumonia, Mycoplasma