Mycoplasma Polymerase Chain Reaction (PCR) testing and sequencing
Respiratory tract specimens, including nasopharyngeal aspirates, sputum samples or gastric lavage were tested for M. pneumoniae by real-time PCR targeting the P1 adhesion protein gene.[16] Samples tested positive for M. pneumoniae were then subjected to real time PCR for the detection of A2063G mutation by the method previously described. The DNA extract prepared from the clinical specimens were analyzed by real time PCR to detect the point mutation from A to G at the position of 2063 (A2063G).(22) Isolates with A2063G mutation is considered to be a strain of genotypic resistant MRMP and negative strains are considered to be wild type (absent of genotypic resistance and assumed to be MSMP in the current study). For strains with no amplification or ambiguous results, the DNA extract is subjected to direct DNA sequencing of the domain V of the 23S rRNA gene to identify A2063G mutation associated with macrolide resistance using techniques previously described by the microbiological department of Tuen Mun Hospital. [16]