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]