عنوان المقالة:Advanced and conventional molecular techniques in the diagnosis of Mycoplasma pneumoniae in patients with respiratory tract infection Advanced and conventional molecular techniques in the diagnosis of Mycoplasma pneumoniae in patients with respiratory tract infection
Background:-M. pneumoniae is an important human pathogen that produces communityacquired respiratory tract infection. Diagnosis of M. pneumoniae infection is challenging
and crucial for the timely initiation of the effective antibiotic therapy.
Objective: This study has been undertaken to detect M. pneumoniae in respiratory
samples (throat swabs, throat wash and sputum) in patients with respiratory tract infection
qualitatively by conventional polymerase chain reaction (PCR). Also, more advanced one,
real time PCR was used to determine mycoplasmal target gene qualitatively and
quantitatively.
Patients and methods: The study was performed on Seventy-five patients and thirty
healthy subjects as control. Human genomic DNA was extracted and M. pneumoniae
target gene (lipoprotein gene) was amplified using conventional PCR. Negative, positive
controls and internal controls were involved in each experimental run. The amplified
products were analyzed in 2% agarose gel and visualized using Red safe staining. In real
time PCR, specific primer and probe mix depending on TaqMan® principle was used to
detect P1 adhesion gene through FAM channel. A fluorogenic probe was included in the
same reaction mixture which consists of a DNA probe labeled with a 5`-dye and a 3`-
quencher. During PCR amplification. Data were analyzed using Smart-cycler software
and M. pneumoniae DNA copy number was estimated from the cross point threshold
relative to positive standard.
Results: Thirty five patients (45.5%) were positive by PCR and Thirty two (42.6 %) were
positive by Real-time PCR. The highest rate of infection by using two molecular methods
was of less than 20 years of age. The quantity of M. pneumoniae DNA target gene in
positive Real-time PCR was ranged between 10-2000copies/µl.
Conclusion: The study concluded that both of molecular techniques conventional and
real-time PCR are a rapid, reliable and ideal in diagnosis of M. pneumoniae using throat
swabs, throat wash and sputum samples
الأستاذ الدكتور مشتاق طالب صالح الندا | Dr. Mushtak T. S. Al-Neda | 6460