Optimization of a polymerase chain reaction (PCR) for increasing its sensitivity to detect Chlamydia pneumoniae specific genome.
Being an intracellular parasite, Chlamydia pneumoniae disseminates to organs outside the respiratory tract and causes chronic diseases in human. Nucleic acid-based method such as polymerase chain reaction (PCR) as diagnostic test has greater sensitivity and specificity than conventional microbiological techniques. The PCR protocol consisting of touchdown technique to detect C. pneumoniae DNA using major outer membrane protein gene (MOMP) was carried out in our laboratory as described in reference paper, but analytical sensitivity reported in it was not reproducible. Hence, the PCR was optimized after modifications in annealing temperature and magnesium ion concentrations. First round PCR profile with annealing at 56 degrees C for 8 cycles followed by 32 cycles with annealing temperature maintained at 54 degrees C and second round profile modified with annealing temperature maintained at 49 degrees C had resulted in 3-fold increase in clinical sensitivity. The present work highlights the importance of optimization of PCR in laboratory settings.
Malathi
J
J
L & T Microbiology Research Center, Vision Research Foundation, Sankara Nethralaya, Chennai, Tamil Nadu.
Shyamala
G
G
Feeba
Varghese
V
Therese
K Lily
KL
Madhavan
H N
HN
D001425:Bacterial Outer Membrane Proteins / Q000235:genetics; D023521:Chlamydophila Infections / Q000175:diagnosis; D016993:Chlamydophila pneumoniae / Q000235:genetics* / Q000302:isolation & purification*; D004269:DNA, Bacterial / Q000032:analysis* / Q000235:genetics*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013696:Temperature
D001425:Bacterial Outer Membrane Proteins; D004269:DNA, Bacterial; C101031:major outer membrane protein, Chlamydia pneumoniae