Optimization of human cytomegalovirus LightCycler real-time PCR.
BACKGROUND
Real-time PCR has been widely considered as a powerful tool for the evaluation of Human Cytomegalovirus (CMV) DNA kinetics. Successful PCR relies on optimization, which is an extremely demanding procedure. Nevertheless, certain values could be optimal for most primers in use.


METHODS
Seventeen CMV primer sets recommended in the literature were selected for optimization in terms of MgCl2 and primers concentrations as well as annealing temperature using the LightCycler instrument and SYBR Green I detection format. Optimal values were considered as those showing the lowest crossing point (Cp), the highest fluorescence intensity, the steepest sigmoid curve slope, and the absence of non-specific PCR products.


RESULTS
Optimal values for most studied primers were found to be 3 mM for MgCl2 concentration, 0.5 microM and 0.6 microM for primers concentration, and 55 degrees C for annealing temperature.


CONCLUSIONS
Adopting the resulting values for CMV-specific primers generally used in single-target real-time PCR assays with the same thermal cycler may guarantee their efficient performance minimizing cost and time needed for optimization.
Habbal
Wafa
W
Clinical Laboratories Department, Al-Assad Hospital, Damascus University, Damascus, Syria. wafahabbal@hotmail.com
Monem
Fawza
F
Gärtner
Barbara Christine
BC
D052160:Benzothiazoles; D003587:Cytomegalovirus / Q000235:genetics* / Q000302:isolation & purification; D003586:Cytomegalovirus Infections / Q000175:diagnosis / Q000821:virology*; D017931:DNA Primers; D004279:DNA, Viral / Q000032:analysis / Q000235:genetics; D003959:Diamines; D006801:Humans; D015636:Magnesium Chloride; D009930:Organic Chemicals; D016133:Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D011804:Quinolines; D013696:Temperature
D052160:Benzothiazoles; D017931:DNA Primers; D004279:DNA, Viral; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; D015636:Magnesium Chloride; C098022:SYBR Green I