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Comparative evaluation of published Cytomegalovirus (CMV) primers for rapid real-time PCR: which is the most sensitive?

¹ Laboratories, Al-Assad Hospital, Damascus University, PO Box 10769, Damascus, Syria;

² University of the Saarland

³ E-mail: vibgae{at}uniklinikum-saarland.de

Received February 25, 2009
Accepted March 16, 2009

Objective: Standardization of Cytomegalovirus (CMV) PCR is highly recommended. Since primers are essential for PCR, we evaluated all published CMV primers to identify the most sensitive single-round real-time PCR primer pairs. Methods: PubMed (1993-2004) was searched for original papers aiming at CMV PCR. Fifty-seven papers were identified revealing 82 different primer pairs. Of these, 17 primer sets were selected for empirical study as they were either used in real-time PCR or comparatively evaluated by conventional PCRs. After optimizing PCR conditions these primer sets were evaluated by real-time PCR using SYBR Green format. Analytical sensitivities were assessed by testing CMV strain AD169 reference standard. A BLAST was performed to identify mismatches with published sequences. Additionally, 60 clinical samples were tested with the three primers showing highest analytical sensitivity and best match to all CMV strains. Results: Three primer sets selected from glycoprotein B (UL55) gene region were the most sensitive using AD strain. However, two of them showed a considerable number of mismatches with clinical isolates in BLAST search. Instead, two other pairs from the lower matrix phosphoprotein (UL83) gene and DNA polymerase (UL54) gene showed fair sensitivity and no mismatches with clinical isolates in BLAST search. These three pairs were further tested with clinical samples indicating that the two primer sets from UL55 und UL54 were the most sensitive. Interestingly, the analytical sensitivity of the PCR was inversely correlated to the size of the PCR product. Conclusion: Two primer pairs are recommended for a standardized highly sensitive real-time PCR.