Development of a quadriplex polymerase chain reaction for human cytomegalovirus detection

The development of a quadriplex PCR method with amplification of HCMV in a single-step procedure using primers taken from four different regions of th e viral genome is described. Different concentrations of dNTPs and MgCl2 we re assayed in order to optimize the constitution of the buffer for the mult iplex PCR. The specificity of the PCR was tested with 100ng, 10ng, and 1ng of genomic MRC-5 cell DNA infected with CMV in the presence of 10 mu g of u ninfected MRC-5 cell DNA. The sensitivity of the PCR was evaluated by the a mplification of various amounts (100ng, 10ng, 1ng, and 0.1ng) of genomic MR C-5 cell DNA infected with CMV. The specificity and sensitivity assays were performed for each pair of primers and for the combined four primer pairs in the multiplex PCR. CMV was consistently detected from 10ng of genomic MR C-5 cell DNA with each primer pair. When all four sets of primers were comb ined in a single reaction tube, the sensitivity of the assay was equivalent to 10ng of genomic MRC-5 cell DNA, whereas amplification from 1ng genomic MRC-5 cell DNA produced only a subset of the amplimers. By amplifying four target-sequences of HCMV simultaneously with minimum incubation time at eac h temperature, a quadriplex, highly sensitive PCR assay was performed. The use of four primer sets designed in different genomic regions of HCMV allow ed the detection of variants and achieved maximal sensitivity and specifici ty which are essential for a diagnostic utilization. J, Clin. Lab. Anal. 13 :99-105, 1999. (C) 1999 Wiley-Liss, Inc.