A multiplex, single-step PCR protocol for the detection of human cytomegalovirus (HCMV) DNA is described. The protocol amplifies regions of the viral LA and IE genes and employs elevated temperatures for both reagent mixing and primer annealing together with product detection by silver staining on polyacrylamide gels. This assay detects one to five HCMV genomes in clinical samples containing up to 100 ng of human DNA, a level of sensitivity equivalent to that of more complex assays involving either nested PCR or postamplification hybridization. As well as being of importance in clinical situations where high-sensitivity qualitative diagnosis is required, this assay is also applicable to the monitoring of HCMV infection in renal transplant recipients. Due to its multiplex format the assay provides quantitative information, in that samples from which a single target is amplified contain on average sevenfold fewer viral genomes per 10 6 leukocytes than those from which both targets are amplified. When weekly blood leukocyte DNA preparations from renal transplant patients were assayed, findings of three consecutive tests in which both HCMV targets were amplified were highly indicative of patients who had developed very high loads of HCMV (100% sensitivity, 88% specificity). We thus show that the same simple PCR assay which permits highly sensitive HCMV diagnosis can also be used for the efficient identification of transplant recipients at risk of clinically significant infection.The diagnostic application of PCR presents challenges in response to which sophisticated modifications of the basic technique are continually being developed. For example, to ensure very-high-sensitivity qualitative diagnosis (one to five target genes in a clinical sample), nested PCR or a postamplification hybridization protocol has become routine (1,9). Where viral load is an issue, quantification is normally undertaken with external competitive standards and/or limiting dilution and multiple replicates (7,22). In general, such quantitative assays do not exhibit the levels of sensitivity of nested PCR or hybridization and are thus not appropriate where initial qualitative diagnosis is required. The plethora of alternative PCR strategies and the diverse requirements of the assay have led to a situation in which the clinical application of PCR has become both complex and costly.We have investigated whether a single, simple, carefully applied PCR assay might be able to substitute both for the use of nested PCR and postamplification hybridization for highsensitivity qualitative diagnosis and for complex quantification protocols for viral load assessment of the same infection. The model with which we chose to work is human cytomegalovirus (HCMV). High-sensitivity detection of HCMV is required for screening of blood donors, evaluation of amniotic fluid in anti-HCMV immunoglobulin M (IgM)-positive pregnant women, and analysis of spinal fluid samples. On the other hand, quantitative viral load assessment is required for monitoring of immunosuppress...
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