Detection of cytomegalovirus using 'boosted' nested PCR

As a part of a study of an outbreak of CMV infections in a neonatal care intensive care unit, a modified nested PCR was developed for detection of CMV DNA in clinical specimens. Standard nested PCR involves a critical step; passage of PCR products from the first reaction round to the second round. We have adapted a 'boosted' nested PCR which implies amplification in one single step, thus reducing the contamination problems. Nasopharyngeal aspirates and urine samples from patients with perinatal CMV infections, breast milk from some of their mothers, amniotic fluids, urine samples and lymphocytes from seropositive healthy adults were examined by PCR and culture. In the total of 614 of clinical specimens, the PCR test yielded positive results in 51 samples from 14 patients, whereas CMV was isolated in 25 samples from 11 cases only. All samples from healthy individuals were negative. CMV DNA was detected in all culture-positive samples, but all samples from healthy adults were negative. 29/68 culture negative specimens were positive by PCR. No cross-reactivity to other herpes viruses or to human DNA was observed. Our findings show a high sensitivity and a high specificity of the 'boosted' nested PCR. We conclude that the described PCR method can be used for the rapid detection of CMV in clinical specimens with a greatly reduced risk of contamination, and it has proved to be a very useful tool in diagnostic work.