Use of a commercial Real-time PCR (Argene) for the detection of CMV-DNA in Dried Saliva Swabs

Objectives. Ease of collection, handling and storage of Dry Saliva Swabs (DSS) collected with COPAN flocked swabs, together with inexpensive pre-PCR treatments could be key factors for the neonatal screening for congenital CMV (cCMV) infection. Previously we showed that a commercial Real-time PCR (Argene) could efficiently detect viral DNA on mock dry samples eluted in water. In the present study we verified if the same analysis was equally accurate on biological samples and so potentially suitable for screening. Methods. We tested DSS collected from 64 children: a) 14 (<1 week) with suspected cCMV, b) 45 (6-36 months) in follow-up because of cCMV, c) 5 (24-30 months) with postnatal CMV infection. Saliva was eluted in PCR grade water and tested by means of both “CMV R-gene” Real-time PCR (ARGENE) and a in-house nested PCR (our reference method). Samples were examined both after simple vortexing (DSS-V) and after vortexing followed by thermal shock (DSS-TS, 45’’ at 70°C, fast cooling and storage at -80°C). c) children had an extra DSS collected which was eluted in MEM and tested as described above. Results. DSS of 4/14 (29%) newborns and 35/45 (77%) children tested positive for CMV by both nested and RT- PCR, with 93, 95 and 100% of agreement in a), b) and c) subjects respectively. Sensitivity, specificity and concordance rates of RT-PCR with n-PCR were 96, 100 and 97% for DSS-V, and 94, 100 and 95% for DSS-TS. Real-time PCR didn’t detect CMV-DNA in all MEM DSS, while gave positive results for water DSS of 5 CMV c) subjects. Conclusion. Regardless treatment pre-PCR, adding molecular biology grade water to DSS was optimal for downstream testing, as commercial Real-time PCR (ARGENE). Being simple and cheap, the method proposed could be suitable for a neonatal screening for cCMV infection, especially only vortexing before PCR.