Preliminary study of a Real-time PCR analysis for detecting CMV-DNA on Dried Saliva Swab (DSS)

Objectives: The identification of infected newborns at birth is necessary to prevent, or at least reduce, possible serious damages due to congenital CMV infection (cCMV). Easy and inexpensive collection, handling and processing of samples are important for implementation of neonatal screening. We previously obtained encouraging results in diagnosing CMV infection testing for CMV DNA by means of nested PCR saliva specimens collected as dry samples (DSS) on COPAN nylon-flocked swabs (sensitivity, specificity and concordance >95% versus n-PCR on liquid samples). The assay comprised: I) elution in MEM, II) vortexing/vortexing plus thermal shock, III) n-PCR amplification. Preliminary results of RT-PCR on some samples were disappointing. In the aim of verifying the possibility of substituting the cumbersome n-PCR with the more high-throughput RT-PCR in this study we tested as mock samples serial dilutions, either in MEM or in water, of cell-grown CMV. For comparison we tested serial dilutions of the Qiagen extract of the same viral suspension by means of the two amplification methods. Methods: A suspension of cell-grown CMV Towne containing 1E07 copies/ml of viral DNA was diluted on a 10-fold basis either in MEM or in molecular biology grade water. Specimens in each series were treated either with vortexing (about 15’’) or vortexing plus thermal shock (45’’ at 70°C, fast cooling and storage at -80°C). Viral DNA was amplified in each sample by means of both in-house n-PCR and commercial RT-PCR (“CMV R-gene”, ARGENE). A Qiagen extract (QIAamp DNA Mini kit) of the original viral suspension was analyzed following the above protocol. Results There was a perfect concordance between n-PCR results (water vs MEM). RT-PCR detected CMV DNA in water diluted samples down to 1E03 CMV DNA copies/ml, as did n-PCR, but it gave negative results in all MEM dilutions of the CMV suspension, even when viral DNA was purified by means of Qiagen. Conclusion Results showed that: a) the choice of eluent can be critical for the RT-PCR analysis as MEM in some way inhibited the amplification while it didn’t affect n-PCR amplification; b) pre-PCR treatment by means of vortexing, only or followed by thermal shock, is valid as we previously demonstrated. Confirmation of these results on clinical samples will indicate that DSS testing by means of RT-PCR, after adding water and vortexing, could be the optimal method for routine diagnostic activity and neonatal screening.