Reverse transcription polymerase chain reaction (RT-PCR) for diagnosis of respiratory syncytial virus infection in adults: Use of a single-tube ?hanging droplet? nested PCR

Rapid diagnosis of Respiratory Syncytial virus (RSV) infection is difficult in elderly persons due to the low quantities of virus shed. Therefore, reverse transcription-polymerase chain reaction (RT-PCR) was used to detect viral RNA in respiratory secretions. A single-tube nested RT-PCR that used primers from a conserved F gene sequence was developed using a "hanging droplet" to physically separate outer and inner primer pairs during the first round of the PCR reaction. This was accomplished by placing the inner primers in a 5 microL droplet on the underside on the reaction tube cap and mixing after the first round of PCR. As few as 0.05 pfu of virus could be detected and gave positive results with RSV strains that represented the major groups and subgroups of RSV grown in tissue culture. The nested PCR was approximately 100-fold more sensitive than standard single primer PCR reactions and equivalent to standard two-tube nested PCR. Viral RNA was detected in nasopharyngeal samples from 12 of 15 culture positive illnesses and in 5 of 17 culture-negative, seropositive illnesses despite specimen volumes less than 1 microL in some samples. The method was also positive in 14 of 25 elderly volunteers inoculated with a live attenuated RSV vaccine candidate, only one of whom was culture positive. Use of a nested RT-PCR significantly improves the ability to detect RSV in respiratory samples and should improve the ability to rapidly diagnose RSV infection in adults, especially in the elderly.